The term “subject” refers to any healthy or diseased animal, including any mammal, ruminant, canine, feline, or human.

Methanogens

The diversity of the ruminal methanogens is much smaller, and their diversity is much lower than that of rumen bacteria, with archaeal SSU rRNA only accounting for 6.8% of rumen total SSU rRNA. Archaea in the rumen is represented by <3.3% of the total rRNA (both 16S and 18S) therein. Representative family of methanogens includes Methanobacteriaceae. Rumen methanogens typically comprises 2-3% of the total microbial biomass in the rumen.

Representative genera of methanogens include Methanobrevibacter, Methanosphaera, Methanomicrobium, Methanobacterium, Methanocorpusulum, Methanosaeta, Methanoculleus, Methanosarcina, and Thermoplasmatales.

Certain species of ruminal methanogens have been isolated into pure cultures: Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanobrevibacter gottschalkii, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanoculleus olentangyi, and Methanosarcina barkeri. Additional species have been recently isolated, including Methanobrevibacter boviskoreani (isolated from the rumen of Korean native cattle), Methanobacterium beijingense (isolated from the rumen of goat), Methanoculleus marisnigri (isolated from the rumen of Indian crossbred cattle), Methanoculleus bourgensis (isolated from the rumen of Holstein cattle), and Methanosarcina mazei (isolated from the rumen of Korean Hanwoo cattle) (based on the RDP database). A Thermoplasmatales-like pyrrolysine-dependent archaeon BRNA1 was also isolated from bovine (GenBank access number: CP002916).

Collectively, 16S rRNA gene sequences from cultured methanogens only accounted for approximately 0.7% of the total archaeal sequences of rumen origin, and several taxa do not have a single cultured representative. Most of the isolates are members of the family Methanobacteriaceae. Compared to other anaerobic habitats where >100 species of methanogens of 28 genera have been isolated, the diversity and species richness of ruminal methanogens are quite low, reflecting the highly selective ruminal environment for methanogens. In addition, sequenced ruminal 16S rRNA gene clones shared >95% sequence similarity with that of Methanobrevibacter gottschalkii, Methanobrevibacter thaueri, Methanobrevibacter smithii and Methanosphaera stadtmanae, indicating that these species may be common ruminal methanogens.

Much of the ruminal methanogen diversity was characterized by 16S rRNA gene sequences. The RDP Release 11 (Update 3) contains 8,623 archaeal 16S rRNA gene sequences of rumen origin. These sequences were generated using the Sanger sequencing technology, which produces higher sequence accuracy than NGS technologies, in 96 separate studies including 48 unpublished studies. About 90% of these sequences were assigned to methanogens. These sequences were classified to 10 known genera, with Methanobrevibacter being represented by 63.2% of all the sequences followed by Methanosphaera (9.8%), Methanomicrobium (7.7%), and Methanobacterium (1.2%). The order Thermoplasmatales, which was previously referred to as the rumen cluster C (RCC) group, is represented by 7.4% of the total archaeal sequences.

Cell Surface Proteins of Methanogens

Provided herein is at least one cell surface protein or a fragment thereof (e.g., an antigenic fragment, e.g., a fragment comprising an epitope, e.g., a fragment comprising an extracellular domain or a portion thereof) of at least one methanogen that can be used in a vaccine composition, which can elicit immune response, antibody production, and antibody-mediated neutralization of the growth of methanogens and/or production of methane. Further provided herein are nucleic acid(s) encoding the at least one cell surface protein or a fragment thereof.

In certain aspects, the at least one cell surface protein or a fragment thereof (e.g., an antigenic fragment, e.g., a fragment comprising an epitope, e.g., a fragment comprising an extracellular domain or a portion thereof) of at least one methanogen is of a family Methanobacteriaceae.

In some embodiments, the at least one methanogen is of a genus selected from: Methanobrevibacter, Methanosphaera, Methanomicrobium, Methanobacterium, Methanocorpusulum, Methanosaeta, Methanoculleus, Methanosarcina, and Thermoplasmatales.

In some embodiments, the at least one methanogen comprises Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanoculleus olentangyi, Methanosarcina barkeri, Methanobrevibacter boviskoreani, Methanobacterium beijingense, Methanoculleus marisnigri, Methanoculleus bourgensis, Methanosarcina mazei, Thermoplasmatales archaeon BRNA1, Methanobrevibacter gottschalkii, Methanobrevibacter thaueri, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanococcoides burtonii, Methanolobus psychrophilus R15, Methanobacterium paludism, Methanohalobium evestigatum, Methanomethylovorans hollandica, Methanothrix soehngenii, Methanocaldococcus vulcanius, Methanosalsum zhilinae, Methanocorpusculum labreanum, Methanoregula formicica, Methanoculleus marisnigri, Methanocella arvoryzae, Methanoculleus bourgensis, Methanolacinia petrolearia, Methanospirillum hungatei, Methanoplanus limicola, Methanohalophilus mahii, Methanococcus aeolicus, Methanosphaerula palustris, Methanocaldococcus fervens, Methanocaldococcus jannaschii, Methanocaldococcus sp. FS406-22, Methanoregula boonei, Methanobrevibacter sp. AbM4, Methanobrevibacter ruminantium, Methanosphaera, Methanobacterium formicicum, Methanocaldococcus villosus, Methanosarcina barkeri, Methanobacterium lacus, Methanotorris igneus, Methanotorris formicicus, Methanocaldococcus infernus, Methanofollis liminatans, Methanothermococcus okinawensis, Methanobrevibacter smithii, Methanobrevibacter, Methanocella conradii, Methanothermococcus thermolithotrophicus, Methanococcus maripaludis, Methanococcus maripaludis, Methanococcus vannielii, Methanothermus fervidus, Methanosarcina acetivorans, Methanosarcina mazei, Methanosaeta harundinacea 6Ac, Methanococcus maripaludis, Methanococcus voltae, Methanolinea tarda, Methanolobus psychrophilus, Methanosaeta harundinacea, or any combination thereof.

In some embodiments, the at least one methanogen comprises Methanobrevibacter ruminantium. In some embodiments, the at least one methanogen comprises Methanobrevibacter ruminantium M1 (DSM 1093).

In preferred embodiments, the at least one methanogen comprises Methanobrevibacter gottschalkii. In some embodiments, the at least one methanogen comprises Methanobrevibacter gottschalkii DSM11977.

In some embodiments, a vaccine composition comprises a polypeptide or a fragment thereof (e.g., an antigenic fragment, an epitope) of at least one cell surface protein. In some embodiments, the polypeptide comprises the extracellular domain or a fragment thereof of at least one cell surface protein. In some embodiments, the polypeptide or a fragment thereof does not comprise the transmembrane and/or intracellular domains or a fragment thereof of the at least one cell surface protein.

In certain aspects, provided herein are vaccine compositions comprising at least one cell surface protein or a fragment thereof (e.g., an antigenic peptide fragment, e.g., a peptide fragment comprising an epitope, e.g., a peptide fragment comprising an extracellular domain or a portion thereof, e.g., a polypeptide fragment, e.g., peptide fragment) of at least one methanogen.

In some embodiments, a vaccine composition comprises at least one polypeptide and/or at least one peptide of at least one methanogen.

In some embodiments, the at least one polypeptide or a fragment thereof does not comprise a signal peptide.

In some embodiments, the at least one polypeptide or a fragment thereof does not comprise a transmembrane domain.

In some embodiments, the at least one polypeptide or a fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100% sequence identity to an amino acid sequence presented herein. In some embodiments, the at least one polypeptide or a fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100% sequence identity to an amino acid sequence set forth in any one of Tables C-F, 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, and 6A-6G, or a fragment thereof.

In some embodiments, the at least one polypeptide or a fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by at least one nucleic acid presented herein. In some embodiments, the at least one polypeptide or a fragment thereof is encoded by a nucleic acid comprising the nucleotide sequence set forth in any one of Tables C-F, 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, and 6A-6G, or a fragment thereof.

In some embodiments, the at least one polypeptide or a fragment thereof further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 heterologous amino acid residues that are not native to the cell surface protein of a methanogen.

In some embodiments, the at least one polypeptide or a fragment thereof further comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 heterologous amino acid residues that are not native to the cell surface protein of a methanogen.

In some embodiments, the at least one polypeptide or a fragment thereof further comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 heterologous amino acid residues that are not native to the cell surface protein of a methanogen.

In some embodiments, the heterologous amino acid residues comprise a heterologous signal peptide and/or a heterologous transmembrane domain.

In some embodiments, a vaccine composition comprises at least one fragment (e.g., at least one polypeptide fragment and/or a peptide fragment) of at least one cell surface protein of at least one methanogen. In some embodiments, a vaccine composition comprises at least one fragment (e.g., polypeptide fragment and/or a peptide fragment), which is not a full-length cell surface protein of at least one methanogen.

In some embodiments, the at least one fragment of at least one cell surface protein lacks at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues from its native full-length protein sequence.

In some embodiments, the at least one fragment of at least one cell surface protein lacks about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues from its native full-length protein sequence.

In some embodiments, the at least one fragment of at least one cell surface protein lacks no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues from its native full-length protein sequence.

In some embodiments, the at least one fragment of at least one cell surface protein lacks at least, about, or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues from the N-terminus of its native full-length protein sequence.

In some embodiments, the at least one fragment of at least one cell surface protein lacks at least, about, or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acid residues from the C-terminus of its native full-length protein sequence.

In preferred embodiments, the at least one fragment of at least one cell surface protein comprises one or more extracellular domain or a portion thereof. A person of ordinary skill in the art can readily predict an extracellular portion of any given sequence by using a suitable tool known in the art, e.g., TMbed or TMHMM.

In some embodiments, the at least one fragment of at least one cell surface protein lacks a native signal peptide.

In some embodiments, the at least one fragment of at least one cell surface protein lacks a native transmembrane domain.

In certain aspects, certain classes of methanogen cell surface proteins are particularly useful in generating an effective vaccine composition of the present disclosure (e.g., comprising at least one cell surface protein or a fragment thereof of at least one methanogen).

In some embodiments, the at least one cell surface protein comprises at least one of the following structures and/or functions: adhesin-like, ATP-processing, cell wall biosynthesis, cofactor biosynthesis, CRISPR (provides methanogens an immunity against viruses), energy metabolism, enzyme, fatty acid synthesis, general metabolism, membrane protein, metal-binding, methanogenesis, methanogenesis Mtr proteins, methanogenesis MtrE proteins, phage related, proteolysis, transcription regulation, ribosomal, substrate binding, transcription, transport, and a protein whose gene expression changes in response to lauric acid stress (see Table 6G below).

A person of ordinary skill in the art can determine the polypeptide sequences from the nucleic acid sequences, or determine the nucleic acid sequences from the polypeptide sequences presented herein or those known in the art.

The nucleic acid and amino acid sequence information for nucleic acid and polypeptide molecules useful in the present invention are well-known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI).

The representative cell surface antigens of various methanogens and their nucleic acid sequences and amino acid sequences are provided in the U.S. application Ser. No. 18/350,526 (e.g., Table 2A, Table 2B, Table 3, Table 17A, Table 19, Table 20, and Table 21), U.S. Application No. 63/359,978, or U.S. Application No. 63/524,513, the entire contents of each of which are incorporated herein by reference in its entirety.

Certain representative antigens and their amino acid and nucleic acid sequences are also presented in Tables C-F, TA, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, and 6A-6G of the present disclosure.

Sequences

As used herein, coding region refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas noncoding region refers to regions of a nucleotide sequence that are not translated into amino acids (e.g., 5′ and 3′ untranslated regions).

Complement [to] or complementary refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (base pairing) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. In some embodiments, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. In other embodiments, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination.

There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE

Alanine (Ala, A)
GCA, GCC, GCG, GCT

Arginine (Arg, R)
AGA, ACG, CGA, CGC, CGG, CGT

Asparagine (Asn, N)
AAC, AAT

Aspartic acid (Asp, D)
GAC, GAT

Cysteine (Cys, C)
TGC, TGT

Glutamic acid (Glu, E)
GAA, GAG

Glutamine (Gln, Q)
CAA, CAG

Glycine (Gly, G)
GGA, GGC, GGG, GGT

Histidine (His, H)
CAC, CAT

Isoleucine (Ile, I)
ATA, ATC, ATT

Leucine (Leu, L)
CTA, CTC, CTG, CTT, TTA, TTG

Lysine (Lys, K)
AAA, AAG

Methionine (Met, M)
ATG

Phenylalanine (Phe, F)
TTC, TTT

Proline (Pro, P)
CCA, CCC, CCG, CCT

Serine (Ser, S)
AGC, AGT, TCA, TCC, TCG, TCT

Threonine (Thr, T)
ACA, ACC, ACG, ACT

Tryptophan (Trp, W)
TGG

Tyrosine (Tyr, Y)
TAC, TAT

Valine (Val, V)
GTA, GTC, GTG, GTT

Termination signal (end)
TAA, TAG, TGA

An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

In making the changes in the amino sequences of polypeptide, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophane (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (<RTI 3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e. still obtain a biological functionally equivalent protein.

As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take various of the foregoing characteristics into consideration are well-known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

In view of the foregoing, the nucleotide sequence of a DNA or RNA encoding a cell surface antigen nucleic acid (or any portion thereof) can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for polypeptide amino acid sequences, corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

- Table 1A: Representative Antigens (full-length proteins) of Methanobrevibacter gottschalkii Set 1 (amino acid sequences (SEQ ID NOS 71070-71706) and nucleic acid sequences (SEQ ID NOS 71707-72343))
- Table 1B: Representative Antigens (full-length proteins) of Methanobrevibacter gottschalkii Set 1 (codon-optimized nucleic acid sequences) Left column discloses SEQ ID NOS 72344-72980 and right column discloses SEQ ID NOS 72981-73617.
- Table 2A: Representative Antigens (full-length proteins) of Methanobrevibacter gottschalkii Set 2 (amino acid sequences (SEQ ID NOS 73618-73993) and nucleic acid sequences (SEQ ID NOS 73994-74369))
- Table 2B: Representative Antigens (full-length proteins) of Methanobrevibacter gottschalkii Set 2 (codon-optimized nucleic acid sequences) Left column discloses SEQ ID NOS 74370-74745 and right column discloses SEQ ID NOS 74746-75121.
- Table 3A: Representative Antigens (full-length proteins) of Methanobrevibacter gottschalkii Set 3 (amino acid sequences (SEQ ID NOS 75122-75220) and nucleic acid sequences (SEQ ID NOS 75221-75319)
- Table 3B: Representative Antigens (full-length proteins) of Methanobrevibacter gottschalkii Set 3 (codon-optimized nucleic acid sequences) Left column discloses SEQ ID NOS 75320-75418 and right column discloses SEQ ID NOS 75419-75517.
- Table 4A: Representative Antigens (fragments) of Methanobrevibacter gottschalkii Set 4 (amino acid sequences (SEQ ID NOS 75518-76049) and nucleic acid sequences (SEQ ID NOS 76050-76581))
- Table 4B: Representative Antigens (fragments) of Methanobrevibacter gottschalkii Set 4 (codon-optimized nucleic acid sequences) Left column discloses SEQ ID NOS 76582-77112 and right column discloses SEQ ID NOS 77113-77643.
- Table 5A: Representative Antigens (fragments) of Methanobrevibacter gottschalkii Set 5 (amino acid sequences (SEQ ID NOS 77644-77967) and nucleic acid sequences (SEQ ID NOS 77968-78291))
- Table 5B: Representative Antigens (fragments) of Methanobrevibacter gottschalkii Set 5 (codon-optimized nucleic acid sequences) Left column discloses SEQ ID NOS 78292-78615 and right column discloses SEQ ID NOS 78616-78939.
- Table 6A: Representative Antigens (fragments) of Methanobrevibacter gottschalkii Set 6 (amino acid sequences (SEQ ID NOS 78940-79032) and nucleic acid sequences (SEQ ID NOS 79033-79125))
- Table 6B: Representative Antigens (fragments) of Methanobrevibacter gottschalkii Set 6 (codon-optimized nucleic acid sequences) Left column discloses SEQ ID NOS 79126-79218 and right column discloses SEQ ID NOS 79219-79311.
- Table 6C: Representative sequences of the cell surface proteins of Methanobrevibacter ruminantium M1 (GenBank: CP001719.1)
  - See SEQ ID NO. 1 to SEQ ID NO. 2217 for the native nucleic acid sequences. See SEQ ID NO. 2218 to SEQ ID NO. 4434 for the bovine codon-optimized nucleic acid sequences. See SEQ ID NO. 4435 to SEQ ID NO. 6651 for the amino acid sequences.
- Table 6D: Representative sequences of the cell surface proteins of Methanobrevibacter gottschalkii
  - See SEQ ID NO. 6652 to SEQ ID NO. 8451 for the native nucleic acid sequences. See SEQ ID NO. 8452 to SEQ ID NO. 10251 for the bovine codon-optimized nucleic acid sequences. See SEQ ID NO. 10252 to SEQ ID NO. 12051 for the amino acid sequences.
- Table 6E: Representative mtrE sequences of methanogens
  - See SEQ ID NO. 12100 to SEQ ID NO. 12147 for the nucleic acid sequences. See SEQ ID NO. 12052 to SEQ ID NO. 12099 for the amino acid sequences.

TABLE 6F

Genes and Bos taurus codon-optimized nucleic acid sequence of the genes

Bos taurus optimized nucleic acid

Name
Species
sequence (SEQ ID NO)

WP_004031278.1
Methanobacterium formicicum
SEQ ID NO: 16969

WP_012956721.1
Methanobrevibacter ruminantium
SEQ ID NO: 16970

WP_019264904.1
Methanobrevibacter smithii
SEQ ID NO: 16971

WP_016359091.1
Methanobrevibacter sp. AbM4
SEQ ID NO: 16972

WP_011033493.1
Methanosarcina mazei
SEQ ID NO: 16973

mru0143
Methanobrevibacter ruminantium
SEQ ID NO: 16974

mru0842
Methanobrevibacter ruminantium
SEQ ID NO: 16975

mru2048
Methanobrevibacter ruminantium
SEQ ID NO: 16976

mru1222
Methanobrevibacter ruminantium
SEQ ID NO: 16977

mru2047
Methanobrevibacter ruminantium
SEQ ID NO: 16978

*The vaccine compositions of the present disclosure may comprise a polypeptides or a fragment thereof encoded by any one of the nucleic acids above.

TABLE 6G

Exemplary structural/functional groups of representative cell surface proteins

This table contains SEQ ID NOs of amino acid sequences of proteins; or SEQ ID NOs of nucleic acid

sequences that encode the proteins that belong to the indicated structural and/or functional groups.

(1) Adhesin-like proteins (SEQ ID NOs)

4438
4449
4453
4454
4465
4472
4482
4498
4506
4510
4511
4513
4516

4517
4518
4519
4520
4524
4577
4594
4618
4656
4679
4689
4747

4751
4752
4760
4761
4765
4772
4850
4851
4852
4883
4884
4922

4926
5137
5156
5160
5205
5208
5244
5257
5261
5272
5275
5276

5329
5335
5395
5396
5403
5409
5410
5411
5412
5509
5510
5521

5556
5563
5639
5642
5654
5679
5753
5774
5790
5808
5818
5819

5848
5849
5856
5897
5929
5930
5931
5932
5938
5945
6036
6038

6082
6083
6158
6230
6387
6403
6428
6475
6480
6481
6484
6485

6486
6487
6491
6522
6555
6556
6566
6579
6610
6621
6628
10279

10280
10281
10282
10336
10337
10341
10342
10357
10369
10514
10522
10539

10540
10591
10613
10614
10616
10617
10618
10627
10637
10638
10639
10640

10665
10671
10677
10697
10723
10731
10810
10935
11091
11311
11331
11528

11594
11595
11596
11646
11902
11903
11904
11909
11961
11987
16974
16975

16976
16977
16774
16777
16780
16784
16785
16786
16787
16789
16791
16792

16793
16794
16795
16797
16799
16800
16801
16802
16803
16804
16805
16806

16807
16808
16857
16861
16863
16864
16865
16868
16869
16871
16872
53218

53219
53220
53221
53222
53223
53224
53225
53226
53227
53228
53229
53230

53231
53232
53233
53234
53235
53236
53237
53238
53239
53240
53241
53242

53250
53251
53252
53253
53254
53255
53256
53257
53258
53259
53260
53261

53262
53263
53264
53265
53266
53267
53268
53269
53270
53271
53272
53273

53274
53275
53276
53277
53278
53279
53280
53281
53282
53283
53284
53285

53286
53287
53288
53289
53290
53291
53292
53293
53294
53295
53296
53297

53298
53299
53300
53301
53302
53303
53304
53305
53306
53307
53308
53309

53310
53311
53312
53313
53314
53315
53316
53317
53318
53319
53320
53321

53322
53323
53324
53325
53326
53327
53328
53329
53330
53331
53332
53333

53334
53335
53336
53337
53338
53339
53340
53341
53342
53343
53344
53345

53346
53347
53348
53349
53350
53351
53352
53353
53354
53355
53356
53357

53358
53359
53360
53361
53362
53363
53364
53365
53366
53367
53368
53370

53371
53372
53373
53374
53375
53376
53377
53378
53379
53380
53381
53382

53383
53384
53385
53386
53387
53388
53389
53390
53391
53392
53393
53394

53395
53396
53397
53398
53399
53400
53401
53402
53403
53406
53407
53408

53409
53410
53411
53412
53413
53414
53415
53416
53417
53418
53419
53420

53421
53422
53423
53424
53425
53426
53427
53428
53429
53430
53431
53432

53433
53434
53435
53436
53437
53438
53439
53440
53441
53442
53443
53444

53445
53446
53447
53448
53449
53450
53451
53452
53453
53454
53455
53456

53457
53458
53459
53460
53461
53462
53463
53464
53465
53466
53467
53468

53477
53478
53479
53480
53481
53482
53483
53484
53485
53486
53487
53488

53489
53490
53491
53492
53493
53494
53495
53496
53497
53498
53499
53500

53501
53502
53503
53504
53505
53508
53509
53510
53511
53512
53522
53523

53524
53525
53526
53527
53528
53529
53530
53531
53532
53533
53534
53535

53536
53537
53538
53539
53540
53541
53542
53546
53547
53548
53549
53550

53551
53552
53553
53554
53555
53556
53557
53558
53559
53560
53561
53562

53563
53564
53571
53572
53573
53574
53575
53576
53577
53578
53579
53580

53581
53582
53583
53584
53585
53586
53587
53594
53595
53596
53597
53598

53599
53603
53604
53605
53606
53607
53608
53609
53610
53611
53612
53613

53614
53615
53616
53617
53618
53619
53620
53621
53622
53623
53624
53625

53626
53627
53628
53629
53630
53631
53632
53633
53634
53635
53636
53637

53638
53639
53640
53641
53642
53643
53644
53645
53646
53647
53648
53649

53657
53658
53659
53660
53661
53662
53663
53664
53665
53666
53667
53668

53669
53670
53671
53672
53673
53674
53675
53684
53685
53694
53695
53696

53697
53698
53699
53700
53701
53702
53703
53704
53705
53706
53707
53708

53709
53710
53711
53712
53713
53714
53715
53716
53717
53718
53719
53720

53721
53722
53723
53724
53725
53726
53727
53728
53729
53730
53731
53732

53733
53734
53735
53736
53737
53738
53739
53740
53741
53742
53743
53744

53745
53746
53747
53748
53749
53750
53751
53752
53753
53754
53755
53756

53757
53758
53759
53760
53761
53762
53763
53764
53765
53766
53767
53768

53769
53770
53771
53772
53773
53774
53775
53776
53777
53778
53779
53780

53781
53782
53783
53784
53785
53786
53787
53788
53789
53790
53791
53792

53793
53794
53796
53797
53798
53799
53800
53801
53802
53803
53804
53805

53806
53807
53808
53809
53810
53811
53812
53813
53820
53821
53822
53823

53824
53825
53826
53827
53828
53829
53830
53831
53832
53833
53834
53835

53836
53837
53838
53839
53840
53841
53842
53843
53844
53845
53846
53847

53848
53849
53850
53851
53852
53853
53854
53855
53856
53857
53858
53859

53860
53861
53862
53866
53867
53868
53869
53870
53871
53872
53873
53874

53875
53876
53877
53878
53879
53880
53906
53907
53908
53909
53910
53911

53916
53917
53918
53919
53920
53921
53922
53923
53924
53925
53926
53927

53928
53929
53930
53931
53932
53934
53935
53936
53937
53938
53939
53940

53941
53942
53943
53944
53945
53946
53947
53948
53949
53950
53951
53952

53953
53954
53955
53956
53957
53958
53959
53960
53961
53962
53963
53964

53965
53966
53967
53968
53969
53970
53972
53973
53974
53976
53977
53978

53979
53980
53981
53982
53983
53984
53986
53987
53988
53989
53990
54010

54011
54034
54035
54036
54037
54038
54039
54040
54041
54042
54043
54044

54045
54046
54047
54048
54049
54050
54051
54052
54053
54054
54055
54056

54057
54058
54059
54060
54061
54062
54063
54064
54065
54066
54067
54068

54069
54070
54071
54072
54075
54076
54077
54078
54079
54081
54082
54093

54099
54100
54110
54111
54112
54113
54114
54115
54116
54117
54118
54122

54132
54133
54134
54135
54136
54137
54138
54139
54140
54149
54150
54151

54152
54155
54156
54188
54189
54190
54191
54192
54193
54194
54195
54196

54197
54198
54199
54200
54201
54202
54203
54204
54205
54222
54223
54224

54225
54242
54243
54247
54248
54249
54250
54251
54252
54253
54262
54263

54264
54265
54266
54267
54268
54269
54270
54271
54272
54283
54284
54285

54286
54287
54288
54289
54290
54291
54292
54293
54296
54297
54313
54314

54315
54316
54318
54328
54329
54330
54331
54332
54333
54334
54335
54336

54337
54353
54371
54372
54373
54374
54375
54376
54377
54378
54387
54392

54393
54417
54462
54463
54464
54465
54466
54467
54468
54469
54472
54473

54474
54475
54493
54494
54495
54496
54497
54498
54499
54500
54501
54502

54503
54504
54505
54506
54519
54520
54521
54526
54527
54542
54543
54544

54545
54646
54647
54648
54649
54650
54651
54652
54653
54654
54655
54656

54657
54699
54707
54708
54709
54710
54711
54712
54713
54716
54732
54733

54734
54735
54736
54737
54738
54739
54740
54748
54749
54750
54751
54755

54756
54757
54758
54773
54774
54775
54826
54827
54828
54829
54830
54831

54832
54833
54834
54835
54836
54837
54838
54854
54887
55009
55075
55076

55077
55078
55079
55080
55081
55082
55083
55084
55088
55089
55096
55097

55102
55103
55104
55105
55106
55114
55115
55116
55117
55118
55125
55126

55127
55128
55129
55130
55131
55142
55161
55162
55166
55167
55168
55169

55188
55189
55190
55191
55258
55259
55260
55261
55285
55286
55287
55288

55290
55291
55329
55370
55375
55376
55462
55463
55464
55465
55466
55467

55468
55469
55470
55471
55472
55473
55725
55726
55727
55732
55733
55797

55798
55799
55800
55805
55810
55811
55812
55813
55843
55844
55894
55895

55896
55897
55898
55899
55970
55971
56160
56161
56162
56163
56164
56165

56166
56180
56471
56472
56473
56552
56553
56554
56555
56591
56640
56641

56668
56669
56704
56705
56747
56748
56757
56758
56763
56764
56772
56773

56774
56775
56776
56777
56778
56779
57110
57111
57112
57144
57145
57153

57154
57181
57199
57200
57201
57202
57206
57221
57225
57250
57251
57252

57265
57266
57318
57319
57343
57344
57345
57375
57376
57381
57382
57383

57384
57407
57421
57422
57428
57429
57448
57449
57483
57484
57485
57486

57529
57530
57546
57619
57641
57642
57643
57644
57645
57689
57696
57697

57814
57815
57841
57842
57843
57844
57848
57922
58229
58230
58231
58232

58266
58267
58279
58280
58281
58282
58283
58284
58311
58312
58313
58314

58315
58351
58352
58353
58354
58355
58356
58357
58358
58578
58579
58703

58704
58705
58706
58707
58708
58709
58740
58741
58742
58743
58746
58747

58752
58761
58762
58768
58769
58841
58842
58940
58941
59909
59910
60356

60357
60445
60446
60468
60569
60663
60664
60676
60677
60751
60754
60755

60784
60785
60786
60787
60788
60789
60790
60791
60792
60814
60815
60940

60941
60942
60943
60944
60945
60946
60947
60997
60998
60999
61000
61001

61002
61006
61083
61093
61094
61095
61131
61136
61137
61138
61139
61140

61141
61142
61143
61162
61378
61379
61380
61492
61493
61494
61633
61679

61680
61681
61735
61736
61737
61738
61778
61779
61780
61911
61912
61913

61914
61915
61959
62019
62054
62055
62060
62061
62076
62077
62078
62079

62080
62081
62082
62083
62084
62103
62104
62107
62108
62137
62261
62262

62263
62270
62292
62293
62294
62295
62311
62332
62345
62384
62385
62540

62541
62542
62624
62625
62626
62627
62645
62646
62647
62648
62649
62650

62651
62652
62916
62917
62942
62957
63054
63066
63203
63204
63205
63206

63207
63211
63212
63213
63214
63215
63497
63551
63552
63553
63554
53555

63556
63557
63558
63632
63703
63706
63724
63735
63899
64177
64178
64179

64180
6418
64182
64183
64184
64200
64216
64252
64253
64254
64296
64297

64298
64299
64314
64337
64338
64339
64340
64341
64342
64343
64390
64471

64515
64516
64517
64528
64529
64535
64536
64537
64569
64570
64633
64634

64635
64636
64715
64716
64723
64724
64732
64733
64734
64739
64740
64741

64742
64755
64756
64776
64777
64778
64779
64780
64952
64953
64985
65050

65051
65127
65170
65320
65323
65506
65507
65508
65509
65510
65523
65531

65571
65586
65618
65642
65643
65664
65736
65810
65811
65812
65833
65948

65949
65950
65951
65952
66006
66144
66145
66393
66394
66396
66438
66439

66458
66538
66656
66670
66687
66716
66839
66892
66907
66908
66909
66952

67160
67161
67221
67281
67520
67774
67775
67776
67880
67924
68140
68141

68284
68402
68409
68421
68436
68476
68482
68571
68648
68649
68650
68690

68695
68698
68722
68723
68741
68742
68743
68744
68911
68989
69071
69116

69124
69160
69161
69162
69201
69202
69203
69204
69233
69278
69299
69300

69301
69333
69343
69344
69425
69464
69508
69523
69524
69574
69577
69585

69603
69613
69734
69839
69896
69897
69958
70062
70078
70081
70107
70114

70142
70143
70145
70154
70162
70204
70214
70231
70241
70249
70256
70289

70316
70320
70326
70334
70370
70402
70444
70446
70475
70500
70509
70511

70519
70521
70524
70537
70538
70539
70543
70546
70548
70558
70559
70569

70576
70585
70586
70587
70590
70591
70599
70601
70603
70613
70614
70616

70617
70619
70625
70631
70646
70648
70649
70658
70659
70666
70667
70668

70669
70677
70679
70692
70693
70696
70707
70731
70737
70741
70752
70757

70768
70796
70807
70808
70811
70819
70820
70821
70829
70837
70841
70843

70851
70852
70860
70865
70879
70880
70881
70882
70890
70891
70895
70896

70905
70906
70907
70918
70920
70926
70931
70951
70993
71009
71014
71015

71017
71020
71022
71031
71035
71038
71055
71062
71082
71083
71085
71104

71105
71106
71107
71116
71121
71182
71186
71195
71196
71214
71217
71218

71219
71220
71221
71226
71232
71233
71234
71235
71248
71251
71253
71255

71262
71265
71300
71343
71383
71461
71470
71531
71560
71561
71562
71580

71653
71654
71655
71658
71674
71682
75524
75525
75527
75544
75545
75546

75547
75548
75549
75550
75551
75552
75553
75554
75555
75566
75571
75572

75615
75620
75626
75627
75637
75638
75639
75640
75641
75642
75643
75644

75645
75646
75654
75658
75659
75660
75661
75679
75681
75682
75683
75695

75698
75725
75726
75727
75728
75729
75730
75731
75766
75798
75852
75858

75859
75860
75861
75862
75863
75864
75865
75866
75867
75868
75869
75870

75871
75914
75937
75938
75939
75948
76002
76003
76004
76006
76019
76020

76021
76022
76023
76024
76025
76026
76030

(2) ATP-processing proteins (SEQ ID NOs)

5132
5619
6156
6234
6293
6369
10378
10839
10840
11388
11452
53881
53882

53883
53884
53933
53991
53992
53993
53994
53995
54005
54006
54007
54008

54009
54073
54074
54172
54173
54174
54212
54213
54238
54239
54277
54278

54279
54280
54281
54282
54294
54295
54298
54299
54302
54303
54306
54320

54321
54324
54325
54369
54370
54388
54389
54390
54391
54394
54395
54396

54397
54398
54399
54400
54401
54402
54403
54404
54405
54406
54407
54408

54409
54410
54411
54412
54413
54414
54415
54416
54418
54419
54420
54421

54422
54423
54424
54425
54426
54427
54428
54429
54430
54431
54440
54442

54443
54536
54537
54844
54845
55542
55559
55560
55561
55562
55567
55577

55615
55616
55705
55724
55791
55793
55795
55796
55804
55806
55807
55809

56212
56213
56585
56586
56725
56726
57045
57046
57196
57207
57208
57363

57364
58862
58970
59114
59115
60701
61682
61994
61997
61998
61999
62000

62001
62002
62003
62008
62009
62010
62014
62015
62016
62017
62018
62022

62117
62279
62280
62281
62282
62308
62318
62333
62614
62615
62616
62617

63506
66812
66813
66814
67056
67761
68729
68731
69049
69050
69051
69081

69082
69168
69356
69595
69608
69614
69640
69659
69663
69665
69671
69715

69718
69726
69732
69735
69736
69741
69745
69747
69748
69781
69794
69960

70169
70180
70185
70350
70508
70609
70726
70761
70970
71047
71081
71095

71097
71125
71143
71149
71150
71210
71237
71245
71286
71305
71306
71309

71310
71386
71389
71394
71403
71409
71445
71457
71476
71493
71504
71505

71507
71508
71583
71584
71606
71608
71684
71686
71695
75573
75732
75733

75889

(3) Cell wall biosynthesis proteins (SEQ ID NOs)

16876
69386
69387
70804

(4) Cofactor biosynthesis proteins (SEQ ID NOs)

55348
55349
55350
56938
56939
56940
57423
57424
58518
58519
58520
58521
65125

68706
71029

(5) CRISPR proteins (provides methanogens an immunity against viruses) (SEQ ID NOs)

70636
71045

(6) Energy metabolism proteins (SEQ ID NOs)

58756
58757
61126
61127
61128

(7) Enzymes (SEQ ID NOs)

4617
4774
4986
6013
6559
11013
11468
11485
11506
11773
11845
16771
53045

53046
53047
53048
53049
53050
53051
53052
53053
53054
53055
53056
53057

53058
53059
53060
53061
53062
53063
53064
53065
53066
53067
53068
53069

53070
53071
53072
53073
53074
53075
53076
53077
53078
53079
53080
53081

53082
53083
53084
53085
53086
53087
53088
53089
53090
53091
53092
53093

53094
53095
53096
53097
53098
53099
53100
53101
53102
53103
53104
53105

53106
53107
53108
53109
53110
53111
53112
53113
53114
53115
53116
53117

53118
53119
53120
53121
53122
53123
53124
53125
53126
53127
53128
53129

53130
53131
53132
53133
53134
53135
53136
53137
53138
53139
53140
53141

53142
53143
53144
53145
53146
53147
53148
53149
53150
53151
53152
53153

53154
53155
53156
53157
53158
53159
53160
53161
53162
53163
53164
53165

53166
53167
53168
53169
53170
53171
53172
53173
53174
53175
53176
53177

53178
53179
53180
53181
53182
53183
53184
53185
53186
53187
53188
53189

53190
53191
53192
53193
53194
53195
53196
53197
53198
53199
53200
53201

53202
53203
53204
53205
53206
53207
53208
53209
53210
53211
53212
53213

53214
53215
53216
53217
53243
53244
53245
53246
53247
53248
53249
53404

54379
54380
54441
54517
54518
54798
54799
54800
54801
54807
54808
54809

54810
55017
55018
55019
55257
55434
55694
55714
55738
55767
55772
55781

55782
55792
55794
55808
56331
56332
56333
56606
56734
56735
56744
56745

56746
57077
57078
57120
57121
57122
57173
57174
57175
57176
57182
57183

57184
57332
57333
57430
57431
57432
57433
57434
57439
57440
57560
57561

57599
57600
57822
57823
57870
57871
58169
58170
58316
58436
58437
58438

58439
58588
58712
58713
58714
58720
58721
58729
58730
58731
58855
58893

58894
58901
58902
58909
58910
58959
58960
59122
59123
59124
59958
59959

59960
60337
60338
60339
60361
60362
60363
60364
60507
60508
60509
60510

60511
60512
60629
60630
60631
60632
60633
60641
60642
60643
60644
60645

60646
60647
60648
60649
60650
60651
60652
60653
60654
60655
60656
60657

60658
60675
60717
60718
60719
60763
60764
60765
60766
60896
60897
60898

61029
61030
61201
61202
61203
61204
61205
61206
61207
61208
61256
61257

61258
61259
61284
61285
61286
61287
61288
61289
61295
61383
61384
61385

61386
61387
61388
61389
61390
61391
61392
61393
61394
61395
61396
61397

61398
61399
61400
61401
61402
61403
61404
61409
61410
61411
61412
61413

61414
61417
61418
61419
61420
61421
61422
61423
61424
61425
61426
61427

61428
61429
61430
61431
61432
61433
61434
61435
61436
61437
61438
61439

61440
61444
61445
61446
61447
61448
61449
61709
61710
61711
61752
61753

61754
61830
61831
61832
61855
61856
61924
62056
62057
62085
62089
62099

62110
62133
62148
62208
62209
62210
62211
62212
62228
62389
62390
62391

62392
62393
62394
62395
62396
62405
62406
62420
62421
62422
62463
62464

62465
62481
62482
62483
62484
62493
62494
62495
62496
62497
62498
62499

62509
62510
62511
62512
62513
62514
62518
62519
62520
62521
62522
62523

62524
62525
62526
62527
62535
62536
62537
62547
62548
62549
62557
62558

62559
62565
62566
62567
62571
62572
62573
62574
62575
62576
62577
62578

62579
62590
62591
62641
62642
62673
62674
62675
62676
62677
63385
63502

63564
63565
63965
64067
64278
64563
64589
64604
64605
64606
64607
64608

64626
64627
64628
64629
64637
64651
64660
64679
64682
64687
64688
64693

64750
64784
64785
64786
64920
64921
65076
65237
65449
65479
65480
65481

65499
65532
65537
65589
65740
65775
65776
65992
65993
65994
65995
65996

65997
65998
66200
66201
66202
66203
66204
66205
66206
66207
66208
66229

66230
66231
66359
66477
66523
66536
66561
67804
67884
67885
67886
67910

67911
68392
68393
68394
68395
68396
68412
68413
68414
68661
68662
68664

68665
68701
68702
68719
68720
68762
68763
68825
68843
69060
69061
69167

69210
69217
69367
69368
69369
69398
69422
69423
69514
69556
69570
69600

69616
69617
69618
69623
69638
69639
69677
69708
69723
69956
69972
70038

70128
70171
70194
70284
70317
70318
70351
70354
70412
70413
70421
70466

70608
70633
70650
70651
70685
70686
70714
70733
70748
70758
70765
70925

71026
71036
71046
71058
71432
71078
71094
71117
71119
71131
71167
71177

71187
71193
71197
71200
71205
71207
71212
71215
71229
71230
71246
71250

71254
71258
71284
71293
71296
71331
71333
71339
71347
71350
71356
71373

71375
71376
71378
71379
71385
71410
71417
71431
71432
71434
71435
71444

71447
71448
71451
71458
71477
71509
71517
71526
71530
71538
71539
71556

71566
71567
71587
71602
71603
71609
71611
71623
71624
71625
71629
71635

71637
71639
71642
71643
71644
71656
71678
71702
71703
75778
75779
75780

75781
75782
75783
75784
75897
75905
75906
75907
75908
75909
75910
75985

75998

(8) Fatty acid synthesis (SEQ ID NOs)

68659
68660

(9) General metabolism (SEQ ID NOs)

5480
5955
11908
16866
16867
16874
53369
53405
54450
54451
54452
54453
54454

54888
54889
54890
54891
54892
55649
56541
56542
56550
56551
56556
56557

56558
56559
56560
56561
56562
56563
56749
56750
56759
56760
56761
56762

56801
56802
56826
56827
57308
57309
57902
57903
58386
58387
58388
58389

58413
58414
58415
58416
61231
61232
61233
62215
62216
62217
62223
62224

62963
63030
63031
63582
63916
63917
63918
63919
63920
63972
63973
63974

64450
64451
65272
65273
65539
65722
65723
65724
65766
65767
65786
66530

66616
66655
67200
67201
67253
67934
67946
67947
68134
68135
68306
68434

68435
68669
68670
68673
68682
68683
68709
68759
68784
68787
68840
68841

69175
69176
69226
69586
69591
69664
69672
69690
70033
70295
70310
70381

70485
70486
70610
70675
70921
71103
71136
71168
71192
71202
71324
71328

71341
71374
71388
71405
71419
71422
71466
71471
71485
71573
71613
71657

71688
71691
76005

(10) Membrane proteins (SEQ ID NOs)

4452
4478
4481
4483
4500
4505
4515
4529
4547
4580
4581
4584
4601

4606
4621
4627
4630
4657
4658
4659
4664
4665
4667
4668
4671

4682
4712
4713
4740
4749
4762
4764
4790
4791
4795
4809
4810

4811
4812
4842
4845
4848
4857
4861
4866
4879
4893
4903
4921

4923
4924
4928
4932
4942
4943
4945
4948
4954
4957
4975
4976

4978
4980
5000
5014
5029
5030
5031
5035
5037
5047
5055
5058

5064
5067
5072
5096
5103
5104
5116
5119
5126
5127
5139
5149

5150
5151
5153
5159
5169
5178
5179
5180
5188
5192
5193
5202

5235
5238
5264
5265
5266
5271
5273
5310
5334
5363
5373
5397

5400
5401
5408
5422
5433
5454
5455
5460
5463
5474
5487
5492

5513
5517
5529
5534
5549
5557
5576
5624
5626
5627
5628
5638

5645
5651
5664
5665
5666
5721
5734
5754
5762
5763
5768
5775

5807
5817
5855
5874
5883
5912
5925
5966
5967
5974
5982
5987

6010
6017
6030
6037
6039
6067
6068
6073
6074
6085
6086
6089

6125
6126
6170
6191
6217
6226
6227
6235
6264
6265
6266
6290

6314
6316
6321
6324
6332
6378
6382
6384
6401
6422
6423
6447

6455
6476
6482
6488
6489
6497
6538
6540
6550
6562
6568
6575

6578
6588
6604
6607
6612
6616
6617
6634
10272
10291
10306
10322

10325
10329
10347
10349
10350
10353
10368
10373
10383
10385
10391
10392

10408
10432
10438
10439
10443
10460
10462
10465
10497
10501
10502
10503

10504
10510
10512
10513
10515
10517
10538
10543
10545
10559
10564
10573

10586
10626
10641
10653
10667
10703
10721
10727
10734
10735
10736
10743

10744
10745
10752
10770
10792
10793
10797
10799
10848
10939
10940
10941

10944
10945
10978
10983
10984
11021
11024
11041
11042
11056
11057
11115

11126
11137
11138
11146
11158
11165
11177
11183
11186
11199
11217
11218

11219
11233
11234
11238
11247
11248
11268
11272
11276
11277
11301
11305

11312
11322
11326
11338
11351
11357
11358
11365
11385
11410
11428
11432

11434
11457
11474
11475
11476
11482
11537
11561
11565
11597
11599
11600

11622
11623
11631
11636
11637
11639
11643
11647
11679
11682
11683
11685

11691
11692
11701
11703
11709
11724
11752
11753
11763
11766
11778
11779

11784
11803
11874
11914
11916
11931
11932
11935
11937
11944
11954
11962

11963
11967
11976
12005
12010
12020
12021
12031
12049
53513
53514
53515

53516
53517
53518
53519
53520
53521
53601
53602
53650
53651
53652
53653

53654
53655
53656
53676
53677
53678
53679
53680
53681
53682
53683
53686

53687
53688
53689
53690
53691
53692
53693
53795
53912
53913
53914
53915

53971
53975
54014
54015
54016
54017
54080
54083
54084
54085
54086
54087

54088
54089
54090
54091
54092
54101
54102
54103
54104
54105
54106
54107

54123
54124
54125
54126
54127
54128
54129
54130
54131
54305
54447
54448

54449
54470
54471
54491
54492
54625
54626
54627
54660
54661
54662
54677

54693
54694
54746
54747
54761
54762
54763
54764
54765
54766
54767
54768

54769
54770
54771
54772
54776
54781
54782
54783
54784
54879
54880
54881

55015
55016
55065
55074
55090
55100
55101
55110
55111
55112
55113
55119

55120
55121
55122
55123
55124
55133
55134
55135
55136
55137
55138
55139

55140
55141
55143
55144
55145
55146
55147
55148
55149
55150
55151
55152

55153
55154
55155
55156
55157
55158
55159
55160
55163
55164
55165
55170

55171
55172
55173
55175
55176
55177
55197
55198
55199
55200
55201
55202

55205
55206
55207
55208
55209
55210
55211
55212
55213
55214
55215
55216

55217
55218
55219
55220
55221
55222
55223
55224
55225
55226
55227
55228

55229
55230
55231
55232
55233
55235
55236
55237
55239
55240
55241
55242

55243
55244
55245
55246
55247
55248
55249
55250
55251
55252
55253
55254

55255
55256
55262
55263
55264
55265
55266
55267
55268
55269
55270
55271

55272
55273
55274
55275
55276
55277
55278
55280
55281
55282
55283
55284

55293
55294
55295
55297
55298
55299
55300
55301
55303
55304
55305
55309

55310
55312
55313
55314
55315
55316
55317
55318
55319
55320
55321
55322

55323
55324
55325
55326
55327
55328
55330
55331
55332
55333
55334
55335

55336
55337
55338
55339
55340
55341
55342
55343
55344
55345
55346
55347

55351
55352
55353
55354
55355
55362
55363
55364
55365
55366
55367
55368

55377
55378
55379
55380
55381
55382
55388
55391
55392
55393
55394
55395

55396
55397
55407
55408
55409
55412
55418
55419
55420
55422
55424
55425

55427
55428
55429
55430
55431
55432
55433
55435
55436
55437
55438
55439

55440
55441
55442
55443
55444
55451
55452
55453
55454
55455
55456
55457

55458
55460
55461
55474
55475
55476
55478
55479
55483
55484
55485
55492

55493
55496
55497
55505
55506
55507
55508
55509
55510
55511
55513
55514

55515
55516
55521
55522
55523
55524
55525
55529
55530
55531
55532
55533

55534
55535
55536
55538
55539
55540
55541
55543
55544
55545
55548
55549

55550
55551
55552
55553
55554
55555
55556
55557
55563
55564
55568
55569

55570
55571
55572
55573
55574
55575
55576
55578
55579
55580
55581
55582

55583
55584
55587
55588
55589
55593
55594
55595
55597
55598
55600
55601

55602
55603
55604
55605
55606
55607
55608
55609
55610
55611
55612
55613

55614
55617
55618
55619
55620
55621
55622
55623
55625
55626
55627
55628

55635
55639
55640
55641
55642
55643
55644
55645
55646
55647
55648
55652

55653
55656
55657
55658
55659
55660
55661
55662
55663
55664
55665
55666

55667
55668
55669
55670
55671
55672
55673
55674
55675
55676
55677
55678

55679
55680
55681
55682
55683
55684
55685
55686
55687
55691
55692
55693

55701
55702
55703
55704
55706
55707
55708
55709
55710
55739
55740
55741

55742
55743
55744
55745
55746
55747
55748
55749
55757
55758
55759
55783

55784
55830
55831
55832
55969
55986
55987
56001
56002
56003
56004
56005

56006
56007
56012
56013
56014
56015
56081
56082
56083
56107
56108
56109

56110
56111
56112
56130
56131
56132
56133
56134
56135
56136
56153
56154

56155
56156
56157
56170
56171
56172
56173
56184
56185
56186
56187
56188

56197
56198
56199
56206
56207
56208
56209
56210
56214
56215
56216
56217

56218
56221
56222
56223
56224
56225
56259
56260
56261
56262
56271
56272

56273
56274
56275
56276
56277
56280
56281
56282
56283
56284
56288
56289

56290
56295
56296
56297
56298
56299
56300
56301
56302
56303
56307
56308

56309
56310
56311
56312
56313
56314
56315
56316
56317
56326
56327
56328

56329
56330
56334
56335
56336
56337
56338
56339
56340
56341
56347
56348

56349
56350
56412
56413
56414
56419
56420
56421
56449
56450
56451
56452

56465
56466
56476
56477
56478
56486
56500
56501
56502
56504
56505
56506

56507
56525
56526
56527
56528
56564
56565
56566
56567
56568
56569
56570

56571
56576
56577
56587
56588
56593
56594
56595
56596
56597
56598
56601

56654
56655
56682
56684
56685
56686
56687
56688
56689
56690
56691
56698

56699
56710
56711
56724
56736
56737
56738
56739
56740
56751
56752
56753

56754
56768
56769
56770
56771
56780
56781
56806
56807
56808
56830
56831

56832
56848
56849
56850
56866
56867
56868
56903
56904
56905
56947
56948

56960
56961
56962
56963
56996
56997
56998
56999
57016
57017
57018
57032

57033
57034
57035
57053
57054
57055
57097
57098
57099
57100
57101
57102

57103
57104
57105
57106
57113
57114
57115
57116
57117
57118
57119
57123

57124
57125
57126
57127
57128
57129
57137
57138
57148
57149
57150
57151

57165
57166
57211
57212
57294
57295
57298
57299
57300
57377
57378
57397

57398
57402
57403
57404
57405
57406
57425
57426
57427
57441
57442
57443

57444
57450
57451
57452
57453
57480
57481
57482
57492
57493
57497
57498

57508
57509
57524
57525
57550
57551
57552
57553
57554
57574
57575
57656

57657
57658
57684
57685
57698
57699
57724
57725
57726
57727
57795
57796

57797
57798
57799
57800
57801
57802
57806
57807
57837
57893
57894
58162

58163
58164
58168
58218
58222
58226
58239
58240
58241
58328
58329
58330

58406
58407
58408
58429
58430
58431
58641
58642
58643
58644
58645
58646

58661
58701
58702
58732
58733
58734
58735
58736
58737
58738
58739
58744

58745
58758
58759
58760
58763
58764
58765
58766
58770
58771
58772
58773

58774
58775
58776
58777
58778
58779
58780
58781
58782
58783
58784
58785

58786
58787
58798
58799
58800
58801
58806
58807
58808
58816
58817
58825

58826
58853
58854
58859
58860
58861
58864
58865
58866
58867
58868
58869

58870
58871
58872
58873
58878
58879
58880
58881
58882
58883
58884
58885

58886
58887
58888
58889
58890
58891
58892
58895
58896
58897
58898
58899

58900
58903
58904
58905
58906
58907
58908
58918
58919
58920
58921
59018

59169
59170
59179
59180
59184
59185
59216
59217
59236
59237
59244
59300

59301
59333
59334
59335
59336
59344
59345
59367
59368
59387
59388
59389

59400
59401
59436
59437
59438
60082
60083
60084
60085
60222
60268
60383

60384
60385
60414
60415
60416
60417
60476
60477
60478
60479
60491
60543

60599
60600
60601
60602
60662
60668
60671
60672
60673
60674
60678
60679

60685
60694
60695
60697
60698
60727
60728
60729
60747
60748
60749
60843

60844
60875
60876
60894
60895
60903
60904
60908
60909
60917
60918
60919

60920
60921
60922
60923
60924
60925
60926
60927
60928
60929
60930
60931

60932
60933
60934
60935
60936
60937
60938
60939
60952
60953
60954
60955

60956
60957
60958
60959
60960
60966
60967
60968
60971
60972
60973
60974

60975
60976
60977
60978
60979
60980
60981
60982
60983
60984
60985
60986

60987
60988
60989
60990
60994
61016
61023
61027
61028
61031
61099
61106

61112
61113
61114
61115
61116
61117
61118
61119
61144
61151
61152
61156

61157
61158
61159
61167
61168
61169
61170
61171
61175
61176
61177
61181

61182
61183
61184
61185
61186
61187
61188
61190
61191
61192
61193
61194

61195
61196
61197
61209
61210
61211
61212
61213
61214
61215
61216
61217

61221
61222
61223
61224
61225
61226
61227
61228
61229
61230
61234
61235

61236
61237
61238
61239
61250
61251
61252
61253
61254
61255
61263
61264

61265
61266
61267
61268
61269
61270
61276
61279
61280
61296
61315
61316

61317
61328
61331
61374
61375
61415
61416
61456
61462
61464
61474
61475

61476
61477
61478
61479
61480
61499
61500
61501
61528
61538
61590
61604

61608
61630
61645
61649
61650
61656
61657
61658
61659
61663
61664
61665

61671
61690
61695
61696
61697
61698
61699
61700
61701
61702
61716
61717

61741
61742
61743
61746
61747
61755
61756
61757
61760
61761
61762
61763

61764
61771
61772
61773
61781
61800
61801
61802
61811
61812
61813
61818

61819
61820
61821
61822
61829
61836
61837
61838
61839
61840
61841
61842

61843
61862
61863
61864
61865
61866
61882
61895
61898
61901
61902
61918

61919
61920
61930
61931
61935
61936
61939
61940
61941
61969
61970
61971

61989
62004
62005
62006
62007
62049
62050
62051
62052
62053
62058
62059

62065
62066
62067
62068
62072
62073
62074
62086
62087
62088
62090
62091

62092
62094
62095
62096
62097
62098
62100
62106
62109
62111
62112
62113

62122
62128
62129
62134
62135
62136
62144
62145
62146
62147
62155
62156

62157
62231
62232
62233
62234
62235
62236
62237
62240
62241
62242
62243

62252
62265
62275
62283
62284
62285
62286
62287
62288
62289
62298
62299

62300
62301
62302
62303
62325
62326
62327
62334
62335
62336
62337
62338

62339
62340
62341
62342
62343
62344
62349
62350
62351
62352
62353
62354

62355
62356
62358
62359
62360
62407
62408
62409
62410
62411
62412
62413

62414
62415
62439
62440
62441
62442
62443
62448
62449
62450
62451
62452

62453
62454
62467
62468
62469
62470
62471
62472
62473
62474
62475
62485

62486
62487
62488
62489
62490
62491
62492
62500
62501
62502
62503
62528

62529
62538
62539
62550
62551
62552
62553
62554
62555
62556
62583
62584

62585
62586
62587
62588
62589
62607
62608
62609
62610
62611
62620
62621

62622
62628
62630
62631
62632
62633
62643
62660
62661
62662
62663
62664

62665
62684
62685
62686
62687
62688
62689
62690
62691
62692
62693
62694

62695
62696
62701
62702
62703
62704
62710
62711
62714
62715
62716
62717

62718
62719
62720
62722
62723
62724
62725
62726
62727
62728
62729
62730

62731
62732
62733
62734
62735
62736
62737
62741
62742
62743
62744
62745

62750
62751
62752
62753
62754
62755
62756
62757
62758
62759
62760
62761

62763
62764
62769
62770
62775
62776
62777
62780
62781
62782
62783
62784

62785
62786
62787
62788
62789
62790
62791
62792
62793
62796
62797
62798

62800
62801
62802
62803
62804
62805
62806
62807
62808
62809
62810
62811

62812
62813
62814
62815
62816
62817
62818
62819
62823
62824
62825
62826

62830
62831
62832
62833
62834
62838
62839
62840
62841
62842
62843
62844

62845
62846
62847
62848
62849
62850
62851
62852
62853
62854
62855
62856

62857
62858
62859
62860
62865
62866
62867
62868
62869
62870
62871
62872

62873
62874
62875
62876
62877
62878
62879
62883
62884
62885
62886
62887

62888
62889
62890
62891
62892
62893
62894
62895
62896
62897
62898
62899

62900
62901
62902
62903
62905
62906
62907
62911
62912
62918
62919
62920

52921
62922
62923
62924
62925
62926
62927
62928
62930
62933
62934
62937

62938
62939
62940
62941
62947
62948
62952
62956
62962
62964
62966
62970

62973
62974
62983
62984
62985
62991
62992
62993
62994
62995
63009
63010

63011
63015
63016
63017
63019
63020
63021
63022
63023
63024
63027
63032

63033
63034
63037
63038
63039
63040
63041
63042
63046
63047
63048
63055

63056
63057
63060
63061
63062
63063
63064
63065
63067
63068
63069
63080

63081
63082
63083
63084
63087
63088
63089
63090
63091
63092
63093
63094

63098
63099
63100
63101
63102
63103
63104
63105
63106
63107
63108
63109

63110
63111
63112
63116
63123
63124
63125
63126
63127
63128
63134
63135

63136
63137
63138
63139
63146
63147
63148
63149
63150
63151
63161
63162

63163
63167
63168
63169
63170
63171
63172
63173
63174
63175
63198
63202

63225
63226
63227
63233
63234
63235
63236
63237
63238
63239
63240
63241

63242
63243
63244
63257
63258
63259
63266
63267
63268
63269
63270
63278

63279
63280
63281
63282
63288
63289
63290
63291
63292
63293
63297
63298

63299
63300
63301
63305
63306
63307
63308
63309
63311
63312
63313
63314

63315
63316
63317
63318
63319
63320
63321
63322
63323
63333
63334
63335

63336
63337
63338
63339
63340
63341
63342
63343
63344
63345
63346
63347

63348
63349
63350
63351
63352
63353
63354
63355
63356
63357
63358
63359

63363
63364
63365
63366
63367
63368
63369
63370
63371
63372
63373
63374

63375
63376
63377
63378
63379
63380
63381
63382
63383
63384
63386
63387

63388
63389
63390
63391
63392
63393
63394
63395
63396
63397
63398
63399

63400
63401
63402
63403
63411
63412
63413
63414
63415
63416
63417
63418

63420
63421
63422
63423
63424
63425
63426
63431
63432
63433
63442
63443

63444
63445
63448
63449
63450
63451
63452
63453
63454
63455
63456
63457

63471
63475
63476
63477
63478
63479
63480
63481
63483
63486
63487
63488

63489
63490
63491
63492
63493
63496
63499
63503
63507
63508
63509
63510

63511
63512
63513
63514
63515
63516
63517
63518
63519
63520
63524
63525

63526
63527
63528
63529
63530
63531
63532
63533
63534
63539
63540
63541

63545
63546
63547
63548
63549
63550
63561
63562
63578
63579
63580
63581

63583
63584
63585
63586
63590
63591
63592
63593
63594
63595
63596
63597

63598
63604
63605
63606
63607
63608
63625
63626
63627
63634
63635
63636

63637
63638
63644
63654
63655
63656
63657
63660
63661
63662
63663
63664

63667
63669
63670
63678
63679
63680
63681
63682
63683
63684
63687
63691

63692
63693
63694
63698
63699
63700
63701
63702
63704
63705
63710
63711

63712
63713
63714
63727
63728
63729
63730
63744
63745
63748
63757
63761

63763
63775
63779
63782
63783
63784
63785
63787
63798
63800
63803
63812

63815
63816
63817
63818
63819
63820
63821
63822
63832
63837
63838
63839

63840
63842
63859
63860
63861
63862
63863
63864
63866
63867
63868
63873

63874
63875
63876
63907
63908
63909
63910
63914
63915
63926
63927
63928

63930
63931
63934
63959
63960
63961
63975
63976
63997
63998
63999
64000

64004
64042
64047
64048
64049
64050
64070
64078
64091
64101
64125
64142

64149
64150
64153
64154
64155
64156
64157
64158
64159
64160
64161
64162

64163
64164
64166
64167
64168
64171
64173
64174
64175
64176
64186
64187

64194
64195
64196
64197
64198
64201
64202
64203
64207
64208
64212
64217

64218
64219
64220
64228
64229
64230
64231
64235
64236
64237
64238
64239

64240
64241
64242
64243
64247
64248
64250
64251
64255
64257
64258
64259

64261
64262
64263
64264
64267
64268
64274
64277
64279
64280
64288
64289

64290
64293
64304
64305
64306
64307
64308
64309
64310
64313
64315
64316

64317
64321
64322
64323
64324
64325
64326
64327
64328
64329
64330
64336

64347
64349
64353
64354
64355
64356
64357
64358
64359
64363
64365
64367

64368
64369
64370
64371
64372
64373
64374
64375
64376
64380
64381
64382

64383
64385
64386
64387
64388
64389
64394
64395
64396
64397
64398
64400

64401
64402
64406
64407
64408
64409
64410
64411
64412
64419
64420
64421

64423
64424
64425
64435
64441
64443
64464
64465
64466
64467
64468
64469

64480
64489
64490
64491
64495
64508
64514
64545
64558
64559
64565
64566

64573
64575
64576
64577
64583
64590
64594
64595
64596
64597
64609
64610

64611
64612
64613
64617
64618
64619
64630
64631
64632
64642
64655
64656

64661
64662
64663
64664
64665
64666
64670
64671
64672
64676
64677
64678

64683
64684
64685
64690
64691
64712
64713
64717
64718
64719
64729
64731

64743
64744
64745
64748
64749
64751
64752
64753
64754
64762
64763
64764

64765
64766
64768
64769
64770
64771
64772
64773
64774
64775
64783
64787

64793
64798
64799
64800
64801
64802
64803
64804
64805
64806
64817
64822

64823
64824
64825
64829
64831
64832
64833
64852
64853
64854
64866
64867

64868
64884
64885
64901
64902
64903
64912
64913
64936
64937
64938
64954

64955
64956
64969
64970
64972
64973
64974
64975
64979
64980
64981
64982

64988
64990
64991
64992
64993
64998
64999
65000
65002
65003
65004
65008

65009
65010
65011
65015
65016
65017
65020
65021
65022
65024
65025
65026

65034
65035
65036
65037
65038
65039
65040
65041
65042
65043
65044
65045

65046
65047
65048
65052
65053
65054
65055
65056
65057
65058
65059
65060

65061
65062
65063
65068
65069
65070
65071
65072
65073
65074
65077
65078

65079
65080
65081
65085
65086
65100
65104
65105
65106
65108
65109
65110

65111
65112
65122
65123
65124
65131
65132
65133
65134
65135
65137
65141

65142
65144
65145
65146
65147
65148
65149
65154
65155
65156
65157
65158

65159
65162
65163
65164
65165
65166
65171
65172
65173
65174
65175
65176

65177
65178
65179
65180
65181
65184
65185
65186
65187
65191
65192
65193

65194
65195
65196
65197
65198
65199
65200
65202
65203
65204
65205
65206

65207
65208
65209
65214
65215
65216
65219
65220
65221
65226
65227
65228

65229
65231
65232
65233
65234
65239
65240
65241
65242
65250
65251
65252

65253
65254
65255
65259
65260
65261
65265
65266
65267
65279
65280
65281

65282
65284
65285
65286
65287
65288
65289
65290
65296
65297
65298
65307

65308
65309
65313
65314
65317
65318
65325
65326
65327
65328
65330
65331

65336
65337
65338
65339
65340
65341
65342
65343
65344
65345
65346
65347

65348
65349
65350
65351
65352
65355
65356
65359
65360
65361
65362
65363

65364
65365
65366
65372
65374
65377
65378
65383
65384
65393
65394
65395

65396
65397
65398
65399
65400
65401
65402
65403
65404
65405
65410
65411

65412
65413
65415
65416
65420
65421
65422
65423
65431
65432
65433
65434

65435
65436
65439
65440
65442
65443
65446
65447
65448
65461
65462
65463

65465
65466
65468
65475
65476
65482
65483
65484
65485
65487
65490
65491

65492
65493
65498
65500
65504
65505
65514
65515
65516
65518
65524
65525

65526
65527
65528
65529
65533
65541
65542
65543
65544
65549
65551
65556

65557
65558
65564
65566
65567
65576
65577
65580
65585
65612
65613
65627

65628
65629
65634
65635
65636
65637
65646
65647
65648
65649
65650
65654

65655
65657
65658
65661
65662
65663
65680
65681
65684
65691
65692
65710

65711
65716
65719
65720
65721
65732
65735
65742
65743
65754
65755
65759

65760
65764
65768
65769
65770
65772
65784
65787
65791
65792
65793
65794

65795
65796
65797
65801
65802
65807
65808
65809
65816
65817
65818
65821

65822
65823
65827
65831
65832
65834
65835
65836
65837
65843
65844
65845

65846
65847
65856
65857
65858
65859
65860
65861
65862
65875
65883
65884

65885
65886
65888
65889
65890
65895
65913
65914
65915
65917
65919
65920

65921
65922
65923
65927
65930
65931
65932
65935
65936
65938
65939
65944

65945
65946
65953
65954
65955
65956
65957
65958
65966
65968
65970
65971

65972
65973
65974
65975
65976
65977
65978
65979
65980
65981
65982
65985

65986
65987
65988
65989
65990
65991
65999
66000
66003
66004
66005
66011

66012
66017
66018
66019
66020
66021
66027
66028
66037
66043
66044
66050

66051
66052
66055
66058
66083
66084
66086
66087
66088
66096
66103
66104

66105
66106
66107
66108
66109
66111
66112
66113
66114
66117
66118
66119

66122
66123
66124
66125
66126
66127
66128
66129
66130
66131
66132
66134

66135
66136
66137
66140
66141
66148
66151
66152
66155
66156
66157
66158

66159
66160
66162
66163
66164
66165
66169
66182
66183
66185
66188
66190

66192
66193
66194
66211
66225
66226
66227
66232
66233
66236
66237
66238

66243
66246
66247
66263
66267
66273
66274
66279
66280
66284
66285
66286

66288
66293
66294
66295
66296
66297
66302
66303
66304
66305
66306
66307

66308
66309
66311
66312
66313
66315
66316
66317
66318
66319
66320
66321

66322
66323
66324
66325
66326
66327
66328
66329
66330
66331
66333
66334

66335
66336
66337
66338
66339
66340
66341
66342
66343
66344
66345
66346

66347
66348
66349
66350
66351
66352
66353
66354
66355
66356
66357
66358

66360
66361
66362
66363
66364
66365
66366
66367
66368
66369
66372
66373

66374
66375
66376
66377
66378
66379
66380
66381
66382
66383
66384
66385

66388
66392
66395
66400
66403
66404
66410
66411
66412
66414
66415
66416

66417
66418
66419
66420
66421
66422
66423
66424
66425
66427
66428
66429

66430
66431
66432
66433
66434
66435
66436
66437
66440
66441
66442
66443

66444
66445
66447
66451
66452
66453
66454
66455
66456
66457
66459
66460

66461
66462
66466
66467
66468
66469
66470
66471
66472
66474
66475
66478

66479
66481
66482
66483
66484
66485
66486
66487
66488
66489
66494
66495

66496
66497
66498
66499
66502
66505
66506
66507
66508
66509
66513
66516

66517
66519
66520
66521
66522
66524
66526
66527
66528
66529
66533
66534

66535
66541
66542
66543
66544
66547
66550
66555
66556
66557
66559
66562

66563
66564
66565
66566
66568
66569
66570
66571
66574
66575
66576
66579

66582
66583
66584
66585
66586
66587
66588
66589
66590
66592
66593
66594

66595
66596
66597
66598
66599
66600
66601
66602
66603
66604
66605
66606

66607
66608
66609
66610
66612
66613
66614
66615
66617
66618
66619
66620

66621
66624
66625
66626
66627
66631
66634
66639
66642
66652
66657
66658

66659
66665
66671
66672
66673
66674
66675
66676
66677
66678
66679
66680

66681
66682
66683
66686
66688
66691
66694
66695
66696
66697
66698
66699

66702
66703
66704
66705
66706
66707
66710
66711
66712
66713
66714
66715

66717
66720
66721
66722
66727
66728
66730
66731
66732
66733
66734
66735

66738
66742
66743
66748
66749
66750
66751
66752
66755
66756
66757
66760

66761
66764
66765
66771
66772
66773
66774
66775
66776
66777
66778
66779

66780
66781
66782
66783
66785
66788
66792
66793
66799
66809
66817
66818

66819
66820
66821
66837
66838
66840
66841
66842
66849
66850
66851
66854

66855
66856
66859
66867
66868
66871
66872
66879
66880
66881
66882
66883

66884
66885
66893
66894
66895
66896
66897
66898
66899
66900
66902
66903

66919
66920
66922
66923
66924
66925
66926
66927
66930
66939
66941
66942

66943
66946
66947
66949
66953
66954
66955
66956
66957
66958
66959
66961

66962
66963
66964
66965
66966
66967
66968
66972
66974
66975
66976
66978

66979
66980
66983
66984
66991
66992
66993
66994
66995
66996
66997
66998

67002
67003
67004
67005
67006
67018
67019
67023
67024
67025
67029
67030

67031
67032
67033
67034
67035
67036
67037
67038
67042
67043
67045
67046

67047
67059
67062
67063
67070
67089
67090
67091
67092
67093
67098
67099

67106
67112
67116
67117
67121
67122
67138
67142
67143
67144
67145
67146

67147
67150
67154
67155
67156
67159
67162
67165
67166
67167
67168
67169

67170
67172
67173
67174
67178
67179
67180
67181
67182
67183
67184
67185

67186
67187
67188
67191
67193
67194
67195
67196
67197
67198
67199
67202

67203
67204
67205
67206
67207
67209
67210
67211
67213
67214
67215
67216

67217
67218
67219
67220
67222
67223
67224
67225
67226
67229
67230
67231

67234
67235
67236
67237
67238
67239
67240
67241
67242
67243
67244
67245

67247
67248
67249
67251
67252
67254
67257
67258
67260
67261
67262
67263

67264
67265
67266
67267
67268
67269
67270
67271
67272
67273
67274
67282

67283
67284
67285
67286
67290
67296
67302
67305
67306
67307
67317
67318

67319
67320
67332
67340
67344
67359
67365
67366
67370
67371
67372
67382

67392
67393
67397
67398
67399
67404
67405
67406
67410
67417
67421
67431

67435
67436
67444
67445
67446
67447
67451
67455
67456
67457
67458
67459

67460
67461
67462
67463
67464
67465
67466
67467
67468
67469
67470
67472

67473
67474
67477
67478
67482
67483
67484
67485
67490
67491
67492
67493

67494
67501
67502
67506
67507
67508
67513
67514
67515
67516
67517
67518

67519
67521
67522
67523
67524
67525
67526
67527
67528
67529
67530
67533

67534
67535
67536
67537
67538
67539
67541
67543
67544
67545
67546
67547

67548
67550
67551
67552
67553
67554
67555
67559
67560
67562
67563
67564

67567
67570
67574
67581
67583
67584
67585
67587
67588
67589
67590
67592

67597
67598
67603
67604
67605
67606
67607
67608
67609
67611
67614
67615

67616
67617
67618
67619
67620
67621
67622
67626
67627
67628
67629
67633

67634
67635
67636
67637
67638
67639
67642
67646
67650
67651
67655
67660

67663
67673
67674
67675
67676
67679
67680
67681
67684
67695
67708
67709

67716
67717
67733
67734
67736
67737
67740
67741
67750
67751
67758
67764

67771
67779
67780
67799
67813
67814
67815
67816
67817
67818
67825
67826

67827
67830
67833
67836
67837
67838
67839
67840
67843
67844
67850
67851

67852
67853
67854
67857
67858
67861
67862
67866
67867
67878
67879
67887

67898
67899
67908
67915
67916
67919
67922
67923
67925
67927
67928
67929

67943
67948
67951
67955
67964
67965
67969
67970
67973
67974
67975
67978

67979
67980
67982
67983
67984
67990
67991
67992
67998
68000
68001
68003

68004
68005
68006
68010
68019
68029
68030
68033
68040
68041
68042
68043

68044
68045
68049
68060
68063
68071
68072
68078
68079
68091
68092
68095

68096
68097
68098
68103
68104
68105
68114
68115
68116
68148
68149
68150

68194
68195
68210
68211
68212
68213
68216
68219
68220
68221
68238
68239

68240
68241
68260
68261
68262
68266
68279
68289
68292
68293
68298
68301

68302
68303
68308
68309
68312
68313
68314
68315
68319
68324
68325
68334

68335
68340
68341
68342
68346
68347
68348
68358
68360
68361
68366
68367

68368
68376
68377
68378
68379
68380
68381
68384
68385
68386
68391
68399

68400
68401
68403
68404
68410
68411
68415
68416
68417
68419
68422
68423

68424
68428
68429
68430
68437
68438
68439
68440
68441
68442
68443
68444

68445
68446
68447
68460
68465
68466
68469
68470
68471
68472
68473
68477

68478
68480
68481
68483
68484
68485
68486
68487
68488
68489
68493
68494

68496
68497
68498
68499
68503
68504
68505
68506
68507
68508
68509
68512

68513
68514
68515
68516
68517
68525
68536
68537
68538
68542
68543
68547

68548
68549
68550
68567
68572
68573
68594
68595
68603
68604
68611
68629

68632
68633
68634
68642
68644
68645
68646
68647
68652
68653
68654
68655

68656
68657
68663
68668
68674
68676
68677
68680
68681
68684
68685
68686

68687
68688
68689
68691
68692
68696
68697
68699
68700
68703
68704
68705

68707
68708
68713
68714
68715
68716
68717
68718
68726
68728
68730
68738

68745
68746
68747
68750
68753
68754
68764
68765
68766
68767
68768
68770

68771
68772
68775
68776
68777
68781
68782
68783
68785
68786
68788
68789

68790
68791
68792
68793
68796
68797
68798
68799
68800
68801
68802
68804

68805
68806
68807
68808
68809
68812
68813
68814
68815
68816
68817
68818

68819
68820
68821
68822
68823
68824
68826
68827
68828
68832
68834
68835

68836
68846
68847
68848
68851
68852
68853
68854
68855
68859
68860
68861

68862
68868
68869
68873
68880
68881
68882
68883
68884
68885
68886
68887

68890
68891
68892
68893
68894
68895
68896
68897
68900
68901
68902
68903

68905
68906
68907
68908
68909
68910
68912
68913
68914
68915
68917
68918

68920
68921
68922
68925
68926
68930
68931
68932
68933
68937
68940
68941

68943
68944
68945
68946
68952
68956
68967
68968
68970
68971
68975
68976

68979
68984
68987
68988
68992
68993
69005
69006
69012
69013
69014
69016

69022
69023
69025
69026
69027
69028
69029
69032
69038
69039
69042
69044

69045
69047
69052
69054
69055
69062
69063
69065
69066
69069
69070
69074

69075
69076
69077
69083
69084
69086
69087
69088
69089
69090
69093
69094

69096
69097
69098
69099
69105
69106
69115
69126
69133
69137
69138
69139

69143
69144
69146
69147
69148
69149
69150
69151
69153
69154
69155
69156

69157
69158
69159
69163
69164
69169
69170
69173
69174
69177
69178
69181

69186
69187
69188
69191
69192
69193
69194
69195
69196
69197
69205
69207

69209
69211
69213
69214
69216
69223
69224
69227
69228
69229
69230
69231

69232
69234
69235
69238
69239
69240
69243
69245
69246
69247
69248
69249

69250
69251
69252
69254
69255
69256
69257
69258
69259
69262
69263
69268

69269
69270
69271
69272
69273
69275
69276
69277
69279
69280
69281
69282

69284
69289
69291
69292
69293
69296
69298
69302
69306
69307
69315
69316

69317
69318
69319
69323
69327
69330
69331
69332
69336
69337
69341
69342

69345
69346
69347
69348
69351
69354
69355
69357
69358
69359
69363
69364

69365
69370
69372
69373
69374
69377
69378
69382
69385
69388
69390
69391

69393
69394
69395
69396
69397
69399
69401
69402
69403
69404
69405
69407

69408
69409
69410
69411
69412
69413
69414
69415
69416
69417
69418
69419

69420
69421
69426
69427
69428
69429
69431
69432
69433
69434
69435
69438

69442
69443
69444
69445
69448
69449
69461
69462
69463
69465
69468
69473

69474
69477
69478
69479
69480
69482
69483
69485
69492
69493
69495
69496

69503
69504
69509
69512
69513
69517
69518
69519
69520
59521
69526
69530

69531
69535
69536
69538
69539
69540
69543
69546
69547
69548
69549
69551

69552
69553
69554
69555
69560
69563
69564
69566
69568
69569
69571
69572

69573
69575
69578
69579
69580
69581
69587
69588
69589
69590
69592
69596

69597
69598
69599
69604
69605
69606
69607
69609
69611
69619
69620
69622

69624
69625
69626
69627
69628
69630
69631
69635
69636
69641
69643
69645

69646
69647
69648
69649
69651
69653
69657
69660
69661
69662
69666
69670

69673
69674
69675
69676
69680
69684
69685
69686
69687
69689
69691
69692

69693
69694
69697
69698
69699
69700
69701
69702
69703
69704
69706
69710

69711
69713
69714
69716
69717
69720
69721
69724
69725
69727
69728
69729

69730
69733
69737
69738
69742
69743
69746
69749
69750
69751
69753
69754

69755
69756
69757
69758
69759
69760
69762
69763
69764
69765
69766
69767

69768
69770
69771
69772
69774
69775
69776
69777
69782
69783
69784
69785

69786
69788
69789
69790
69793
69800
69801
69802
69806
69807
69808
69809

69811
69814
69815
69821
69824
69827
69833
69835
69837
69838
69841
69842

69844
69845
69848
69851
69852
69853
69855
69857
69858
69865
69866
69868

69869
69870
69871
69872
69873
69875
69879
69880
69881
69884
69887
69888

69890
69892
69894
69898
69900
69901
69904
69907
69908
69909
69911
69912

69914
69915
69916
69917
69919
69925
69926
69929
69931
69933
69937
69945

69946
69950
69952
69959
69985
69988
69989
69991
69992
69993
69994
69998

69999
70002
70003
70004
70005
70006
70009
70011
70012
70013
70014
70016

70017
70019
70023
70026
70027
70028
70029
70030
70032
70034
70036
70037

70042
70043
70045
70046
70047
70049
70051
70052
70054
70055
70057
70059

70061
70063
70064
70065
70072
70073
70075
70076
70079
70080
70083
70085

70086
70089
70090
70091
70092
70093
70094
70095
70096
70098
70099
70100

70102
70103
70105
70106
70108
70109
70110
70111
70113
70115
70116
70117

70118
70119
70122
70127
70129
70133
70134
70135
70138
70139
70141
70144

70147
70148
70149
70150
70151
70155
70159
70160
70161
70163
70165
70170

70174
70176
70182
70187
70188
70191
70192
70195
70197
70198
70199
70200

70201
70202
70203
70205
70206
70207
70208
70209
70212
70213
70215
70216

70217
70219
70220
70221
70222
70223
70224
70225
70226
70229
70233
70234

70235
70236
70238
70239
70240
70243
70244
70245
70247
70248
70252
70254

70255
70257
70259
70260
70262
70264
70266
70267
70270
70271
70272
70273

70274
70275
70276
70277
70279
70280
70281
70282
70283
70286
70287
70290

70291
70292
70294
70296
70297
70298
70299
70300
70301
70303
70304
70307

70308
70311
70312
70313
70314
70315
70319
70321
70322
70328
70330
70331

70335
70337
70338
70342
70344
70345
70347
70348
70349
70352
70353
70356

70357
70358
70359
70360
70361
70363
70364
70365
70366
70367
70368
70369

70373
70374
70375
70376
70377
70378
70380
70382
70384
70385
70386
70387

70390
70391
70392
70393
70394
70396
70399
70400
70401
70409
70415
70416

70417
70422
70425
70429
70430
70436
70443
70450
70469
70473
70474
70476

70477
70479
70481
70483
70484
70488
70489
70492
70493
70494
70495
70498

70501
70504
70522
70526
70531
70536
70555
70562
70563
70564
70574
70577

70579
70580
70583
70593
70594
70605
70611
70618
70624
70630
70632
70638

70639
70640
70641
70642
70644
70647
70656
70660
70662
70663
70664
70665

70676
70678
70691
70701
70704
70708
70709
70712
70715
70717
70718
70719

70720
70722
70723
70727
70728
70729
70730
70732
70734
70738
70739
70740

70743
70744
70745
70746
70747
70749
70750
70751
70756
70759
70760
70762

70766
70767
70771
70772
70773
70774
70775
70776
70777
70779
70780
70781

70782
70783
70785
70786
70787
70788
70789
70790
70791
70792
70793
70794

70795
70797
70798
70799
70800
70802
70803
70805
70806
70809
70812
70813

70814
70815
70816
70818
70822
70824
70825
70827
70828
70833
70834
70835

70836
70838
70840
70844
70845
70846
70847
70848
70849
70853
70854
70855

70856
70857
70858
70859
70861
70862
70863
70864
70866
70867
70868
70869

70874
70875
70877
70878
70883
70884
70885
70886
70888
70889
70892
70897

70898
70899
70901
70902
70903
70904
70909
70910
70911
70912
70913
70915

70917
70919
70922
70923
70927
70930
70932
70934
70935
70937
70938
70939

70940
70941
70942
70943
70944
70946
70947
70949
70950
70952
70953
70955

70956
70957
70958
70960
70961
70962
70963
70964
70965
70966
70967
70968

70969
70971
70972
70975
70976
70977
70978
70979
70981
70982
70983
70984

70985
70986
70987
70988
70989
70990
70991
70995
70996
70998
70999
71000

71001
71002
71003
71004
71006
71007
71008
71010
71011
71013
71016
71018

71019
71021
71023
71024
71027
71033
71042
71044
71051
71053
71056
71057

71061
71064
71065
71066
71679
71070
71077
71088
71093
71096
71099
71100

71109
71110
71111
71113
71120
71123
71128
71130
71132
71133
71140
71145

71146
71147
71148
71151
71152
71154
71156
71157
71159
71160
71161
71162

71163
71165
71166
71170
71173
71174
71175
71176
71179
71180
71181
71183

71185
71194
71198
71199
71203
71204
71206
71213
71225
71236
71241
71249

71257
71261
71263
71266
71267
71268
71271
71272
71273
71276
71280
71285

71291
71292
71294
71295
71313
71344
71345
71346
71348
71349
71352
71353

71354
71361
71362
71367
71368
71371
71372
71391
71396
71398
71399
71404

71406
71408
71412
71415
71418
71421
71428
71429
71430
71438
71439
71440

71442
71443
71449
71450
71453
71454
71459
71460
71462
71464
71467
71472

71480
71482
71483
71486
71488
71489
71492
71495
71498
71500
71502
71506

71512
71513
71514
71516
71534
71540
71542
71543
71544
71547
71548
71551

71563
71564
71565
71569
71570
71572
71574
71575
71577
71578
71579
71581

71588
71591
71592
71594
71595
71596
71597
71598
71600
71604
71617
71618

71620
71621
71626
71627
71630
71631
71632
71636
71648
71662
71664
71666

71667
71668
71669
71670
71672
71673
71675
71676
71677
71679
71689
71692

71699
71700
71701
71705
71706
75529
75539
75540
75542
75543
75557
75560

75561
75562
75570
75576
75577
75578
75582
75586
75587
75588
75589
75592

75593
75594
75595
75596
75600
75611
75612
75613
75614
75616
75619
75628

75629
75630
75631
75653
75669
75680
75690
75694
75699
75700
75705
75706

75743
75767
75768
75772
75773
75774
75775
75776
75777
75787
75788
75791

75807
75808
75812
75814
75816
75818
75819
75827
75828
75829
75830
75837

75838
75839
75840
75841
75843
75844
75848
75849
75850
75851
75853
75872

75880
75885
75886
75888
75891
75892
75893
75901
75902
75903
75904
75917

75923
75924
75925
75926
75928
75940
75941
75943
75945
75946
75947
75952

75957
75958
75960
75961
75962
75963
75964
75969
75970
75978
75979
75980

75981
75982
75983
75984
75986
75987
75992
76011
76013
76015
76016
76017

76018
76027
76028
76029
76033
76035
76044
76045
76046
76047
76048

(11) Metal-binding proteins (SEQ ID NOs)

4825
4936
4973
5174
5220
5224
5382
6020
6050
6142
6317
6337
6468

6605
10463
10856
10857
10862
10885
11032
11034
11431
11869
11994
12018

16978
16769
16778
16779
16781
16782
16783
16858
16862
16873
16877
16878

16879
16880
54097
54098
54143
54144
54145
54146
54147
54148
54153
54154

54157
54158
54159
54160
54161
54162
54163
54164
54165
54166
54167
54168

54169
54170
54171
54175
54176
54177
54178
54179
54180
54181
54182
54183

54184
54185
54186
54187
54209
54210
54211
54214
54215
54216
54217
54218

54219
54226
54227
54228
54229
54230
54231
54232
54233
54234
54235
54236

54237
54240
54241
54244
54245
54246
54307
54308
54455
54456
54457
54458

54459
54460
54511
54512
54513
54514
54665
54666
54667
54802
55005
55480

55566
55630
55631
55632
55842
56663
56664
56700
56701
56702
56703
56731

56732
56733
56815
56816
56828
56829
57141
57142
57143
57159
57160
57161

57162
57213
57214
58180
58181
58262
58263
58264
58265
58268
58269
58270

58271
58299
58300
58301
58302
58305
58306
58307
58308
58317
58318
58319

58320
58394
58395
58396
58397
58458
58459
58460
58461
58544
58545
58546

58547
59096
59097
59098
59099
59100
60610
60611
60612
60613
60614
61600

61601
62043
62044
62045
62166
62167
62367
62368
62369
62370
62438
63120

63121
63122
63179
63180
63277
63629
63630
63631
64185
64350
64351
64352

65143
65188
65225
65324
65545
65559
65581
65582
65591
65592
65607
65828

65829
65830
65841
65842
65848
65849
65850
65865
65918
65964
65983
65984

66007
66008
66059
66060
66061
66062
66066
66075
66076
66077
66078
66079

66092
66093
66094
66095
66097
66098
66177
66184
66386
66387
66532
66560

66567
66646
66647
67110
67111
67212
67540
67586
67692
67950
67987
68345

68510
68511
68535
68592
68593
68671
68672
69056
69057
69104
69182
69183

69253
69265
69266
69267
69557
69558
69612
69632
69744
69797
69798
69863

69944
69971
70020
70021
70041
70070
70104
70156
70346
70371
70427
70442

70447
70448
70449
70451
70453
70455
70458
70459
70463
70465
70467
70468

70470
70471
70487
70490
70491
70497
70506
70507
70510
70514
70530
70535

70540
70554
70556
70557
70561
70626
70653
70873
71012
71054
71067
71072

71086
71153
71320
71321
71325
71329
71332
71334
71357
71359
71364
71365

71366
71420
71441
71456
71499
71601
71610
71622
71647
71685
71697
75590

75753
75754
75755
75762
75785
75786
75999
76000
76001
76042

(12) Methanogenesis proteins (SEQ ID NOs)

5833
5835
5836
5837
5838
5839
5840
5841
5842
5843
5844
6360
6432

6436
6437
6438
6439
6440
6441
6442
6443
6444
6445
10474
10475

10476
10477
10478
10479
10480
10481
10482
10483
10484
10485
11511
11549

11554
11555
11556
11557
11558
11559
11560
11562
16859
54741
54742
54743

54744
54745
54786
54787
54788
54789
54790
54871
54872
54873
54874
54875

56843
56844
57209
57210
57215
57216
57217
57218
57219
57220
57230
57231

57232
57233
57234
57235
57267
57268
57269
57270
57271
57272
57273
57274

57275
57276
57277
57346
57347
57348
57349
57350
57351
57365
57366
57367

57368
57416
57417
57418
57461
57462
57463
57464
57499
57500
57501
57510

57511
57512
57706
57707
57708
57721
57722
57723
57734
57735
57736
57737

57738
57739
57979
57980
57981
57982
57983
57984
57998
57999
58000
58001

58002
58003
58011
58012
58013
58017
58018
58019
58023
58024
58025
58029

58030
58031
58035
58036
58037
58047
58048
58049
58065
58066
58067
58077

58078
58079
58080
58081
58082
58104
58105
58106
58107
58108
58109
58113

58114
58115
58119
58120
58121
58144
58145
58146
58185
58186
58187
58188

58189
58190
58191
58192
58193
58194
58195
58196
58200
58201
58202
58203

58204
58205
58206
58209
58210
58211
58212
58213
58214
58242
58243
58244

58245
58246
58843
58844
58845
58846
59842
59843
59844
59845
59846
59847

60615
60616
60617
60618
60624
60625
60626
60627
60628
60634
60635
60636

60637
60659
61096
61097
61098
61240
61241
61242
61243
61511
61531
61532

61533
61534
61712
61713
61714
61715
61790
61793
61796
61806
61833
61846

61851
61854
61857
61871
61874
61883
61888
61891
61925
61944
61945
61956

61962
62778
62779
63283
63284
63844
63845
63846
63851
63903
63911
63924

63925
63929
63932
63933
63935
63936
63937
63938
63939
63940
63941
63942

63943
63944
63945
63949
63951
63955
63956
63957
63958
63962
63966
63967

63968
63977
63981
63982
63983
63984
63985
63986
64041
64141
64165
65295

65303
65305
65310
65315
65316
65329
65334
65467
65469
65486
65503
65511

65517
65519
65530
65534
65538
65540
65546
65550
65656
65880
66628
67044

67561
67566
67568
67569
67571
67572
67575
67578
67580
67582
67669
67670

67682
67683
67694
67700
67711
67712
67718
67719
67720
67723
67724
67729

67732
67735
67738
67739
67742
67743
67754
67755
67756
67757
67783
67784

67789
67790
67795
67796
67802
67803
67805
67806
67809
67810
67811
67812

67821
67822
67823
67824
67831
67832
67841
67842
67845
67846
67863
68637

68712
68721
68732
68733
68734
68735
68736
68737
68739
68740
68748
68749

68751
68752
68755
68757
68758
68760
68761
68769
68856
58857
68871
68874

68878
68916
69112
69113
69114
69140
69145
69215
69244
69290
69297
69303

69310
69329
69335
69338
69350
69360
69361
69366
69389
69392
69406
69439

69455
69469
69475
69476
69481
69484
69489
69491
69494
69497
69498
69500

69501
69502
69505
69506
69525
69527
69534
69582
69920
69924
69934
69939

69940
69941
69942
69943
69948
69949
69951
69953
69954
69961
69962
69964

69967
69968
69970
69973
69974
69975
69977
69978
69979
69980
69981
69982

69983
69986
69987
69990
69995
69997
70022
70040
70048
70056
70068
70074

70097
70112
70124
70146
70153
70175
70193
70261
70309
70339
70343
70355

70389
70395
70408
70428
70452
70457
70464
70520
70523
70529
70534
70544

70547
70550
70553
70560
70565
70566
70573
70578
70581
70584
70588
70592

70595
70597
70598
70600
70606
70607
70670
70687
70689
70695
70697
70699

70702
70703
70705
70706
70711
70713
70736
70769
70770
70870
70945
71358

71336
71337
71358
71527
75913

(13) Methanogenesis Mtr proteins (SEQ ID NOs)

6349
6350
6351
6352
6353
6354
11495
11496
11498
11499
11500
65965
66149

66150
66166
66173
66174
66948
67048
67049
67153
67192
67246
67443
67479

67480
67481
67486
67489
67495
67500
67512
67542
67601
68002
70323
70324

70332
70333
70397
70398
70671
70673
70680
70688
70690
70710
70980
71521

71282
71282
71503
71518
71519
71520
71521
71522
71523
71524
71651
75911

(14) Methanogenesis MtrE proteins (SEQ ID NOs)

6355
11501
12052
12053
12054
12055
12056
12057
12058
12059
12060
12061
12062

12063
12064
12065
12066
12067
12068
12069
12070
12071
12072
12073
12074

12075
12076
12077
12078
12079
12080
12081
12082
12083
12084
12085
12086

12087
12088
12089
12090
12091
12092
12093
12094
12095
12096
12097
12098

12099
16969
16970
16971
16972
16973
64574
65049
65136
65190
65201
65238

65294
65312
65962
65963
66049
66091
66635
66636
66808
71525
75912

(15) Phage related (SEQ ID NOs)

4742
4744
4745
4750
57399
57400
57401
69072

(16) Proteolysis (SEQ ID NOs)

4466
4564
4672
4754
5032
5120
5314
5461
5560
5678
5695
5744
5936

6069
6109
6130
6600
10411
10525
10580
10737
11111
11232
11426
11638

11652
11798
16770
16788
16790
53469
53470
53471
53472
53473
53474
53475

53476
53506
53507
53814
53815
53816
53817
53818
53819
53863
53864
53865

53885
53886
53887
53888
53889
53890
53891
53892
53893
53894
53895
53896

53897
53898
53899
53900
53901
53902
53903
53904
53905
53996
53997
53998

53999
54000
54001
54002
54003
54004
54012
54013
54022
54023
54024
54025

54026
54027
54028
54029
54030
54031
54032
54033
54141
54254
54300
54301

54322
54323
55371
55372
55373
55374
55500
56158
56159
57152
57223
57224

57359
57410
57411
57412
57885
57886
57887
57888
57889
58584
58585
60294

60295
60296
60297
60690
60691
60692
60693
60773
60774
60775
60776
60777

60778
60779
60845
60874
61622
61623
61909
61910
61934
61976
61979
61982

61983
61990
62063
62064
62093
62105
62165
62175
62176
62183
62189
62190

62191
62192
62193
62196
62197
62202
62203
62204
62213
62214
62222
62227

62229
62230
62255
62256
62361
62362
62386
62433
62434
62436
62437
62444

62445
62446
62447
62944
62945
62946
63152
63153
63467
63950
64060
64061

64062
64063
64064
64065
64066
64204
64205
64206
64265
64270
64272
64273

64294
64295
64331
64332
64333
64379
64391
64392
64393
64403
64404
64405

64413
64414
64415
64427
64428
64429
64436
64437
64438
64452
64453
64454

64458
64459
64460
64473
64474
64475
64482
64483
64484
64488
64506
64511

64513
64520
64521
64522
64564
64591
64592
64593
64598
64599
64600
64620

64621
64622
64643
64644
64645
64646
64647
64648
64652
64653
64654
64667

64668
64669
64699
64700
64701
64720
64929
64930
64957
64958
64959
65012

65013
65014
65120
65121
65138
65139
65140
65277
65278
65283
65301
65302

65357
65358
65367
65368
65379
65380
65381
65382
65385
65386
65387
65388

65389
65390
65391
65406
65407
65408
65409
65418
65419
65424
65425
65426

65427
65429
65430
65437
65438
65444
65445
65450
65451
65453
65454
65455

65456
65457
65458
65459
65460
65470
65565
65788
65789
65790
65798
65799

65800
65813
65814
65815
65874
65891
65892
66033
66034
66038
66133
66161

66171
66172
66191
66212
66228
66239
66240
66264
66700
66886
66887
66888

67060
67061
67325
67326
67327
67531
67532
67666
67667
67705
67706
67707

67807
67808
67855
67856
67859
67860
67936
67995
68017
68018
68022
68023

68076
68077
68110
68111
68112
68113
68131
68132
68142
68143
68146
68147

68166
68167
68168
68169
68170
68171
68176
68177
68182
68183
68186
68187

68196
68197
68202
68203
68204
68205
68214
68215
68222
68223
68226
68227

68228
68229
68230
68231
68242
68243
68244
68245
68250
68251
68254
68255

68269
68270
68271
68272
68273
68274
68326
68327
68328
68329
68362
68373

68374
68418
68602
68628
68635
68636
68643
68675
69127
69128
69129
69179

69180
69199
69200
69236
69237
69328
69446
69447
69593
69787
69862
69882

69883
70035
70157
70184
70288
70340
70379
70383
70411
70461
70462
70533

70654
70655
70894
71084
71135
71142
71188
71211
71269
71390
71411
71437

71497
71576
71582
71634
75526
75584
75585
75621
75622
75623
75636
75701

75835
75836
75890
75990
75991

(17) Transcription regulation (SEQ ID NOs)

54777
54778
54779
54780
70620

(18) Ribosomal (SEQ ID NOs)

61896
61897
62721
66723
66724
66725
66726
66729
67400
71216
71355
71652

(19) Substrate binding (SEQ ID NOs)

16860

(20) Transcription (SEQ ID NOs)

57620
57621
59813
65150
65151
65152
68305
68658
69018
69019
69020
70177
70179

70285
70621
70622
71134
71231
71360
71427
71607
71646

(21) Transport (SEQ ID NOs)

4437
4442
4450
4451
4480
4503
4527
4550
4573
4574
4575
4632
4634

4635
4640
4641
4649
4650
4673
4685
4686
4699
4717
4763
4766

4786
4792
4800
4813
4839
4840
4841
4900
4915
4929
4930
4967

4970
4972
4974
4991
4992
5003
5004
5040
5042
5049
5069
5092

5093
5129
5130
5138
5157
5222
5241
5254
5260
5308
5316
5317

5318
5383
5419
5426
5435
5503
5514
5515
5516
5623
5633
5634

5653
5717
5757
5764
5765
5772
5802
5805
5815
5860
5869
5871

5888
5942
5954
5956
6029
6044
6048
6049
6059
6060
6090
6134

6140
6141
6145
6153
6154
6167
6183
6197
6208
6209
6218
6221

6272
6273
6312
6377
6385
6400
6416
6452
6456
6477
6478
6479

6483
6608
6609
6629
6638
6640
6650
10256
10271
10275
10276
10284

10295
10296
10302
10303
10343
10352
10354
10355
10365
10380
10381
10405

10406
10407
10409
10425
10496
10499
10516
10527
10528
10578
10623
10624

10625
10629
10630
10648
10651
10652
10655
10656
10657
10663
10701
10718

10728
10738
10753
10841
10842
10845
10847
10850
10851
10854
10855
10863

10864
10870
10905
10932
10976
11054
11073
11083
11095
11121
11124
11134

11140
11141
11142
11159
11205
11214
11215
11227
11274
11329
11341
11342

11343
11355
11371
11421
11433
11469
11470
11513
11514
11530
11544
11564

11572
11574
11575
11583
11626
11671
11677
11680
11681
11684
11705
11725

11742
11751
11787
11813
11817
11839
11912
11980
11991
12007
12013
12017

12019
16772
16773
16775
16776
16796
16798
16870
16875
53543
53544
53545

53565
53566
53567
53568
53569
53570
53588
53589
53590
53591
53592
53593

53600
53985
54018
54019
54020
54021
54094
54095
54096
54119
54120
54121

54142
54206
54207
54208
54220
54221
54255
54256
54257
54258
54259
54260

54261
54273
54274
54275
54276
54304
54309
54310
54311
54312
54317
54319

54326
54327
54338
54339
54340
54341
54342
54343
54344
54345
54346
54347

54348
54349
54350
54351
54352
54354
54355
54356
54357
54358
54359
54360

54361
54362
54363
54364
54365
54366
54367
54368
54381
54382
54383
54384

54385
54386
54432
54433
54434
54435
54436
54437
54438
54439
54444
54445

54446
54461
54476
54477
54507
54508
54509
54510
54515
54516
54522
54523

54524
54525
54528
54529
54530
54531
54532
54533
54534
54535
54538
54539

54540
54541
54546
54547
54548
54549
54550
54551
54552
54553
54554
54555

54556
54557
54558
54559
54560
54561
54562
54563
54564
54565
54566
54567

54568
54569
54570
54571
54572
54573
54574
54575
54576
54577
54578
54579

54580
54581
54582
54583
54584
54585
54586
54587
54588
54589
54590
54591

54592
54593
54594
54595
54596
54597
54598
54599
54600
54601
54602
54603

54604
54605
54606
54607
54608
54609
54610
54611
54612
54613
54614
54615

54616
54617
54618
54619
54620
54621
54622
54623
54624
54628
54629
54630

54631
54632
54633
54634
54635
54636
54637
54638
54639
54640
54641
54642

54643
54644
54645
54658
54659
54663
54664
54668
54669
54670
54671
54672

54673
54674
54675
54676
54678
54679
54680
54681
54682
54683
54684
54685

54686
54687
54688
54689
54690
54691
54692
54695
54696
5469
54698
54700

54701
54702
54703
54704
54705
54714
54715
54717
54718
54719
54720
54721

54722
54723
54724
54725
54726
54727
54728
54729
54730
54731
54752
54753

54754
54759
54760
54785
54795
54796
54797
54803
54804
54805
54806
54815

54816
54817
54841
54842
54843
54846
54847
54848
54849
54850
54851
54852

54853
54855
54856
54857
54858
54859
54860
54861
54862
54863
54864
54865

54866
54867
54868
54869
54870
54876
54877
54878
54882
54883
54884
54885

54886
54893
54894
54895
54896
54897
54898
54899
54900
54901
54902
54903

54904
54905
54906
54907
54908
54909
54910
54911
54912
54913
54914
54915

54916
54917
54918
54919
54920
54921
54922
54923
54924
54925
54926
54927

54928
54929
54930
54931
54932
54933
54934
54935
54936
54937
54938
54939

54940
54941
54942
54943
54944
54945
54946
54947
54948
54949
54950
54951

54952
54953
54954
54955
54956
54957
54958
54959
54960
54961
54962
54963

54964
54965
54966
54967
54968
54969
54970
54971
54972
54973
54974
54975

54976
54977
54978
54979
54980
54981
54982
54983
54984
54985
54986
54987

54988
54989
54990
54991
54992
54993
54994
54995
54996
54997
54998
54999

55000
55001
55002
55003
55004
55006
55007
55008
55010
55011
55012
55013

55014
55020
55021
55022
55023
55024
55025
55026
55027
55028
55029
55030

55031
55032
55033
55034
55035
55036
55037
55038
55039
55040
55041
55042

55043
55044
55045
55046
55047
55048
55049
55050
55051
55052
55053
55054

55055
55056
55057
55058
55059
55060
55061
55062
55063
55064
55066
55067

55068
55069
55070
55071
55072
55073
55085
55086
55087
55091
55092
55093

55094
55095
55098
55099
55107
55108
55109
55132
55174
55178
55179
55180

55181
55182
55192
55193
55194
55195
55196
55203
55204
55234
55238
55279

55289
55292
55296
55302
55306
55307
55308
55311
55356
55357
55358
55359

55360
55361
55369
55389
55390
55398
55399
55400
55401
55402
55403
55404

55405
55406
55410
55411
55413
55414
55415
55416
55417
55421
55423
55426

55445
55446
55447
55448
55449
55450
55459
55477
55481
55482
55486
55487

55488
55489
55490
55491
55494
55495
55498
55499
55501
55502
55503
55504

55512
55517
55518
55519
55520
55526
55527
55528
55537
55546
55547
55558

55565
55585
55586
55590
55591
55592
55596
55599
55624
55629
55633
55634

55636
55637
55638
55650
55651
55654
55655
55688
55689
55690
55695
55696

55697
55698
55699
55700
55711
55712
55713
55715
55716
55717
55718
55719

55720
55721
55722
55723
55728
55729
55730
55731
55734
55735
55736
55737

55750
55751
55752
55753
55754
55755
55756
55760
55761
55762
55763
55764

55765
55766
55768
55769
55770
55771
55773
55774
55775
55776
55777
55778

55779
55780
55785
55786
55787
55788
55789
55790
55801
55802
55803
55814

55815
55816
55817
55818
55819
55820
55821
55822
55823
55824
55825
55826

55827
55828
55829
55833
55834
55835
55836
55837
55838
55839
55840
55841

55845
55846
55847
55848
55849
55850
55851
55852
55853
55854
55855
55856

55857
55858
55859
55860
55861
55862
55863
55864
55865
55866
55867
55868

55869
55870
55871
55872
55873
55874
55875
55876
55877
55878
55879
55880

55881
55882
55883
55884
55885
55886
55887
55888
55889
55890
55891
55892

55893
55900
55901
55902
55903
55904
55905
55906
55907
55908
55909
55910

55911
55912
55913
55914
55915
55916
55917
55918
55919
55920
55921
55922

55923
55924
55925
55926
55927
55928
55929
55930
55931
55932
55938
55939

55940
55941
55942
55943
55944
55945
55946
55947
55948
55949
55950
55951

55952
55953
55954
55955
55956
55957
55958
55959
55960
55961
55962
55963

55964
55965
55966
55967
55968
55972
55973
55974
55975
55976
55977
55978

55979
55980
55981
55982
55983
55984
55985
55988
55989
55990
55991
55992

55993
55994
55995
55996
55997
55998
55999
56000
56008
56009
56010
56011

56016
56017
56018
56019
56020
56021
56022
56023
56024
56025
56026
56027

56028
56029
56030
56031
56032
56033
56034
56035
56036
56037
56038
56039

56040
56041
56042
56043
56044
56045
56046
56047
56048
56049
56050
56051

56052
56053
56054
56055
56056
56057
56058
56059
56060
56061
56062
56063

56064
56065
56066
56067
56068
56069
56070
56071
56072
56073
56074
56075

56076
56077
56078
56079
56080
56084
56085
56086
56087
56088
56089
56090

56091
56092
56093
56094
56095
56096
56097
56098
56099
56100
56101
56102

56103
56104
56105
56106
56113
56114
56115
56116
56117
56118
56119
56120

56121
56122
56123
56124
56125
56126
56127
56128
56129
56137
56138
56139

56140
56141
56142
56143
56144
56145
56146
56147
56148
56149
56150
56151

56152
56167
56168
56169
56174
56175
56176
56177
56178
56179
56181
56182

56183
56189
56190
56191
56192
56193
56194
56195
56196
56200
56201
56202

56203
56204
56205
56211
56219
56220
56226
56227
56228
56229
56230
56231

56232
56233
56234
56235
56236
56237
56238
56239
56240
56241
56242
56243

56244
56245
56246
56247
56248
56249
56250
56251
56252
56253
56254
56255

56256
56257
56258
56263
56264
56265
56266
56267
56268
56269
56270
56278

56279
56285
56286
56287
56291
56292
56293
56294
56304
56305
56306
56318

56319
56320
56321
56322
56323
56324
56325
56342
56343
56344
56351
56352

56353
56354
56355
56356
56357
56358
56359
56360
56361
56362
56363
56364

56365
56366
56367
56368
56369
56370
56371
56372
56373
56374
56375
56376

56377
56378
56379
56380
56381
56382
56383
56384
56385
56386
56387
56388

56389
56390
56391
56392
56393
56394
56395
56396
56397
56398
56399
56400

56401
56402
56403
56404
56405
56406
56407
56408
56409
56410
56411
56415

56416
56417
56418
56422
56423
56424
56425
56426
56427
56428
56429
56430

56431
56432
56433
56434
56435
56436
56437
56440
56441
56442
56443
56444

56445
56446
56447
56448
56455
56456
56457
56458
56459
56462
56463
56464

56467
56468
56469
56470
56474
56475
56479
56480
56481
56482
56483
56484

56485
56487
56488
56489
56490
56491
56492
56493
56494
56495
56496
56497

56498
56499
56503
56508
56509
56510
56511
56512
56513
56514
56515
56516

56517
56518
56519
56520
56521
56522
56523
56524
56529
56530
56531
56532

56533
56534
56535
56536
56537
56538
56539
56540
56543
56544
56545
56546

56547
56548
56549
56572
56573
56574
56575
56578
56579
56580
56581
56582

56583
56584
56589
56590
56592
56602
56603
56604
56605
56607
56608
56609

56610
56611
56612
56613
56614
56615
56616
56617
56618
56619
56620
56621

56622
56623
56624
56625
56626
56627
56628
56629
56630
56631
56632
56633

56634
56635
56636
56637
56638
56639
56642
56643
56644
56645
56646
56647

56648
56649
56650
56651
56652
56653
56658
56659
56660
56661
56662
56665

56666
56667
56670
56671
56672
56673
56674
56675
56676
56677
56678
56679

56683
56692
56693
56694
56695
56696
56697
56706
56707
56708
56709
56712

56713
56714
56715
56716
56717
56727
56728
56729
56730
56741
56742
56743

56765
56766
56767
56782
56783
56784
56785
56786
56787
56788
56798
56799

56800
56803
56804
56805
56809
56810
56811
56812
56813
56814
56817
56818

56819
56820
56821
56822
56823
56824
56825
56833
56834
56835
56836
56837

56838
56839
56840
56841
56842
56845
56846
56847
56851
56852
56853
56854

56855
56856
56857
56858
56859
56860
56861
56862
56863
56864
56865
56869

56870
56871
56872
56873
56874
56875
56876
56877
56878
56879
56880
56881

56882
56883
56884
56885
56886
56887
56888
56889
56890
56891
56892
56893

56894
56895
56896
56897
56898
56899
56900
56901
56902
56906
56907
56908

56909
56910
56911
56912
56913
56914
56915
56916
56917
56918
56919
56920

56921
56922
56923
56924
56925
56926
56927
56928
56929
56930
56931
56932

56933
56934
56935
56936
56937
56941
56942
56943
56944
56945
56946
56949

56950
56951
56952
56953
56954
56955
56956
56957
56958
56959
56964
56965

56966
56967
56968
56969
56970
56971
56972
56973
56974
56975
56976
56977

56978
56979
56980
56981
56982
56983
56984
56985
56986
56987
56988
56993

56994
56995
57000
57001
57002
57003
57004
57005
57006
57007
57008
57009

57010
57011
57012
57013
57014
57015
57019
57020
57021
57022
57023
57024

57025
57026
57027
57028
57029
57030
57031
57036
57037
57038
57039
57040

57041
57042
57043
57044
57047
57048
57049
57050
57051
57052
57056
57057

57058
57059
57060
57061
57062
57063
57064
57065
57066
57067
57068
57069

57070
57071
57072
57073
57074
57075
57076
57079
57080
57081
57082
57083

57084
57085
57086
57087
57088
57089
57090
57091
57092
57093
57094
57095

57096
57107
57108
57109
57130
57131
57132
57133
57134
57135
57136
57139

57140
57146
57147
57155
57156
57157
57158
57163
57164
57167
57168
57169

57170
57171
57172
57177
57178
57179
57180
57185
57186
57187
57188
57189

57190
57191
57192
57193
57194
57195
57197
57198
57203
57204
57205
57222

57226
57227
57228
57229
57236
57237
57238
57239
57240
57241
57242
57243

57244
57245
57246
57247
57248
57249
57253
57254
57255
57256
57257
57258

57259
57260
57261
57262
57263
57264
57278
57279
57280
57281
57286
57287

57288
57289
57290
57291
57292
57293
57296
57297
57301
57302
57303
57304

57305
57306
57307
57310
57311
57312
57313
57314
57315
57316
57317
57322

57323
57324
57325
57326
57327
57328
57329
57330
57331
57334
57335
57336

57337
57338
57339
57340
57341
57342
57352
57353
57354
57355
57356
57357

57358
57369
57370
57371
57372
57373
57374
57408
57409
57413
57414
57415

57435
57436
57437
57438
57445
57446
57447
57454
57455
57456
57457
57458

57459
57460
57465
57466
57467
57468
57469
57470
57471
57472
57473
57474

57475
57476
57477
57478
57479
57487
57488
57489
57490
57491
57494
57495

57496
57505
57506
57507
57513
57514
57515
57516
57517
57518
57519
57520

57521
57522
57523
57526
57527
57528
57531
57532
57533
57534
57535
57536

57537
57538
57539
57540
57541
57542
57543
57544
57545
57547
57548
57549

57555
57556
57557
57558
57559
57562
57563
57564
57565
57566
57567
57568

57569
57570
57571
57572
57573
57576
57577
57578
57579
57580
57581
57582

57583
57584
57585
57586
57587
57588
57589
57590
57591
57592
57593
57594

57595
57596
57597
57598
57601
57602
57603
57604
57605
57606
57607
57608

57609
57610
57611
57612
57613
57614
57615
57616
57617
57618
57622
57623

57624
57625
57626
57627
57628
57629
57630
57631
57632
57633
57634
57635

57636
57637
57638
57639
57640
57646
57647
57648
57649
57650
57653
57654

57655
57659
57660
57661
57662
57663
57664
57665
57666
57667
57668
57669

57670
57671
57672
57673
57674
57675
57676
57677
57678
57679
57680
57681

57682
57683
57686
57687
57688
57690
57691
57692
57693
57694
57695
57700

57701
57702
57703
57704
57705
57709
57710
57711
57712
57713
57714
57715

57716
57717
57718
57719
57720
57728
57729
57730
57731
57740
57741
57742

57743
57744
57745
57746
57747
57748
57749
57750
57751
57752
57753
57754

57755
57756
57757
57758
57759
57760
57763
57764
57765
57766
57767
57768

57769
57770
57771
57772
57773
57774
57775
57776
57777
57778
57779
57780

57781
57782
57783
57784
57785
57786
57787
57788
57789
57790
57791
57792

57793
57794
57803
57804
57805
57808
57809
57810
57811
57812
57813
57816

57817
57818
57819
57820
57821
57824
57825
57826
57827
57828
57829
57830

57834
57835
57836
57838
57839
57840
57845
57846
57847
57849
57850
57851

57852
57853
57854
57855
57856
57857
57858
57859
57860
57861
57862
57863

57864
57865
57866
57867
57868
57869
57872
57873
57874
57875
57876
57877

57878
57879
57880
57881
57882
57883
57884
57890
57891
57892
57895
57896

57897
57898
57899
57900
57901
57904
57905
57906
57907
57908
57909
57910

57911
57912
57913
57914
57915
57916
57917
57918
57919
57920
57921
57923

57924
57925
57926
57927
57928
57929
57930
57931
57932
57933
57934
57935

57936
57937
57938
57939
57940
57941
57942
57943
57944
57945
57946
57947

57948
57949
57950
57951
57952
57953
57954
57955
57956
57957
57958
57959

57960
57961
57962
57963
57964
57965
57966
57967
57968
57969
57970
57971

57972
57973
57974
57975
57976
57977
57978
57985
57986
57987
57988
57989

57990
57991
57992
57993
57994
57995
57996
57997
58004
58005
58006
58007

58008
58009
58010
58014
58015
58016
58020
58021
58022
58026
58027
58028

58032
58033
58034
58038
58039
58040
58041
58042
58043
58044
58045
58046

58050
58051
58052
58053
58054
58055
58056
58057
58058
58059
58060
58061

58062
58063
58064
58068
58069
58070
58071
58072
58073
58074
58075
58076

58083
58084
58085
58086
58087
58088
58089
58090
58091
58092
58093
58094

58095
58096
58097
58098
58099
58100
58101
58102
58103
58110
58111
58112

58116
58117
58118
58122
58123
58124
58125
58126
58127
58128
58129
58130

58131
58132
58133
58134
58135
58136
58137
58138
58139
58140
58141
58142

58143
58147
58148
58149
58150
58151
58152
58153
58154
58155
58156
58157

58158
58159
58160
58161
58165
58166
58167
58171
58172
58173
58174
58175

58176
58177
58178
58179
58182
58183
58184
58197
58198
58199
58215
58216

58217
58219
58220
58221
58223
58224
58225
58227
58228
58233
58234
58235

58236
58237
58238
58247
58248
58249
58250
58251
58252
58253
58254
58255

58256
58257
58258
58259
58260
58261
58272
58273
58274
58275
58276
58277

58278
58285
58286
58287
58288
58289
58290
58291
58292
58293
58294
58295

58296
58297
58298
58309
58310
58321
58322
58323
58324
58325
58326
58327

58331
58332
58333
58334
58335
58336
58337
58338
58339
58340
58341
58342

58343
58344
58345
58346
58347
58348
58349
58350
58359
58360
58361
58362

58363
58364
58365
58366
58367
58368
58369
58370
58371
58372
58373
58374

58375
58376
58377
58378
58379
58380
58381
58382
58383
58384
58385
58390

58391
58392
58393
58398
58399
58400
58401
58402
58403
58404
58405
58409

58410
58411
58412
58417
58418
58419
58420
58421
58422
58423
58424
58425

58426
58427
58428
58432
58433
58434
58435
58440
58441
58442
58443
58444

58445
58446
58447
58448
58449
58450
58451
58452
58453
58454
58455
58456

58457
58462
58463
58464
58465
58466
58467
58468
58469
58470
58471
58472

58473
58474
58475
58476
58477
58478
58479
58480
58481
58482
58483
58484

58485
58486
58487
58488
58489
58490
58491
58492
58493
58494
58495
58496

58497
58498
58499
58500
58501
58502
58503
58504
58505
58506
58507
58508

58509
58510
58511
58512
58513
58514
58515
58516
58517
58522
58523
58524

58525
58526
58527
58528
58529
58530
58531
58532
58533
58534
58535
58536

58537
58538
58539
58540
58541
58542
58543
58548
58549
58550
58551
58552

58553
58554
58555
58556
58557
58558
58559
58560
58561
58562
58563
58564

58565
58566
58567
58568
58569
58570
58571
58572
58573
58574
58575
58576

58577
58580
58581
58582
58583
58586
58587
58589
58590
58591
58592
58593

58594
58595
58596
58597
58598
58599
58600
58601
58602
58603
58604
58605

58606
58607
58608
58609
58610
58611
58612
58613
58614
58615
58616
58617

58618
58619
58620
58621
58622
58623
58624
58625
58626
58627
58628
58629

58630
58631
58632
58633
58634
58635
58636
58637
58638
58639
58640
58647

58648
58649
58650
58651
58652
58653
58654
58655
58656
58657
58658
58659

58660
58662
58663
58664
58665
58666
58667
58668
58669
58670
58671
58672

58673
58674
58675
58676
58677
58678
58679
58680
58681
58682
58683
58684

58685
58686
58687
58688
58689
58690
58691
58692
58693
58694
58695
58696

58697
58698
58699
58700
58710
58711
58715
58716
58717
58718
58719
58722

58723
58724
58725
58726
58727
58728
58748
58749
58750
58751
58753
58754

58755
58767
58788
58789
58790
58791
58792
58793
58794
58795
58796
58797

58802
58803
58804
58805
58809
58810
58811
58812
58813
58814
58815
58818

58819
58820
58821
58822
58823
58824
58827
58828
58829
58830
58831
58832

58833
58834
58835
58836
58837
58838
58839
58840
58847
58848
58849
58850

58851
58852
58856
58857
58858
58863
58874
58875
58876
58877
58911
58912

58913
58914
58915
58916
58917
58922
58923
58924
58925
58926
58927
58928

58929
58930
58931
58932
58933
58934
58935
58936
58937
58938
58939
58942

58943
58944
58945
58946
58947
58948
58949
58950
58951
58952
58953
58954

58955
58956
58957
58958
58961
58962
58963
58964
58965
58966
58967
58968

58969
58971
58972
58973
58974
58975
58976
58977
58978
58979
58980
58981

58982
58983
58984
58985
58986
58987
58988
58989
58990
58991
58992
58993

58994
58995
58996
58997
58998
58999
59000
59001
59002
59003
59004
59005

59006
59007
59011
59012
59013
59014
59015
59016
59017
59019
59020
59021

59022
59023
59024
59025
59026
59027
59028
59029
59030
59031
59032
59033

59034
59035
59036
59037
59038
59039
59040
59041
59042
59043
59044
59045

59046
59047
59048
59049
59050
59051
59052
59053
59054
59055
59056
59057

59058
59059
59060
59061
59062
59063
59064
59065
59066
59067
59068
59069

59070
59071
59072
59073
59074
59075
59076
59077
59078
59079
59080
59081

59082
59083
59084
59085
59086
59087
59088
59089
59090
59091
59092
59093

59094
59095
59101
59102
59103
59104
59105
59106
59107
59108
59109
59110

59111
59112
59113
59116
59117
59118
59119
59120
59121
59125
59126
59127

59128
59129
59130
59131
59132
59133
59134
59135
59136
59137
59138
59139

59140
59141
59142
59143
59144
59145
59146
59147
59148
59149
59150
59151

59152
59153
59154
59155
59156
59157
59158
59159
59160
59161
59162
59163

59164
59165
59166
59167
59168
59171
59172
59173
59174
59175
59176
59177

59178
59181
59182
59183
59186
59187
59188
59189
59190
59191
59192
59193

59194
59195
59196
59197
59198
59199
59200
59201
59202
59203
59204
59205

59206
59207
59208
59209
59210
59211
59212
59213
59214
59215
59218
59219

59220
59221
59222
59223
59224
59225
59226
59227
59228
59229
59230
59231

59232
59233
59234
59235
59238
59239
59240
59241
59242
59243
59245
59246

59247
59248
59249
59250
59251
59252
59253
59254
59255
59256
59257
59258

59259
59260
59261
59262
59263
59264
59265
59266
59267
59268
59269
59270

59271
59272
59273
59274
59275
59276
59277
59278
59279
59280
59281
59282

59283
59284
59285
59286
59287
59288
59289
59290
59291
59292
59293
59294

59295
59296
59297
59298
59299
59302
59303
59304
59305
59306
59307
59308

59309
59310
59311
59312
59313
59314
59315
59316
59317
59318
59319
59320

59321
59322
59323
59324
59325
59326
59327
59328
59329
59330
59331
59332

59337
59338
59339
59340
59341
59342
59343
59346
59347
59348
59349
59350

59351
59352
59353
59354
59355
59356
59357
59358
59359
59360
59361
59362

59363
59364
59365
59366
59369
59370
59371
59372
59373
59374
59375
59376

59377
59378
59379
59380
59381
59382
59383
59384
59385
59386
59393
59394

59395
59396
59397
59398
59399
59402
59403
59404
59405
59406
59407
59408

59409
59410
59411
59412
59413
59414
59415
59416
59417
59418
59419
59420

59421
59422
59423
59424
59425
59426
59427
59428
59429
59430
59431
59432

59433
59434
59435
59439
59440
59441
59442
59443
59444
59445
59446
59447

59448
59449
59450
59451
59452
59453
59454
59455
59456
59457
59458
59459

59460
59461
59462
59463
59464
59465
59466
59467
59468
59469
59470
59471

59472
59473
59474
59475
59476
59477
59478
59479
59480
59481
59482
59483

59484
59485
59486
59487
59488
59489
59490
59491
59492
59493
59494
59495

59496
59497
59498
59499
59500
59501
59502
59503
59504
59505
59506
59507

59508
59509
59510
59511
59512
59513
59514
59515
59516
59517
59518
59519

59520
59521
59522
59523
59524
59525
59526
59527
59528
59529
59530
59531

59532
59533
59534
59535
59536
59537
59538
59539
59540
59541
59542
59543

59544
59545
59546
59547
59548
59549
59550
59551
59552
59553
59554
59555

59556
59557
59558
59559
59560
59561
59562
59563
59564
59565
59566
59567

59568
59569
59570
59571
59572
59573
59574
59575
59576
59577
59578
59579

59580
59581
59582
59583
59584
59585
59586
59587
59588
59589
59590
59591

59592
59593
59594
59595
59596
59597
59598
59599
59600
59601
59602
59603

59604
59605
59606
59607
59608
59609
59610
59611
59612
59613
59614
59615

59616
59617
59618
59619
59620
59621
59622
59623
59624
59625
59626
59627

59628
59629
59630
59631
59632
59633
59634
59635
59636
59637
59638
59639

59640
59641
59642
59643
59644
59645
59646
59647
59648
59649
59650
59651

59652
59653
59654
59655
59656
59657
59658
59659
59660
59661
59662
59663

59664
59665
59666
59667
59668
59669
59670
59671
59672
59673
59674
59675

59676
59677
59678
59679
59680
59681
59682
59683
59684
59685
59686
59687

59688
59689
59690
59691
59692
59693
59694
59695
59696
59697
59698
59699

59700
59701
59702
59703
59704
59705
59706
59707
59708
59709
59710
59711

59712
59713
59714
59715
59716
59717
59718
59719
59720
59721
59722
59723

59724
59725
59726
59727
59728
59729
59730
59731
59732
59733
59734
59735

59736
59737
59738
59739
59740
59741
59742
59743
59744
59745
59746
59747

59748
59749
59750
59751
59752
59753
59754
59755
59756
59757
59758
59759

59760
59761
59762
59763
59764
59765
59766
59767
59768
59769
59770
59771

59772
59773
59774
59775
59776
59777
59778
59779
59780
59781
59782
59783

59784
59785
59786
59787
59788
59789
59790
59791
59792
59793
59794
59795

59796
59797
59798
59799
59800
59801
59802
59803
59804
59805
59806
59807

59808
59809
59810
59811
59812
59814
59815
59816
59817
59818
59819
59820

59821
59822
59823
59824
59825
59826
59827
59828
59829
59830
59831
59832

59833
59834
59835
59836
59837
59838
59839
59840
59841
59848
59849
59850

59851
59852
59853
59854
59855
59856
59857
59858
59859
59860
59861
59862

59863
59864
59865
59866
59867
59868
59869
59870
59871
59872
59873
59874

59875
59876
59877
59878
59879
59880
59881
59882
59883
59884
59885
59886

59887
59888
59889
59890
59891
59892
59893
59894
59895
59896
59897
59898

59899
59900
59901
59902
59903
59904
59905
59906
59907
59908
59911
59912

59913
59914
59915
59916
59917
59918
59919
59920
59921
59922
59923
59924

59925
59926
59927
59928
59929
59930
59931
59932
59933
59934
59935
59936

59937
59938
59939
59940
59941
59942
59943
59944
59945
59946
59947
59948

59949
59950
59951
59952
59953
59954
59955
59956
59957
59961
59962
59963

59964
59965
59966
59967
59968
59969
59970
59971
59972
59973
59974
59975

59976
59977
59978
59979
59980
59981
59982
59983
59984
59985
59986
59987

59988
59989
59990
59991
59992
59993
59994
59995
59996
59997
59998
59999

60000
60001
60002
60003
60004
60005
60006
60007
60008
60009
60010
60011

60012
60013
60014
60015
60016
60017
60018
60019
60020
60021
60022
60023

60024
60025
60026
60027
60028
60029
60030
60031
60032
60033
60034
60035

60036
60037
60038
60039
60040
60041
60042
60043
60044
60045
60046
60047

60048
60049
60050
60051
60052
60053
60054
60055
60056
60057
60058
60059

60060
60061
60062
60063
60064
60065
60066
60067
60068
60069
60070
60071

60072
60073
60074
60075
60076
60077
60078
60079
60080
60081
60086
60087

60088
60089
60090
60091
60092
60093
60094
60095
60096
60097
60098
60099

60100
60101
60102
60103
60104
60105
60106
60107
60108
60109
60110
60111

60112
60113
60114
60115
60116
60117
60118
60119
60120
60121
60122
60123

60124
60125
60126
60127
60128
60129
60130
60131
60132
60133
60134
60135

60136
60137
60138
60139
60140
60141
60142
60143
60144
60145
60146
60147

60148
60149
60150
60151
60152
60153
60154
60155
60156
60157
60158
60159

60160
60161
60162
60163
60164
60165
60166
60167
60168
60169
60170
60171

60172
60173
60174
60175
60176
60177
60178
60179
60180
60181
60182
60183

60184
60185
60186
60187
60188
60189
60190
60191
60192
60193
60194
60195

60196
60197
60198
60199
60200
60201
60202
60203
60204
60205
60206
60207

60208
60209
60210
60211
60212
60213
60214
60215
60216
60217
60218
60219

60220
60221
60223
60224
60225
60226
60227
60228
60229
60230
60231
60232

60233
60234
60235
60236
60237
60238
60239
60240
60241
60242
60243
60244

60245
60246
60247
60248
60249
60250
60251
60252
60253
60254
60255
60256

60257
60258
60259
60260
60261
60262
60263
60264
60265
60266
60267
60269

60270
60271
60272
60273
60274
60275
60276
60277
60278
60279
60280
60281

60282
60283
60284
60285
60286
60287
60288
60289
60290
60291
60292
60293

60298
60299
60300
60301
60302
60303
60304
60305
60306
60307
60308
60309

60310
60311
60312
60313
60314
60315
60316
60317
60318
60319
60320
60321

60322
60323
60324
60325
60326
60327
60328
60329
60330
60333
60334
60335

60336
60340
60341
60342
60343
60344
60345
60346
60347
60348
60349
60350

60351
60352
60353
60354
60355
60358
60359
60360
60365
60366
60367
60368

60369
60370
60371
60372
60373
60374
60375
60376
60377
60378
60379
60380

60381
60382
60386
60387
60388
60392
60393
60394
60395
60396
60397
60398

60399
60400
60401
60402
60403
60404
60405
60406
60407
60408
60409
60410

60411
60412
60413
60418
60419
60420
60421
60422
60423
60424
60425
60426

60427
60428
60429
60430
60431
60432
60433
60434
60435
60436
60437
60438

60439
60440
60441
60442
60443
60444
60447
60448
60449
60450
60451
60452

60453
60454
60455
60456
60457
60458
60459
60460
60469
60470
60471
60472

60473
60474
60475
60480
60481
60482
60483
60484
60485
60486
60487
60488

60489
60490
60492
60493
60494
60495
60496
60497
60498
60499
60500
60501

60502
60503
60504
60505
60506
60513
60514
60515
60516
60517
60518
60519

60520
60521
60522
60523
60524
60525
60526
60527
60528
60529
60530
60531

60532
60533
60534
60535
60536
60537
60538
60539
60540
60541
60542
60544

60545
60546
60547
60548
60549
60550
60551
60552
60553
60554
60555
60556

60557
60558
60559
60560
60561
60562
60563
60564
60565
60566
60567
60568

60570
60571
60572
60573
60574
60575
60576
60577
60578
60579
60580
60581

60582
60583
60584
60585
60586
60587
60588
60589
60590
60591
60592
60593

60594
60595
60596
60597
60598
60603
60604
60605
60606
60607
60608
60609

60619
60620
60621
60622
60623
60638
60639
60640
60660
60661
60665
60666

60667
60669
60670
60680
60681
60682
60683
60684
60686
60687
60688
60689

60696
60699
60700
60702
60703
60704
60705
60706
60707
60708
60709
60710

60711
60712
60713
60714
60715
60716
60720
60721
60722
60723
60724
60725

60726
60730
60731
60732
60733
60734
60735
60736
60737
60738
60739
60740

60741
60742
60743
60744
60745
60746
60750
60752
60753
60756
60757
60758

60759
60760
60761
60762
60767
60768
60769
60770
60771
60772
60780
60781

60782
60783
60793
60794
60795
60796
60797
60798
60799
60800
60801
60802

60803
60804
60805
60806
60807
60808
60809
60810
60811
60812
60813
60816

60817
60818
60819
60820
60821
60822
60823
60824
60825
60826
60827
60828

60829
60830
60831
60832
60833
60834
60835
60836
60837
60838
60839
60840

60841
60842
60846
60847
60848
60849
60850
60851
60852
60853
60854
60855

60856
60857
60858
60859
60860
60861
60862
60863
60864
60865
60866
60867

60868
60869
60870
60871
60872
60873
60877
60878
60879
60880
60881
60882

60883
60884
60885
60886
60887
60888
60889
60890
60891
60892
60893
60899

60900
60901
60902
60905
60906
60907
60910
60911
60912
60913
60914
60915

60916
60948
60949
60950
60951
60961
60962
60963
60964
60965
60969
60970

60991
60992
60993
60995
60996
61003
61004
61005
61007
61008
61009
61010

61011
61012
61013
61014
61015
61017
61018
61019
61020
61021
61022
61024

61025
61026
61032
61033
61034
61035
61036
61037
61038
61039
61040
61041

61042
61043
61044
61045
61046
61047
61048
61049
61050
61051
61052
61053

61054
61055
61056
61057
61058
61059
61060
61061
61062
61063
61064
61065

61066
61067
61068
61069
61070
61071
61072
61073
61074
61075
61076
61077

61078
61079
61080
61081
61082
61084
61085
61086
61087
61088
61089
61090

61091
61092
61100
61101
61102
61103
61104
61105
61107
61108
61109
61110

61111
61122
61123
61124
61125
61132
61133
61134
61135
61145
61146
61147

61148
61149
61150
61153
61154
61155
61160
61161
61163
61164
61165
61166

61172
61173
61174
61178
61179
61180
61189
61198
61199
61200
61218
61219

61220
61244
61245
61246
61247
61248
61249
61260
61261
61262
61271
61272

61273
61274
61275
61277
61278
61281
61282
61283
61290
61291
61292
61293

61294
61297
61298
61299
61300
61301
61302
61303
61304
61305
61306
61307

61308
61309
61310
61311
61312
61313
61314
61318
61319
61320
61321
61322

61323
61324
61325
61326
61327
61329
61330
61332
61333
61334
61335
61336

61337
61338
61339
61340
61341
61342
61343
61344
61345
61346
61347
61348

61349
61350
61351
61352
61353
61354
61355
61356
61357
61358
61359
61360

61361
61362
61363
61364
61365
61366
61367
61368
61369
61370
61371
61372

61373
61376
61377
61381
61382
61405
61406
61407
61408
61441
61442
61443

61450
61451
61452
61453
61454
61455
61457
61458
61459
61460
61461
61463

61465
61466
61467
61468
61469
61470
61471
61472
61473
61481
61482
61483

61484
61485
61488
61489
61490
61491
61495
61496
61497
61498
61502
61503

61504
61505
61506
61507
61508
61509
61510
61512
61513
61514
61515
61516

61517
61518
61519
61520
61521
61522
61523
61524
61525
61526
61527
61529

61530
61535
61536
61537
61539
61540
61541
61542
61543
61544
61545
61546

61547
61548
61549
61550
61551
61552
61553
61554
61555
61556
61557
61558

61559
61560
61561
61562
61563
61564
61565
61566
61567
61568
61569
61570

61571
61572
61573
61574
61575
61576
61577
61578
61579
61580
61581
61582

61583
61584
61585
61586
61587
61588
61589
61591
61592
61593
61594
61595

61596
61597
61598
61599
61602
61603
61605
61606
61607
61609
61610
61611

61612
61613
61614
61615
61616
61617
61618
61619
61620
61621
61624
61625

61626
61627
61628
61629
61631
61632
61634
61635
61636
61637
61638
61639

61640
61641
61642
61643
61644
61646
61647
61648
61651
61652
61653
61654

61655
61660
61661
61662
61666
61667
61668
61669
61670
61672
61673
61674

61675
61676
61677
61678
61683
61684
61685
61686
61687
61688
61689
61691

61692
61693
61694
61703
61704
61705
61706
61707
61708
61718
61719
61720

61721
61722
61723
61724
61725
61726
61727
61728
61729
61730
61731
61732

61733
61734
61739
61740
61744
61745
61748
61749
61758
61759
61765
61766

61767
61768
61769
61770
61774
61775
61782
61783
61784
61785
61786
61787

61788
61789
61791
61792
61794
61795
61797
61798
61799
61803
61804
61805

61807
61808
61809
61810
61814
61815
61816
61817
61823
61824
61825
61826

61827
61828
61834
61835
61844
61845
61847
61848
61849
61850
61852
61853

61858
61859
61860
61861
61867
61868
61869
61870
61872
61873
61875
61876

61877
61878
61879
61880
61881
61884
61885
61886
61887
61889
61890
61892

61893
61894
61899
61900
61903
61904
61905
61906
61907
61908
61916
61917

61921
61922
61923
61932
61933
61937
61938
61942
61943
61946
61947
61948

61949
61950
61951
61952
61953
61954
61955
61957
61958
61960
61961
61963

61964
61965
61966
61967
61968
61972
61973
61974
61975
61977
61978
61980

61981
61984
61985
61986
61987
1988
61991
61992
61993
61995
61996
52011

62012
62013
62020
62021
62023
62024
62025
62026
62027
62028
62029
62030

62031
62032
62033
62034
62035
62036
62037
62038
62039
62040
62041
62042

62046
62047
62048
62062
62069
62070
62071
62075
62101
62102
62114
62115

62116
62118
62119
62120
62121
62123
62124
62125
62126
62127
62130
62131

62132
62138
62139
62140
62141
62142
62143
62149
62150
62151
62152
62153

62154
62158
62159
62160
62161
62162
62163
62164
62170
62171
62172
62173

62174
62177
62178
62179
62180
62181
62182
62184
62185
62186
62187
62188

62194
62195
62198
62199
62200
62201
62205
62206
62207
62225
62226
62238

62239
62244
62245
62246
62247
62248
62249
62250
62251
62253
62254
62264

62266
62267
62268
62269
62271
62272
62273
62274
62276
62277
62278
62290

62291
62296
62297
62304
62305
62306
62307
62309
62310
62312
62313
62314

62315
62316
62317
62319
62320
62321
62322
62323
62324
62328
62329
62330

62331
62346
62347
62348
62357
62363
62364
62365
62366
62371
62372
62373

62374
62375
62376
62377
62378
62379
62380
62381
62382
62383
62387
62388

62397
62398
62399
62400
62401
62402
62403
62404
62416
62417
62418
62419

62423
62424
62425
62426
62427
62428
62429
62430
62431
62432
62435
62455

62456
62457
62458
62459
62460
62461
62462
62466
62476
62477
62478
62479

62480
62504
62505
62506
62507
62508
62515
62516
62517
62530
62531
62532

62533
62534
62543
62544
62545
62546
62560
62561
62562
62563
62564
62568

62569
62570
62580
62581
62582
62592
62593
62594
62595
62596
62597
62598

62599
62600
62601
62602
62603
62604
62605
62606
62612
62613
62618
62619

62623
62629
62634
62635
62636
62637
62638
62639
62640
62644
62653
62654

62655
62656
62657
62658
62659
62666
62667
62668
62669
62670
62671
62672

62678
62679
62680
62681
62682
62683
62697
62698
62699
62700
62705
62706

62707
62708
62709
62712
62713
62738
62739
62740
62746
62747
62748
62749

62762
62765
62766
62767
62768
62771
62772
62773
62774
62794
62795
62799

62820
62821
62822
62827
62828
62829
62835
62836
62837
62861
62862
62863

62864
62880
62881
62882
62904
62908
62909
62910
62913
62914
62915
62929

62931
62932
62935
62936
62943
62949
62950
62951
62953
62954
62955
62958

62959
62960
62961
62965
62967
62968
62969
62971
62972
62975
62976
62977

62978
62979
62980
62981
62982
62986
62987
62988
62989
62990
62996
62997

62998
62999
63000
63001
63002
63003
63004
63005
63006
63007
63008
63018

63025
63026
63028
63029
63035
63036
63043
63044
63045
63049
63050
63051

63052
63053
63058
63059
63070
63071
63072
63073
63085
63086
63095
63096

63097
63113
63114
63115
63117
63118
63119
63129
63130
63131
63132
63133

63140
63141
63142
63143
63144
63145
63154
63155
63156
63157
63158
63159

63160
63164
63165
63166
63176
63177
63178
63181
63182
63183
63184
63185

63186
63187
63188
63189
63190
63191
63192
63193
63194
63195
63196
63197

63199
63200
63201
63208
63209
63210
63216
63217
63218
63219
63220
63221

63222
63223
63224
63228
63229
63230
63231
63232
63245
63246
63247
63248

63249
63250
63251
63252
63253
63254
63255
63256
63260
63261
63262
63263

63264
63265
63271
63272
63273
63274
63275
63276
63285
63286
63287
63294

63295
63296
63302
63303
63304
63310
63324
63325
63326
63327
63328
63329

63330
63331
63332
63360
63361
63362
63404
63405
63406
63407
63408
63409

63410
63419
63427
63428
63429
63430
63434
63435
63436
63437
63438
63439

63440
63441
63446
63447
63458
63459
63460
63461
63462
63463
63464
63465

63466
63468
63469
63470
63472
63473
63474
63482
63484
63485
63494
63495

63498
63500
63501
63504
63505
63521
63522
63523
63535
63536
63537
63538

63542
63543
63544
63559
63560
63563
63566
63567
63568
63569
63570
63571

63572
63573
63574
63575
63576
63577
63587
63588
63589
63599
63600
63601

63602
63603
63609
63610
63611
63612
63613
63614
63615
63616
63617
63618

63619
63620
63621
63622
63623
63624
63628
63633
63639
63640
63641
63642

63643
63645
63646
63647
63648
63649
63650
63651
63652
63653
63658
63659

63665
63666
63668
63671
63672
63673
63674
63675
63676
63677
63685
63686

63688
63689
63690
63695
63696
63697
63707
63708
63709
63715
63716
63717

63718
63719
63720
63721
63722
63723
63725
63726
63731
63732
63733
63734

63736
63737
63738
63739
63740
63741
63742
63743
63746
63747
63749
63750

63751
63752
63753
63754
63755
63756
63758
63759
63760
63762
63764
63765

63766
63767
63768
63769
63770
63771
63772
63773
63774
63776
63777
63778

63780
63781
63786
63788
63789
63790
63791
63792
63793
63794
63795
63796

63797
63799
63801
63802
63804
63805
63806
63807
63808
63809
63810
63811

63813
63814
63823
63824
63825
63826
63827
63828
63829
63830
63831
63833

63834
63835
63836
63841
63843
63847
63848
63849
63850
63852
63853
63854

63855
63856
63857
63858
63865
63869
63870
63871
63872
63877
63878
63879

63880
63881
63882
63883
63884
63885
63886
63887
63888
63889
63890
63891

63892
63893
63894
63895
63896
63897
63898
63900
63901
63902
63904
63905

63906
63912
63913
63921
63922
63923
63946
63947
63948
63952
63953
53954

63963
63964
63969
63970
63971
63978
63979
63980
63987
63988
63989
63990

63991
63992
63993
63994
63995
63996
64001
64002
64003
64005
64006
64007

64008
64009
64010
64011
64012
64013
64014
64015
64016
64017
64018
64019

64020
64021
64022
64023
64024
64025
64026
64027
64028
64029
64030
64031

64032
64033
64034
64035
64036
64037
64038
64039
64040
64043
64044
64045

64046
64051
64052
64053
64054
64055
64056
64057
64058
64059
64068
64069

64071
64072
64073
64074
64075
64076
64077
64079
64080
64081
64082
64083

64084
64085
64086
64087
64088
64089
64090
64092
64093
64094
64095
64096

64097
64098
64099
64100
64102
64103
64104
64105
64106
64107
64108
64109

64110
64111
64112
64113
64114
64115
64116
64117
64118
64119
64120
64121

64122
64123
64124
64126
64127
64128
64129
64130
64131
64132
64133
64134

64135
64136
64137
64138
64139
64140
64143
64144
64145
64146
64147
64148

64151
64152
64169
64170
64172
64188
64189
64190
64191
64192
64193
64199

64209
64210
64211
64213
64214
64215
64221
64222
64223
64224
64225
64226

64227
64232
64244
64245
64246
64249
64256
64260
64266
64269
64271
64275

64276
64281
64282
64283
64284
64285
64286
64287
64291
64292
64300
64301

64302
64303
64311
64312
64318
64319
64320
64334
64335
64344
64345
64346

64348
64360
64361
64362
64364
64366
64377
64378
64384
64399
64416
64417

64418
64422
64426
64430
64431
64432
64433
64434
64439
64440
64442
64444

64445
64446
64447
64448
64449
64455
64456
64457
64461
64462
64463
64470

64472
64476
64477
64478
64479
64481
64485
64486
64487
64492
64493
64494

64496
64497
64498
64499
64500
64501
64502
64503
64504
64505
64507
64509

64510
64512
64518
64519
64523
64524
64525
64526
64527
64530
64531
64532

64533
64534
64538
64539
64540
64541
64542
64543
64544
64546
64547
64548

64549
64550
64551
64552
64553
64554
64555
64556
64557
64560
64561
64562

64567
64568
64571
64572
64578
64579
64580
64581
64582
64584
64585
64586

64587
64588
64601
64602
64603
64614
64615
64616
64623
64624
64625
64638

64639
64640
64641
64649
64650
64657
64658
64659
64673
64674
64675
64680

64681
64686
64689
64692
64694
64695
64696
64697
64698
64702
64703
64704

64705
64706
64707
64708
64709
64710
64711
64714
64721
64722
64725
64726

64727
64728
64730
64735
64746
64747
64757
64758
64759
64761
64767
64781

64782
64788
64789
64790
64791
64792
64794
64795
64796
64797
64807
64808

64809
64810
64811
64812
64813
64814
64815
64816
64818
64819
64820
64821

64826
64827
64828
64830
64834
64835
64836
64837
64838
64839
64840
64841

64842
64843
64844
64845
64846
64847
64848
64849
64850
64851
64855
64856

64857
64858
64859
64860
64861
64862
64863
64864
64865
64869
64870
64871

64872
64873
64874
64875
64876
64877
64878
64879
64880
64881
64882
54883

64886
64887
64888
64889
64890
64891
64892
64893
64894
64895
64896
64897

64898
64899
64900
64904
64905
64906
64907
64908
64909
64910
64911
64914

64915
64916
64917
64918
64919
64922
64923
64924
64925
64926
64927
64928

64931
64932
64933
64934
64935
64939
64940
64941
64942
64943
64944
64945

64946
64947
64948
64949
64950
64951
64960
64961
64962
64963
64964
64965

64966
64967
64968
64971
64976
64977
64978
64983
64984
64986
64987
64989

64994
64995
64996
64997
65001
65005
65006
65007
65018
65019
65023
65027

65028
65029
65030
65031
65032
65033
65064
65065
65066
65067
65075
65082

65083
65084
65087
65088
65089
65090
65091
65092
65093
65094
65095
65096

65097
65098
65099
65101
65102
65103
65107
65113
65114
65115
65116
65117

65118
65119
65126
65128
65129
65130
65153
65160
65161
65167
65168
65169

65182
65183
65189
65210
65211
65212
65213
65222
65223
65224
65230
65235

65236
65243
65244
65245
65246
65247
65248
65249
65256
65257
65258
65262

65263
65264
65268
65269
65270
65271
65274
65275
65276
65291
65292
65293

65299
65300
65304
65306
65311
65319
65321
65322
65332
65333
65335
65369

65370
65371
65373
65375
65376
65392
65414
65417
65428
65441
65452
65464

65473
65474
65477
65478
65488
65489
65494
65495
65496
65497
65501
65502

65512
65513
65520
65521
65522
65535
65536
65547
65548
65552
65553
65554

65555
65560
65561
65562
65563
65568
65569
65570
65572
65573
65574
65575

65578
65579
65583
65584
65587
65588
65590
65593
65594
65595
65596
65597

65598
65599
65600
65601
65602
65603
65604
65605
65606
65608
65609
65610

65611
65614
65615
65616
65617
65619
65620
65621
65622
65623
65624
65625

65626
65630
65631
65632
65633
65640
65641
65644
65645
65659
65660
65665

65666
65667
65668
65669
65670
65671
65672
65673
65674
65675
65676
65677

65678
65679
65682
65683
65685
65686
65687
65688
65689
65690
65693
65694

65695
65696
65699
65700
65701
65702
65703
65704
65705
65706
65707
65708

65709
65712
65713
65714
65715
65717
65718
65725
65726
65727
65728
65729

65730
65731
65733
65734
65737
65739
65741
65744
65745
65746
65747
65748

65749
65750
65751
65752
65753
65756
65757
65758
65761
65762
65763
65765

65771
65773
65774
65777
65778
65779
65780
65781
65782
65783
65785
65803

65804
65805
65806
65819
65820
65824
65825
65826
65838
65839
65840
65851

65852
65853
65854
65855
65863
65864
65866
65867
65868
65869
65870
65871

65872
65873
65876
65877
65878
65879
65881
65882
65887
65893
65894
65896

65897
65898
65899
65900
65901
65902
65903
65904
65905
65906
65907
65908

65909
65910
65911
65912
65916
65924
65925
65926
65928
65929
65933
65934

65937
65940
65941
65942
65943
65947
65959
55960
65961
65967
65969
66001

66002
66013
66014
66015
66016
66022
66023
66024
66025
66026
66029
66030

66031
66032
66035
66036
66039
66040
66041
66042
66045
66046
66047
66048

66053
66054
66063
66064
66065
66067
66068
66069
66070
66071
66072
66073

66074
66080
66081
66082
66085
66089
66090
66099
66100
66101
66102
66115

66116
66120
66121
66138
66139
66142
66143
66146
66147
66153
66154
66167

66168
66175
66176
66178
66179
66180
66181
66186
66187
66189
66195
66196

66197
66198
66199
66209
66210
66213
66214
66215
66216
66217
66218
66219

66220
66221
66222
66223
66224
66234
66235
66241
66242
66244
66245
66248

66249
66250
66251
66252
66253
66254
66255
66256
66257
66258
66259
66265

66266
66268
66269
66270
66271
66272
66275
66276
66277
66278
66281
66282

66283
66287
66289
66290
66298
66299
66310
66314
66332
66370
66371
66389

66390
66391
66397
66398
66399
66401
66402
66405
66406
66407
66408
66409

66413
66426
66446
66448
66449
66450
66463
66464
66465
66473
66476
66480

66490
66491
66492
66493
66500
66501
66503
66504
66510
66511
66512
66514

66515
66518
66525
66531
66537
66539
66540
66545
66546
66548
66549
66551

66552
66553
66554
66558
66572
66573
66577
66578
66580
66581
66591
66611

66622
66623
66629
66630
66632
66633
66637
66638
66640
66641
66643
66644

66645
66648
66649
66650
66651
66653
66654
66660
66661
66662
66663
66664

66666
66667
66668
66669
66684
66685
66689
66690
66692
66693
66701
66708

66709
66718
66719
66736
66737
66744
66745
66746
66747
66753
66754
66758

66759
66762
66763
66766
66767
66768
66769
66770
66786
66787
66789
66790

66791
66794
66795
66796
66797
66798
66800
66801
66802
66803
66804
66805

66806
66807
66810
66811
66815
66816
66822
66823
66824
66825
66826
66827

66828
66829
66830
66831
66832
66833
66834
66835
66836
66843
66844
66845

66846
66847
66848
66852
66853
66857
66858
66860
66861
66862
66863
66864

66865
66866
66869
66870
66873
66874
66875
66876
66877
66878
66889
66890

66891
66901
66904
66905
66906
66910
66911
66914
66915
66916
66917
66918

66921
66928
66929
66931
66932
66933
66937
66938
66940
66944
66945
66950

66951
66960
66969
66970
66971
66973
66977
66981
66982
66985
66986
66987

66988
66989
66990
66999
67000
67001
67007
67008
67009
67010
67011
67012

67013
67014
67015
67016
67017
67020
67021
67022
67026
67027
67028
67039

67040
67041
67050
67051
67052
67053
67054
67055
67057
67058
67064
67065

67066
67067
67068
67069
67071
67072
67073
67074
67075
67076
67077
67078

67079
67080
67081
67082
67083
67084
67085
67086
67087
67088
67094
67095

67096
67097
67100
6710
67102
67103
67104
67105
67107
67108
67109
67113

67114
67115
67118
67119
67120
67123
67124
67125
67126
67127
67128
67129

67130
67131
67132
67133
67134
67135
67136
67137
67139
67140
67141
67148

67149
67151
67152
67157
67158
67163
67164
67171
67175
67176
67177
67189

67190
67208
67227
67228
67232
67233
67250
67255
67256
67259
67275
67276

67277
67278
67279
67280
67287
67288
67289
67291
67292
67293
67294
67295

67297
67298
67299
67300
67301
67303
67304
67308
67309
67310
67311
67312

67313
67314
67315
67316
67321
67322
67323
67324
67328
67329
67330
67331

67333
67334
67335
67336
67337
67338
67339
67341
67342
67343
67345
67346

67347
67348
67349
67350
67351
67352
67353
67354
67355
67356
67357
67358

67360
67361
67362
67363
67364
67367
67368
67369
67373
67374
67375
67376

67377
67378
67379
67380
67381
67383
67384
67385
67386
67387
67388
67389

67390
67391
67394
67395
67396
67401
67402
67403
67407
67408
67409
67411

67412
67413
67414
67415
67416
67418
67419
67420
67422
67423
67424
67425

67426
67427
67428
67429
67430
67432
67433
67434
67437
67438
67439
67440

67441
67442
67448
67449
67450
67452
67453
67454
57471
67475
67476
67487

67488
67496
67497
67498
67499
67503
67504
67505
67509
67510
67511
67549

67556
67557
67558
67573
67576
67577
67579
67591
67593
67594
67595
67596

67599
67600
67602
67610
67612
67613
67623
67624
67625
67630
67631
67632

67640
67641
67643
67644
67645
67647
67648
67649
67652
67653
67654
67656

67657
67658
67659
67661
67662
67664
67665
67668
67671
67672
67677
67678

67685
67686
67687
67688
67689
67690
67691
67693
67696
67697
67698
67699

67701
67702
67703
67704
67710
67713
67714
67715
67721
67722
67725
67726

67727
67728
67730
67731
67744
67745
67746
67747
67748
67749
67752
67753

67759
67760
67762
67763
67765
67766
67767
67768
67769
67770
67772
67773

67777
67778
67781
67782
67785
67786
67787
67788
67791
67792
67793
67794

67797
67798
67800
67801
67819
67820
67828
67829
67834
67835
67847
67848

67849
67864
67865
67868
67869
67870
67871
67872
67873
67874
67875
67876

67877
67881
67882
67883
67888
67889
67890
67891
67892
67893
67894
67895

67896
67897
67900
67901
67902
67903
67904
67905
67906
67907
67909
67912

67913
67914
67917
67918
67920
67921
67926
67930
67931
67932
67933
67935

67937
67938
67939
67940
67941
67942
67944
67945
67953
67954
67956
67957

67958
67959
67960
67961
67962
67963
67967
67968
67971
67972
67976
67977

67981
67985
67986
67988
67989
67993
67994
67996
67997
67999
68007
68008

68009
68011
68012
68013
68014
68015
68016
68020
68021
68024
68025
68026

68027
68028
68031
68032
68034
68035
68036
68037
68038
68039
68046
68047

68048
68050
68051
68052
68053
68054
68055
68056
68057
68058
68059
68061

68062
68064
68065
68066
68067
68068
68069
68070
68073
68074
68075
68080

68081
68082
68083
68084
68085
68086
68087
68088
68089
68090
68093
68094

68099
68100
68101
68102
68106
68107
68108
68109
68117
68118
68119
68120

68121
68122
68123
68124
68125
68126
68127
68128
68129
68130
68133
68136

68137
68138
68139
68144
68145
68151
68152
68153
68154
68155
68156
68157

68158
68159
68160
68161
68162
68163
68164
68165
68172
68173
68174
68175

68178
68179
68180
68181
68184
68185
68188
68189
68190
68191
68192
68193

68198
68199
68200
68201
68206
68207
68208
68209
68217
68218
68224
68225

68232
68233
68234
68235
68236
68237
68246
68247
68248
68249
68252
68253

68256
68257
68258
68259
68263
68264
68265
68267
68268
68275
68276
68277

68278
68280
68281
68282
68283
68285
68286
68287
68288
68290
68291
68294

68295
68296
68297
68299
68300
68304
68307
68310
68311
68316
68317
68318

68320
68321
68322
68323
68330
68331
68332
68333
68336
68337
68338
68339

68343
68344
68349
68350
68351
68352
68353
68354
68355
68356
68357
68359

68363
68364
68365
68369
68370
68371
68372
68375
68382
68383
68387
68388

68389
68390
68397
68398
68405
68406
68407
68408
68420
68425
68426
68427

68431
68432
68433
68448
68449
68450
68451
68452
68453
68454
68455
68456

68457
68458
68459
68461
68462
68463
68464
68467
68468
68474
68475
68479

68490
68491
68492
68495
68500
68501
68502
68518
68519
68520
68521
68522

68523
68524
68526
68527
68528
68529
68530
68531
68532
68533
68534
68539

68540
68541
68544
68545
68546
68551
68552
68553
68554
68555
68556
68557

68559
68560
68561
68562
68563
68564
68565
68566
68568
68569
68570
68574

68575
68576
68577
68578
68579
68580
68581
68582
68583
68584
68585
68586

68587
68588
68589
68590
68591
68596
68597
68598
68599
68600
68601
68605

68606
68607
68608
68609
68610
68612
68613
68614
68615
68616
68617
68618

68619
68620
68621
68622
68623
68624
68625
68626
68627
68630
68631
68638

68639
68640
68641
68651
68666
68667
68678
68679
68693
68694
68710
68711

68724
68725
68727
68756
68773
68774
68778
68779
68780
68794
68795
68803

68810
68811
68829
68830
68831
68837
68838
68839
68842
68844
68845
68849

68850
68858
68863
68864
68865
68866
68867
68870
68872
68875
68876
68877

68879
68888
68889
68898
68899
68904
68919
68923
68924
68927
68928
68929

68936
68938
68939
68942
68947
68948
68949
68950
68951
68953
68954
68955

68957
68958
68959
68960
68961
68962
68963
68964
68965
68966
68969
68972

68973
68974
68977
68978
68980
68981
68982
68983
68985
68986
68990
68991

68994
68995
68996
68997
68998
68999
69000
69001
69002
69003
69004
69007

69008
69009
69010
69011
69015
69017
69021
69030
69031
69033
69034
69035

69036
69037
69040
69041
69043
69046
69048
69053
69058
69064
69073
69078

69079
69080
69085
69091
69092
69095
69100
69101
69102
69103
69107
69108

69109
69110
69111
69117
69120
69121
69125
69130
69131
69132
69134
69135

69136
69141
69142
69152
69184
69185
69189
69190
69198
69206
69208
69218

69219
69220
69221
69222
69225
69260
69261
69274
69283
69285
69286
69287

69288
69294
69295
69304
69305
69308
69309
69311
69312
69313
69314
69320

69321
69322
69325
69326
69334
69349
69352
69353
69362
69371
69375
69376

69379
69380
69381
69383
69384
69400
69424
69430
69436
69437
69440
69441

69450
69451
69452
69453
69454
69456
69457
69458
69459
69460
69466
69467

69470
69471
69472
69486
69487
69488
69490
69499
69507
69510
69511
69515

69516
69522
69528
69529
69532
69533
69537
69541
69542
69544
69545
69550

69559
69561
69562
69565
69567
69583
69584
69594
69601
69610
69615
69621

69629
69633
69634
69637
69642
69644
69650
69652
69654
69655
69656
69658

69667
69668
69678
69679
69681
69682
69683
69688
69695
69696
69705
69707

69709
69712
69719
69722
69731
69739
69740
69761
69769
69773
69778
69779

69780
69791
69792
69795
69796
69799
69803
69804
69805
69810
69812
69813

69816
69817
69818
69819
69820
69822
69823
69825
69826
69828
69829
69830

69831
69832
69834
69836
69840
69843
69846
69847
69849
69850
69854
69856

69859
69860
69861
69864
69867
69874
69876
69877
69878
69885
69886
69889

69891
69893
69895
69899
69903
69905
69906
69910
69913
69918
69921
69922

69923
69927
69928
69930
69932
69935
69936
69938
69947
69957
69965
69966

69969
69976
69996
70000
70001
70007
70008
70010
70015
70024
70025
70031

70039
70044
70050
70053
70058
70060
70069
70082
70084
70087
70088
70101

70120
70121
70123
70125
70126
70130
70131
70136
70137
70152
70164
70166

70167
70168
70172
70183
70186
70189
70196
70210
70211
70218
70227
70228

70230
70232
70237
70242
70246
70250
70251
70253
70258
70263
70265
70268

70269
70278
70302
70305
70306
70325
70327
70329
70336
70341
70362
70372

70388
70403
70404
70405
70406
70407
70410
70418
70419
70420
70423
70424

70426
70431
70432
70433
70434
70435
70437
70438
70439
70440
70441
70445

70456
70460
70472
70478
70480
70482
70499
70502
70503
70505
70512
70513

70515
70516
70517
70518
70525
70527
70528
70532
70541
70542
70545
70549

70551
70552
70567
70568
70570
70571
70572
70575
70582
70589
70596
70602

70604
70612
70615
70623
70627
70628
70629
70634
70635
70637
70643
70645

70652
70661
70672
70674
70681
70682
70683
70694
70700
70721
70724
70725

70735
70742
70753
70754
70755
70763
70764
70778
70784
70801
70810
70817

70823
70826
70831
70832
70842
70850
70871
70872
70887
70893
70900
70908

70914
70916
70924
70928
70929
70933
70936
70948
70954
70959
70973
70974

70992
70994
70997
71005
71028
71030
71032
71034
71037
71039
71040
71041

71043
71048
71049
71050
71052
71059
71060
71063
71469
71124
71694
71189

71071
71076
71079
71080
71087
71089
71090
71091
71092
71108
71112
71114

71115
71118
71124
71126
71127
71137
71138
71139
71141
71144
71155
71158

71164
71169
71172
71184
71189
71190
71191
71209
71222
71223
71224
71227

71228
71238
71239
71240
71242
71243
71244
71247
71256
71260
71264
71270

71277
71307
71308
71311
71312
71314
71315
71316
71317
71318
71319
71322

71323
71326
71327
71330
71335
71342
71351
71370
71377
71381
71384
71387

71393
71395
71397
71400
71401
71402
71407
71423
71425
71426
71433
71452

71469
71473
71474
71475
71479
71481
71490
71491
71496
71501
71510
71511

71528
71529
71532
71536
71537
71541
71545
71546
71549
71550
71552
71553

71554
71555
71557
71558
71571
71585
71586
71589
71590
71593
71599
71605

71612
71615
71616
71628
71633
71638
71640
71641
71660
71681
71683
71690

71694
71696
71698
75518
75519
75520
75521
75522
75523
75528
75530
75531

75532
75533
75534
75535
75536
75537
75538
75556
75558
75559
75563
75564

75565
75567
75568
75569
75574
75575
75579
75580
75581
75583
75591
75597

75598
75599
75607
75608
75609
75610
75617
75618
75624
75625
75633
75634

75635
75647
75648
75649
75650
75651
75652
75655
75656
75657
75662
75663

75664
75665
75666
75667
75668
75670
75671
75672
75673
75674
75675
75676

75677
75678
75684
75685
75686
75687
75688
75689
75691
75692
75693
75696

75697
75702
75703
75704
75734
75735
75736
75737
75738
75739
75740
75741

75742
75744
75745
75746
75747
75748
75749
75750
75751
75752
75756
75757

75758
75759
75760
75761
75763
75765
75769
75770
75771
75789
75790
75792

75793
75794
75795
75796
75799
75800
75802
75803
75804
75805
75806
75809

75810
75811
75813
75820
75821
75822
75831
75832
75833
75834
75845
75846

75847
75857
75873
75874
75875
75876
75877
75878
75879
75881
75882
75883

75884
75887
75894
75895
75896
75898
75899
75900
75915
75918
75919
75920

75921
75922
75927
75929
75930
75931
75932
75933
75934
75935
75944
75949

75950
75951
75953
75954
75955
75956
75959
75965
75966
75967
75968
75971

75972
75973
75975
75976
75977
75988
75989
75993
75994
75995
75996
75997

76008
76009
76010
76031
76032
76034
76037
76038
76039
76040
76041
76043

(22) Protein whose gene expression changes in response to lauric acid stress (SEQ ID NOs)

4449
4453
4454
4465
4503
4518
4594
4617
4618
4634
4635
4665
4679

4685
4686
4742
4744
4745
4747
4749
4750
4751
4760
4761
4763

4765
4766
4772
4786
4850
4972
4973
5120
5396
5556
5695
5815

5840
5842
5849
5897
5932
5938
5942
6013
6049
6082
6140
6141

6142
6158
6208
6218
6293
6350
6468
6475
6481
6522
6579
10271

10275
10276
10280
10281
10295
10296
10357
10369
10476
10478
10498
10514

10516
10539
10540
10580
10614
10618
10638
10640
10656
10657
10677
10723

10731
10745
10752
10855
10862
10863
10864
10905
11057
11091
11238
11311

11343
11452
11496
11594
11622
11623
11682
11684
11839
11904
11987
12018

12019
16769
16770
16771
16772
16773
16774
16775
16776
16777
16780
16782

16783
16784
16785
16786
16787
16788
16789
16790
16791
16792
16793
16794

16795
16799
16800
16801
16802
16803
16804
16805
16806
16807
16808
71282

71076
71079
71080
71081
71083
71084
71089
71090
71116
71121
71157
71159

71171
71177
71182
71184
71195
71196
71211
71218
71221
71233
71235
71243

71244
71245
71253
71262
71265
71273
71276
71282
71287
71293
71317
71319

71323
71325
71326
71327
71335
71360
71372
71383
71413
71440
71445
71461

71463
71475
71476
71494
71505
71508
71520
71560
71569
71570
71591
71593

71602
71628
71641
71650
71655
71682
71697
71698
75518
75519
75520
75521

75522
75523
75525
75526
75530
75531
75532
75566
75571
75572
75601
75602

75603
75604
75605
75606
75615
75617
75618
75626
75627
75636
75639
75646

75659
75661
75671
75672
75673
75674
75675
75682
75695
75698
75706
75750

75751
75752
75755
75756
75757
75758
75759
75760
75763
75791
75798
75839

75852
75877
75878
75879
75937
75943
75957
75959
75996
75997
76004
76030

76042
76043

Included in all nucleic acid sequences disclosed herein are DNA nucleic acid molecules, RNA nucleic acid molecules (e.g., thymidine replaced with uridine), nucleic acid molecules encoding orthologs of the encoded proteins, as well as DNA or RNA nucleic acid sequences or any variant thereof (a structural variant or a chemical variant (e.g., chemically modified nucleotide)) comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the nucleic acid sequence of any SEQ ID NO presented herein, or a portion thereof. Such nucleic acid molecules can have a function of the full-length nucleic acid (e.g., for the intended function of inducing an immune response) as described further herein.

Included in all amino acid sequences disclosed herein are amino acid sequences or any variant thereof (a structural variant or a chemical variant) comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the amino acid sequence of any SEQ ID NO listed in presented herein, or a portion thereof. Such polypeptides can have a function of the full-length polypeptide (e.g., for the intended function of inducing an immune response) as described further herein.

Homology

Function-conservative variants are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Amino acids other than those indicated as conserved may differ in a protein so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm. A function-conservative variant also includes a polypeptide which has at least 60% amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, more preferably at least 85%, still preferably at least 90%, and even more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein to which it is compared.

Homology, as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotide sequence 5′-TATGGC-3′ share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

For nucleic acids, the term “substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more of the nucleotides, and more preferably at least about 97%, 98%, 99% or more of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=#of identical positions/total #of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available on the world wide web at the GCG company website), using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11 17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444 453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available on the world wide web at the GCG company website), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used (available on the world wide web at the NCBI website).

Codon Optimization

In preferred embodiments, the nucleic acid (e.g., DNA or RNA) vaccines of the present disclosure comprise those that are codon-optimized for expression in a host cell or a subject.

Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at the “Codon Usage Database,” available World Wide Web at kazusa.or.jp/codon/, and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). In preferred embodiments, codon tables from the following website are used: World Wide Web at kazusa.or.jp/codon/.

Accordingly, the term “codon-optimized” encompasses any modification of the nucleic acid sequence to comprise at least one codon that is more frequently used in a given host cell or subject. The term “codon-optimized” is not intended to mean that all codons in the nucleic acid are optimized for expression in a given host cell or subject.

Polypeptide and/or Peptide Vaccines

In certain aspects, provided herein are vaccines that utilize peptides and/or polypeptides that comprise the sequence of at least one cell surface protein of at least one methanogen, or any portion thereof. Such peptides and/or polypeptides can be chemically synthesized, or produced using an expression vector (e.g., bacteria, yeast, insect cells, mammalian cells) or in vitro translated, e.g., via methods described herein and/or those known in the art.

The polypeptide and/or peptide vaccines of the present disclosure may comprise a single peptide or a single polypeptide. Alternatively, the polypeptide and/or peptide vaccines of the present disclosure may comprise a mixture of various peptides and/or polypeptides that target at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell surface protein of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 methanogens.

In some embodiments, the polypeptide and/or peptide vaccine of the present disclosure comprises at least two different fragments of the same protein. In some embodiments, the polypeptide and/or peptide vaccine comprises at least two different fragments and/or two different polypeptides of different proteins. In some embodiments, the polypeptide and/or peptide vaccine comprises multiple fragments and/or polypeptides of different proteins of different methanogens. In some embodiments, the polypeptide and/or peptide vaccine comprises multiple fragments and/or polypeptides of different proteins of the same methanogen.

In some embodiments, the polypeptide(s)/peptide(s) concentration in a vaccine composition is from about 0.001 mg to about 500 mg per mL. In some embodiments, the concentration of the polypeptide(s)/peptide(s) in a vaccine composition is from about 0.01 mg to about 50 mg per mL. In some embodiments, the concentration range may be between about 0.1 mg and about 5 mg per mL.

In some embodiments, the polypeptide(s)/peptide(s) concentration in a vaccine composition is at least about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, or 50 mg per mL.

The polypeptide and/or peptide vaccines of the present disclosure may be administered in a pharmaceutical composition described herein. The protein vaccines of the present disclosure may be administered with an adjuvant and/or other agents that enhance immune response. Multiple dosings of the protein vaccine is contemplated herein as described.

Production of Polypeptides and/or Peptides

Methanogen proteins can be produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector, the expression vector is introduced into a host cell (as described above), and the methanogen protein is expressed in the host cell. The methanogen protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a methanogen protein, polypeptide, or peptide can be synthesized chemically using standard peptide synthesis techniques. Accordingly, the amino acid sequences disclosed herein will enable those of skill in the art to produce polypeptides corresponding peptide sequences and sequence variants thereof. Such polypeptides can be produced in host cells by expression of polynucleotides encoding the peptide sequence, frequently as part of a larger polypeptide. A host cell for expression can be any prokaryotic or eukaryotic cell. For example, protein can be expressed in bacterial cells such as E. coli, insect cells (e.g., SF9, SF21, etc.), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, etc.) or mammalian cells (such as Fao hepatoma cells, primary hepatocytes, Chinese hamster ovary cells (CHO), COS cells, etc.). Other suitable host cells are known to those skilled in the art. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are described further in Maniatis et al. Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J. Am. Chem. Soc. 91:501; Chaiken I. M. (1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference).

Peptides can be produced, typically by direct chemical synthesis. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus. In certain preferred embodiments, either the carboxy-terminus or the amino-terminus, or both, are chemically modified. The most common modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively. Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, can be incorporated into various embodiments of the invention. Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others.

Pharmaceutical Composition

Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, etc.), or other compositions of the present disclosure may be in a pharmaceutical composition, and thus further comprise at least one excipient and/or carrier described herein or those known in the art.

The vaccine, antibody, milk, animal feed, or agent of the present disclosure (e.g., polypeptide and/or peptide vaccines) may comprise at least one excipient that (1) increases stability; (2) permits the sustained or delayed release (e.g., from a depot formulation); and/or (3) alters the biodistribution (e.g., target to specific tissues or cell types). In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, excipients of the present disclosure include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, hyaluronidase, nanoparticle mimics, and combinations thereof.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.

A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient (e.g., the vaccine). The amount of the active ingredient may be generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.01% and 99% (w/w) of the active ingredient. By way of example, the composition may comprise between 0.01% and 100%, e.g., between 0.05 and 50%, between 0.1-30%, between 5-80%, at least 80% (w/w) active ingredient.

Pharmaceutical compositions may comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21^stEdition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surface active agents and/or emulsifiers, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in the pharmaceutical formulations of the invention.

Controlled/Sustained Release

In some embodiments, the compositions or agents of the present invention can be formulated for controlled release and/or targeted delivery. As used herein, “controlled release” refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a specific outcome.

In some embodiments, the compositions or agents may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term “encapsulate” means to enclose, surround or encase. As it relates to the formulation of the compounds of the invention, encapsulation may be substantial, complete or partial. The term “substantially encapsulated” means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. “Partially encapsulation” means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the invention using fluorescence and/or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the delivery agent.

In some embodiments, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (International Pub. No. WO2012131104 and WO2012131106; the contents of each of which is herein incorporated by reference in its entirety).

Excipients

Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, etc.), or other compositions of the present disclosure may comprise at least one excipient and/or carrier described herein or those known in the art (e.g., a pharmaceutically acceptable excipient and/or carrier).

A pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, antioxidants, osmolality adjusting agents, pH adjusting agents and the like, as suited to the particular dosage form desired.

In some embodiments, one or more excipients or accessory ingredients may make up greater than 50% of the total mass or volume of a pharmaceutical composition. In some embodiments, the one or more excipients or accessory ingredients may make up at least about 50%, 60%, 70%, 80%, 90%, or more of a pharmaceutical convention.

Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21^stEdition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety).

In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical compositions. The composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRU®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids (e.g., glycine); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and/or combinations thereof.

Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Oxidation is a potential degradation pathway for many compounds. In order to prevent oxidation, antioxidants can be added to the formulation. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®, NEOLONE™, KATHON™, and/or EUXYL®.

In some embodiments, the pH of the vaccine solutions are maintained between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and/or sodium malate. In another embodiment, the exemplary buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions.

Exemplary buffering agents may also include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and/or combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.

Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition.

Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), addition of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In addition, antioxidants and suspending agents can be used.

Cryoprotectants

In some embodiments, the lipid nanoparticles and/or pharmaceutical compositions of the disclosure are refrigerated or frozen for storage and/or shipment (e.g., being stored at a temperature of 4° C. or lower, such as a temperature between about −150° C. and about 0° C. or between about −80° C. and about −20° C. (e.g., about −5° C., −10° C., −15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C., −80° C., −90° C., −130° C. or −150° C.). For example, the pharmaceutical composition comprising one or more lipid nanoparticles is a solution or solid (e.g., via lyophilization) that is refrigerated for storage and/or shipment at, for example, about −20° C., −30° C., −40° C., −50° C., −60° C., −70° C., or −80° C. In certain embodiments, the disclosure also relates to a method of increasing stability of the lipid nanoparticles and by storing the lipid nanoparticles and/or pharmaceutical compositions thereof at a temperature of 4° C. or lower, such as a temperature between about −150° C. and about 0° C. or between about −80° C. and about −20° C., e.g., about −5° C., −10° C., −15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C., −80° C., −90° C., −130° C. or −150° C.).

In some embodiments, vaccine formulations may comprise cryoprotectants. As used herein, there term “cryoprotectant” refers to one or more agent that when combined with a given substance, helps to reduce or eliminate damage to that substance that occurs upon freezing. In some embodiments, cryoprotectants are combined with vaccines in order to stabilize them during freezing. Frozen storage of vaccines between −20° C. and −80° C. may be advantageous for long term (e.g., 36 months) storage. In some embodiments, cryoprotectants are included in vaccine formulations through freeze/thaw cycles and under frozen storage conditions. Cryoprotectants of the present invention may include, but are not limited to sucrose, trehalose, lactose, glycerol, dextrose, raffinose and/or mannitol. Trehalose is listed by the Food and Drug Administration as being generally regarded as safe (GRAS) and is commonly used in commercial pharmaceutical formulations.

Inactive Ingredients

In some embodiments, vaccine formulations may comprise at least one excipient which is an inactive ingredient. As used herein, the term “inactive ingredient” refers to one or more inactive agents included in formulations. Exemplary non-exhaustive lists of inactive ingredients and the routes of administration the inactive ingredients may be formulated in are described in Tables 7-8.

TABLE 7

Exemplary inactive ingredients

Inactive Ingredient
Route of Administration

Alpha-Terpineol
Topical

Alpha-Tocopherol
Intravenous; Topical

Alpha-Tocopherol Acetate, DI-
Topical

Alpha-Tocopherol, DI-
Intravenous; Topical

1,2,6-Hexanetriol
Topical

1,2-Dimyristoyl-Sn-Glycero-3-(Phospho-S-
Intravenous; Infusion (IV)

(1-Glycerol))

1,2-Dimyristoyl-Sn-Glycero-3-
Intravenous; Infusion (IV)

Phosphocholine

1,2-Dioleoyl-Sn-Glycero-3-Phosphocholine
Epidural

1,2-Dipalmitoyl-Sn-Glycero-3-(Phospho-
Epidural

Rac-(1-Glycerol))

1,2-Distearoyl-Sn-Glycero-3-(Phospho-Rac-
Intravenous

(1-Glycerol))

1,2-Distearoyl-Sn-Glycero-3-Phosphocholine
Intravenous

1-O-Tolylbiguanide
Topical

TABLE 8

Exemplary inactive ingredients

Route of Administration
Inactive Ingredient

Intrathecal (AN, CNBLK)
Acetone Sodium Bisulfite; Citric Acid; Hydrochloric Acid; Sodium

Chloride; Sodium Hydroxide; Sodium Metabisulfite

Infiltration (AN)
Acetic Acid; Acetone Sodium Bisulfite; Ascorbic Acid; Benzyl

Alcohol; Calcium Chloride; Carbon Dioxide; Chlorobutanol; Citric

Acid; Citric Acid Monohydrate; Edetate Calcium Disodium; Edetate

Disodium; Hydrochloric Acid; Hydrochloric Acid, Diluted; Lactic Acid;

Methylparaben; Monothioglycerol; Nitrogen; Potassium Chloride;

Potassium Metabisulfite; Potassium Phosphate, Monobasic;

Propylparaben; Sodium Bisulfite; Sodium Carbonate; Sodium Chlorate;

Sodium Chloride; Sodium Citrate; Sodium Hydroxide; Sodium Lactate;

Sodium Metabisulfite; Sodium Phosphate, Dibasic, Heptahydrate

Sympathetic NBLK (AN)
Hydrochloric Acid; Sodium Chloride; Sodium Hydroxide

Auricular (Otic)
Acetic Acid; Aluminum Acetate; Aluminum Sulfate Anhydrous;

Benzalkonium Chloride; Benzethonium Chloride; Benzyl Alcohol;

Boric Acid; Calcium Carbonate; Cetyl Alcohol; Chlorobutanol;

Chloroxylenol; Citric Acid; Creatinine; Cupric Sulfate; Cupric Sulfate

Anhydrous; Edetate Disodium; Edetic Acid; Glycerin; Glyceryl

Stearate; Hydrochloric Acid; Hydrocortisone; Hydroxyethyl Cellulose;

Isopropyl Myristate; Lactic Acid; Lecithin, Hydrogenated;

Methylparaben; Mineral Oil; Petrolatum; Petrolatum, White;

Phenylethyl Alcohol; Polyoxyl 40 Stearate; Polyoxyl Stearate;

Polysorbate 20; Polysorbate 80; Polyvinyl Alcohol; Potassium

Metabisulfite; Potassium Phosphate, Monobasic; Povidone K901;

Povidones; Propylene Glycol; Propylene Glycol Diacetate;

Propylparaben; Sodium Acetate; Sodium Bisulfite; Sodium Borate;

Sodium Chloride; Sodium Citrate; Sodium Hydroxide; Sodium

Phosphate, Dibasic, Anhydrous; Sodium Phosphate, Dibasic,

I Teptahydrate; Sodium Phosphate, Monobasic, Anhydrous; Sodium

In Table 8, “AN” means anesthetic, “CNBLK” means cervical nerve block, “NBLK” means nerve block, and “IV” means intravenous.

Naked Delivery

The compositions or agents of the present invention may be delivered to a subject naked or in saline. The naked compositions or agents may be administered to a subject using routes of administration known in the art and described herein.

Administration

Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure (e.g., those reducing methane production in a subject) may be administered to a subject by any route which results in a therapeutically effective outcome. These include, but are not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracisternal (within the cisterna magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the auras media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique, ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. In specific embodiments, compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.

Non-limiting routes of administration for the compositions or agents of the present disclosure are described below.

Parenteral and Injectable Administration

Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

A pharmaceutical composition for parenteral administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for parenteral administration includes hydrochloric acid, mannitol, nitrogen, sodium acetate, sodium chloride and sodium hydroxide. Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. The sterile formulation may also comprise adjuvants such as local anesthetics, preservatives and buffering agents.

Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

Injectable formulations may be for direct injection into a region of a tissue, organ and/or subject. As a non-limiting example, a tissue, organ and/or subject may be directly injected a formulation by intramyocardial injection into the ischemic region. (See e.g., Zangi et al. Nature Biotechnology 2013; the contents of which are herein incorporated by reference in its entirety).

In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

Rectal and Vaginal Administration

Compositions for rectal or vaginal (e.g., transvaginal) administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.

As a non-limiting example, the formulations for rectal and/or vaginal administration may be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and/or vagina to release the drug. Such materials include cocoa butter and polyethylene glycols.

A pharmaceutical composition for rectal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for rectal administration includes alcohol, alcohol, dehydrated, aluminum subacetate, anhydrous citric acid, aniseed oil, ascorbic acid, ascorbyl palmitate, balsam peru, benzoic acid, benzyl alcohol, bismuth subgallate, butylated hydroxyanisole, butylated hydroxytoluene, butylparaben, caramel, carbomer 934, carbomer 934p, carboxypolymethylene, cerasynt-se, cetyl alcohol, cocoa butter, coconut oil, hydrogenated, coconut oil/palm kernel oil glycerides, hydrogenated, cola nitida seed extract, d&c yellow no. 10, dichlorodifluoromethane, dichlorotetrafluoroethane, dimethyldioctadecylammonium bentonite, edetate calcium disodium, edetate disodium, edetic acid, epilactose, ethylenediamine, fat, edible, fat, hard, fd&c blue no. 1, fd&c green no. 3, fd&c yellow no. 6, flavor fig 827118, flavor raspberry pfc-8407, fructose, galactose, glycerin, glyceryl palmitate, glyceryl stearate, glyceryl stearate/peg stearate, glyceryl stearate/peg-40 stearate, glycine, hydrocarbon, hydrochloric acid, hydrogenated palm oil, hypromelloses, lactose, lanolin, lecithin, light mineral oil, magnesium aluminum silicate, magnesium aluminum silicate hydrate, methylparaben, nitrogen, palm kernel oil, paraffin, petrolatum, white, polyethylene glycol 1000, polyethylene glycol 1540, polyethylene glycol 3350, polyethylene glycol 400, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polysorbate 60, polysorbate 80, potassium acetate, potassium metabisulfite, propylene glycol, propylparaben, saccharin sodium, saccharin sodium anhydrous, silicon dioxide, colloidal, simethicone, sodium benzoate, sodium carbonate, sodium chloride, sodium citrate, sodium hydroxide, sodium metabisulfite, sorbitan monooleate, sorbitan sesquioleate, sorbitol, sorbitol solution, starch, steareth-10, steareth-40, sucrose, tagatose, d-, tartaric acid, dl-, trolamine, tromethamine, vegetable oil glyceride, hydrogenated, vegetable oil, hydrogenated, wax, emulsifying, white wax, xanthan gum and zinc oxide.

A pharmaceutical composition for vaginal administration may comprise at least one inactive ingredient. Any or none of the inactive ingredients used may have been approved by the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for vaginal administration includes adipic acid, alcohol, denatured, allantoin, anhydrous lactose, apricot kernel oil peg-6 esters, barium sulfate, beeswax, bentonite, benzoic acid, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, calcium lactate, carbomer 934, carbomer 934p, cellulose, microcrystalline, ceteth-20, cetostearyl alcohol, cetyl alcohol, cetyl esters wax, cetyl palmitate, cholesterol, choleth, citric acid, citric acid monohydrate, coconut oil/palm kernel oil glycerides, hydrogenated, crospovidone, edetate disodium, ethylcelluloses, ethylene-vinyl acetate copolymer (28% vinyl acetate), ethylene-vinyl acetate copolymer (9% vinylacetate), fatty alcohols, fd&c yellow no. 5, gelatin, glutamic acid, dl-, glycerin, glyceryl isostearate, glyceryl monostearate, glyceryl stearate, guar gum, high density polyethylene, hydrogel polymer, hydrogenated palm oil, hypromellose 2208 (15000 mpa·s), hypromelloses, isopropyl myristate, lactic acid, lactic acid, dl-, lactose, lactose monohydrate, lactose, hydrous, lanolin, lanolin anhydrous, lecithin, lecithin, soybean, light mineral oil, magnesium aluminum silicate, magnesium aluminum silicate hydrate, magnesium stearate, methyl stearate, methylparaben, microcrystalline wax, mineral oil, nitric acid, octyldodecanol, peanut oil, peg 6-32 stearate/glycol stearate, peg-100 stearate, peg-120 glyceryl stearate, peg-2 stearate, peg-5 oleate, pegoxol 7 stearate, petrolatum, white, phenylmercuric acetate, phospholipon 90g, phosphoric acid, piperazine hexahydrate, poly(dimethylsiloxane/methylvinylsiloxane/methylhydrogensiloxane) dimethylvinyl or dimethylhydroxy or trimethyl endblocked, polycarbophil, polyester, polyethylene glycol 1000, polyethylene glycol 3350, polyethylene glycol 400, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyglyceryl-3 oleate, polyglyceryl-4 oleate, polyoxyl palmitate, polysorbate 20, polysorbate 60, polysorbate 80, polyurethane, potassium alum, potassium hydroxide, povidone k29/32, povidones, promulgen d, propylene glycol, propylene glycol monopalmitostearate, propylparaben, quaternium-15 cis-form, silicon dioxide, silicon dioxide, colloidal, silicone, sodium bicarbonate, sodium citrate, sodium hydroxide, sodium lauryl sulfate, sodium metabisulfite, sodium phosphate, dibasic, anhydrous, sodium phosphate, monobasic, anhydrous, sorbic acid, sorbitan monostearate, sorbitol, sorbitol solution, spermaceti, stannous 2-ethylhexanoate, starch, starch 1500, pregelatinized, starch, corn, stearamidoethyl diethylamine, stearic acid, stearyl alcohol, tartaric acid, dl-, tert-butylhydroquinone, tetrapropyl orthosilicate, trolamine, urea, vegetable oil, hydrogenated, wecobee fs, white ceresin wax and white wax.

Oral Administration

Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents and/or excipients commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

Suspensions for oral dosage may contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be suspending agents, as a non-limiting example the suspending agents may be sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate; or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions for oral dosage can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavoring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.

The oral dosage may also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil or mixtures of these. Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g. paraffin), absorption accelerators (e.g. quaternary ammonium compounds), wetting agents (e.g. cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents. The solid dosage forms may also dissolve once they come in contact with liquid such as, but not limited to, salvia and bile.

Compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.

Solid dosage forms may be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Dosage forms for oral delivery may also be chewable. The chewable dosages forms may be sustained release formulations such as, but not limited to, the sustained release compositions described in International Publication No WO2013082470 and US Publication No US20130142876, each of which is herein incorporated by reference in its entirety. The chewable dosage forms may comprise amphipathic lipids such as, but not limited to, those described in International Publication No WO2013082470 and US Publication No US20130142876, each of which is herein incorporated by reference in its entirety.

Topical or Transdermal Administration

As described herein, compositions of the present disclosure may be formulated for administration transdermally. The skin may be an ideal target site for delivery as it is readily accessible. Gene expression may be restricted not only to the skin, potentially avoiding nonspecific toxicity, but also to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered compositions will depend on the route of delivery. Two routes are commonly considered to deliver compositions to the skin: (ii) intradermal injection; and (iii) systemic delivery (e.g. for treatment of dermatologic diseases that affect both cutaneous and extracutaneous regions). Compositions can be delivered to the skin by several different approaches known in the art.

In some embodiments, the invention provides for the compositions or agents to be delivered in more than one injection.

In some embodiments, before transdermal administration at least one area of tissue, such as skin, may be subjected to a device and/or solution which may increase permeability. In one embodiment, the tissue may be subjected to an abrasion device to increase the permeability of the skin (see U.S. Patent Publication No. 20080275468, herein incorporated by reference in its entirety). In another embodiment, the tissue may be subjected to an ultrasound enhancement device. An ultrasound enhancement device may include, but is not limited to, the devices described in U.S. Publication No. 20040236268 and U.S. Pat. Nos. 6,491,657 and 6,234,990; each of which are herein incorporated by reference in their entireties. Methods of enhancing the permeability of tissue are described in U.S. Publication Nos. 20040171980 and 20040236268 and U.S. Pat. No. 6,190,315; each of which are herein incorporated by reference in their entireties.

In some embodiments, a device may be used to increase permeability of tissue before delivering formulations of compositions described herein. The permeability of skin may be measured by methods known in the art and/or described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety. As a non-limiting example, a formulation may be delivered by the drug delivery methods described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety.

In another non-limiting example tissue may be treated with a eutectic mixture of local anesthetics (EMLA) cream before, during and/or after the tissue may be subjected to a device which may increase permeability. Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated by reference in its entirety) showed that using the EMLA cream in combination with a low energy, an onset of superficial cutaneous analgesia was seen as fast as 5 minutes after a pretreatment with a low energy ultrasound.

In some embodiments, enhancers may be applied to the tissue before, during, and/or after the tissue has been treated to increase permeability. Enhancers include, but are not limited to, transport enhancers, physical enhancers, and cavitation enhancers. Non-limiting examples of enhancers are described in U.S. Pat. No. 6,190,315, herein incorporated by reference in its entirety.

In some embodiments, a device may be used to increase permeability of tissue before delivering formulations of compositions described herein, which may further contain a substance that invokes an immune response. In another non-limiting example, a formulation containing a substance to invoke an immune response may be delivered by the methods described in U.S. Publication Nos. 20040171980 and 20040236268; each of which are herein incorporated by reference in their entireties.

Dosage forms for transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.

Additionally, the compositions of the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.

A pharmaceutical composition for transdermal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for transdermal administration includes acrylates copolymer, acrylic acid-isooctyl acrylate copolymer, acrylic adhesive 788, adcote 72a103, aerotex resin 3730, alcohol, alcohol, dehydrated, aluminum polyester, bentonite, butylated hydroxytoluene, butylene glycol, butyric acid, caprylic/capric triglyceride, carbomer 1342, carbomer 940, carbomer 980, carrageenan, cetylpyridinium chloride, citric acid, crospovidone, daubert 1-5 pestr (matte) 164z, diethylene glycol monoethyl ether, diethylhexyl phthalate, dimethicone copolyol, dimethicone mdx4-4210, dimethicone medical fluid 360, dimethylaminoethyl methacrylate-butyl methacrylate-methyl methacrylate copolymer, dipropylene glycol, duro-tak 280-2516, duro-tak 387-2516, duro-tak 80-1196, duro-tak 87-2070, duro-tak 87-2194, duro-tak 87-2287, duro-tak 87-2296, duro-tak 87-2888, duro-tak 87-2979, edetate disodium, ethyl acetate, ethyl oleate, ethylcelluloses, ethylene vinyl acetate copolymer, ethylene-propylene copolymer, fatty acid esters, gelva 737, glycerin, glyceryl laurate, glyceryl oleate, heptane, high density polyethylene, hydrochloric acid, hydrogenated polybutene 635-690, hydroxyethyl cellulose, hydroxypropyl cellulose, isopropyl myristate, isopropyl palmitate, lactose, lanolin anhydrous, lauryl lactate, lecithin, levulinic acid, light mineral oil, medical adhesive modified s-15, methyl alcohol, methyl laurate, mineral oil, nitrogen, octisalate, octyldodecanol, oleic acid, oleyl alcohol, oleyl oleate, pentadecalactone, petrolatum, white, polacrilin, polyacrylic acid (250000 mw), polybutene (1400 mw), polyester, polyester polyamine copolymer, polyester rayon, polyethylene terephthalates, polyisobutylene, polyisobutylene (1100000 mw), polyisobutylene (35000 mw), polyisobutylene 178-236, polyisobutylene 241-294, polyisobutylene 35-39, polyisobutylene low molecular weight, polyisobutylene medium molecular weight, polyisobutylene/polybutene adhesive, polypropylene, polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride, polyvinyl chloride-polyvinyl acetate copolymer, polyvinylpyridine, povidone k29/32, povidones, propylene glycol, propylene glycol monolaurate, ra-2397, ra-3011, silicon, silicon dioxide, colloidal, silicone, silicone adhesive 4102, silicone adhesive 4502, silicone adhesive bio-psa q7-4201, silicone adhesive bio-psa q7-4301, silicone/polyester film strip, sodium chloride, sodium citrate, sodium hydroxide, sorbitan monooleate, stearalkonium hectorite/propylene carbonate, titanium dioxide, triacetin, trolamine, tromethamine, union 76 amsco-res 6038 and viscose/cotton.

A pharmaceutical composition for intradermal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for intradermal administration includes benzalkonium chloride, benzyl alcohol, carboxymethylcellulose sodium, creatinine, edetate disodium, glycerin, hydrochloric acid, metacresol, methylparaben, phenol, polysorbate 80, protamine sulfate, sodium acetate, sodium bisulfite, sodium chloride, sodium hydroxide, sodium phosphate, sodium phosphate, dibasic, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, anhydrous and zinc chloride.

Depot Administration

As described herein, in some embodiments, the composition is formulated in depots for extended release. Generally, a specific organ or tissue (a “target tissue”) is targeted for administration.

In some aspects, the compositions or agents are spatially retained within or proximal to a target tissue. Provided are method of providing a composition to a target tissue of a mammalian subject by contacting the target tissue (which contains one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the composition is retained in the target tissue.

Aspects of the invention are directed to methods of providing a composition to a target tissue of a subject, by contacting the target tissue (comprising one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue.

In some embodiments, the compositions may be retained near target tissue using a small disposable drug reservoir, patch pump or osmotic pump. Non-limiting examples of patch pumps include those manufactured and/or sold by BD® (Franklin Lakes, NJ), Insulet Corporation (Bedford, MA), SteadyMed Therapeutics (San Francisco, CA), Medtronic (Minneapolis, MN) (e.g., MiniMed), UniLife (York, PA), Valeritas (Bridgewater, NJ), and SpringLeaf Therapeutics (Boston, MA). A non-limiting example of an osmotic pump include those manufactured by DURECT® (Cupertino, CA) (e.g., DUROS® and ALZET®).

Pulmonary Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are suitably in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having a boiling point of below 65° F. at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1% to 20% (w/w) of the composition. A propellant may further comprise additional ingredients such as a liquid non-ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient). As a non-limiting example, the compositions described herein may be formulated for pulmonary delivery by the methods described in U.S. Pat. No. 8,257,685; herein incorporated by reference in its entirety.

Pharmaceutical compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.

The compositions and formulations provided herein which may be used for pulmonary delivery may further comprise one or more surfactants. Suitable surfactants or surfactant components for enhancing the uptake of the compositions of the invention include synthetic and natural as well as full and truncated forms of surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant Protein E, di-saturated phosphatidylcholine (other than dipalmitoyl), dipalmitoylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine; phosphatidic acid, ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols, sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate; as well as natural and artificial lamellar bodies which are the natural carrier vehicles for the components of surfactant, omega-3 fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid, non-ionic block copolymers of ethylene or propylene oxides, polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomelic and polymeric, poly(vinyl amine) with dextran and/or alkanoyl side chains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf, Survan and Atovaquone, among others. These surfactants can be used either as single or part of a multiple component surfactant in a formulation, or as covalently bound to a component of a pharmaceutical composition herein.

Intranasal, Nasal and Buccal Administration

Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2μιη to 500μιη. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods and may, for example, 0.10% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

A pharmaceutical composition for inhalation (respiratory) administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for inhalation (respiratory) administration includes acetone sodium bisulfite, acetylcysteine, alcohol, alcohol, dehydrated, ammonia, apaflurane, ascorbic acid, benzalkonium chloride, calcium carbonate, carbon dioxide, cetylpyridinium chloride, chlorobutanol, citric acid, d&c yellow no. 10, dichlorodifluoromethane, dichlorotetrafluoroethane, edetate disodium, edetate sodium, fd&c yellow no. 6, fluorochlorohydrocarbons, gelatin, glycerin, glycine, hydrochloric acid, hydrochloric acid, diluted, lactose, lactose monohydrate, lecithin, lecithin, hydrogenated soy, lecithin, soybean, lysine monohydrate, mannitol, menthol, methylparaben, nitric acid, nitrogen, norflurane, oleic acid, polyethylene glycol 1000, povidone k25, propylene glycol, propylparaben, saccharin, saccharin sodium, silicon dioxide, colloidal, sodium bisulfate, sodium bisulfite, sodium chloride, sodium citrate, sodium hydroxide, sodium lauryl sulfate, sodium metabisulfite, sodium sulfate anhydrous, sodium sulfite, sorbitan trioleate, sulfuric acid, thymol, titanium dioxide, trichloromonofluoromethane, tromethamine and zinc oxide.

A pharmaceutical composition for nasal administration may comprise at least one inactive ingredient. Any or none of the inactive ingredients used may have been approved by the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for nasal administration includes acetic acid, alcohol, dehydrated, allyl .alpha.-ionone, anhydrous dextrose, anhydrous trisodium citrate, benzalkonium chloride, benzethonium chloride, benzyl alcohol, butylated hydroxyanisole, butylated hydroxytoluene, caffeine, carbon dioxide, carboxymethylcellulose sodium, cellulose, microcrystalline, chlorobutanol, citric acid, citric acid monohydrate, dextrose, dichlorodifluoromethane, dichlorotetrafluoroethane, edetate disodium, glycerin, glycerol ester of hydrogenated rosin, hydrochloric acid, hypromellose 2910 (15000 mpa·s), methylcelluloses, methylparaben, nitrogen, norflurane, oleic acid, petrolatum, white, phenylethyl alcohol, polyethylene glycol 3350, polyethylene glycol 400, polyoxyl 400 stearate, polysorbate 20, polysorbate 80, potassium phosphate, monobasic, potassium sorbate, propylene glycol, propylparaben, sodium acetate, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, sodium phosphate, dibasic, sodium phosphate, dibasic, anhydrous, sodium phosphate, dibasic, dihydrate, sodium phosphate, dibasic, dodecahydrate, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, anhydrous, sodium phosphate, monobasic, dihydrate, sorbitan trioleate, sorbitol, sorbitol solution, sucralose, sulfuric acid, trichloromonofluoromethane and trisodium citrate dihydrate.

Ophthalmic and Auricular (Otic) Administration

A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for delivery to and/or around the eye and/or delivery to the ear (e.g., auricular (otic) administration). Non-limiting examples of route of administration for delivery to and/or around the eye include retrobulbar, conjuctival, intracorneal, intraocular, intravitreal, ophthlamic and subconjuctiva. Such formulations may, for example, be in the form of eye drops or ear drops including, for example, a 0.1/1.0% (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein. Other ophthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this invention. A multilayer thin film device may be prepared to contain a pharmaceutical composition for delivery to the eye and/or surrounding tissue.

A pharmaceutical composition for ophthalmic administration may comprise at least one inactive ingredient. Any or none of the inactive ingredients used may have been approved by the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for ophthalmic administration includes acetic acid, alcohol, alcohol, dehydrated, alginic acid, amerchol-cab, ammonium hydroxide, anhydrous trisodium citrate, antipyrine, benzalkonium chloride, benzethonium chloride, benzododecinium bromide, boric acid, caffeine, calcium chloride, carbomer 1342, carbomer 934p, carbomer 940, carbomer homopolymer type b (allyl pentaerythritol crosslinked), carboxymethylcellulose sodium, castor oil, cetyl alcohol, chlorobutanol, chlorobutanol, anhydrous, cholesterol, citric acid, citric acid monohydrate, creatinine, diethanolamine, diethylhexyl phthalate, divinylbenzene styrene copolymer, edetate disodium, edetate disodium anhydrous, edetate sodium, ethylene vinyl acetate copolymer, gellan gum (low acyl), glycerin, glyceryl stearate, high density polyethylene, hydrocarbon gel, plasticized, hydrochloric acid, hydrochloric acid, diluted, hydroxyethyl cellulose, hydroxypropyl methylcellulose 2906, hypromellose 2910 (15000 mpa·s), hypromelloses, jelene, lanolin, lanolin alcohols, lanolin anhydrous, lanolin nonionic derivatives, lauralkonium chloride, lauroyl sarcosine, light mineral oil, magnesium chloride, mannitol, methylcellulose (4000 mpa·s), methylcelluloses, methylparaben, mineral oil, nitric acid, nitrogen, nonoxynol-9, octoxynol-40, octylphenol polymethylene, petrolatum, petrolatum, white, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric nitrate, phosphoric acid, polidronium chloride, poloxamer 188, poloxamer 407, polycarbophil, polyethylene glycol 300, polyethylene glycol 400, polyethylene glycol 8000, polyoxyethylene-polyoxypropylene 1800, polyoxyl 35 castor oil, polyoxyl 40 hydrogenated castor oil, polyoxyl 40 stearate, polypropylene glycol, polysorbate 20, polysorbate 60, polysorbate 80, polyvinyl alcohol, potassium acetate, potassium chloride, potassium phosphate, monobasic, potassium sorbate, povidone k29/32, povidone k30, povidone k90, povidones, propylene glycol, propylparaben, soda ash, sodium acetate, sodium bisulfate, sodium bisulfite, sodium borate, sodium borate decahydrate, sodium carbonate, sodium carbonate monohydrate, sodium chloride, sodium citrate, sodium hydroxide, sodium metabisulfite, sodium nitrate, sodium phosphate, sodium phosphate dihydrate, sodium phosphate, dibasic, sodium phosphate, dibasic, anhydrous, sodium phosphate, dibasic, dihydrate, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, sodium phosphate, monobasic, anhydrous, sodium phosphate, monobasic, dihydrate, sodium phosphate, monobasic, monohydrate, sodium sulfate, sodium sulfate anhydrous, sodium sulfate decahydrate, sodium sulfite, sodium thiosulfate, sorbic acid, sorbitan monolaurate, sorbitol, sorbitol solution, stabilized oxychloro complex, sulfuric acid, thimerosal, titanium dioxide, tocophersolan, trisodium citrate dihydrate, triton 720, tromethamine, tyloxapol and zinc chloride.

A pharmaceutical composition for retrobulbar administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for retrobulbar administration includes hydrochloric acid and sodium hydroxide.

A pharmaceutical composition for intraocular administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for intraocular administration includes benzalkonium chloride, calcium chloride, citric acid monohydrate, hydrochloric acid, magnesium chloride, polyvinyl alcohol, potassium chloride, sodium acetate, sodium chloride, sodium citrate and sodium hydroxide.

A pharmaceutical composition for intravitreal administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for intravitreal administration includes calcium chloride, carboxymethylcellulose sodium, cellulose, microcrystalline, hyaluronate sodium, hydrochloric acid, magnesium chloride, magnesium stearate, polysorbate 80, polyvinyl alcohol, potassium chloride, sodium acetate, sodium bicarbonate, sodium carbonate, sodium chloride, sodium hydroxide, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate and trisodium citrate dehydrate.

A pharmaceutical composition for subconjunctival administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for subconjunctival administration includes benzyl alcohol, hydrochloric acid and sodium hydroxide.

A pharmaceutical composition for auricular administration may comprise at least one inactive ingredient. A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for auricular administration includes acetic acid, aluminum acetate, aluminum sulfate anhydrous, benzalkonium chloride, benzethonium chloride, benzyl alcohol, boric acid, calcium carbonate, cetyl alcohol, chlorobutanol, chloroxylenol, citric acid, creatinine, cupric sulfate, cupric sulfate anhydrous, edetate disodium, edetic acid, glycerin, glyceryl stearate, hydrochloric acid, hydrocortisone, hydroxyethyl cellulose, isopropyl myristate, lactic acid, lecithin, hydrogenated, methylparaben, mineral oil, petrolatum, petrolatum, white, phenylethyl alcohol, polyoxyl 40 stearate, polyoxyl stearate, polysorbate 20, polysorbate 80, polyvinyl alcohol, potassium metabisulfite, potassium phosphate, monobasic, povidone k90f, povidones, propylene glycol, propylene glycol diacetate, propylparaben, sodium acetate, sodium bisulfite, sodium borate, sodium chloride, sodium citrate, sodium hydroxide, sodium phosphate, dibasic, anhydrous, sodium phosphate, dibasic, heptahydrate, sodium phosphate, monobasic, anhydrous, sodium sulfite, sulfuric acid and thimerosal.

Dosing

Provided herein are methods comprising administering a vaccine composition to a subject. The specific dose level for any particular subject will depend upon a variety of factors including the species, the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the arts. Compositions in accordance with the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure may be decided by the attending administrator within the scope of sound judgment.

In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 25 mg/kg, from about 0.1 mg/kg to about 250 mg/kg, or any range in between or any value in between, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.

In some embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.001 mg to about 500 mg of polypeptide(s) and/or peptide(s), or any range in between or any value in between. In some embodiments, compositions may be administered at dosage levels sufficient to deliver from about 0.01 to about 50 mg, or any range in between or any value in between. For example, in some embodiments, the range may be between about 0.1 and about 5 mg, or any range in between or any value in between, e.g., between about 0.1 and about 2 mg, or any range in between or any value in between.

The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, twenty, thirty, forty, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.

In some embodiments, an animal is administered with a prime (initial dose), followed by a boost (second dose) at least about 1, 1.5, 2, 2.5, 3, 3.5, or 4 weeks after the prime. In some embodiments, at least one additional dose is given after the boost, optionally after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months after the boost. In some embodiments, the at least one additional dose is repeated every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 months. In preferred embodiments, an animal is administered with a prime, followed by a boost about 2 or 3 weeks after the prime. In yet other preferred embodiments, an animal is administered with (a) a prime, (b) a boost about 2 or 3 weeks after the prime, and (c) at least one additional dose every 3, 4, 5, or 6 months. Each dose in the dosing schedule may comprise the same or different amount of the vaccine composition.

According to the present disclosure, the compositions of the present disclosure may be administered in split-dose regimens. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose. In some embodiments, the compositions of the present disclosure are administered to a subject in split doses. The compositions may be formulated in buffer only or in a formulation described herein.

Dosage Forms

Vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, etc.), or other compositions of the present disclosure may be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).

Liquid Dosage Forms

Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain embodiments for parenteral administration, compositions may be mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, water, Ringer's solution, U.S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In order to prolong the effect of an active ingredient, it may be desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compositions then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compositions may be accomplished by dissolving or suspending the compositions in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compositions in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compositions to polymer and the nature of the particular polymer employed, the rate of a compound release can be controlled. Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the compositions in liposomes or microemulsions which are compatible with body tissues.

Pulmonary Formulation

Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2μιη to 500μιη. Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21^sted., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

Coatings or Shells

Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

Multi-Dose and Repeat-Dose Administration

In some embodiments, vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure may be administered in two or more doses (referred to herein as “multi-dose administration”). Such doses may comprise the same components or may comprise components not included in a previous dose. Such doses may comprise the same mass and/or volume of components or an altered mass and/or volume of components in comparison to a previous dose. In some embodiments, multi-dose administration may comprise repeat-dose administration. As used herein, the term “repeat-dose administration” refers to two or more doses administered consecutively or within a regimen of repeat doses comprising same or different components. In some embodiments, the repeat dose may comprise substantially the same components provided at substantially the same mass and/or volume. In other embodiments, the repeat dose may comprise different components (e.g., different adjuvant for a vaccine composition).

Adjuvant

Adjuvants or immune potentiators, may also be administered with or in combination with one or more vaccine composition of the present disclosure.

The term “adjuvant” refers to an agent that when administered in conjunction with or as part of a composition described herein augments, enhances, and/or boosts the immune response to a methanogen, but when the agent is administered alone does not generate an immune response. In some embodiments, the adjuvant generates an immune response to a methanogen and does not produce an allergy or other adverse reaction. Adjuvants can enhance an immune response by several mechanisms including, e.g., lymphocyte recruitment, stimulation of B and/or T cells, and stimulation of macrophages.

In some embodiments, an adjuvant acts as a co-signal to prime T-cells and/or B-cells and/or NK cells as to the existence of the cell surface protein of a methanogen in a vaccine composition of the present disclosure.

Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccines. These may be co-administered by any route, e.g., intramusculary, subcutaneous, IV or intradermal injections.

Adjuvants useful in the present invention may include, but are not limited to, natural or synthetic. They may be organic or inorganic.

When a vaccine or immunogenic composition of the invention comprises adjuvants or is administered together with one or more adjuvants, the adjuvants that can be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, particulate adjuvants, microparticulate adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 De-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (Glaxo SmithKline), AS04 (Glaxo SmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT/US2007/064857, published as International Publication No. WO2007/109812), imidazoquinoxaline compounds (see International Application No. PCT/US2007/064858, published as International Publication No. WO2007/109813) and saponins, such as QS21 (see Kensil et al, in Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540). In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil in water emulsions (such as squalene or peanut oil), optionally in combination with immune stimulants, such as monophosphoryl lipid A (see Stoute et al, N. Engl. J. Med. 336, 86-91 (1997)).

Adjuvants may be selected from any of the classes (1) mineral salts, e.g., aluminium hydroxide and aluminium or calcium phosphate gels; (2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidised detergent stabilised oil-in-water emulsion, purified saponin, oil-in-water emulsion, stabilised water-in-oil emulsion; (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and/or (6) inert vehicles, such as gold particles; (7) microorganism derived adjuvants; (8) tensoactive compunds; (9) carbohydrates; or combinations thereof.

Other adjuvants which may be utilized in the vaccines of the present disclosure include any of those listed on the web-based vaccine adjuvant database, Vaxjo; World Wide Web at violinet.org/vaxjo/ and described in for example Sayers, et al., J. Biomedicine and Biotechnology, volume 2012 (2012), Article ID 831486, 13 pages, the content of which is incorporated herein by reference in its entirety.

Selection of appropriate adjuvants will be evident to one of ordinary skill in the art. Specific adjuvants may include, without limitation, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, alhydrogel, ISCOM(s)™, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium-derb/ed P40 Vaccine Adjuvant, MPL™ Adjuvant, AS04, AS02, Lipopolysaccharide Vaccine Adjuvant, Muramyl Dipeptide Adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine Adjuvant, Montanide ISA 51, Bordetella pertussis component Vaccine Adjuvant, Cationic Liposomal Vaccine Adjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, Arlacel A, VSA-3 Adjuvant, Aluminum vaccine adjuvant, Polygen Vaccine Adjuvant, Adjumer™, Algal Glucan, Bay R1005, Theramide®, Stearyl Tyrosine, Specol, Algammulin, Avridine®, Calcium Phosphate Gel, CTA1-DD gene fusion protein, DOC/Alum Complex, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Recombinant hlFN-gamma/Interferon-g, Interleukin-{umlaut over (ι)}β, Interleukin-2, Interleukin-7, Sclavo peptide, Rehydragel LV, Rehydragel HPA, Loxoribine, MF59, MTP-PE Liposomes, Murametide, Murapalmitine, D-Murapalmitine, NAGO, Non-Ionic Surfactant Vesicles, PMMA, Protein Cochleates, QS-21, SPT (Antigen Formulation), nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR192G Vaccine Adjuvant, E. coli heat-labile toxin, LT, amorphous aluminum hydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant, Montanide Incomplete Seppic Adjuvant, Imiquimod, Resiquimod, AF03, Flagellin, Poly(LC), ISCOMATRIX®, Abisco-100 vaccine adjuvant, Albumin-heparin microparticles vaccine adjuvant, AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, Immunoliposomes Containing Antibodies to Costimulatory Molecules, SAF-1, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Threonyl muramyl dipeptide (TMDP), Ty Particles vaccine adjuvant, Bupivacaine vaccine adjuvant, DL-PGL (Polyester poly (DL-lactide-co-glycolide)) vaccine adjuvant, IL-15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic mutant E1 12K of Cholera Toxin mCT-E1 12K, and/or Matrix-S.

In some embodiments, the at least one adjuvant comprises oil emulsions, e.g., comprising at least (a) mineral oil lipid and (b) aqueous phase (e.g., Freund's complete adjuvant, Freund's incomplete adjuvant, Montanide ISA series (e.g., ISA70, ISA61, ISA206, ISA50), squaline-based emulsion, (e.g., MF59 and/or AS03), saponins, (e.g., Quil-A, Spikoside, QS21, or ISCOMs, e.g., ISCOPREP 703), aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate), also known to a skilled artisan as ‘alum’, (e.g., Imject Alum), dextran sulfate, chitosan thermogel, (e.g., monophosphoryl lipid A), Carbol, PLGA, MDP, CpG ODN, cytokine-based adjuvants such as IL-12 and/or GM-CSF, a lipid nanoparticle/cationic liposome adjuvant, an immune stimulating complex, or any combination of two or more thereof. In preferred embodiments, the at least one adjuvant comprises Freund's complete adjuvant and/or Freund's incomplete adjuvant. See Spickler and Roth (2003) J Vet Intern Med, 17:273-281, which is incorporated herein by reference.

In some embodiments, the at least one adjuvant comprises Emulsigen-D, Emulsigen, Emulsigen-P, and/or Polygen (MVP adjuvant, Omaha, NE). In some embodiments, the at least one adjuvant comprises ENABL 06 (HuvePharma, Peachtree City, GA). In some embodiments, the at least one adjuvant comprises Montainde ISA 201 and/or Montanide Gel 02 (Seppic Inc., New Jersey).

Other adjuvants which may be co-administered with the vaccine compositions of the invention include, but are not limited to interferons, TNF-alpha, TNF-beta, chemokines such as CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD-40L, CD28 agonists, PD-1, soluble PD1, L1 or L2, or interleukins such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-21, IL-23, IL-15, IL-17, and IL-18.

In some embodiments, the adjuvant comprises Glucopyranosyl Lipid Adjuvant (GLA), CpG oligodeoxynucleotides (e.g., Class A or B), poly(LC), aluminum hydroxide, or Pam3CSK4.

In some embodiments, the adjuvant comprises: (a) (±)—N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(syn-9-tetradeceneyloxy)-1-propanaminium bromide (GAP-DMORIE) and a neutral lipid; (b) a cytokine; (c) mono-phosphoryl lipid A and trehalosedicorynomycolateAF (MPL+TDM); (d) a solubilized mono-phosphoryl lipid A formulation; and/or (e) CRL1005/BAK.

In some embodiments, the neutral lipid in (a) comprises (a) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); (b) 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPyPE); and/or (c) 1,2-dimyristoyl-glycer-3-phosphoethanolamine (DMPE).

In some embodiments, the adjuvant comprises saponin, Montanide ISA61, a chitosan thermogel, a lipid nanoparticle/cationic liposome adjuvant, or any combination thereof. In preferred embodiments, the adjuvant comprises Montanide ISA61.

Antibody

Unless otherwise specified here within, the terms “antibody” and “antibodies” broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE); and recombinant antibodies, such as single-chain antibodies, chimeric antibodies, and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site. Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.

The term “antibody” as used herein also includes an “antigen-binding portion” of an antibody (or simply “antibody portion”). The term “antigen-binding portion”, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., at least one cell surface protein or fragment thereof of at least one methanogen). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osboum et al. 1998, Nature Biotechnology 16: 778). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. Any VH and VL sequences of specific scFv can be linked to immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes (e.g., IgGA). VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

Still further, an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, biomarker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv polypeptides (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab′)₂fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.

Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. chimeric, etc.). Antibodies may also be fully specific to the subject, e.g., the antibodies may be fully ruminant or fully human. The terms “monoclonal antibodies” and “monoclonal antibody composition,” as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

The present disclosure includes a monoclonal antibody that works particularly well in binding and neutralizing at least one methanogen. Upon immunizing a subject population (e.g., of milk-producing subject), the milk comprising the antibody can be screened for antibodies with superior activity (e.g., specific binding, neutralizing at least one methanogen, etc.). The amino acid sequence of such antibodies can be determined (e.g., mass spec-based sequencing, Next Gen Sequencing, or other methods known in the art), their expressing DNA vectors can be synthesized, and monoclonal antibodies can be produced. One or a combination of at least two or more antibodies can be added to the drinking water and/or animal feed, and be given to a subject population.

Alternatively, such monoclonal antibodies can be generated by immunizing a vehicle animal (e.g., mouse, rabbit, etc.), and hybridomas expressing the animals can be recovered. Standard hybridoma methods for producing antibodies are described in, e.g., Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), Immunobiology, 5^thEd., Garland Publishing, New York, NY (2001)). Antibodies produced by hybridomas can be screened and utilized according to the methods described above and herein.

In some embodiments, the antibody is a polyclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an IgG or IgA. In some embodiments, the antibody is IgA. The IgA isoform, at least in cattle, may be more stable in the rumen. For example, IgA levels in cattle saliva were reduced by only 40% after 8 h exposure to rumen contents while IgG levels were reduced by 80%.

In some embodiments, the antibody is lyophilized. In some embodiments, the antibody is in a pharmaceutical composition of the present disclosure or those known in the art. In some embodiments, a composition comprising an antibody further comprises at least one excipient and/or carrier. In some embodiments, the antibody is in the animal feed. In some embodiments, the antibody is in the solid animal feed. In other embodiments, the antibody is in the liquid animal feed. In some embodiments, the antibody is in the drinking water or milk. In preferred embodiments, the antibody is orally consumed by a subject such that the antibody comes in contact with at least one methanogen present in the gut of the subject. In preferred embodiments, oral administration of the antibody reduces the number and/or type of at least one methanogen.

Methods for Detection of Antibody

In certain embodiments, an efficacious vaccine produces an antibody titer of greater than 1:40, greater that 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:500, greater than 1:6000, greater than 1:7500, greater than 1:10000. In some embodiments, the antibody titer is produced or reached by 10 days following vaccination, by 20 days following vaccination, by 30 days following vaccination, by 40 days following vaccination, or by 50 or more days following vaccination. In some embodiments, the titer is produced or reached following a single dose of vaccine administered to the subject. In other embodiments, the titer is produced or reached following multiple doses, e.g., following a first and a second dose (e.g., a repeat dose.).

In certain aspects, antigen-specific antibodies are measured in units of pg/ml or are measured in units of IU/L (International Units per liter) or mIU/ml (milli International Units per ml). In some embodiments of the invention, an efficacious vaccine produces >0.5 pg/ml, >0.1 pg/ml, >0.2 pg/ml, >0.35 pg/ml, >0.5 pg/ml, >1 pg/ml, >2 pg/ml, >5 pg/ml or >10 pg/ml. In some embodiments, an efficacious vaccine produces >10 mIU/ml, >20 mIU/ml, >50 mIU/ml, >100 mIU/ml, >200 mIU/ml, >500 mIU/ml or >1000 mIU/ml.

Methods of detecting the presence of antibodies are well known in the art.

In some embodiments, antibody level or concentration is determined or measured by neutralization assay, e.g., neutralization of at least one methanogen.

Other exemplary methods include, but are not limited to, immunodiffusion, immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs; including variants such as competitive ELISA, sandwich ELISA, etc.), immunofluorescent assays, Western blotting, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn. pp 217-262, 1991 which is incorporated by reference).

For example, ELISA and RIA procedures may be conducted such that a desired protein standard (e.g., an extracellular domain of at least one cell surface protein or a fragment thereof of at least one methanogen) is labeled (with a radioisotope such as ¹²⁵I or ³⁵S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and is brought into contact with a sample comprising the antibody, whereon the amount of the labeled protein standard bound to the antibody is measured.

Enzymatic and radiolabeling of a protein and/or the antibodies may be effected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzymes are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.

It may be desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support.

Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

Milk

In certain aspects, provided herein are milk and derivatives thereof. Milk produced by vaccinated female subjects (e.g., dairy cows) comprises antibodies that bind at least one cell surface protein or a fragment thereof of at least one methanogen. Such milk can be orally consumed by subjects such that the antibodies therein can come in contact with at least one methanogen present in the gut of the subjects. Upon contact, the antibodies in the milk can neutralize the at least one methanogen and contribute to reducing methane production by the subjects.

Thus, milk from vaccinated female subject can be used to treat breast-feeding animals, thereby reducing methane production and/or methanogen colonization in young animals. This can be especially important when vaccination of young animals occurs concurrently with weaning.

In some embodiments, the milk is pasteurized and/or homogenized. In some embodiments, the milk is lyophilized, filtered, concentrated, evaporated, or processed to form dry milk powder (e.g., boiling at low pressure at low temperature). In some embodiments, said processing may allow longer shelf life of the milk/milk product and the antibodies present therein. In some embodiments, the fat content is removed/reduced from the milk. Processing of milk and/or preparation of derivatives of milk are well known in the art.

Appropriate care is taken to preserve the structural and functional (e.g., binding a methanogen) aspects of the antibodies. For example, in some embodiments, high pressure (˜200 MPa) and low temperature (−4° C.) are used throughout the process as described at least by Kim et al. (2008) Journal of Dairy Science, 91:4176-4182. In other embodiments, milk may be pasteurized at low-temperature of 60° C. for 10 minutes at standard pressure. These conditions may pasteurize milk without significantly altering the antibody function.

Alternatively, milk can be filtered to remove microorganisms instead of pasteurizing. Microfiltration is a process that replaces pasteurization and produces milk with fewer microorganisms and longer shelf life without a change in the quality of the milk. In this process, cream is separated from the skimmed milk and the skimmed milk is forced through ceramic microfilters that trap 99.9% of microorganisms in the milk (as compared to 99.999% killing of microorganisms in standard high temperature short time pasteurization).

Ultrafiltration uses finer filters than microfiltration, which allow lactose and water to pass through while retaining fats, calcium and protein. As with microfiltration, the fat may be removed before filtration and added back in afterwards. Ultrafiltered milk is used widely in the industry in cheesemaking.

Colostrum may similarly be used in the milk embodiments disclosed herein.

Animal Feed

Provided herein are animal feeds that are useful in reducing methane production by a subject. Animal feed encompasses any edible consumables that are suitable for consumption by the subject of the present disclosure. Accordingly, animal feed also includes drinking water or other food items that may be consumed by the subject including but not limited to humans, canines, felines, and ruminants.

Animal feed may be used in combination with or comprise any one of vaccines, antibodies, milk, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure (e.g., those reducing methane production in a subject). Animal feed may comprise at least one agent, which reduces the methane production in a subject.

In some embodiments, the animal feed comprises an antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen.

In some embodiments, the animal feed comprises a composition comprising an antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen. For example, the animal feed may comprise milk or derivatives thereof comprising said antibody.

In some embodiments, the animal feed is liquid (e.g., drinking water, milk). An antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen or a composition comprising same may be added to the liquid animal feed (e.g., drinking water) before being given to the subject. In some embodiments, said antibody or a composition comprising same may be added to the drinking water. In other embodiments, the milk or derivatives thereof comprising said antibody may be given directly to the subject, or added to other liquid animal feed (e.g., drinking water).

In other embodiments, the animal feed is solid. In some such embodiments, the animal feed may comprise hay, straw, silage, compressed and pelleted feeds, oils and mixed rations, and sprouted grains and legumes. An antibody that binds at least one cell surface antigen or a fragment thereof of at least one methanogen or a composition comprising same may be added to the solid animal feed before being given to the subject.

In some embodiments, an animal feed may comprise fats and fatty acids that further aid in reducing methane production in subjects. Based on a meta-analysis, fat supplementation reduced CH₄by 3.77% in cattle and 4.30% in sheep per 1% dietary fats. Fat decreases CH₄production (expressed as g/kg digestible dry matter (DM)) more from sheep than from cattle, which was attributed to the comparatively lower depression of DM digestion together with numerically larger depression of CH₄production (g/kg DM) by fat in sheep. Among fatty acids, C12:0, C18:3 and other polyunsaturated fatty acids (PUFA) are more potent than saturated fatty acids. The CH₄-suppressing efficacy of fats generally persists, with persistent suppression being noted for 72 days and longer in cattle.

Fats supplemented up to 6% of the diet (DM) can also improve milk production while appreciably decreasing CH₄emissions (15%) in cattle, but higher concentrations decreased production efficiency due to a reduction of feed digestion and fermentation. Medium-chain fatty acids (MCFA) and PUFA can lower abundance and metabolic activities of rumen methanogens and change their species composition. PUFA can also directly inhibit protozoa and serve as hydrogen sink through biohydrogenation. Both MCFA and PUFA appear to damage the cell membrane, thereby abolishing the selective permeability of cell membrane, which is required for survival and growth of methanogens and other microbes. The inhibitory effect of fat on methanogenesis is more pronounced in cattle fed concentrate-based diets than in cattle fed forage-based diets. Because C12; and C14:0 is more inhibitory to M. ruminantium at pH 5 than at pH 7, the concentrate level-dependent anti-methanogenic efficacy of MCFA and PUFA is probably attributed to the lower pH associated with high-concentrate diets.

In some embodiments, the animal feed comprises fat and/or fatty acid. In some embodiments, the animal feed comprises fat and/or fatty acid that is at least about 1%, 2%, 3%, 4%, 5%, or 6% of the diet (e.g., diet based on dry matter).

Numerous animal feed and feed additives are known in the art. Any agent that reduce methane production in a subject (e.g., small molecule inhibitors, e.g., Table 9, probiotic bacterial strain, etc.; see below) described herein or those known in the art may be used as a feed additive. Certain exemplary feed additives include: berberine, nitrate, eucalyptus oil, alliin, diallyl disulfide (DADS), flavanone glycoside (e.g., neohesperidin, isonaringin, poncirin, hesperidin), 3-nitrooxypropanol, rac-4-Phenylbutane-1,2-diyl dinitrate, 2-(hydroxymethyl)-2-(nitrooxymethyl)-1,3-propanediol, N-ethyl-3-nitro-oxy-propionic sulfonyl amide, 5-nitrooxy-pentanenitrile, 5-nitrooxy-pentane, 3-nitro-oxy-propyl propionate, 1,3-bis-nitrooxypropane, 1,4-bis-nitrooxybutane, 1,5-bis-nitrooxypentane, 3-nitro-oxy-propyl benzoate, 3-nitro-oxy-propyl hexanoate, 3-nitro-oxy-propyl 5-nitro-oxy-hexanoate, benzylnitrate, isosorbid-dinitrate, N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide, 3-nitrooxy propionic acid, methyl-3-nitrooxy propionate, ethyl-3-nitrooxy propionate, ethyl-4-nitrooxy butanoate, ethyl-3-nitrooxy butanoate, 5-nitrooxy pentanoic acid, ethyl-5-nitrooxy pentanoate, 6-nitrooxy hexanoic acid, ethyl-6-nitrooxy hexanoate, ethyl-4-nitrooxy-cyclohexylcarboxylate, 8-nitrooxy octanoic acid, ethyl-8-nitrooxy octanoate, 11-nitrooxy undecanoic acid, ethyl-11-nitrooxy undecanoate, 5-nitrooxy-pentanoic amide, 5-nitrooxy-N-methyl-pentanoic amide, lauric acid, and haloform (e.g., bromoform, chloroform, iodoform).

Agents that Reduce Methane and/or Hydrogen Production in Ruminants

Combination Treatment

Vaccines, antibodies, milk, animal feed, and agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, etc.) may be administered to a subject in any combination. By “in combination with,” it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent (vaccines, antibodies, milk, animal feed, agents that reduce methane production in a subject) will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of combinations that may improve immune response against at least one methanogen, and/or reduce methane production by a subject.

The combinations can conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical compositions comprising a combination as defined above together with a pharmaceutically acceptable diluent or carrier represent a further aspect of the invention.

The individual compounds of such combinations can be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations.

It will further be appreciated that (vaccines, antibodies, milk, animal feed, agents that reduce methane production in a subject) in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. In some embodiments, the combinations, each or together may be administered according to the split dosing regimens described herein.

The term “conjoint” or “combination” administration, as used herein, refers to the administration of two or more agents that aid in reducing methane production in a subject. The different agents comprising the combination may be administered concomitant with, prior to, or following the administration of one or more agents.

In certain embodiments, combination administration can demonstrate synergisms between the two or more agents resulting in a greater methane reduction in subject than either agent alone.

Synergistic effects, also known as synergy, refer to the phenomenon where the combined effect of two or more factors or components is greater than the sum of their individual effects. In other words, when these factors or components interact, they create an amplified or enhanced effect that is greater than what would be expected based on their individual contributions.

In certain cases, the agents may have different modes of action or mechanisms by which they exert their effects, for example targeting different methanogens or different methanogen enzymes. When these agents are combined, their actions can complement each other, targeting different aspects of a problem or working on multiple pathways simultaneously. This complementary action enhances their overall effectiveness, resulting in a better outcome than either agent could achieve alone.

In certain cases, synergism can significantly enhance the efficacy of the agents involved. For example, the agents may interact in a way that enhances their absorption, distribution, or bioavailability, increasing their effectiveness in treating a particular condition.

In certain cases, one agent may enhance the effects of the other without contributing much individually. This is known as potentiation. The presence of one agent can increase the uptake, binding affinity, or sensitivity of the other, making it more potent and effective. The combined effect is greater than what would be achieved by either agent on its own.

In certain cases, one or more agents may have inherent weaknesses or face resistance from target organisms or systems. By combining them with another agent, the synergistic interaction can bypass or counteract these obstacles, leading to a more effective outcome. For example, antibodies generated via vaccination of a first vaccine may face resistance to ruminal proteases, thus, combining with protease inhibitors or one or more additional vaccine towards certain ruminal proteases may reduce the resistance of the first vaccine.

In certain cases, combining two agents can amplify the positive effects or benefits they provide individually. For example, combining a vaccine for a methanogen with a vaccine towards a separate microorganism that is syntrophic with the methanogen can amplify the positive effects or benefits of the vaccine by further reducing the fitness of the methanogen.

The level of synergism achieved when combining agents depends on numerous factors and is typically assessed through experimental studies or empirical observations specific to the agents and desired outcomes. Generally, combination of agents can lead to different degrees of synergisms ranging from No synergism to Supra-additive synergism.

No Synergism: In some cases, the combined effect of two or more agents may simply be additive or even less than additive. This means that the combined effect is equal to the sum of their individual effects or even lower. In such instances, no synergism is observed, and the agents may not interact in a way that amplifies their effects.

Mild to Moderate Synergism: A common outcome when combining agents is a mild to moderate level of synergism. This implies that the combined effect is greater than the sum of their individual effects, but not dramatically so. The degree of synergism may vary depending on the specific agents and the conditions of their interaction.

Strong Synergism: In some cases, the combination of agents can lead to a strong synergistic effect. This means that the combined effect is significantly greater than the sum of their individual effects. Strong synergism often results in an amplified and more potent effect, exceeding what would be expected based on the additive effects of the individual agents.

Supra-additive Synergism: In rare instances, the combined effect of two or more agents can be supra-additive, meaning it surpasses even strong synergism. Supra-additive synergism results in an exceptionally powerful effect that far exceeds the sum of the individual effects. Such cases are usually considered highly beneficial, as they can provide remarkable outcomes in terms of efficacy, efficiency, or other desired parameters.

Any suitable degree of synergism (%_Synergism) can be demonstrated with a combination therapy comprising any two or more compositions or agents of the present disclosure (e.g., a vaccine and another agent), such as an improvement of at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400% and/or not more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000%, for example 5-1000%, preferably 10-500%, more preferably 30-300%.

In certain embodiments, the improvement is measured in the amount of methane reduced when administered a combination therapy as compared to either agent alone, for example %_Synergism=Methane_combo*(Methane_vaccine)⁻¹. In certain embodiments, the %_Synergismis measured in a herd of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, or 1000 animals and/or not more than 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, or 2000 animals and the statistical significance, e.g., coefficient of variation of %_Synergismwithin the herd is at least 50, 60, 70, 75, 80, 85, 90, 95, 99, 99.5, or 100%.

In certain embodiments, the synergism resulting from the combinatorial therapy results in a prolonged efficacy of the treatment as compared to either alone. In certain embodiments, the combinatorial therapy is effective for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, or 20 months and/or not more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, or 24 months, for example 1-24 months. In certain embodiments, the length of efficacy of the treatment is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, or 400% and/or not more than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000% greater than either agent alone, for example 5-1000%, preferably 10-500%, more preferably 30-300%.

An exemplary combinatorial therapy includes vaccination with a first vaccine encoding one or more methanogen surface proteins in combination with at least one additional vaccine encoding one or more of different methanogen surface proteins.

Another exemplary combinatorial therapy includes vaccination with a vaccine encoding one or more methanogen surface proteins in combination with administration of a small molecule inhibitor of methanogenesis. Without wishing to be bound to theory, it is hypothesized that the small molecule inhibitor of methanogenesis may remove the plurality of ruminal methanogens, and antibodies generated from the vaccination prevent new methanogens for colonizing the methanogen-deficient rumen.

Accordingly, in certain aspects, a vaccine of the present disclosure is administered to a subject conjointly or in a combination with at least one inhibitor of methane production described herein or those known in the art. In some embodiments, the at least one inhibitor is selected from Tables 9-13.

In some embodiments, the at least one inhibitor is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 times. In some embodiments, the at least one inhibitor is administered to a subject daily, semiweekly, weekly, biweekly (every 2 weeks), monthly, semiannually, or annually. In some embodiments, the at least one inhibitor is administered to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days. In some embodiments, the at least one inhibitor is administered to a subject for a duration of at least, about, or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 weeks. In some embodiments, the at least one inhibitor is administered to a subject for at least 1 week but no more than 1 month. In some embodiments, the at least one inhibitor is administered to a subject orally, intravenously, intramuscularly, or subcutaneously. In preferred embodiments, the at least one inhibitor is administered to a subject orally. In some embodiments, the at least one inhibitor is administered to a subject as a feed additive.

In some embodiments, the at least one inhibitor is administered to a subject concomitant with, prior to, or following the vaccination with a vaccine of the present disclosure. In some embodiments, the at least one inhibitor is administered to a subject on the same day as the subject is vaccinated. In some embodiments, a subject is administered with the at least one inhibitor one or more times to reduce the methane production by the subject, and said subject is vaccinated as a maintenance regimen.

In some embodiments, the at least one inhibitor comprises 3NOP. In some embodiments, a subject is administered at least about or no more than 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 grams (g) of 3NOP per dose. In some embodiments, 3NOP is administered to a subject every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 days.

In some embodiments, at least about 0.5 g but no more than 25 g of 3NOP is administered to a subject in a given day. In preferred embodiments, about 2.5 g of 3NOP is administered to a subject in a given day. In some embodiments, about 2.5 g of 3NOP is administered to a subject per day for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.

TABLE 9

Exemplary methanogenesis inhibitor

Chemical
Reference

1,3-bis-nitrooxypropane
US10154981B2

1,3-bis-nitrooxypropane
US9266814B2

1,4-bis-nitrooxybutane
US10806706B2

1,4-bis-nitrooxybutane
US10154981B2

1,4-bis-nitrooxybutane
US9266814B2

1,5-bis-nitrooxypentane
US10806706B2

1,5-bis-nitrooxypentane
US10154981B2

1,5-bis-nitrooxypentane
US9266814B2

11-nitrooxy undecanoic acid
US9365489B2

11-nitrooxy undecanoic acid
US10154981B2

2 mercaptoethanesulfonate
EP2747181A1

2-bromoethane sulfonic acid (2-BESA)
EP2747181A1

2-bromoethane sulphonate
US6251879B1

2-chloroethanesulfonate
EP2747181A1

2-nitropropanol
EP2747181A1

3-nitro-oxy-propyl 5-nitro-oxy-hexanoate
US10154981B2

3-nitro-oxy-propyl benzoate
US10154981B2

3-nitro-oxy-propyl hexanoate
US10154981B2

3-nitro-oxy-propyl propionate
US10154981B2

3-nitrooxy propionic acid
US9365489B2

3-nitrooxy propionic acid
US10154981B2

3-nitrooxy-propyl 5-nitrooxy-hexanoate
US9266814B2

3-nitrooxy-propyl benzoate
US9266814B2

3-nitrooxy-propyl hexanoate
US9266814B2

3-nitrooxy-propyl propionate
US9266814B2

3-nitrooxypropanol
US10806706B2

3-nitrooxypropanol
US10154981B2

3-nitrooxypropanol
US9266814B2

5-nitrooxy hexanoic acid
US10806706B2

5-nitrooxy pentanoic acid
US10806706B2

5-nitrooxy pentanoic acid
US9365489B2

5-nitrooxy pentanoic acid
US10154981B2

5-nitrooxy-N-methyl-pentanoic amide
US9365489B2

5-nitrooxy-N-methyl-pentanoic amide
US10154981B2

5-nitrooxy-N-pentanoic amide
US9365489B2

5-nitrooxy-pentane
US10154981B2

5-nitrooxy-pentane
US9266814B2

5-nitrooxy-pentanenitrile
US10154981B2

5-nitrooxy-pentanenitrile
US9266814B2

5-nitrooxy-pentanoic amide
US10154981B2

6-nitrooxy hexanoic acid
US9365489B2

6-nitrooxy hexanoic acid
US10154981B2

8-nitrooxy octanoic acid
US9365489B2

8-nitrooxy octanoic acid
US10154981B2

9-nitrooxynonanol
US10806706B2

abietic acid
US10799544B2

acetylene
EP2747181A1

ajoene
US20220256890A1

Alcaligenes faecalis
US20140072535A1

allicin
US20220256890A1

alliin
US20220256890A1

allylpropyl disulfide
US20220256890A1

Asparagopsis armata
US10881697B2

Bacillus amyloliquefaciens
WO2021211548A1

Bacillus strain 300 (NRRL No. B-50943)
US20210360945A1

Bacillus strain 86 (NRRL No. B-50944)
US20210360945A1

Bacillus, Lactobacillus, Strptocoecus, Candida and Pichia
WO2007072935A1

bambermycin
AU2018229465A1

benzylnitrate
US10154981B2

berberine, nitrate, and eucalyptus oil
US10440975B2

bis-(2-nitrooxyethyl) ether
US9266814B2

bis(2-hydroxyethyl)amine dinitrate
US10806706B2

bromochloromethane
US6251879B1

Bromoforms
US20220175670A1

chloroform
EP2747181A1

Chloroforms
US20220175670A1

Cysteine
US5843498A

dehydoabietic acid
US10799544B2

diallyl disulfide
US20180093308A1

diallyl disulphide
US20220256890A1

diallyl trisulfide
US20180093308A1

diallyl trisulfide
US20220256890A1

egg powder from immunizing hens with methane-producing M. stadtmaniae
US7820171B2

methanogens

Eggs laid by hens innoculated with methanogen antigens
US20200121730A1

essential oil
US20180001358A1

ethyl 2 butynoate
EP2747181A1

ethyl propionate
US20180093308A1

ethyl-11-nitrooxy undecanoate
US9365489B2

ethyl-11-nitrooxy undecanoate
US10154981B2

ethyl-3-nitrooxy butanoate
US10154981B2

ethyl-3-nitrooxy propionate
US9365489B2

ethyl-3-nitrooxy propionate
US10154981B2

ethyl-3-nitrooxy-butanoate
US9365489B2

ethyl-4-nitrooxy butanoate
US10154981B2

ethyl-4-nitrooxy-butanoate
US9365489B2

ethyl-4-nitrooxy-cyclohexylcarboxylate
US9365489B2

ethyl-4-nitrooxy-cyclohexylcarboxylate
US10154981B2

ethyl-5-nitrooxy pentanoate
US9365489B2

ethyl-5-nitrooxy pentanoate
US10154981B2

ethyl-6-nitrooxy hexanoate
US9365489B2

ethyl-6-nitrooxy hexanoate
US10154981B2

ethyl-8-nitrooxy octanoate
US9365489B2

ethyl-8-nitrooxy octanoate
US10154981B2

ethylene
EP2747181A1

eugenol
US7763273B2

fluoroacetate
EP2747181A1

fluvastatin
EP2747181A1

halothane
US6251879B1

hesperidin
EP2838376B1

hop acid
US8197863B2

hydrogen peroxide
WO2010071222A1

hydroxycinnamic acid
WO2021038832A1

Impatiens balsamina
NZ562783A

Iodoforms
US20220175670A1

isonaringin
EP2838376B1

isopimaric acid
US10799544B2

isosorbid-dinitrate
US10154981B2

isosorbid-dinitrate
US9266814B2

laidlomycin
AU2018229465A1

lasalocid
AU2018229465A1

limonene
US7763273B2

lovastatin
EP2747181A1

lumazine
EP2747181A1

medium and long chain fatty acids
EP2747181A1

Megasphaera sp. & Coprococcus catus
US10961559B2

methyl chloride
EP2747181A1

methyl fluoride
EP2747181A1

methyl-3-nitrooxy propionate
US9365489B2

methyl-3-nitrooxy propionate
US10154981B2

mevastatin
EP2747181A1

Monascus sp.
US20130011384A1

monensin
AU2018229465A1

N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide
US10154981B2

N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide
US9266814B2

N-ethyl-3-nitro-oxy-propionic sulfonyl amide
US10154981B2

neoabietic acid
US10799544B2

neohesperidin
EP2838376B1

nitrate
EP2747181A1

nitrite
EP2747181A1

nitroethane
EP2747181A1

palustric acid
US10799544B2

Peroxide source, peroxidase
US20210227850A1

phosphate
EP2747181A1

pimaric acid
US10799544B2

poncirin
EP2838376B1

Propionibacterium
US20120276058A1

propynoic acid
EP2747181A1

protease-resistant bacteriocin
EP1673983A1

R(OC2H4)n(OH); R = C12-15; n = 7
AU2013288441B2

R1(CH2)nONO2; n = 1-15; R1 = H, C1-C6alkyl, phenyl, —OH, —NH2, —CN,
US10806706B2

—COOH, —O(C═O)R8, —NHC(═O)R8, SO2NHR8, or —ONO2; R8 = C1-C6alkyl,

phenyl, pyridyl; when n is >3 the hydrocarbon chain may be interrupted by —O— or

—NH—

rac-4-Phenylbutane-1,2-diyl dinitrate, 2-(hydroxymethyl)-2-(nitrooxymethyl)-1,3-
US10154981B2

propanediol

red yeast rice
US20140322798A1

s-allyleysteine
US20220256890A1

saponin
US20180001358A1

Saponin & medium chain fatty acid
US10695393B2

sodium bromoethenesulfonate (BES)
EP2747181A1

vinyldithiines
US20220256890A1

ω-nitrooxyC3-10alkane-1-ol
US20210392922A1

[Image Omitted]

US9365489B2

[Image Omitted]

WO2022103280A1

[Image Omitted]

US20160183564A1

[Image Omitted]

EP3628169A1

[Image Omitted]

US20210163397A1

[Image Omitted]

WO2020221805A1

[Image Omitted]

AU2018383221A1

[Image Omitted]

US20030219467A1

Asparagopsis taxiformis
USPP34607P3

Lauric acid

*Each of the references in Table 9 is incorporated herein by reference.

Other exemplary inhibitors of methane production are discussed below.

Probiotics that Reduce Methane Production in Animals

Probiotics are a class of beneficial active microorganisms or their cultures. Probiotics are useful in reducing CH4 emissions in animals (Table 10). There are many types of probiotics, and different strains have different inhibitory effects on CH4 emissions. For example, the GA03 strain of Acetobacter is more effective at inhibiting CH4 production than other isolated strains. Most probiotics reduce CH4 production by influencing the activities of ruminal microorganisms, with no adverse effects on animals. In addition, probiotics enhance ruminal fermentation.

Lactic acid bacteria, which have been used as feed additives for a long time, not only reduce CH4 emissions per unit volatile fatty acid (VFA) output, but also improve the fermentation quality and fiber digestibility of silage. In addition, the denitrifying bacterium Bacillus 79R4 could prevent NO-2-N poisoning and microbial ecosystems from impairing fermentation efficiency. Furthermore, Bacillus licheniformis reduces CH4 production and increases feed energy and protein utilization.

TABLE 10

Exemplary probiotic bacteria that reduce methane production

Addition amount; maximum

Types of probiotics
Inhibitory effect
suppression methane amount
Inhibition mechanism

Propionic acid bacillus
[Image Omitted]

(Most propionic bacteria)
100 μL of the propionic acid bacteria
Unknown

[Image Omitted]

(P. jenscenii LMGT282
culture (2 × 10 [Image Omitted]

to 4 × 10

and P. thoenii LMGT2827 or T [Image Omitted]

)
colony forming units), Propioni-

bacterium thoenii T159; 20%

Lactic acid bacteria
[Image Omitted]

cfu/g fresh weight, Lacto-
Hydrogen consumption

bacillus plantarum, 8.8 ml/g(72 h)

Acetic acid bacteria
[Image Omitted]

1% Proteiniphilum acetatigenes
Reduced the number of

GA [Image Omitted]

methanogens

Enterococcus faccium
[Image Omitted]

0.1%; 2.08 mM/mL
Alters microbial flora

SROD

Probiotic products
[Image Omitted]

2 g probiotic products in
Reduce the number of

of Ruminococcus

powder 1.2 ml/g of dry matter
rumen protozoa

flavefaciens

10 ml probiotic products in liquid;

1.2 ml/g of dry matter

Baccillus licheniformis
[Image Omitted]

2.5 × 10

2.7

Unknown

Saccharomyces
[Image Omitted]

(

et al., 2019)
—
Affects rumen microbes

cerevistae
( [Image Omitted]

et al., 2019)

[Image Omitted]

indicates data missing or illegible when filed

Prebiotics that Reduce Methane Production

Prebiotics are substances that are not easily digested or absorbed by the host. They selectively stimulate the growth and activity of one or several ruminal microorganisms with a positive effect on ruminal fermentation. Prebiotics suppress ruminal CH4 production in subjects. Prebiotics mainly reduce rumen CH4 production by altering the bacterial community structure, influencing the permeability of the cell walls of methanogenic archaea, and stimulating other bacteria to compete with methanogens for H2 (Table 11).

The prebiotic chitosan can influence bacterial community structures by altering microbial population compositions, for example, by replacing fibrinolytic enzyme-producing microbes (Firmicutes and Fibrobacteres) with amylolytic enzyme-producing microbes (Bacteroides and Proteus); in turn, reducing CH4 production. Chitosan could influence the ruminal fermentation process by altering VFA distributions and increasing propionic acid concentrations, which reduces CH4 production in turn. However, the reduction in CH4 is associated with the degree of chitosan deacetylation, which could alter the permeability of the methanogen cell wall. In addition, various yeast products could reduce CH4 emissions by stimulating acetic acid-producing bacteria to compete with methanogens or metabolize hydrogen.

TABLE 11

Exemplary prebiotic bacteria that reduce methane production

Addition amount; maximum

Types of prebiotics
Inhibitory effects
methane suppression amount
Inhibition mechanismi

Chitosan
—
3000 (molecular weights)
(1) Alters microbial

dry matter; 22.9%
community structure

ml/day (Tong et al., 2020)
(Tong et al., 2020)

2% Chitosan + 21% of crude
(2) Alters fermentation

glycerin; 53.679% ( [Image Omitted]

pathway (

2020)

2020)

(3) Influences

methanogenic bacteria

cell wall permeability

(Zanferari et al., 2018)

Yeast products
—
4 mg/1 g dry matter; [Image Omitted]

Indirect consumption of

hydrogen

[Image Omitted]

indicates data missing or illegible when filed

Other Agents that Reduce Methane in Animals

Among the CH4 mitigation options, inhibiting the growth or the metabolic activity of methanogens is the most effective approach. Another strategy is to modulate rumen microbiome so that fermentation is shifted toward decreased H₂production and increased production of reduced VFA (e.g., propionate). Provided herein are exemplary and non-exhaustive descriptions of anti-methanogenic compounds evaluated with a focus on their impact rumen methanogens.

Coenzyme M Analogs

Methyl-CoM reductase (Mcr) mediates the final step of all the methanogenesis pathways and CoM (2-mercaptoethanesulfonic acid) is an essential cofactor serving as the methyl group carrier. Mcr reduces methyl-CoM to CH₄. CoM is found in all known methanogens but not in other archaea or bacteria. Several halogenated sulfonated compounds, including 2-bromoethanesulfonate (BES), 2-chloroethanesulfonate (CES), and 3-bromopropanesulfonate (BPS), are structural analogs of CoM, and they can competitively and specifically inhibit Mcr activity, lowering CH₄production at relatively low concentrations. Different species of methanogens vary in sensitivity to these inhibitors. Of three species tested on BES, Mbb. Ruminantium was the most sensitive, while Methanosarcina mazei was the least sensitive, with Methanomicrobium mobile being intermediate. All three species appeared to be resistant to BPS up to 250 μmol/L in pure cultures. The different sensitivity to these CoM analogs has been attributed to varying ability to uptake these inhibitors into the cells. Methanogens able to synthesize their own CoM are less dependent on external CoM and are thus less sensitive. Mbb. Ruminantium is the only ruminal methanogen that requires CoM synthesized by other methanogens.

Halogenated Aliphatic C₁-C₂Hydrocarbon

Halogenated aliphatic compounds with 1 or 2 carbons, such as chloroform, bromochloromethane (BCM), bromoform, bromodichloromethane, dibromochloromethane, carbon tetrachloride, trichloroacetamide, and trichloroethyladipate, can lower ruminal CH₄production. These halogenated compounds block the function of corrinoid enzymes and inhibit cobamide-dependent methyl group transfer in methanogenesis. These halogenated compounds also competitively inhibit CH₄production by serving as terminal electron (e) acceptors. Drenching chloroform to cattle inhibited methanogenesis substantially for up to 32 days without affecting feed digestion or basic rumen function. The addition of BCM depressed CH₄production both in vitro and in vivo. In steers fed grain-based diets, BCM decreased CH₄production by 50 to 60% with no signs of toxicity or residues in meat. It was also reported that the abundance of total bacteria and protozoa was not changed, but methanogenesis and growth of methanogens were drastically inhibited by BCM in both batch cultures and continuous fermenters. While the commercial use of chloroform, a recognized carcinogen, is not practical, it provides validation for the class of BCM compounds in reducing methane production.

Some marine plants such as red seaweed, and algae, lichen, and fungi can contain high concentrations of organobromine compounds such as bromomethane and bromoform. A recent in vitro study showed that red seaweed Asparagopsis taxiformis lowered CH₄production by 99% at a dose of 2% of organic matter substrate. No adverse effect on feed digestion or fermentation was noted at ≤5% (of dry matter) inclusion. Thus, red seaweed, and probably other organobromine-rich plants, may offer a potentially practical natural approach to mitigate CH₄emission.

Nitrooxy Compounds

3-Nitrooxypropanol (3NOP) and ethyl-3NOP, two new synthetic compounds, have been shown to have specific anti-methanogenic properties. 3NOP appears to inactive Mcr by competitively binding to the Mcr active site and then oxidizing the Ni¹⁺ that is required for Mcr activity. Feeding of 3NOP at a dose rate of 2.5 g/day/cow mixed in diets decreased CH₄emission by 60% per kg of DM intake. In a study using beef cattle, 3NOP fed at 2.0 g/day/cow decreased CH₄yield by 59%, and the inhibition persisted for up to 112 days without much effect on feed intake, nutrient digestibility or total VFA concentrations. In one recent study, 3NOP fed at 40-80 mg/kg feed DM in dairy cows decreased CH₄production by about 30% persistently for up to 84 days. Similarly, 3NOP fed at 2.5 g/day/cow decreased CH₄yield by 37% in dairy cows. In sheep, 3NOP at 0.5 g/day also decreased CH₄production by 29% without adverse effect on digestion or rumen fermentation. However, when 3NOP was directly added to the rumen through rumen cannula at a daily dose of 0.50 or 2.5 g per cow (equivalent to 25 to 125 mg/kg feed dry mailer), the degree of CH₄suppression declined to 7-10%. The later study suggests that 3NOP needs to be fed together with the diet to achieve higher efficacy. Thus, 3NOP could be used to lower CH₄emission from cows and sheep without adverse effects on nutrient utilization or animal performance. It has been demonstrated that 3NOP indeed decreased methanogen abundance while increasing the abundance of protozoa.

TABLE 12

Exemplary nitrogen-containing compounds that reduce methane production in animals

Inhibitory
Addition amount; maximum

Types of nitrogenous compounds
effect
methane suppression amount
Inhibition mechanism

Nitrate
—
20 mg/g dry matter; 21%
(1) Hydrogen consumption;

(Alvarez- [Image Omitted]

et al., 2019)
(2) Inhibits the proliferation

5 mmol/L; 32.92%
of methanogen [Image Omitted]

reduces their

(Wa et al., 2019)
activity and abundance

5 mM; 43.26% Lin rt al., 2017)

Encapsulated nitrate (EN)
—
70 g/100 kg of body
Reduces methane reducing

weight; 18.59% CH₄/kg of
bacteria

forage dry matter intake

(Granja- [Image Omitted]

et al., 2019)

2.5%; 9.37 mM/d

(Capelari et al., 2018)

2.5%, 2.8 g/kg Dry matter intake

(Alema et al., 2019)

Urea and nitrate mixture
—
34 g/kg straw dry matter + 6 g/kg
Indirect consumption of hydrogen

dry matter of ammonium nitrate;

10.2% (Zhang et al., 2019)

Urea + ammonium nitrate (34 +

6 g/kg of dry matter, respectively);

3.1 mL/g dry matter (Zhang et al.,

2018)

Nitroethane (NE), 2-Nitroethanol
—
10 mmol/L; 96.7% (NE), 96.7%
(1) Inhibits the activity

(NEOH), 2-Nitro-1-Propanol

(NEOH), 41.7% (NPOH)
of methanogens;

(NPOH)

(2) Inhibits methyl-

coenzyme M gene expression;

(3) Reduces the content of

coenzyme F420 and F430

3-Nitrooxypropanol (NOP)
—
0.08 mg/g dry matter; 44%
Inhibits methyl-Coenzyme M

(Alvarez- [Image Omitted]

et al., 2019)
activity

2.5 g/animal/day; 38%/kg

dry matter intake (Martinez-

Fernandez et al., 2018)

60 mg/kg of feed dry matter;

26%/day (Melgar et al., 2020)

1.6 g; 28%(roughage), 23%

(concentrate pellet) (Van

[Image Omitted]

et al., 2019)

[Image Omitted]

indicates data missing or illegible when filed

Pterin Compounds

Pterin is a group of structural analogs of deazaflavin (F₄₂₀), which is a coenzyme involved in two steps of the hydrogenotrophic methanogenesis pathway. Therefore, pterin compounds can competitively inhibit CH₄production. In one study, CH₄production by Mbb. ruminantium, Ms. mazei, and Mm. mobile was significantly decreased by lumazin (2,4-pteridinedione) in a dose-dependent manner from 0.06 to 0.24 mmol/L. As expected, pterin is much less efficacious in mixed rumen cultures than in pure methanogen cultures. It was suggested that lumazine could be degraded or transformed by some microbes in mixed cultures or adsorbed to solid particles where it becomes unavailable to methanogens. Some N-substituted derivatives of p-aminobenzoic acid, which are inhibitors of methanopterin synthesis in methanogens, decreased methanogenesis in ruminal cultures without inhibiting VFA production.

Hydroxymethylglutaryl-CoA (HMG-S-CoA) Reductase Inhibitors

All archaea contain long-chain isoprenoid alcohols as the major component of their cell membrane. Isoprenoid alcohols are unique to archaea. They are synthesized from mevalonate that is formed by reduction of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-S-CoA) catalyzed by HMG-S-CoA reductase. This enzyme is also used for the synthesis of the same precursor ultimately used in cholesterol synthesis in humans. As an inhibitor of HMG-S-CoA reductase, statins (e.g., lovastatin and mevastatin) can inhibit the growth of methanogens by inhibiting the synthesis of mevalonate. Lovastatin and mevastatin may also act as a potential inhibitor of F₄₂₀-dependent NADP oxidoreductase as shown in the model structure of that enzyme. In the earliest reported study, mevastatin at 5.6 μmol/L inhibited the growth of all three strains of rumen Methanobrevibacter, but not rumen bacteria in vitro. In studies using a rumen simulation technique (Rusitec), lovastatin (150 mg/L) reduced CH₄production by approximately 42% without altering bacterial counts or nutrient fermentation.

The high cost of pure statins promoted a search for natural sources of statins as agents to mitigate CH₄production. Lovastatin is a secondary metabolite of idiophase of several fungal species (e.g., Penicillium spp., Aspergillus terreus, Monascus purpureus, and Pleurotus ostreatus), and it can reach a concentration up to 2.8% of the dry weight of P. ostreatus (oyster mushrooms) and 261 mg/kg fermented rice straw. Lovastatin extracted from fermented rice straw significantly reduced total CH₄production by rumen methanogens by nearly 28% after 48 h in vitro incubation. Extract from A. terreus-fermented rice straw containing lovastatin (97 mg/g dry mass) also significantly reduced CH₄production and abundance of methanogens, especially Methanobacteriales, and aerobic fungi, but increased several fiber-degrading bacteria. Lovastatin also altered the morphology of M. smithii significantly, resulting in abnormal membrane formation and asymmetric cell divisions and increased HMG-S-CoA reductase gene expression. Fermented rice straw extract also modulated expression of several genes associated with methanogenesis, increasing expression of mtr, mta, and mcr while decreasing expression of hmd and fno. Supernatant fractions containing statins produced by Mortierella wolfii also appeared promising to inhibit methanogenesis without reducing overall fermentation. In another study using sheep, fermented rice straw containing metabolites (possibly pravastatin and mevastatin) produced by Monascus spp. Decreased CH₄emission (by 30%), the abundance of methanogens, and ruminal acetate:propionate ratio compared with the unfermented rice straw.

Diallyl disulfide, the main ingredient of garlic oil, is known to inhibit HMG-S-CoA reductase. Garlic oil (300 mg/L) was more effective than lovastatin as an inhibitor of CH₄production (by up to 91% reduction); however, garlic oil also inhibited bacterial growth, which likely reduces the availability of methanogenesis substrates. Garlic oil lowered CH₄production in vitro and growth of methanogens, altered community structure of methanogens after 24 h incubation. Moreover, interestingly, the anti-methanogenic efficacy increased over time up to 18 days of incubation.

Plant Secondary Metabolites

Plants secondary metabolites (PSM), such as saponins, tannins, flavonoids, organosulphur compounds, and essential oils, have anti-microbial activities against several types of microorganisms. Numerous PSM extracts have been recognized as potential inhibitors of rumen methanogens and CH₄production. Some forage plants rich in tannins and saponins have also shown promise at mitigating CH₄emission from animals.

TABLE 13

Exemplary plant extracts that reduce methane production in animals

Inhibitory
Addition amount; maximum

Types of probiotics

effects
suppression methane amount
Inhibition mechanisms

Plant extracts
Corymbia [Image Omitted]

leaf
—
10 ml/calf/day; —
(1) Protozoa number

reduced (1.84 × 10 [Image Omitted]

/ml)

(2) Ratios of volatile fatty

acids altered

Aloe vera, [Image Omitted]

—
25 mg/L and 50 mg/L Axadirachta
Unknown

papaya, [Image Omitted]

indica, Carica papaya, Tithonia

indica, Moringa oleifern,

diversifolia; 15% Jatropha

Tithania diversifolia,

curcas and Moringa oleifera pods;

Jatropha curcas, and

30% ( [Image Omitted]

Hassen, 2018)

Moringa oleifera pod

Eragrostis substrate, 4 ml plant

extracts

extracts; 50% reduction in methane/

Total gas production ( [Image Omitted]

Hassen

2020)

Pomegranate peel extract
—
2% of dry matter intake,
Unknown

and desert teak

Punica granatum; 40%

extract

T [Image Omitted]

mella undulata; 42%

Rhus [Image Omitted]

extract
—
50 mg/L; lowest
Unknown

[Image Omitted]

and Acacia
—
2% dry matter basis, [Image Omitted]

Unknown

extract

Catechu; 21% Acacia [Image Omitted]

23%

—
1.0%; 67.2% (Roque et al., 2019)
Unknown

Garlic extract
—
[Image Omitted]

(Kim et al., 2018b)
Decreased abundance of

1 g the experimental mixture; 6.9 ±
methanogenic archaea

10.7 ml/d ( [Image Omitted]

et al., 2018)

Plant extract [Image Omitted]

—
25 mg, high-forage diets; 60%
Decreased abundance of

high-concentrate diets; 41%
Methanobacter

Plant extract [Image Omitted]

—
12 mM,

acid; 37

%
Unknown

acid and p-coumaric

p-coumaric acid; 28 [Image Omitted]

%

acid

[Image Omitted]

indicates data missing or illegible when filed

1. Tannins

Tannins decrease CH₄production by directly inhibiting methanogens and indirectly decreasing H₂production as a result of decreased fiber digestion and protozoal population in the rumen. The inhibitory activity of tannins extracted from Lotus pedunculatus was demonstrated on pure cultures of methanogens. The inhibition of methanogen populations was also shown by tannins in the rumen of goats fed diets containing tannins. Studies on structure-activity relationships have shown that types and molecular weights of tannins are important in determining their potency in lowering CH₄production and abundance and diversity of rumen methanogens, with high molecular weight condensed tannins (CT) being more potent. Such structure-activity relationships have been demonstrated using members of Methanobacteriales including Methanobrevibacter.

2. Flavonoids

Flavonoids have not been extensively evaluated with respect to rumen methanogenesis. It was reported that inclusion of flavone, myricetin, naringin, rutin, quercetin, or kaempferol decreased in-vitro CH₄production by 5 to 9 mL/g DM. Their potency ranked as follows: myricetin≥kaempferol≥flavone>quercetin≥naringin>rutin≥catechin. Catechin decreased CH₄production both in vitro and in vivo. All the flavonoids, when fed at 0.2 g/kg DM, noticeably decreased relative abundances of hydrogenotrophic methanogens, and citrus (Citrus aurantium) extract rich in mixed flavonoids and its pure flavonoid components, neohesperidin and naringin, appeared to result in the greatest inhibition. Methanosarcina spp. Were also inhibited by poncirin, neohesperidin, naringin and their mixture. Flavonoids directly inhibit methanogens and also likely acts as H₂sinks via cleavage of ring structures (e.g., catechin) and reductive dihydroxylation.

3. Saponins

The effects of saponins on rumen fermentation, rumen microbial populations, and ruminant productivity have been examined extensively. Quillaja saponin at 1.2 g/L, but not at 0.6 g/L, lowered CH₄production in vitro and the abundance of methanogens (by 0.2-0.3 log) and altered their composition. Ivy fruit saponin decreased CH₄production by 40%, modified the structure of the methanogen community, and decreased its diversity. Saponins from Saponaria officinalis decreased CH₄and abundance of both methanogens and protozoa in vitro. It is hypothesized that saponins lower H2 production, thereby reduce CH4 production.

4. Essential Oils

The effects, mostly beneficial, of essential oils (EO) on rumen fermentation, microbial populations, and ruminant productivity have frequently been reviewed. Several EO compounds, either in pure form or in mixtures, are anti-methanogenic. The effects of EO on CH₄production and methanogens are variable depending on dose, types, and diet. Five EO (clove, eucalyptus, peppermint, origanum, and garlic oil) that have different chemical structures in vitro at three different doses (0.25, 0.50 and 1.0 g/L) were tested for their effect on CH₄production and archaeal abundance and diversity. Overall, all these EO suppressed CH₄production and abundance of archaea and protozoa in a dose-dependent manner, but they differed in potency. Thyme oil or cinnamon oil fed to Holstein steers at 0.5 g/day decreased the relative abundance of total protozoa and methanogens. However, feeding beef cattle a blend of EO (CRINA®) did not affect CH₄production, methanogen abundance or its diversity. Overall, methanogens may be directly inhibited or indirectly inhibited by Eos via inhibition of protozoa and H₂-producing bacteria in the rumen.

Alternative Hydrogen Sinks

Compounds with a redox potential higher than CO₂can thermodynamically outcompete CO₂for reducing equivalents produced during rumen fermentation. These compounds, thus, can be used as alternative e⁻ acceptors to redirect e⁻ flux away from methanogenesis. The commonly evaluated alternative e⁻ acceptors are discussed below.

1. Nitrate and Sulfate

Nitrate (NO₃¹⁻) decreased CH₄production both in vitro and in vivo. Mechanistically, nitrate decreases CH₄production by outcompeting CO₂as an e⁻ acceptor, and its reduction intermediates, nitrite (NO₂¹⁻) and nitrous oxide (N₂O), also directly inhibit methanogens as well as some H₂producers. Sulfate also lowers CH₄production, but much less effectively than nitrate. Archaeal abundance declined in goats receiving nitrate. While nitrate is not toxic to methanogens, it is toxic to protozoa, fungi and to a lesser extent to select bacterial species, suggesting a more general toxicity of nitrate. Nitrate can replace a portion of the dietary nitrogen as it is reduced to ammonia.

2. Nitrocompounds

A few organic nitrocompounds have been evaluated for their efficacy to decrease methanogens and CH₄production. These compounds can serve as e⁻ acceptors by some bacteria competing with methanogens for reducing equivalents. This is demonstrated by nitroethane that can be used as a terminal e⁻ acceptor by Dentitrobacterium detoxificans, thereby indirectly decreasing CH₄production. Nitrocompounds may also inhibit methanogenesis by directly inhibiting the activity of formate dehydrogenase/formate hydrogen lyase and hydrogenase, all of which are involved in the early step(s) of the hydrogenotrophic methanogenesis pathway, or inhibiting e⁻ transfer between ferredoxin and hydrogenase.

Nitrocompounds generally are quite effective in lowering CH₄production, with 3-nitro-propionate, 2-nitropropanol, 2-nitroethanol and nitroethane being able to decrease CH₄production by 57 to 98% in vitro. Using sheep, it was shown that nitroethane decreased CH₄production by up to 45% and 69%, respectively, when orally administrated at 24 and 72 mg/kg body weight daily for 5 days. Although less effective than nitroethane, 2-nitropropanol also significantly lowered CH₄production (by 37%) in steers.

3. Propionate and Butyrate Enhancers

Malate, acrylate, oxaloacetate, and fumarate are intermediates of carbohydrate fermentation. They can be converted to propionate or used in anabolism for the synthesis of amino acids or other molecules. They can accept reducing equivalents and thus stoichiometrically lower H₂available for CH₄production. When added at a concentration of 3.5 g/L, fumarate decreased CH₄production by 38% in continuous fermenters with forages as a substrate. Types of forages and their combinations appeared to affect the anti-methanogenic efficacy of fumarate, ranging from 6 to 27% inhibition at 10 mmol/L. Acrylate also depresses CH₄production in the rumen, but to a lesser extent than an equimolar level of fumarate. Malate was found to decrease CH₄production by beef cattle in a dose-dependent manner, with a 16% decrease being noted when fed at 7.5% of DM intake, which corresponds to a 9% reduction per unit of DM intake. Different studies reported different anti-methanogenic potencies of this type of e⁻ acceptors. Fumarate fed to goats at 10 g/day/goat was found to decrease the abundance of methanogens and CH₄production only by 11.9% while increasing concentrations of total VFA, acetate and propionate. Some of the intermediates of pyruvate conversion to butyrate can act as e⁻ acceptors, which could also decrease CH₄production.

4. Unsaturated Organic Acids

Unsaturated fatty acids can act as hydrogen sinks during their biohydrogenation and thereby lower CH₄production. Propynoic acid (an unsaturated analog of propionic acid), 3-butenoic acid and 2-butynoic acid (both unsaturated analogs of butyric acid), and ethyl 2-butynoate each at 6 to 18 mmol/L have been evaluated as alternative e⁻ sinks to lower methanogenesis in vitro. Only propynoic acid and ethyl 2-butynoate markedly lowered CH₄production, by 65 to 76% and 24 to 79%, respectively. In another study, propynoic acid lowered CH₄production by 67% and 78% at 6 and 12 mmol/L, respectively and decreased methanogen abundance. Propynoic acid and ethyl 2-butynoate are directly toxic to methanogens, and species of methanogens vary in their sensitivity to these two inhibitors, with Mbb. ruminantium being most sensitive, Ms. mazei least sensitive, and Mm. mobile intermediate.

Inhibitors to Hydrogen-Producing Bacteria
1. Ionophores

Ionophores, such as monensin and lasalocid, are commonly used to improve rumen microbial metabolism. Being highly lipophilic ion carriers, they pass through the cell wall of Gram-positive bacteria and penetrate into the cell membrane. Therein, they serve as H⁺/Na⁻ and H⁺/K⁻ antiporters, dissipating ion gradients that are needed for ATP synthesis, nutrient transport, and other essential cellular activities and ultimately resulting in delayed cell division and even cell death. Ionophores preferentially inhibit Gram-positive bacteria, including members of class Clostridia, including Ruminococcus species that produce acetate and H₂. Ionophores can also inhibit some Gram-negative rumen bacteria, including bacteria that produce formate and H₂. Therefore, ionophores may lower CH₄emission by decreasing H₂production. For examples, monensin fed at 24-35 mg/kg diet lowered CH₄production by up to 10% (g/kg DM intake), though no CH₄suppression was observed at 10-15 ppm. In a recent in vivo study, however, monensin at 60 mg/day/cow did not lower CH₄production by tropical cattle, though it decreased CH₄production by about 30% when fed at 250 mg/day/cow. As repeatedly noted, at such high supplementation level, DM intake was lowered, which explains most of the observed decrease in CH₄emission. Ionophores are not known to directly inhibit methanogens, but they can change the population dynamics of methanogen species. For example, monensin decreased the population of Methanomicrobium spp. While increasing that of Methanobrevibacter spp. Total methanogens were also decreased in cattle fed monensin. These can be explained by reduced availability of H₂and differences in affinity for H₂and growth kinetics among methanogen species.

2. Bacteriocins

Bacteriocins are proteins or peptides produced by bacteria and inhibit select microbial species in the rumen and other habitats. There are only a few studies investigating the effect of bacteriocins on CH₄emission. Bovicin HC5, a bacteriocin produced by Streptococcus spp. From the rumen, was reported to suppress CH₄by 50% in vitro. Nisin, a bacteriocin produced by Lactobacillus lactis subsp. Lactis, has also been shown to decrease CH₄production in vitro by up to 40% depending upon its concentration. Similar to monensin, bacteriocins probably modulate rumen fermentation leading towards increased propionate, thereby decreasing CH₄production.

Additional Agents that Reduce Methane in Animals

Biochemical Pathways

In certain embodiments, the one or more deleterious atmospheric gases and/or precursors thereof are microbially derived through one or more biosynthetic pathway. The deleterious atmospheric gas can be any suitable deleterious atmosphere gas, such as carbon dioxide, methane, nitrous oxide, or a combination thereof. The deleterious atmospheric gas precursor can be any suitable precursor, such as acetate, hydrogen, carbon, methanol, monomethylamine, dimethylamine, trimethylamine, nitric oxide, or a combination thereof. In preferred embodiments, the deleterious atmosphere gas comprises carbon dioxide, hydrogen, or methane more preferably methane. In certain embodiments, wherein the resultant deleterious atmospheric gas comprises methane, the one or more biosynthetic pathways include the acetoclastic, hydrogenotrophic, and methylotrophic pathways, which differ based on the starting substrates, i.e., precursor, more preferably the acetoclastic or hydrogenotrophic pathways, even more preferably the acetoclastic pathway.

The acetoclastic pathway comprises a series of enzymes that convert the precursor acetate through a series of enzymatic conversions to methane. Starting from acetate, (1) acetate is converted to acetyl phosphate by acetate kinase (ack); (2) acetyl phosphate is converted to acetyl-CoA by phosphotransacetylase (pta); (3) the acetyl group from acetyla-CoA is transferred to a protein intermediate by acetyl-CoA decarbonylase; (4) the acetyl group is then transferred to tetrhydrosarcinapterin to form 5-methyl-tetrahydrosarcinapterin by methyltetrahydrosarcinapterin methyltransferase; (5) 5-methyl-tetrahydrosarcinapterin is converted to methyl-CoM by methyl-H₄SPT:CoM methyltransferase (Mtr); and (6) methyl-CoM is reduced to methane by methyl-CoM reductase (Mcr) (FIG. 8 and FIG. 9).

The hydrogenotrophic pathway comprises a series of enzymes that convert the precursors hydrogen and carbon dioxide to methane. Starting from carbon dioxide and hydrogen, (1) a formylmethanofuran dehydrogenase (Fwd/Fmd) produces a formylmethanofuran, (2) which is further converted into 5-formyl-tetrahydromethanopterin by a formylmethanofuran:H₄MPT formylatransfer (Ftr); (3) 5-formyl-tetrahydromethanopterin is further converted into 5,10-methenyltetrahydromethanopterin by methyl-H₄MPT cyclohydrolase (Mch); (4) 5,10-methenyltetrahydromethanopterin is converted to N₆-methyltetrahydromethanopterin by F₄₂₀-dependent methylene-H₄MPT reductase (Mer); (5) N₆-methyltetrahydromethanopterin is converted to methyl-CoM by methyl-H₄MPT:coenzyme M methyltransferase (Mtr); and (6) methyl-CoM is reduced to methane by methyl-CoM reductase (Mcr) (FIG. 8 and FIG. 9).

The methylotrophic pathway comprises a series of enzymes that convert one or more of dimethylamine, methanethiol, methanol, methylamine, methylthiopropanoate, tetramethylammonium, and/or trimethylamine into methyl-CoM, wherein methyl-CoM is reduced to methane by methyl-CoM reductase (Mcr) (FIG. 8 and FIG. 9).

In certain embodiments, provided herein are compositions, methods, and/or kits comprising one or more small molecules that reduce the activity of one or more enzymes in one or more methane biosynthetic pathways. The enzyme can be any suitable enzyme, such as 3-(methylthio)propanoate:coenzyme M methyltransferase, acetate kinase, acetyl-CoA decarbonylase, acetyl-CoA decarbonylase/synthase complex α₂ε₂, acetyl-CoA decarbonylase/synthase complex β, acetyl-CoA decarbonylase/synthase complex γδ, acetyl-CoA synthase, carbon monoxide dehydrogenase, carbonic anhydrase, Co-methyltransferase, coenzyme M reductase, cyclohydrolase, dehydrogenase, dimethylamine-[corrinoid protein] Co-methyltransferase, F₄₂₀-dependent methylene-H₄MPT reductase, F₄₂₀-dependent methylene-H₄SPT dehydrogenase, formylmethanofuran dehydrogenase, formylmethanofuran:H₄MPT formyltransferase, formylmethanofuran:H₄SPT formyltransferase, formyltransferase, H₂-forming methylene-H₄MPT dehydrogenase, methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, methenyl-H₄MPT cyclohydrolase, methyl-coenzyme M reductase, methyl-H₄SPT:CoM methyltransferase, methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, methylcobamide:CoM methyltransferase, methylthiol:coenzyme M methyltransferase, methyltransferase, MtaC protein:coenzyme M methyltransferase, phosphotransacetylase, tetrahydromethanopterin S-methyltransferase, tetramethylammonium methyltransferase, trimethylamine-corrinoid protein Co-methyltransferase, or a combination thereof. In a preferred embodiment, the enzyme comprises methyl-CoM reductase (Mcr) (FIG. 10).

Compositions for Reducing Production of Deleterious Atmospheric Gases and/or Precursors Thereof In certain embodiments provided herein are compositions. In certain embodiments, provided herein are compositions comprising one or more small molecules. In preferred embodiments, provided herein are compositions comprising one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors thereof.

The small molecule can be any suitable small molecule for reducing the production of one or more greenhouse gases and/or precursors thereof, for example a small molecule that interferes with the uptake and/or conversion of acetate, hydrogen, carbon dioxide, methanol, monomethylamine, dimethylamine, trimethylamine, nitric oxide, or a combination thereof, and/or a small molecule that interfere with the production of carbon dioxide, hydrogen nitrous oxide, or a combination thereof. In preferred embodiments, the small molecule interferes with the uptake and/or conversion of acetate, hydrogen and/or carbon dioxide and/or the production of carbon dioxide or methane, more preferably with the production of methane.

Small Molecules that Affect Production of Deleterious Atmospheric Gases and/or Precursors Thereof

In certain embodiments, provided herein is a composition for reducing emissions of deleterious atmospheric gasses and/or precursors thereof comprising: one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and/or precursors thereof. The one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and/or precursors can be any suitable molecule.

In certain embodiments, the one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and/or precursors comprises a compound with the formula the formula R¹—[CH₂]_n—ONO₂

- wherein
- n is an integer from 1 to 15;
- R¹is selected from the group consisting of H, C₁-C₆alkyl, phenyl, —OH, —NH₂, —CN, —COOH, —O(C═O)R³, —NHC(═O)R³, SO₂NHR³, or —ONO₂, —SH and R³is C₁-C₆alkyl, phenyl, pyridyl;
- with the proviso that when n is >3 the hydrocarbon chain may be interrupted by —O— or —NH—.

In some embodiments, the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors comprises 3-nitrooxypropanol, 9-nitrooxynonanol, 5-nitrooxy pentanoic acid, 6-nitrooxy hexanoic acid, bis(2-hydroxyethyl)amine dinitrate, 1,4-bis-nitrooxybutane, 1,5-bis-nitrooxypentane, or any combination thereof. Preferably, the one or more small molecules is 3-nitrooxypropanol (3NOP).

In some embodiments, the composition comprises about 1 to about 25% by weight of the small molecule, about 5 to about 20% by weight of the small molecule, or about 5 to about 15% by weight of the small molecule.

Solid Carriers

In certain embodiments, the composition further comprises one or more solid carriers. As used herein, the term “solid carrier” includes additives commonly used in the preparation of powderous formulations such as thickeners, for example gums or cellulose derivatives such as xanthan gum, karaya gum and/or ethylcellulose. The one or more solid carriers can be any agriculturally suitable carrier, such as attapulgite, kaolinite, fuller's earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, a natural or synthetic derivative thereof, or a combination thereof.

In certain embodiments, the one or more solid carriers comprises any carrier suitable for ingestion, such as a saccharide comprising cellulose, xantham gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, or a combination thereof.

In some embodiments, the carrier comprises attapulgite, kaolinite, fuller's earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, a natural or synthetic derivative thereof, or a combination thereof.

In other embodiments, the carrier comprises attapulgite, kaolinite, fuller's earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, silica, propylene glycol, hemp protein, biochar, montmorillonite, activated charcoal, lignin, wood flour, hemp protein, pea protein, soy protein, gelatin, casein, chitosan, talc, calcium phosphate, arginine, lysine, calcium carbonate, carbon black, glutamine, betaine, bismuth phosphate, bismuth citrate, iron phosphate, or any combination thereof.

In some embodiments, the carrier comprises the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, or a combination thereof.

In some embodiments, the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, carrageenan, cellulose acetate, hydroxypropyl cellulose, cellulose acetate phthalate, maltrodextran, dextran, inulin, corn starch, amylopectin, sodium starch glycolate, pentaerthritol, cyclodextrin, or a combination thereof.

In certain preferred embodiments, the solid carrier comprises silica and ethylcellulose, more particularly about 10% to about 50% by weight of the silica and about 50 to about 90% by weight of the ethylcellulose.

In other preferred embodiments, the solid carrier comprises silica and activated charcoal, particularly about 10% to about 90% by weight of the silica and about 10% to about 90% by weight of the activated charcoal.

In certain embodiments, the binder further comprises arginine, lysine, or both arginine and lysine. While not being bound by theory, it is believed that arginine and lysine are capable of forming hydrogen bonds with the small molecule, such as 3NOP, thereby altering the release rate.

In other preferred embodiments, the carrier comprises activated charcoal and ethylcellulose, particularly about 10% to about 50% by weight of the activated charcoal and about 40 to about 90% by weight of the ethylcellulose.

In some embodiments, the carrier further comprises about 1 to about 10% by weight of sodium lignosulfate. While not being bound by theory, it is believed that sodium lignosulfate improves coating adhesion to the tablet resulting in a reduction in release rate of the small molecule.

In other embodiments, the carrier comprises arginine and polycaprolactone, such as about 10 to about 60% by weight of the arginine and about 30 to about 90% by weight of the polycaprolactone.

In other preferred embodiments, the carrier comprises 25% silica, 66% polycaprolactone, such as about 10 to about 60% by weight of the silica and about 30 to about 90% by weight of the polycaprolactone.

In certain embodiments, the composition comprises a granular shape. The composition may comprise any suitable shape, such as a spherical-, square-, rectangular-, capsular-, cylindrical-, conical-, ovular-, triangular-, diamond-, disk-like shape, or a combination thereof. In certain embodiments, the shape of the particle affects the rate of dissolution of the particle.

The granular particle can comprise any suitable texture, for example hard or soft. In certain embodiments, the texture of the particle affects the rate of dissolution of the particle. In certain embodiments, the composition comprises a combination of differently textured pellets each of which release the small molecule at different rates.

In certain embodiments, the granular particles comprise a uniform size distribution, for example about ±20%, ±15%, ±10%, ±5%, ±2%, or ±1% size distribution in the median particle size. In certain embodiments, the granular particles comprise a non-uniform size distribution, for example greater than about ±20%. In certain embodiments, the granular particles comprise a plurality of differently sized populations of granular particles each of which comprise a uniform size distribution.

In certain embodiments, the one or more solid carrier dissolves and thereby releases the one or more small molecules that reduce the production of greenhouse gases and/or precursors thereof. In a preferred embodiment, the one or more solid carriers will dissolve in water.

Extended and Delayed Release

It may be necessary to vary the rate of dissolution of the composition. For example, one may want to produce an extended-release formulation, wherein the composition releases the one or more small molecules over a period of time to maintain a suitable environmental concentration of the one or more small molecules. This can be beneficial to reduce the frequency of applications, for example to reduce labor costs and/or applications in rural and/or hard to reach environments. In certain embodiments, complete dissolution of the composition and full release of the one or more small molecules occurs over at least about 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, and/or nor more than about 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, or 147 days, for example about 1 to about 147 days, preferably about 7 to about 63 days, more preferably about 7 to about 42 days even more preferably about 14 to about 42 days yet even more preferably about 14 to about 28 days.

In certain embodiments, the extended-release formula may comprise any suitable extended-release formula. In certain embodiments, the extended-release formula comprises one or more additives that reduce the rate of dissolution of the composition, one or more additives that reduce the rate of dissolution of the small molecule from the composition, one or more additives that comprise a membrane that dissolves over time, wherein the rate of dissolution of the membrane controls the rate of release of the one or more small molecules, a suitable alternative, or a combination thereof.

In certain embodiments, the population of granular particles comprises a plurality of populations of granular particles wherein each population comprises a different formulation and/or shape. In certain embodiments, the population of granular particles comprises a first population and a second population. In certain embodiments, the population of granular particles further comprises at least 1, 2, 3, 4, 5, 5, 6, 8, or 9 and/or no more than 4, 5, 6, 7, 8, 9, or 10 additional populations, for example a total of 3-10 additional populations, preferably 3-7 additional populations, more preferable 3-5 additional populations. In a preferred embodiment, each of the additional populations comprises a different formulation than the others.

In certain embodiments, the rate of dissolution of the granular particles is modulated by the size of the granular particle. In certain embodiments, smaller granular particles dissolve faster than larger granular particles, such that each successive larger population in the plurality of populations of differently size particles provides a delayed release compared to the smaller populations of particles. In certain embodiments, an increased proportion of larger to smaller granular particles in a population of granular particles results in slower rates of dissolution of the population of granular particles.

In certain embodiments, the first population of particles comprises an immediate release formulation. In certain embodiments, the second population comprises a delayed release formulation, wherein the second population dissolves and/or releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and/or precursors after the first population. In certain embodiments, each additional population comprises a delayed release formulation, wherein each population dissolves and/or releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gasses and/or precursors at a different time than each of the other populations.

In some embodiments, an immediate release formulation releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gases with about 1 day, 2 days, 3 days, 4 days, 5 days, or 6 days. In some embodiments, a delayed release formulation releases the one or small molecules after about 1 week or more, for example up to about 21 weeks.

In some embodiments, the first population of particles provides has a half-life for the one or more small molecules (such as about 1 to 12 hours), the second population has a longer half-life (such as about 24 or more hours), each additional population has a longer half-life than the previous population such that an effective amount of the one or more small molecules is maintained for weeks or months. Thus, the present compositions advantageously do not, in such embodiments, require repeated, frequent applications.

In certain embodiments, the extended release happens within the first 10% of the half-life and then the 1st delayed happens with 1-2 half lives, then the next with 1-2 of the delayed release.

In some embodiments, the particles have a size ranging from about 1 mm to about 20 mm, about 1 to about 15 mm, about 1 to about 10 mm, about 5 to about 20 mm, about 5 to about 15 mm, or about 5 to about 10 mm.

Coatings

In certain embodiments, the composition may comprise a coating, for examples particles or tablet having a coating. The coating can comprise any suitable coating, such as a wax, a fat, or a synthetic polymer. In certain embodiments, the wax comprises organic compounds consisting of long alkyl chains, natural waxes (plant, animal) which are typically esters of fatty acids and long chain alcohols as well as synthetic waxes, which are long-chain hydrocarbons lacking functional groups. In certain embodiments, the fat comprises a wide group of compounds which are soluble in organic solvents and largely insoluble in water such as hydrogenated fats (or saturated fats) which are generally triesters of glycerol and fatty acids. Suitable fats can have natural or synthetic origin. In certain embodiment, the fat comprises glycerine monostearate, carnauba wax, candelilla wax, sugarcane wax, palmitic acid, stearic acid hydrogenated cottonseed oil, hydrogenated palm oil and hydrogenated rapeseed oil, or combinations thereof. Any suitable synthetic polymer can be used, such as poly-L-glutamic acid (PGA) and polylactic acid (PLA). In preferred embodiments, the synthetic polymer is at least partially water soluble.

The coating may be single layer or multiple layers, preferably two layers.

In some embodiments, the coating is selected from cellulose acetate phlalate, ethyl cellulose, hydroxypropyl cellulose, polycaprolactone, alginate, chitosan, polyethylene glycol, cellulose acetate, triacetin, propylene glycol, n-methyl-2-pyrollidone, and any combination thereof.

In certain preferred embodiments, the coating comprises two or more polyelectrolytes, such as polystyrene sulfonate, polyethyleneimine, sodium lignosulfate, polyglutamic acid and poly-L-lysine, poly-L-arginine, polyallylamine hydrochloride, polyacrylic acid, or any combination thereof.

In some preferred embodiments, the polyelectrolytes comprise polyallylamine hydrochloride and sodium lignosulfate.

In some preferred embodiments, the polyelectrolytes comprise polyallylamine hydrochloride and polystyrene sulfonate.

In other preferred embodiments, the polyelectrolytes comprise sodium lignosulfate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine, and sodium lignosulfate.

In still other preferred embodiments, the polyelectrolytes comprise polystyrene sulfonate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine.

The polyelectrolytes may, in certain embodiments, be chemically cross-linked with a cross-linking agent.

In certain embodiments, the composition comprises one or more coatings applied with minimal to no bubbles. Additionally or alternatively, the composition comprises one or more coatings that comprise a foam or a plurality of air bubbles. In certain cases, the foamed coating can temporarily alter the buoyancy of the composition. One such example includes a composition comprising a foamed coating that floats when initially applied, then, after a period of time, the air pockets in the foamed coating fill with water resulting in the composition sinking to the bottom.

Additives with a Density Greater than Water

In certain embodiments, the composition further comprises one or more additives with a density greater than water. For example, the additive may have a density greater than 1.1, preferably about 1.1 mg/mL to about 3 mg/mL, about 1.5 to about 3 mg/mL, about 1.5 to about 2.5 mg/mL, or about 1.5 to about 2 mg/mL. Suitable additives include silica, attapulgite, kaolinite, fuller's earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, or a combination thereof. In certain embodiments, the one or more additives with a density greater than water result in the composition sinking below the surface. In certain embodiments, the one or more additives with a density greater than water result in the composition partially or completely sinking to the bottom. In preferred embodiments, the composition completely sinks to the bottom. In certain embodiments, the composition comprising the additive with a density greater than water has a density of at least 1.1, preferably about 1.1 mg/mL to about 3 mg/mL, about 1.5 to about 3 mg/mL, about 1.5 to about 2.5 mg/mL, or about 1.5 to about 2 mg/mL.

Agriculturally Beneficial Additives

In certain embodiments, the composition further comprises one or more agriculturally beneficial additives. The agriculturally beneficial additive can be any suitable additive depending on the application, such a vitamin, a nutrient, an antibiotic, a fungicide, or a combination thereof.

In certain embodiments, the additive includes one or more suitable components that reduce methanogenesis by methanogens, such as, seaweed (e.g., Asparagopsis taxiformis), kelp, 3-nitrooxypropanol, anthraquinones, ionophores (e.g., monensin and/or lasalocid), polyphenols (e.g., saponins, tannins), organosulfurs (e.g., garlic extract), flavonoids (e.g., quercetin, rutin, kaempferol, naringin, and anthocyanidins; bioflavonoids from green citrus fruits, rose hips and black currants), carboxylic acid, terpenes (e.g., D-limonene, pinene and citrus extracts), or a combination thereof.

Methods for Reducing Production of Deleterious Atmospheric Gases and/or Precursors Thereof

In certain embodiments provided herein are methods. In certain embodiments, provided herein are methods for using one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors thereof. In certain embodiments, provided herein are methods for applying one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors thereof to any suitable environment. The suitable environment can comprise any suitable environment. In some embodiments, the suitable environment comprises an environment in which a ruminant (unvaccinated or vaccinated) lives or occupies, e.g., a habitat for a ruminant. In some embodiments, the suitable environment comprises the rumen.

In certain embodiments, the method for reducing emissions of deleterious atmospheric gasses and/or precursors thereof comprises applying a composition comprising one or more small molecules that reduce the production of the deleterious atmospheric gasses and/or precursors thereof. The composition can comprise any suitable composition. In a preferred embodiment, the composition comprises any one of the compositions as described in the Compositions for reducing production of deleterious atmospheric gases and/or precursors thereof section above. In a more preferred embodiments, the composition comprises 3NOP. In certain embodiments, the composition is applied to a water source, such as a trough or a pond, from which a ruminant ingests the composition or a portion thereof from the water source into the rumen. For example as illustrated in FIGS. 12-13.

In certain cases, the composition needs to be reapplied periodically to maintain a suitable concentration of the one or more small molecules. In certain embodiments, the method further comprises reapplying after a period of time a composition comprising one or more small molecules that reduce the production of the deleterious atmospheric gasses and/or precursors thereof. In certain embodiments, the method further comprises, reapplying again after a period of time a composition comprising one or more small molecules that reduce the production of the deleterious atmospheric gasses and/or precursors thereof. Any suitable number of reapplications may be performed as needed to maintain a an effective amount of the one or more small molecules. In some embodiments, the composition is reapplied after about 7 to about 28 days, about 7 to 46 days, about 7 to 92 days or about 7 to 147 days.

In certain embodiments, the composition is delivered to one or more water sources.

In certain cases, the concentration of the one or more small molecules can be measured to ensure the presence of a suitable concentration of the one or more small molecules. Any suitable method may be used to measure the concentration, such as a strip test, liquid chromatography, or thin layer chromatography. The method can be performed with or without human intervention.

Kits for Reducing Production of Deleterious Atmospheric Gases and/or Precursors Thereof

In certain embodiments, provided herein are kits. In certain embodiments, the kit comprises any one of the compositions as described in the Compositions for reducing production of deleterious atmospheric gases and/or precursors thereof section. In certain embodiments, the kit further comprises a suitable container for shipping.

Methods of Using Vaccines, Animal Feed, Antibodies, Milk

Provided herein are methods of using the vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, etc.), or any combination thereof.

In certain aspects, provided herein are methods of inducing an immune response against at least one methanogen in a subject, the method comprising administering to the subject the vaccines or pharmaceutical compositions of the present disclosure.

In some embodiments, the immune response comprises a B cell response (e.g., to produce the antibodies). The antibodies produced in response to the vaccine are transferred to the saliva of the subjects, which are swallowed by the subjects to enter the rumen. Once in the rumen, the antibodies come in contact with at least one methanogen to bind/neutralize said methanogen.

As used herein, the term “neutralization of a methanogen” encompasses any reduction in one or more activities that are normally carried out by the methanogen in the absence of the antibodies that bind the methanogen.

In some embodiments, the activity of a methanogen includes but is not limited to, the activity that aids in producing methane gas. For example, binding of the antibodies to the methanogen may reduce the ability of the methanogen to carry out biochemical reactions that are necessary to produce methane, e.g., reduce the ability to convert hydrogen (H₂) and carbon dioxide (CO₂) or acetate into methane (CH₄) and ATP. In some embodiments, the reduced ability to produce methane may lower the fitness of methanogen in the rumen.

In some embodiments, the activity of a methanogen includes but is not limited to, the activity that aids in forming a granular colony with other bacteria. In some embodiments, such activity may be disrupted physically—e.g., antibodies binding to the methanogen would prevent physical association and/or film formation of the granular colony of bacteria. In some embodiments, a reduction in the activity of forming a granular colony may lead to the reduced ability of a methanogen to remain in the rumen. In some embodiments, such reduced ability may result in the reduction of the total number of methanogens inside the rumen.

In certain aspects, provided herein are methods of reducing the activity, number, and/or type of methanogens in the gut of a subject, the method comprising administering to the subject the vaccines or pharmaceutical compositions of the present disclosure.

In certain aspects, provided herein are methods of reducing the amount of methane (CH₄) and/or hydrogen (H₂) emitted by a subject, preferably eructated and/or exhaled, the method comprising administering to the subject a vaccine composition, antibody, milk, and/or animal feed of the present disclosure.

In some embodiments, the amount of methane (CH₄) and/or hydrogen (H₂) is reduced by about 5-100%, preferably by about 10-100%, compared to a control.

In some embodiments, the amount of methane (CH₄) and/or hydrogen (H₂) is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

In some embodiments, the amount of methane (CH₄) and/or hydrogen (H₂) is reduced by about 20-100%, preferably by about 30-100%, compared to a control.

In some embodiments, the amount of methane (CH₄) is reduced by (a) about 1 kg-about 50 kg within 8 weeks from the time of first vaccination, or (b) about 5 kg-about 300 kg within a year from the time of first vaccination, compared to a control.

In some embodiments, the amount of methane (CH₄) normalized to an amount of CO₂emitted by the subject (i.e., CH₄/CO₂) is reduced by about 5-100%, preferably by about 10-100%, compared to a control. In some embodiments, the amount of methane (CH₄) normalized to the amount of CO₂is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

In some embodiments, the amount of methane (CH₄) normalized to the amount of CO₂is reduced by about 20-100%, preferably by about 30-100%, compared to a control.

In some embodiments, the amount of hydrogen (H₂) is reduced by (a) about 10 g-about 500 g within 8 weeks from the time of first vaccination, or (b) about 50 g-about 3 kg within a year from the time of first vaccination, compared to a control.

In certain aspects, provided herein are methods of increasing the amount of carbon dioxide (CO₂) emitted by a subject, preferably eructated and/or exhaled, the method comprising administering to the subject a vaccine composition, antibody, milk, and/or animal feed of the present disclosure.

In some embodiments, the amount of carbon dioxide (CO₂) is increased by about 1-100%, preferably by about 1-20%, compared to a control. In some embodiments, the amount of CO₂is increased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

In some embodiments, the amount of carbon dioxide (CO₂) is increased by about 3-10%, preferably by about 3-20%, compared to a control.

In some embodiments, the amount of carbon dioxide (CO₂) is increased by (a) about 29.8 kg-about 1,490 kg within 8 weeks from the time of first vaccination, or (b) about 149 kg-about 8,940 kg within a year from the time of first vaccination, compared to a control.

In some embodiments, the control is: (a) an accepted reference; (b) the amount of methane, CO₂-normalized methane, hydrogen, or carbon dioxide emitted by an unvaccinated subject; or (c) the amount of methane, CO₂-normalized methane, hydrogen, or carbon dioxide emitted by the vaccinated subject prior to vaccination.

In preferred embodiments, any one of the methods produces an antibody against at least one methanogen. In some embodiments, the antibody is an IgM, IgG, or an IgA. In preferred embodiments, the antibody is an IgA or IgM. The IgA isoform, at least in cattle, may be more stable in the rumen. For example, IgA levels in cattle saliva were reduced by only 40% after 8 h exposure to rumen contents while IgG levels were reduced by 80%.

In some embodiments, the antibody is produced in an amount sufficient to: (a) carry the antibody to the gut; (b) reduce the number and/or type of methanogens in the gut; and/or (c) reduce the amount of methane produced by the subject.

In some embodiments, the method reduces the methane production by the subject by at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared with a control.

In some embodiments, the method reduces the H₂emission from the subject from at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% as compared with a control.

In some embodiments, the method increases the feed conversion efficiency of the subject by about 0.50%, 10%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.50%, 50%, 5.50%, 6%, 6.5%, 7%, 7.5%, 80%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

In some embodiments, the method increases the concentration of one or more volatile fatty acids (e.g., propionate, butyrate, acetate) in the rumen of the subject by about 0.5%, 1%, 1.50%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.50%, 6%, 6.5%, 7%, 7.5%, 80%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

In some embodiments, the method increases the average daily gain (ADG) of the subject by about 0.50%, 10%, 1.50%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.50%, 6%, 6.5%, 7%, 7.5%, 80%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

In some embodiments, the method increases the dry matter intake (DMI) of the subject by about 0.50%, 10%, 1.50%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.50%, 6%, 6.5%, 7%, 7.5%, 80%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control. Additionally or alternatively, the method increases the milk production of the subject by about 0.5%, 1%, 1.5%, 2%, 2.50%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.50%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, or 12% as compared with a control.

In some embodiments, the control is an accepted reference, or the amount of methane production in a subject that has not been vaccinated.

Notably, methane emission or production of methane by a subject can occur at any part of its intestinal track, which includes, e.g., a rumen and a lower bowel (lower intestinal track).

The rumen accounts for 90% of all methane production. The rumen has no adaptive immune response. Thus, to be effective in reducing the level of methane production, the rumen or a methanogen therein must be exposed to neutralizing antibodies that bind and inactivate the methanogen. By contrast, the lower bowel, which accounts for 10% of all methane production, has adaptive immune response such that any immune response due to a vaccine can be amplified in the lower bowel. Accordingly, in preferred embodiments, the result of immune response from a vaccine is exposed to the lower bowel of a subject, which then further amplifies the effect of the vaccine. In other words, in preferred embodiments, a vaccine of the present disclosure or the immune response it elicits is exposed to a methanogen in a lower bowel of the subject. Thus, the present disclosure encompasses a method of reducing (i) methane production and/or (ii) activity, number, and/or type of methanogens in the lower intestinal track of a subject, the method comprising administering to the subject a vaccine comprising at least one methanogen cell surface protein or a fragment thereof.

Accordingly, in preferred embodiments, the methods and compositions of the present disclosure elicit immune response that is exposed to a methanogen in a lower bowel of the subject.

In some embodiments, the methods and compositions of the present disclosure reduce the activity, number, and/or type of methanogens in the lower intestinal track (lower bowel) of a subject.

In some embodiments, the methods and compositions of the present disclosure reduce the amount of methane produced by a subject in the lower intestinal track of a subject.

In some embodiments, the methods and compositions of the present disclosure induce an immune response against at least one methanogen in the lower intestinal track of a subject.

In some embodiments, the method of the present disclosure results in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 80%, 9%, 10%, 110%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% reduction in the level of methane produced by the subject.

In some embodiments, the reduction in the level of methane is compared to an untreated subject.

In some embodiments, the subject methane emissions are measured using a respiratory chamber.

As described herein, in some embodiments, the vaccine or the pharmaceutical composition is administered to the subject via a route selected from intramuscular administration, intradermal administration, subcutaneous administration, and nasal administration.

In some embodiments, the subject is administered with at least one dose of the vaccine or pharmaceutical composition.

In some embodiments, the subject is administered with at least one or two repeat doses of the vaccine or pharmaceutical composition (e.g., booster dose).

In some embodiments, the subject is administered with the repeat dose of the vaccine or pharmaceutical composition after at least about 2 weeks, 1 month, 6 months, or 12 months from the time the subject is administered with the preceding dose of the vaccine.

In some embodiments, the subject is administered with the repeat dose of the vaccine or pharmaceutical composition no more than about 3 months, 6 months, 12 months, or 24 months from the time the subject is administered with the preceding dose of the vaccine.

In some embodiments, the subject receives the repeat dose of the vaccine after at least about 2 weeks and no more than about 18 months from the time the subject is administered with the preceding dose of the vaccine.

In some embodiments, the subject receives a repeat dose of the vaccine after at least about 4 weeks and no more than about 12 months from the time the subject is administered with the preceding dose of the vaccine.

In certain embodiments, the methods of the present disclosure further comprises administering to the subject at least one agent (e.g., at least one additional agent) that reduces the level of methane produced by the subject.

The vaccine composition may be administered before, concurrently with, or after, any agent, milk, antibody, animal feed, or any composition of the present disclosure.

In some embodiments, the at least one agent is selected from 3-Nitrooxypropanol (3NOP), ethyl-3NOP, 2-bromoethanesulfonate (BES), 2-chloroethanesulfonate (CES), 3-bromopropanesulfonate (BPS), bromochloromethane (BCM), bromoform, bromodichloromethane, dibromochloromethane, carbon tetrachloride, trichloroacetamide, trichloroethyladipate, lumazin (2,4-pteridinedione), p-aminobenzoic acid, lovastatin, mevastatin, pravastatin, diallyl disulfide, garlic oil, saponins, tannins, flavonoids, nitrate, nitroethane, -nitro-propionate, 2-nitropropanol, 2-nitroethanol, malate, acrylate, oxaloacetate, fumarate, propynoic acid, 3-butenoic acid, 2-butynoic acid, ethyl 2-butynoate, monensin, lasalocid, bovicin HC5, nisin, and any combination thereof.

In preferred embodiments, the agent is 3NOP or ethyl-3NOP.

In certain aspects, provided herein are methods of reducing methane production in a subject, the method comprising orally administering to and/or feeding the subject the antibody, the milk and/or the derivatives thereof, and/or the animal feed of the present disclosure.

In certain aspects, the methods of the present disclosure relate to a subject. In some embodiments, the subject is selected from a cow, cattle, a bull, a bison, a yak, a buffalo, an antelope, a goat, a sheep, a deer, a giraffe, a caribou, a gazelle, a macropod, a llama, a camel, and an alpaca.

In some embodiments, the subject is an offspring (e.g., calf) of the vaccinated female subject that received the milk comprising an antibody that binds at least one methanogen.

In some embodiments, the subject is an adult subject. In other embodiments, the subject is a young subject (e.g., a calf). In some embodiments, a young subject includes a subject from birth to weaning. In some embodiments, a young subject includes a subject from birth up to two years of age, such from birth up to 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Accordingly, in some embodiments, a young subject may be at least, about, or no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months in age. In preferred embodiments, a young subject is administered with a vaccine, antibodies, milk, animal feed, agent (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other composition of the present disclosure prior to establishment of methanogens. In some such embodiments, a lower dose of vaccine or other agents may be required.

In some embodiments, a subject is a subject born from a vaccinated parent(s). In some embodiments, a subject is a subject born from a vaccinated mother such that the subject received a high level of methanogen-neutralizing antibodies in the colostrum and milk fed to the subject at birth. Such a subject or a subject who received an early treatment may have low initial methanogen establishment, thereby enhancing a long term performance of the vaccines, antibodies, milk, animal feed, agents (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), or other compositions of the present disclosure.

In some embodiments, a subject is vaccinated with each change in hands and/or environment, such as from one owner to another, one ranch to another, or one geographical area to another. Typical ranch operations will vaccinate a newly arrived animal to manage disease, and vaccinating with a vaccine of the present disclosure (e.g., a vaccine that targets at least one methanogen cell surface protein) will fall into this standard agronomic practice.

In some embodiments, a subject is vaccinated with a vaccine of the present disclosure (e.g., a vaccine that targets at least one methanogen cell surface protein) at a time when the subject is subject to at least one other vaccination. For example, a subject (e.g., a domesticated ruminant, a dairy cow, a beef cow) is subject to vaccination against infectious bovine rhinotracheitis (IBR), bovine virus diarrhea (BVD), parainfluenza-3 (PI3), bovine respiratory syncytial virus (BRSV), clostridia, E. Coli mastitis, leptospirosis, Mannheimia hemolytica, Brucella, vibriosis, Campylobacter, trichomonas, trichomoniasis, rotavirus, foot and mouth disease virus (FMDV), coronavirus, and/or respiratory disease. An exemplary vaccination regimen for a ruminant throughout the life cycle is shown in Table 14.

Additionally or alternatively, CH₄reducing vaccine compositions may be combined with one or more additional antigens configured to target infectious bovine rhinotracheitis (IBR), bovine virus diarrhea (BVD), parainfluenza-3 (PI3), bovine respiratory syncytial virus (BRSV), clostridia, E. Coli mastitis, leptospirosis, Mannheimia hemolytica, Brucella, vibriosis, Campylobacter, trichomonas, trichomoniasis, rotavirus, foot any mouth disease virus (FMDV) coronavirus, and/or respiratory disease to form a multivalent vaccine composition, which is then administered to the subject as disclosed herein.

TABLE 14

Exemplary administration schedule of USDA-approved vaccinations for a ruminant

Animal
Life Stage
Exemplary Vaccinations

Dairy
Adult
4-way viral-IBR/BVD/PI3/BRSV

Dry-off (e.g., 45-60 days pre-calving)
Leptospirosis (5-way)

Clostridial (7-way)

J-5 (mastitis)

Dairy
Adult
4-way viral-IBR/BVD/PI3/BRSV

Pre-breeding (e.g., 30 days prior to breeding

season)
Leptospirosis (5-way)

Dairy
Calf
4-way viral-IBR/BVD/PI3/BRSV

Weaning (e.g., 6-12 weeks)
Leptospirosis (5-way)

Clostridial (7-way)

Mannheimia hemolytica

Dairy
Calf
4-way viral-IBR/BVD/PI3/BRSV

Yearling (i.e., 12-24 mo)
Leptospirosis (5-way)

Clostridial (7-way)

Mannheimia hemolytica

Brucella

Beef
Adult
4-way viral-IBR/BVD/PI3/BRSV

Leptospirosis (5-way)

Pre-breeding (e.g., 30 days prior to breeding
Vibriosis (Campylobacter)-

season)
natural mating

Trichomonas-natural mating

Beef
Adult
4-way viral-IBR/BVD/PI3/BRSV

Pregnancy examination (e.g., 30-45 days

post-breeding season)
Leptospirosis (5-way)

Clostridial (7-way)

Animal
Life Stage
Exemplary Vaccinations

Scour vaccines-E. coli, possibly

combination with rota and

coronavirus

Beef
Calf
Clostridial vaccines

Newborn (e.g., 0-3 mo)
Possibly respiratory vaccines, if

there is a history of disease in very

young calves

Beef
Calf
4-way viral-IBR/BVD/PI3/BRSV

Leptospirosis (5-way)

Clostridial (7-way)

Weaning (e.g., 6-8 mo)
Mannheimia hemolytica

Brucella

Beef
Adult
4-way viral-IBR/BVD/PI3/BRSV

Arrival at stocker operation (e.g., 6-8 mo)
Mannheimia hemolytica

Beef
Adult
4-way viral-IBR/BVD/PI3/BRSV

14-21d post-arrival at stocker operation
Clostridial (7-way)

Mannheimia hemolytica

Leptospirosis (5-way) (optional)

Beef
Adult
4-way viral-IBR/BVD/PI3/BRSV

Arrival at feedlot operation (e.g., 9-11 mo)
Mannheimia hemolytica

Beef
Adult
4-way viral-IBR/BVD/PI3/BRSV

14-21d post-arrival at feedlot operation
Clostridial (7-way)

Mannheimia hemolytica

Leptospirosis (5-way) (optional

Vaccinating a large number of animals (e.g., in a ranch, e.g., herding ruminants) is a challenging process. Thus, in preferred embodiments, the administration of a vaccine of the present disclosure is incorporated into a pre-existing vaccination program that an animal is already subject to. Such a method reduces the cost, time, and efforts in administering a vaccine of the present disclosure to an animal.

Methods for Detection of Methane Production

Various methods for detecting the level of methane produced by animals are known in the art and can be applied to the methods of the present disclosure.

In some embodiments, portable respiration hoods for tethered and non-tethered animals (Garnsworthy et al. (2012) J. Dairy Sci. 95:3166-3180; Garnsworthy et al. (2019) Animals 9:837; Zimmerman and Zimmerman WO2011130538; each of which is incorporated herein by reference) directly measure the gas concentration of incoming and exhaust air from individual animals.

In some embodiments, tracer-ratio gas releases from the animal (Johnson et al. (1994) Environ. Sci. Technol., 28, 359-362, which is incorporated herein by reference), such as Sulfur hexafluoride (SF6) (Grainger et al. (2007) J. Dairy Sci., 90:2755-2766; Vechi et al. (2022) Agriculture, Ecosystems and Environment 330:107885; each of which is incorporated herein by reference), assumes that the tracer gas and the emitted CH4 have similar transport paths, so that a tracer measurement can establish the CH4 emission rate.

In some embodiments, micrometeorological techniques are typically considered a herd-scale measurement, where the emission rate is calculated from the measurement of enhanced gas concentrations downwind of an animal herd (Harper et al. (2011) Anim. Feed Sci. Tech., 166-167, 227-239, which is incorporated herein by reference), and these include the mass balance technique (Laubach et al. (2008) Aust. J. Exp. Agr., 48:132-137; Lockyer and Jarvis (1995) Environ. Pollut. 90:383-390; each of which is incorporated by reference), eddy covariance (Dengel et al. (2011) Glob. Change Biol., 17:3524-3533; Felber et al. (2015) Biogeosciences, 12:3925-3940; each of which is incorporated herein by reference), and inverse dispersion techniques (Flesch et al., (2005) Atmos. Environ., 39:4863-4874; Todd et al. (2014) J. Environ. Qual., 43:1125-1130; Bai et al. (2021) Atmos. Meas. Tech., 14:3469-3479; each of which is incorporated herein by reference). The main advantage of micrometeorological techniques is that they do not interfere with the animals or the environment.

There are also devices that measure the level of methane (see e.g., Rey et al. (2019) Animals 9:563, Mapfumo et al. (2018) Pastoralism: Research, Policy and Practice 8:15; each of which is incorporated herein by reference). For example, the laser methane detector (LMD) is a hand held open path laser measuring device (e.g., LaserMethaneMini (Tokyo Gas Engineering Co., Ltd. Anritsu Devices Co., Ltd., Tokyo, Japan)). The principle of the LMD measuring technology is described (Chagunda et al. (2013) Animal, 7:394-400; Garnsworthy et al. (2012) J. Dairy Sci. 95:3166-3180; and Chagunda et al. (2009) Comput. Electron. Agric. 68:157-160; each of which is incorporated herein by reference). Briefly, this device is based on infrared absorption spectroscopy using a semiconductor laser for CH4 detection. The device must be pointed towards the nostrils of the cow from a fixed distance. Then, the LMD measures the density of the air column between the device and the animal's nostrils. The reflected laser beam is detected by the device, and its signal is processed and converted to the cumulative CH4 concentration along the laser path in ppm-m. The LMD is connected to a tablet (Samsung Galaxy Tab A6, New Jersey, USA) running GasViewer app (Tokyo Gas Engineering Solutions, Tokyo, Japan) via Bluetooth connection for exporting and storing the data in real time at 0.5 s intervals. The effect of atmospheric ambient CH4 concentration from the measurements is discounted using the offset function of the LMD.

The non-dispersive infrared analyzer CH4 analyzer (NDIR) (Guardian NG Edinburg Instruments Ltd., Livinstong, UK) is one of the so-called sniffer methods that measure CH4 concentration (ppm) in breath or exhaled air. These methods have been previously used (e.g., by Garnsworthy et al. (2012) J. Dairy Sci. 95:3166-3180) to assess the CH4 production of dairy cows at commercial farms. Briefly, a gas sampling tube from the front of a cow's head to a gas analyzer to continuously measure CH4 concentration in the cow's breath is used. Then, air is drawn through the instrument by an integral pump between the gas inlet port and analyzer. The device can have a range of 0 to 10,000 ppm, and air can be sampled continuously at a rate of 1 L/min through an 8 mm polyamide tube, using approximately 2 m of tube from the analyzer to cow's nostrils. Methane concentration can be recorded at 1 s intervals and stored in a datalogger (Data Recorder SRD-99; Simex Sp. Z o.o, Gdansk, Poland). Baseline or ambient CH4 concentration can be calculated as mean CH4 concentration before starting the measurements and subtracted from the measured data. Each day before starting measurements, the NDIR analyzer should be verified using standard mixtures of CH4 in N₂(0.0%, 0.25%, 0.50%, 0.75% and 1.0%; MESA International Technologies INC, Santa Ana, CA, USA).

Certain methods and devices are described further below and in Table 15.

TABLE 15

Features of exemplary methods for measuring methane output by individual animals

Purchase
Running

Behaviour

Method
Cost ²
Costs ²
Labour ²
Repeatability
Alteration ³
Throughput

Respiration chamber
High
High
High
High
High
Low

SF₆technique
Medium
High
High
Medium
Medium
Medium

Breath sampling during
Low ⁴
Low
Low
Medium
None
High

milking and feeding

GreenFeed
Medium
Medium
Low
Medium
Low
Medium

Laser methane detector
Low
Low
High
Low
Low-Medium
Medium

As indicated above and shown in Table 15, exemplary methods include respiration chambers, the sulfur hexafluoride (SF6) tracer technique, breath sampling during milking or feeding, the GreenFeed system, and the laser methane detector. Each method measures different components of methane output. Only respiration chambers measure total emissions from the animal via the oral, nasal and anal routes; all other methods ignore emissions via the anus and only measure methane emitted in breath. Breath measurements are justified because 99% of methane is emitted from the mouth and nostrils, and only 1% via the anus. The SF6 technique samples breath over 24 h, whereas other techniques use spot samples of breath over periods of minutes throughout the day, so diurnal variation has to be considered. The majority of methane (87%) is released by eructation, which provides a clear signal for sample processing.

Respiration Chamber

Respiration chambers for open- or closed-circuit indirect calorimetry are considered the ‘Gold Standard’, and were used extensively in nutrition studies when establishing the Metabolisable Energy system. A single animal (or occasionally more) is confined in a chamber for between 2 and 7 days. Concentration of methane (and other gases if required) is measured at the air inlet and outlet vents of the chamber. The difference between outlet and inlet concentrations is multiplied by airflow to indicate methane emissions rate. In most installations, a single gas analyser is used to measure both inlet and outlet concentrations, often for two or more chambers. This involves switching the analyser between sampling points at set intervals, so concentrations are actually measured for only a fraction of the day.

Respiration chambers vary in construction materials, size of chamber, gas analysis equipment and airflow rate, all of which can influence results. Validation of 22 chambers at six UK research sites revealed an uncertainty of 25.7% between facilities, which was reduced to 2.1% when correction factors were applied to trace each facility to the international standard for methane. The main sources of uncertainty were stability and measurement of airflow, which are crucial for measuring methane emission rate. It was concluded, however, that chambers were accurate for comparing animals measured at the same site. It is an added challenge, when benchmarking alternative methods against respiration chambers, that the respiration chambers themselves have not been benchmarked against respiration chambers at other facilities.

For large-scale evaluation of methane emissions by individual animals, respiration chambers are challenging, with only a single study in growing Angus steers and heifers exceeding 1000 animals, which found methane production to be moderately heritable h²=0.27 0.07. Installation costs and running costs are high, and only one animal can be measured at a time. If the monitoring time is three days per animal, and chambers are run continuously, then maximum throughput would be approximately 100 animals per chamber per year. In practice, throughput is likely to be 30 to 50 animals per year. Cows are social animals, and confinement in a chamber may ultimately influence their feeding behaviour, resulting in less feed being consumed and in a different meal pattern compared with farm conditions. Altered feeding patterns or levels is not a problem for metabolic studies evaluating feeds, but can be a problem when evaluating individual animals. Furthermore, the representativeness of respiration chambers to grazing systems has been called into question. However, promising developments have led to more animal friendly respiration chambers constructed from cheaper, transparent materials. These lower the cost and reduce the stress of confinement with minimal disruptions to accuracy, precision and no drop in feed intake of the cows (Hellwing et al. (2012) J. Dairy Sci. 95:6077-6085, which is incorporated herein by reference).

The SF₆Method

The SF₆tracer gas technique was developed in an attempt to measure methane emissions by animals without confinement in respiration chambers. Air is sampled near the animal's nostrils through a tube attached to a halter and connected to an evacuated canister worn around the animal's neck or on its back. A capillary tube or orifice plate is used to restrict airflow through the tube so that the canister is between 50 and 70% full after approximately 24 h. A permeation tube containing SF₆is placed into the rumen of each animal. The pre-determined release rate of SF₆is multiplied by the ratio of methane to SF₆concentrations in the canister to calculate methane emission rate.

Many research centres have used the SF₆technique with variations in design of sampling and collection equipment, permeation tubes, and gas analysis. Reliable results depend on following standard protocols, with greatest variation coming from accuracy of determining SF6 release rate from permeation tubes and control of sampling rate. With capillary tubes, sampling rate decreases as pressure in the canister increases, whereas an orifice plate gives a steadier sampling rate over 24 h. A source of error that has not been evaluated is that animals might interact and share methane emissions when the sampling tube of one animal is near the head of another animal. There is good agreement between methane emissions measured by the SF6 technique and respiration chambers, although results from the SF₆technique are more variable.

For large-scale evaluation of methane emissions by individual animals, the SF₆technique is more useful than respiration chambers. Animal behaviour and intake might be affected by wearing the apparatus, and by daily handling to exchange canisters, but the technique is considerably less intrusive than respiration chambers, because cows remain in the herd. Labour and monetary costs for changing canisters each day and for lab analysis are high. Throughput is limited by the number of sets of apparatus available, handling facilities, labour, and the capacity of the lab for gas analysis. Animals need to be measured for 5 to 7 days, and it is recommended that group size should be less than 15 animals, so maximum throughput would be about 750 animals per year. Heritability has been estimated for methane production in grazing Holstein cows at h²=0.33±0.15.

Breath Sampling During Milking and Feeding

Several research groups have developed methods to measure methane concentration in breath of cows during milking and/or feeding. These are often referred to as ‘sniffer methods’ because they use devices originally designed to detect dangerous gas leaks. Air is sampled near the animal's nostrils through a tube fixed in a feed bin and connected directly to a gas analyser. The feed bin might be in an automatic milking station or in a concentrate feeding station. Different research centres use different gas analysers (Nondispersive Infrared (NDIR), Fourier-transform infrared (FTIR) or photoacoustic infrared (PAIR)) and different sampling intervals (1, 5, 20 or 90-120 s). Methane concentration during a sampling visit of typically between 3 and 10 min may be specified as the overall mean, or the mean of eructation peaks. Some centres use CO₂as a tracer gas and calculate daily methane output according to ratio of methane to CO₂and daily CO₂output predicted from performance of the cow. Repeatability and rank correlations were higher for eructation peaks than for mean concentrations, and were higher for eructation peaks than for methane to CO₂ratio. However, all methods show good repeatability.

For large-scale evaluation of methane emissions by individual animals, breath-sampling methods have significant advantages compared with other methods. Breath-sampling methods are non-invasive because, once installed, animals are unaware of the equipment and are in their normal environment. Animals follow their normal routine, which includes milking and feeding, so no training of animals, handling, or change of diet is required. Equipment is relatively cheap, although more expensive gas analysers are available, and running costs are negligible.

The compromise for non-invasiveness of breath-sampling is that concentrations of gasses in the sampled air are influenced by cow head position relative to the sampling tube. The use of head position sensors and data filtering algorithms can remove the effects when the cow's head is completely out of the feed bin, but not within the feed bin. Consequently, sniffer measurements are more variable than flux methods, with factors like variable air flow in the barn increasing measurement error (imprecision), and head position, a highly repeatable characteristic, inflating between-cow variability.

Using CO₂as a tracer gas partly addresses the issue but, because CO₂arises from metabolism as well as rumen fermentation, variability of CO₂emissions has to be considered. A further consideration is diurnal variation in breath concentrations of methane and CO₂because animals are spot-sampled at different times of day and night. Diurnal variation can be accounted for either by fitting a model derived from the whole group of animals, or by including time of measurement in the statistical model.

The number of observations per analyser is limited only by number of cows assigned to one automatic milking station or concentrate feeding station and length of time equipment is installed. Typically, each analyser will record 40 to 70 animals 2 to 7 times per day for 7 to 10 days, although the number of sampling stations per analyser can be increased by using an automatic switching system. Throughput per analyser is likely to be 2000 to 3000 animals per year. Estimates of heritability for methane production measured using this method range from h²=0.12 to 0.45 over multiple studies.

GreenFeed

GreenFeed (C-Lock Inc., Rapid City, SD, USA) is a sophisticated sniffer system where breath samples are provided when animals visit a bait station. As with other sniffer systems, GreenFeed samples breath from individual animals several times per day for short periods (3 to 7 min). GreenFeed is a portable standalone system used in bam and pasture applications, and incorporates an extractor fan to ensure active airflow and head position sensing for representative breath sampling. Measurements are pre-processed by the manufacturer, and data are available in real time through a web-based data management system. As GreenFeed captures a high proportion of emitted air and measures airflow, which can be calibrated using a tracer gas, methane emission is estimated as a flux at each visit. Providing visits occur throughout the 24 h, methane emission can be estimated directly as g/day.

A limitation of the GreenFeed system Is that animals require training to use the system, although animals which have been trained to use the system will readily use it again. However, some animals will not use the system or will use it infrequently, and frequency of visits is affected by diet. This can be a challenge when screening commercial herds for methane emission under genetic evaluation.

The manufacturer recommends 15 to 25 animals per GreenFeed unit, and recordings are made typically for 7 days. If all animals visit the unit adequately, throughput per unit is likely to be 750 to 1250 animals per year.

Laser Methane Detector (LMD)

The laser methane detector (LMD) is a highly responsive, hand-held device that is pointed at an animal's nostrils and measures methane column density along the length of the laser beam (ppm·m). In the first implementation of LMD on a farm, measurements for each cow were taken over periods of 15 to 25 s between eructation events, and could detect methane emitted each time the animal breathed out. In a later study with sheep and beef cattle, monitoring periods of 2 to 4 min allowed authors to separate breathing cycles from eructation events. Typically, animals are restrained either manually or in head yokes at a feed fence for the required length of time. The operator has to stand at the same distance (1 to 3 m) from each animal every time and must be careful to keep the laser pointed at the animal's nostrils throughout the measurement period.

The LMD can be used in the animal's normal environment, although for consistency restraint is required during measurement. Because the LMD measures methane in the plume originating from the animal's nostrils, results can be a_ected by factors such as: distance from the animal; pointing angle; animal's head orientation and head movement; air movement and temperature in the barn; adjacent animals; and operator variation. Operator variation is likely to be one of the biggest factors, because the operator controls distance and pointing angle, and is responsible for ensuring that the laser remains on target. The structure of the barn and the resulting ventilation conditions and wind speed at the location of the measurement are also considerable sources of variation in recorded methane.

Assuming operator fatigue does not limit measurements, each LMD could record up to 10 animals per hour. If each animal is recorded 3 times (on 3 consecutive days, for example), throughput is likely to be up to 1000 animals per year.

Methods for Producing of Recombinant Protein and/or Antibodies

The terms“expression vector” mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Thus, a further object of the invention relates to a vector comprising a nucleic acid of the present invention.

Such vectors may comprise regulatory elements, such as a promoter, enhancer, terminator and the like, to cause or direct expression of said polypeptide upon administration to a subject. Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami T. et al. 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana Y et al. 1987), promoter (Mason J O et al. 1985) and enhancer (Gillies S D et al. 1983) of immunoglobulin H chain and the like.

Any expression vector for animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1 beta d2-4-(Miyaji H et al. 1990) and the like. Other representative examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like. Representative examples of viral vector include adenoviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, gPenv-positive cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95/14785, WO 96/22378, U.S. Pat. Nos. 5,882,877, 6,013,516, 4,861,719, 5,278,056 and WO 94/19478.

A further object of the present invention relates to a cell which has been transfected, infected or transformed by a nucleic acid and/or a vector according to the invention. The term “transformation” means the introduction of a “foreign” (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been “transformed.”

The nucleic acids may be used to produce a recombinant polypeptide of the invention in a suitable expression system. The term “expression system” means a host cell and compatible vector under suitable conditions, e.g. for the expression of a protein coded for by foreign DNA carried by the vector and introduced to the host cell.

Common expression systems include E. coli host cells and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, without limitation, prokaryotic cells (such as bacteria) and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures (e.g., produced from lymphoblasts, fibroblasts, embryonic cells, epithelial cells, nervous cells, adipocytes, etc.). Examples also include mouse SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as “DHFR gene”) is defective (Urlaub G et al; 1980), rat YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL 1662, hereinafter referred to as “YB2/0 cell”), and the like. The YB2/0 cell is preferred, since ADCC activity of chimeric or humanized antibodies is enhanced when expressed in this cell.

The present invention also relates to a method of producing a recombinant host cell expressing an antibody or a polypeptide of the invention according to the invention, said method comprising the steps consisting of (i) introducing in vitro or ex vivo a recombinant nucleic acid or a vector as described herein into a competent host cell, (ii) culturing in vitro or ex vivo the recombinant host cell obtained and (iii), optionally, selecting the cells which express and/or secrete said antibody or polypeptide. Such recombinant host cells can be used for the production of antibodies and polypeptides of the invention.

Antibodies and fragments thereof, immunoglobulins, and polypeptides of the present invention may be produced by any technique known in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination.

Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said antibodies or polypeptides, by standard techniques for production of polypeptides. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif) and following the manufacturer's instructions. Alternatively, antibodies and other polypeptides of the present invention can be synthesized by recombinant DNA techniques as is well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly)peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired polypeptide, from which they can be later isolated using well-known techniques.

In particular, the present invention further relates to a method of producing an antibody or a polypeptide of the invention, which method comprises the steps consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said antibody or polypeptide; and (ii) recovering the expressed antibody or polypeptide.

Antibodies and other polypeptides of the present invention are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.

Chimeric antibodies (e.g., mouse-ruminant chimeras, or one ruminant-another ruminant (e.g., goat-cow) chimeras, or ruminant-human chimeras) of the present invention can be produced by obtaining nucleic sequences encoding VL and VH domains as previously described, constructing a human chimeric antibody expression vector by inserting them into an expression vector for animal cell having genes encoding human antibody CH and human antibody CL, and expressing the coding sequence by introducing the expression vector into an animal cell. The CH domain of a human chimeric antibody can be any region which belongs to human immunoglobulin, such as the IgG class or a subclass thereof, such as IgG1, IgG2, IgG3 and IgG4. Similarly, the CL of a human chimeric antibody can be any region which belongs to Ig, such as the kappa class or lambda class. Chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Patent Publication PCT/US86/0226; Akira et al. European Patent Application 184,18; Taniguchi, M. European Patent Application 171,49; Morrison et al. European Patent Application 173,49; Neuberger et al. PCT Application WO 86/0153; Cabilly et al. U.S. Pat. No. 4,816,56; Cabilly et al. European Patent Application 125,02; Better et al. (1988) Science 24:1041-104; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques:21; Winter U.S. Pat. 5,225,53; Jones et al. (1986) Nature 32:552-52; Verhoeyan et al. (1988) Science 239:1534; and Beidler et al. (1988)J Immunol. 141:4053-4060.

In addition, methods for producing antibody fragments are well-known. For example, Fab fragments of the present invention can be obtained by treating an antibody which specifically reacts with a ganglioside with a protease such as papain. Also, Fabs can be produced by inserting DNA encoding Fabs of the antibody into a vector for prokaryotic expression system, or for eukaryotic expression system, and introducing the vector into a procaryote or eucaryote (as appropriate) to express the Fabs.

Similarly, F(ab′)2 fragments of the present invention can be obtained treating an antibody which specifically reacts with a ganglioside with a protease, pepsin. Also, the F(ab′)2 fragment can be produced by binding Fab′ described below via a thioether bond or a disulfide bond.

Fab′ fragments of the present invention can be obtained treating F(ab′)2 which specifically reacts with a ganglioside with a reducing agent, dithiothreitol. Also, the Fab′ fragments can be produced by inserting DNA encoding a Fab′ fragment of the antibody into an expression vector for prokaryote, or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote (as appropriate) to perform its expression.

In addition, scFvs of the present invention can be produced by obtaining cDNA encoding the VH and VL domains as previously described, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote, or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote (as appropriate) to express the scFv.

Methods for Treating Diseases or Conditions

The vaccine compositions and methods of the present disclosure are also useful for treating disease(s) and/or condition(s). It is well documented that methanogens are associated with various diseases, including inflammatory bowel disease (IBD), gingivitis, and bloat. The vaccines of the present disclosure are also useful in treating diseases in subjects (e.g., animals, mammals, ruminants, humans) that are associated with elevated, increased, or severe lactic acidosis. Thus, the vaccine compositions methods of the present disclosure have utility in treating these diseases or conditions in subjects (e.g., humans, felines, canines, ruminants).

Kits

The present invention also encompasses kits. For example, the kit can comprise a vaccine of the present disclosure, any one of pharmaceutical compositions described herein, at least one additional agent that reduces methane production in a subject described herein, or any combination thereof, packaged in a suitable container and can further comprise instructions for using such reagents. The kit may also contain other components, such as administration tools packaged in the same or separate container.

Production of Low Carbon Animal Products

In some aspects, the vaccine compositions and methods disclosed herein may be used to produce low carbon animal products. The production of low carbon animal products may involve administering a vaccine composition to an animal, determining greenhouse gas emissions from the vaccinated animal, harvesting an animal product, and determining the carbon intensity of the harvested product compared to a product from an unvaccinated animal.

In some embodiments, a method of producing a low carbon animal product may comprise:

- (a) Administering to an animal a vaccine composition comprising cells and/or cell parts of at least one methanogen, as described in any of the embodiments herein. The animal may be a mammal, such as a ruminant.
- (b) Determining an amount of emissions of at least one greenhouse gas from the vaccinated animal following administration until animal product harvesting. The greenhouse gas may include, but is not limited to, CO₂, CH₄, N₂O, and/or H₂. In some cases, the emissions may be measured using a GreenFeed system or other suitable emissions measurement technique.
- (c) Harvesting an animal product from the vaccinated animal. The animal product may include, but is not limited to, meat, milk, wool, or other products derived from the animal.
- (d) Determining an amount of the harvested animal product.
- (e) Determining a first carbon intensity as a ratio of the amount of emissions from the vaccinated animal and the amount of harvested animal product.
- (f) Determining a second carbon intensity as a ratio of an amount of carbon emissions from an untreated animal and the same amount of harvested animal product.
- (g) Determining the difference between the first carbon intensity and the second carbon intensity.

In some embodiments, the method may further comprise administering to the animal at least one agent that reduces methane production, in addition to the vaccine composition. This may provide a synergistic effect in reducing greenhouse gas emissions.

In some aspects, the method may include certifying the animal product as a low carbon intensity product based on the determined difference between the first carbon intensity and the second carbon intensity. This certification may provide added value to the animal product in markets where low carbon products are desired.

The methods described herein may allow for the production of animal products with a reduced carbon footprint compared to conventional production methods. This may be beneficial for meeting sustainability goals, reducing environmental impact, and potentially commanding premium prices for low carbon animal products in certain markets.

EXEMPLARY EMBODIMENTS

1. A vaccine composition comprising at least one polypeptide and/or at least one peptide of at least one cell surface protein or a fragment thereof of at least one methanogen.

2. The vaccine composition of embodiment 1, wherein

- (a) the at least one methanogen is of a family Methanobacteriaceae;
- (b) the at least one methanogen is of a genus selected from: Methanobrevibacter, Methanosphaera, Methanomicrobium, Methanobacterium, Methanocorpusulum, Methanosaeta, Methanoculleus, Methanosarcina, and Thermoplasmatales, optionally Methanobrevibacter, Methanomicrobium, and Methanosarcina; and/or
- (c) the at least one methanogen comprises Methanobacterium formicicum, Methanobacterium bryantii, Methanobrevibacter ruminantium, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanomicrobium mobile, Methanoculleus olentangyi, Methanosarcina barkeri, Methanobrevibacter boviskoreani, Methanobacterium beijingense, Methanoculleus marisnigri, Methanoculleus bourgensis, Methanosarcina mazei, Thermoplasmatales archaeon BRNA1, Methanobrevibacter gottschalkii, Methanobrevibacter thaueri, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanococcoides burtonii, Methanolobus psychrophilus R15, Methanobacterium paludism, Methanohalobium evestigatum, Methanomethylovorans hollandica, Methanothrix soehngenii, Methanocaldococcus vulcanius, Methanosalsum zhilinae, Methanocorpusculum labreanum, Methanoregula formicica, Methanoculleus marisnigri, Methanocella arvoryzae, Methanoculleus bourgensis, Methanolacinia petrolearia, Methanospirillum hungatei, Methanoplanus limicola, Methanohalophilus mahii, Methanococcus aeolicus, Methanosphaerula palustris, Methanocaldococcus fervens, Methanocaldococcus jannaschii, Methanocaldococcus sp. FS406-22, Methanoregula boonei, Methanobrevibacter sp. AbM4, Methanobrevibacter ruminantium, Methanosphaera, Methanobacterium formicicum, Methanocaldococcus villosus, Methanosarcina barkeri, Methanobacterium lacus, Methanotorris igneus, Methanotorris formicicus, Methanocaldococcus infernus, Methanofollis liminatans, Methanothermococcus okinawensis, Methanobrevibacter smithii, Methanobrevibacter, Methanocella conradii, Methanothermococcus thermolithotrophicus, Methanococcus maripaludis, Methanococcus maripaludis, Methanococcus vannielii, Methanothermus fervidus, Methanosarcina acetivorans, Methanosarcina mazei, Methanosaeta harundinacea 6Ac, Methanococcus maripaludis, Methanococcus voltae, Methanolinea tarda, Methanolobus psychrophilus, Methanosaeta harundinacea, or any combination thereof.

3. The vaccine composition of embodiment 1 or 2, wherein the at least one methanogen comprises Methanobrevibacter gottschalkii and/or Methanobrevibacter ruminantium.

4. The vaccine composition of any one of embodiments 1-3, wherein the vaccine composition comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides and/or peptides.

5. The vaccine composition of embodiment 4, wherein the at least two polypeptides and/or peptides are of the same cell surface protein or of different cell surface proteins.

6. The vaccine composition of any one of embodiments 1-5, wherein the at least one cell surface protein or a fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100% sequence identity to an amino acid sequence of the present disclosure, optionally wherein the at least one cell surface protein or a fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100% sequence identity to an amino acid sequence set forth in any one of Tables C-F, 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, and 6A-6G, or a fragment thereof.

7. The vaccine composition of any one of embodiments 1-6, wherein the at least one cell surface protein or a fragment thereof comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99%, or 100% sequence identity to an amino acid sequence encoded by at least one nucleic acid of the present disclosure, optionally wherein the at least one nucleic acid comprises the nucleotide sequence set forth in any one of Tables C-F, 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, and 6A-6G, or a fragment thereof.

8. The vaccine composition of any one of embodiments 1-7, wherein the vaccine composition comprises at least one full-length protein of at least one cell surface protein.

9. The vaccine composition of any one of embodiments 1-7, wherein the vaccine composition comprises at least one fragment of at least one cell surface protein, optionally wherein the fragment is a polypeptide or a peptide.

10. The vaccine composition of embodiment 9, wherein the at least one fragment:

- (a) comprises an extracellular domain or a portion thereof;
- (b) lacks a signal peptide, optionally a native signal peptide; and/or
- (c) lacks a transmembrane domain, optionally a native transmembrane domain.

11. The vaccine composition of embodiment 9 or 10, wherein the at least one fragment lacks at least, about, or no more than 1, 5, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 amino acids, optionally from the N-terminus and/or the C-terminus.

12. The vaccine composition of any one of embodiments 1-11, wherein the at least one cell surface protein or a fragment thereof comprises an adhesin-like protein; adhesin-like protein with cysteine protease domain; tetrahydromethanopterin S-methyltransferase subunit; ATP-processing protein; cell wall biosynthesis protein; cofactor biosynthesis protein; CRISPR protein; energy metabolism protein; enzyme; fatty acid synthesis protein; general metabolism protein; membrane protein; metal-binding protein; methanogenesis protein; Mtr protein; MtrE protein; phage-related protein; proteolysis protein; transcription regulation protein; ribosomal protein; substrate binding protein; transcription protein; transport protein; protein whose expression changes in response to lauric acid stress; a fragment thereof; and/or any combination thereof (optionally those listed in e.g., Table 6G).

13. The vaccine composition of any one of embodiments 1-12, wherein the at least one polypeptide and/or at least one peptide is formulated in lipid or saline.

14. The vaccine composition of any one of embodiments 1-13, wherein in the vaccine composition is a pharmaceutical composition, optionally comprising at least one carrier and/or at least one excipient.

15. The vaccine composition of any one of embodiments 1-14, wherein the vaccine composition comprises about 0.01 mg to about 50 mg of protein per mL, optionally about 0.1 mg to about 5 mg of protein per mL.

16. The vaccine composition of any one of embodiments 1-15, wherein the vaccine composition comprises about 0.01 mg to about 50 mg of protein, optionally about 0.1 mg to about 5 mg of protein.

17. The vaccine composition of any one of embodiments 1-16, wherein the at least one polypeptide and/or at least one peptide is lyophilized.

18. The vaccine composition of any one of embodiments 1-16, wherein the at least one polypeptide and/or at least one peptide is in a liquid composition.

19. The vaccine composition of any one of embodiments 1-18, wherein the vaccine composition comprises at least one adjuvant.

20. The vaccine composition of embodiment 19, wherein the at least one adjuvant comprises:

- (a) inorganic salts, preferably aluminum and calcium salts;
- (b) an oil emulsion;
- (c) saponins;
- (d) lipid or liposomes
- (e) an immune-stimulating complex;
- (f) a carrier protein (e.g., KLH, optionally conjugated to the at least one polypeptide and/or at least one peptide);
- (g) a bacterial product or derivatives thereof (e.g., bacterial toxins, lipopolysaccharide, etc.);
- (h) a cytokine; or
- (i) any combination of two or more selected from (a)-(h).

21. The vaccine composition of embodiment 19 or 20, wherein the at least one adjuvant comprises Complete Freund's adjuvant, Incomplete Freund's adjuvant, Montanide ISA70, Montanide ISA61, Saponin, chitosan thermogel, lipid (e.g., monophosphoryl lipid A), a lipid nanoparticle/cationic liposome adjuvant, Emulsigen-D, Emulsigen, Emulsigen-P, Polygen, ENABL 06, Montainde ISA 201, Montanide Gel 02, or any combination of two or more thereof.

22. The vaccine composition of any one of embodiments 1-21, wherein the vaccine composition induces immune response against at least one cell surface protein or a fragment thereof of the at least one methanogen.

23. A method of treating a disease in a subject, the method comprising administering to the subject a vaccine composition of any one of embodiments 1-22.

24. The method of embodiment 23, wherein the disease is a periodontal disease, Inflammatory Bowel Disease (IBD), irritable bowel syndrome (ISB), ISB-C, small intestinal bacterial overgrowth (SIBO), colorectal cancer, obesity, metabolic syndrome, diverticulosis and diverticulitis, liver abscess, gingivitis, and/or bloat.

25. The method of embodiment 23 or 24, wherein the disease is associated with elevated, increased, or severe lactic acidosis.

26. A method of inducing an immune response against at least one methanogen in a subject, the method comprising administering to the subject the vaccine of any one of embodiments 1-22.

27. The method of embodiment 26, wherein the immune response comprises a B cell response and/or a T cell response, preferably a B cell response.

28. A method of reducing rumen lactate, increasing pH, or combination thereof in a subject, the method comprising administering to the subject the vaccine of any one of embodiments 1-22.

29. A method of reducing the activity, number, and/or type of methanogens in a digestive tract of a subject, the method comprising administering to the subject the vaccine composition of any one of embodiments 1-22.

30. The method of embodiment 29, wherein the digestive track comprises rumen, reticulum, omasum, abomasum, small intestine, and/or large intestine, preferably rumen.

31. A method of reducing the amount of methane (CH₄) and/or hydrogen (H₂) emitted by a subject, preferably eructated and/or exhaled, the method comprising administering to the subject the vaccine composition of any one of embodiments 1-22.

32. The method of embodiment 31, wherein the amount of methane (CH₄) and/or hydrogen (H₂) is reduced by about 5-100%, preferably by about 10-100%, compared to a control, optionally wherein the amount of methane (CH₄) and/or hydrogen (H₂) is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

33. The method of embodiment 31 or 32, wherein the amount of methane (CH₄) and/or hydrogen (H₂) is reduced by about 20-100%, preferably by about 30-100%, compared to a control.

34. The method of any one of embodiments 31-33, wherein the amount of methane (CH₄) is reduced by (a) about 1 kg-about 50 kg within 8 weeks from the time of first vaccination, or (b) about 5 kg-about 300 kg within a year from the time of first vaccination, compared to a control.

35. The method of any one of embodiments 31-34, wherein the amount of methane (CH₄) normalized to an amount of CO₂emitted by the subject (i.e., CH₄/CO₂) is reduced by about 5-100%, preferably by about 10-100%, compared to a control, optionally wherein the amount of methane (CH₄) normalized to the amount of CO₂is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

36. The method of embodiment 35, wherein the amount of methane (CH₄) normalized to the amount of CO₂is reduced by about 20-100%, preferably by about 30-100%, compared to a control.

37. The method of any one of embodiments 31-36, wherein the amount of hydrogen (H₂) is reduced by (a) about 10 g-about 500 g within 8 weeks from the time of first vaccination, or (b) about 50 g-about 3 kg within a year from the time of first vaccination, compared to a control.

38. A method of increasing the amount of carbon dioxide (CO₂) emitted by a subject, preferably eructated and/or exhaled, the method comprising administering to the subject the vaccine composition of any one of embodiments 1-22.

39. The method of embodiment 38, wherein the amount of carbon dioxide (CO₂) is increased by about 1-100%, preferably by about 1-20%, compared to a control, optionally wherein the amount of CO₂is increased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to a control.

40. The method of embodiment 38 or 39, wherein the amount of carbon dioxide (CO₂) is increased by about 3-10%, preferably by about 3-20%, compared to a control.

41. The method of any one of embodiments 38-40, wherein the amount of carbon dioxide (CO₂) is increased by (a) about 29.8 kg-about 1,490 kg within 8 weeks from the time of first vaccination, or (b) about 149 kg-about 8,940 kg within a year from the time of first vaccination, compared to a control.

42. The method of any one of embodiments 32-41, wherein the control is:

- (a) an accepted reference;
- (b) the amount of methane, CO₂-normalized methane, hydrogen, or carbon dioxide emitted by an unvaccinated subject; or
- (c) the amount of methane, CO₂-normalized methane, hydrogen, or carbon dioxide emitted by the vaccinated subject prior to vaccination.

43. The method of any one of embodiments 23-42, wherein the subject produces an antibody against at least one methanogen.

44. The method of embodiment 43, wherein the antibody is an IgG, IgM, or an IgA, preferably an IgA or IgM.

45. The method of embodiment 43 or 44, wherein the antibody is produced in an amount sufficient to:

- (a) be detectable in the digestive tract;
- (b) reduce the number and/or alter the relative abundances of methanogens in the digestive tract;
- (c) reduce the amount of methane, the CO₂-normalized methane, and/or hydrogen emitted by the subject; and/or
- (d) increase the amount of CO₂emitted by the subject.

46. The method of any one of embodiments 23-45, wherein the vaccine composition is administered to the subject via a route selected from intramuscular administration, intradermal administration, subcutaneous administration, and nasal administration.

47. The method of any one of embodiments 23-46, wherein the vaccine composition is administered to the subject via intramuscular administration or subcutaneous administration, preferably subcutaneous administration.

48. The method of any one of embodiments 23-47, wherein the subject is administered with at least one repeat dose of the vaccine composition of any one of embodiments 1-22.

49. The method of embodiment 48, wherein the subject is administered with at least two repeat doses of the vaccine composition.

50. The method of embodiment 48 or 49, wherein the subject is administered with the vaccine composition at least 3 times per year.

51. The method of any one of embodiments 48-50, wherein the at least one repeat dose comprises the same dose or a different dose compared to the preceding dose of the vaccine composition.

52. The method of any one of embodiments 48-51, wherein the at least one repeat dose comprises the same adjuvant or a different adjuvant compared to the preceding dose of the vaccine composition.

53. The method of any one of embodiments 48-52, wherein the subject is administered with the repeat dose of the vaccine composition after at least about 2 weeks, about 3 weeks, about 1 month, about 6 months, or about 12 months from the time the subject is administered with the preceding dose of the vaccine composition.

54. The method of any one of embodiments 48-53, wherein the subject is administered with the repeat dose of the vaccine composition no later than about 1 month, about 2 months, about 3 months, 6 months, 12 months, 18 months, or 24 months from the time the subject is administered with the preceding dose of the vaccine composition.

55. The method of any one of embodiments 48-54, wherein the subject receives the repeat dose of the vaccine after at least about 2 weeks and no more than about 18 months from the time the subject is administered with the preceding dose of the vaccine.

56. The method of any one of embodiments 48-55, wherein the subject is administered with at least two repeat doses of the vaccine composition, and the subject receives:

- (a) the first repeat dose (“boost 1”) after about 2 weeks to about 4 weeks from the time the subject is administered with the initial vaccine dose (“prime”); and/or
- (b) the second repeat dose (“boost 2”) after about 3 months to about 6 months from the time the subject is administered with the initial vaccine dose (“prime”).

57. The method of any one of embodiments 23-56, wherein the subject is administered with a dosage of between about 0.1 mg of protein per kg of animal body weight and about 250 mg of protein per kg of animal body weight of the vaccine composition each time of vaccination.

58. The method of any one of embodiments 23-57, further comprising administering to the subject (a) at least one agent that reduces the level of methane and/or hydrogen produced by the subject; and/or (b) at least one agent that increases production efficiency.

59. The method of embodiment 58, wherein the at least one agent is administered to a subject concomitant with, prior to, or after the vaccination.

60. The method of embodiment 58 or 59, wherein the at least one agent is administered to a subject after the vaccination.

61. The method of any one of embodiments 58-60, wherein the at least one agent is administered to a subject daily, semiweekly, weekly, biweekly (every two weeks), or monthly.

62. The method of any one of embodiments 58-61, wherein the at least one agent is administered to a subject for a duration of at least 1 week but no more than 1 month.

63. The method of any one of embodiments 58-62, wherein the at least one agent comprises:

- (a) an agent selected from the agents listed in Tables 9-13;
- (b) 3-Nitrooxypropanol (3NOP), ethyl-3NOP, 2-bromoethanesulfonate (BES), 2-chloroethanesulfonate (CES), 3-bromopropanesulfonate (BPS), bromochloromethane (BCM), bromoform, bromodichloromethane, dibromochloromethane, carbon tetrachloride, trichloroacetamide, trichloroethyladipate, lumazin (2,4-pteridinedione), p-aminobenzoic acid, lovastatin, mevastatin, pravastatin, diallyl disulfide, garlic oil, saponins, tannins, flavonoids, nitrate, nitroethane, -nitro-propionate, 2-nitropropanol, 2-nitroethanol, malate, acrylate, oxaloacetate, fumarate, propynoic acid, 3-butenoic acid, 2-butynoic acid, ethyl 2-butynoate, monensin, lasalocid, bovicin HC5, nisin, or any combination thereof; and/or
- (c) Monensin (Rumensin®), Optigrid® 45 (ractopamine hydrochloride), Amprolium (Corid), Bacitracin (Albac, BMD), Bambermycin (GainPro), Decoquinate (Deccox), Fenbendazole (Safe-Guard), Laidlomycin (Cattlyst), Lasalocid (Bovetec), Melengestrol Acetate (MGA), Methoprene (Altosid), Morantel (Rumatel), Poloxalene (Bloat Guard), Ractopamine (Optaflexx, Actogain), Tetraclovinphos (Rabon), or any combination thereof.

64. The method of any one of embodiments 58-63, wherein the at least one agent is 3NOP or ethyl-3NOP.

65. The method of embodiment 64, wherein the subject is administered with

- (a) at least about 0.5 g but no more than 25 g of 3NOP per day;
- (b) at least about 1 g but no more than 5 g of 3NOP per day; or
- (c) about 2.5 g of 3NOP per day.

66. The method of embodiment 64 or 65, wherein the subject is administered with 3NOP for a duration of at least 1 week but no more than 1 month.

67. The method of any one of embodiments 55-66, wherein the at least one agent is formulated in animal feed.

68. The method of embodiment 58, wherein the at least one agent is a composition comprising one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors thereof and one or more agriculturally suitable carriers.

69. The method of embodiment 68, wherein the one or more agriculturally suitable carriers comprises a solid carrier.

70. The method of embodiment 69, wherein the one or more solid carriers comprises attapulgite, kaolinite, fuller's earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, a natural or synthetic derivative thereof, or a combination thereof.

71. The method of embodiment 69, wherein the one or more solid carriers comprises attapulgite, kaolinite, fuller's earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, a polysaccharide, a disaccharide, a monosaccharide, a gum, silica, propylene glycol, hemp protein, biochar, montmorillonite, activated charcoal, lignin, wood flour, hemp protein, pea protein, soy protein, gelatin, casein, chotsan, talc, calcium phosphate, arginine, lysine, calcium carbonate, carbon black, glutamine, betaine, bismuth phosphate, bismuth citrate, iron phosphate, or any combination thereof.

72. The method of any one of embodiments 69-71, wherein the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, or a combination thereof.

73. The method of any one of embodiments 69-71, wherein the one or more solid carriers comprises a saccharide comprising cellulose, xanthan gum, karaya gum, ethylcellulose, inositol, galactose, arabinose, lactose, lactulose, mannitol, mannose, sorbose, turanose, platinose, carrageenan, cellulose acetate, hydroxypropyl cellulose, cellulose acetate phthalate, maltrodextran, dextran, inulin, corn starch, amylopectin, sodium starch glycolate, pentaerthritol, cyclodextrin, or a combination thereof.

74. The method of any one of embodiments 69-73, wherein the solid carrier comprises silica and ethylcellulose.

75. The method of embodiment 74, wherein the carrier comprises about 10% to about 50% by weight of the silica and about 50 to about 90% by weight of the ethylcellulose.

76. The method of any one of embodiments 69-73, wherein the carrier comprises silica and activated charcoal.

77. The method of any one of embodiments 69-73, wherein the carrier comprises about 10% to about 90% by weight of the silica and about 10% to about 90% by weight of the activated charcoal.

78. The method of any one of embodiments 77, wherein the carrier further comprises arginine, lysine, or both arginine and lysine.

79. The method of any one of embodiments 69-73, wherein the carrier comprises activated charcoal and ethylcellulose.

80. The method of embodiment 79, wherein the carrier comprises about 10% to about 50% by weight of the activated charcoal and about 40% to about 90% by weight of the ethylcellulose.

81. The method of embodiment 79 or 80, wherein the carrier further comprises about 1 to about 10% by weight of sodium lignosulfate or about 1 to about 10% by weight of hydroxyethyl cellulose.

82. The method of any one of embodiments 69-73, wherein the carrier comprises arginine and poly caprolactone.

83. The method of embodiment 82, wherein the carrier comprises about 10 to about 60% by weight of the arginine and about 30 to about 90% by weight of the polycaprolactone.

84. The method of any one of embodiments 69-73, wherein the carrier comprises silica and polycaprolactone.

85. The method of embodiment 84, wherein the carrier comprises about 10 to about 60% by weight of the silica and about 30 to about 90% by weight of the polycaprolactone.

86. The method of any one of embodiments 69-85, wherein the one or more solid carriers is inert.

87. The method of any one of embodiments 69-86, wherein the one or more solid carriers is water soluble.

88. The method of any one of embodiments 69-87, further comprising one or more additives with a density greater than water and/or one or more additives that reduces the rate of dissolution of the composition in water.

89. The method of embodiment 88, wherein the composition has a density of at least 1.1, preferably about 1.1 mg/mL to about 3 mg/mL, about 1.5 to about 3 mg/mL, about 1.5 to about 2.5 mg/mL, or about 1.5 to about 2 mg/mL.

90. The method of embodiment 88 or 89, wherein the one or more additives with a density greater than water comprises silica, attapulgite, kaolinite, fuller's earth, calcium carbonate, perlite, diatomaceous earth, calcium silicate, fly ash, or any combination thereof.

91. The method of any one of embodiments 88-90, wherein the one or more additives that reduces the rate of dissolution of the composition further reduces a rate of release of the one or more small molecules into water.

92. The method of embodiment 91, wherein the composition dissolves over at least about 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, and/or nor more than about 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, 133, 140, or 147 days, about 1 to about 147 days, more preferably 7-63 days, more preferably about 7-42 days, even more preferably 14-42 days yet even more preferably 14-28 days.

93. The method of any one of embodiments 69-92, wherein about 40 to about 80% of the small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors is thereof is released in water after 15 days.

94. The method of any one of embodiments 69-93, wherein the composition comprises particles having a uniform size distribution.

95. The method of any one of embodiments 69-94, wherein the composition comprises particles having a non-uniform size distribution.

96. The method of embodiment 94 or 95, wherein the particles comprise a spherical-, square-, rectangular-, capsular-, cylindrical-, conical-, ovular-, triangular-, diamond-, or disk-like shape.

97. The method of any one of embodiments 69-96, wherein the particles have a size ranging from about 1 mm to about 20 mm, about 1 to about 15 mm, about 1 to about 10 mm, about 5 to about 20 mm, about 5 to about 15 mm, or about 5 to about 10 mm.

98. The method of any one of embodiments 69-97, wherein the particles further comprises a coating.

99. The method of embodiment 98, wherein the coating comprises at least two layers.

100. The method of embodiment 98 or 99, wherein the coating is selected from cellulose acetate phlalate, ethyl cellulose, hydroxypropyl cellulose, polycaprolactone, alginate, chitosan, polyethylene glycol, cellulose acetate, triacetin, propylene glycol, n-methyl-2-pyrollidone, and any combination thereof.

101. The method of embodiment 98 or 99, wherein the coating comprises two or more polyelectrolytes.

102. The method of embodiment 101, wherein the polyelectrolytes comprise polystyrene sulfonate, polyethyleneimine, sodium lignosulfate, polyglutamic acid and poly-L-lysine, poly-L-arginine, polyallylamine hydrochloride, polyacrylic acid, or any combination thereof.

103. The method of embodiment 102, wherein the polyelectrolytes comprise polyallylamine hydrochloride and sodium lignosulfate.

104. The method of embodiment 102, wherein the polyelectrolytes comprise polyallylamine hydrochloride and polystyrene sulfonate.

105. The method of embodiment 102, wherein the polyelectrolytes comprise sodium lignosulfate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine, and sodium lignosulfate.

106. The method of embodiment 102, wherein the polyelectrolytes comprise polystyrene sulfonate and one of polyglutamic acid and poly-L-lysine, or poly-L-arginine.

107. The method of any one of embodiments 101-106, wherein the two or more polyelectrolytes are crosslinked.

108. The method of any one of embodiments 69-107, wherein the one or more small molecules comprise a molecule that interferes with the uptake and/or conversion of acetate, H₂, CO², methanol, monomethylamine, dimethylamine, trimethylamine, nitric oxide, or a combination thereof.

109. The method of any one of embodiments 69-108, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors interact with an enzyme selected from the group consisting of 3-(methylthio)propanoate:coenzyme M methyltransferase, acetate kinase, acetyl-CoA decarbonylase, acetyl-CoA decarbonylase/synthase complex α₂ε₂, acetyl-CoA decarbonylase/synthase complex β, acetyl-CoA decarbonylase/synthase complex γδ, acetyl-CoA synthase, carbon monoxide dehydrogenase, carbonic anhydrase, Co-methyltransferase, coenzyme M reductase, cyclohydrolase, dehydrogenase, dimethylamine-[corrinoid protein] Co-methyltransferase, F₄₂₀-dependent methylene-H₄MPT reductase, F₄₂₀-dependent methylene-H₄SPT dehydrogenase, formylmethanofuran dehydrogenase, formylmethanofuran:H₄MPT formyltransferase, formylmethanofuran:H₄SPT formyltransferase, formyltransferase, H₂-forming methylene-H₄MPT dehydrogenase, methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, methenyl-H₄MPT cyclohydrolase, methyl-coenzyme M reductase, methyl-H₄SPT:CoM methyltransferase, methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, methylcobamide:CoM methyltransferase, methylthiol:coenzyme M methyltransferase, methyltransferase, MtaC protein:coenzyme M methyltransferase, phosphotransacetylase, tetrahydromethanopterin S-methyltransferase, tetramethylammonium methyltransferase, trimethylamine-corrinoid protein Co-methyltransferase, and any combination thereof.

110. The method of embodiment 109, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors interact with methyl-coenzyme M reductase (MCR).

111. The method of any one of embodiments 69-110, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors comprise a compound having the formula R¹—[CH₂]_n—ONO₂wherein

- n is an integer from 1 to 15;
- R¹is selected from the group consisting of H, C₁-C₆alkyl, phenyl, —OH, —NH₂, —CN, —COOH, —O(C═O)R³, —NHC(═O)R³, SO₂NHR³, or —ONO₂, —SH and R³is C₁-C₆alkyl, phenyl, pyridyl;
- with the proviso that when n is >3 the hydrocarbon chain may be interrupted by —O— or —NH—.

112. The method of embodiment 111, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors is selected from 3-nitrooxypropanol, 9-nitrooxynonanol, 5-nitrooxy pentanoic acid, 6-nitrooxy hexanoic acid, bis(2-hydroxyethyl)amine dinitrate, 1,4-bis-nitrooxybutane, 1,5-bis-nitrooxypentane, and any combination thereof.

113. The method of embodiment 111, wherein the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors comprises 3-nitrooxypropanol (3NOP).

114. The method of any one of embodiments 69-113, wherein the composition comprises about 1 to about 25% by weight of the small molecule, about 5 to about 20% by weight of the small molecule, or about 5 to about 15% by weight of the small molecule.

115. The method of any one of embodiments 69-114, wherein the composition comprises a plurality of populations of particles, wherein each population or particles comprises a different formulation, a different shape, and/or a different size distribution.

116. The method of embodiment 115, wherein the plurality of populations of particles comprises a first population and a second population.

117. The method of embodiment 116, wherein the population of granular particles further comprises at least 1, 2, 3, 4, 5, 5, 6, 8, or 9 and/or no more than 4, 5, 6, 7, 8, 9, or 10 additional populations, for example 3-10 additional populations, preferably 3-7 additional populations, more preferably 3-5 additional populations.

118. The method of embodiment 115 or 116, wherein the first population comprises an immediate release formulation.

119. The method of any one of embodiments 116-118, wherein the second population comprises a delayed release formulation.

120. The method of any one of embodiments 117-119, wherein each additional population comprises a delayed release formulation, wherein each population dissolves and/or releases the one or more small molecules that reduce the production of one or more deleterious atmospheric gases and/or precursors at a different time than each of the other populations.

121. An antibody produced by the method of any one of embodiments 23-120, or an antigen-binding fragment thereof.

122. The antibody of embodiment 121, wherein the antibody is a monoclonal antibody.

123. The antibody of embodiment 121 or 122, wherein the antibody is an IgM, an IgG or an IgA, preferably an IgA or an IgM.

124. The antibody of any one of embodiments 121-123, wherein the antibody is lyophilized.

125. The antibody of any one of embodiments 121-124, wherein the antibody is in a pharmaceutical composition, optionally comprising at least one excipient and/or carrier.

126. Milk and/or a derivative thereof produced by the subject of any one of embodiments 23-120.

127. The milk and/or a derivative thereof of embodiment 126, wherein the milk and/or derivatives thereof comprises an antibody that binds at least one methanogen.

128. The milk and/or a derivative thereof of embodiment 126 or 127, wherein the milk and/or derivatives thereof is pasteurized and/or homogenized.

129. The milk and/or a derivative thereof of any one of embodiments 127-128, wherein the milk and/or derivatives thereof is lyophilized or evaporated to form dry milk powder (e.g., boiling at low pressure at low temperature).

130. The milk and/or a derivative thereof of any one of embodiments 126-129, further comprising at least one agent that reduces methane and/or hydrogen production in a subject, optionally wherein the at least one agent is selected from the agents in Table 9.

131. An animal feed comprising:

- (a) the antibody of any one of embodiments 121-125;
- (b) at least one agent that reduces methane and/or hydrogen production in a subject, optionally wherein the at least one agent is selected from the agents in Tables 9-13;
- (c) the milk of any one of embodiments 126-130; or
- (d) any combination of two or more of (a)-(c).

132. The animal feed of embodiment 131, wherein the animal feed is liquid (e.g., drinking water, milk) or solid (e.g., fodder).

133. The animal feed of embodiment 131 or 132, wherein the animal feed comprises fat and/or fatty acid, optionally wherein the animal feed comprises fat and/or fatty acid that is at least about 1%, 2%, 3%, 4%, 5%, or 6% of the diet.

134. A method of reducing methane and/or hydrogen production or increasing CO₂production in a subject, the method comprising orally administering to and/or feeding the subject the antibody of any one of embodiments 121-125, the milk and/or a derivative thereof of any one of embodiments 126-130, the animal feed of any one of embodiments 131-133, or any combination of two or more thereof.

135. The method of embodiment 134, further comprising administering at least one agent that reduces methane and/or hydrogen production in a subject, optionally wherein the at least one agent is selected from the agents in Tables 9-13.

136. The method of embodiment 134 or 135, further comprising administering the subject with the at least one vaccine composition of any one of embodiments 1-22.

137. The method of any one of embodiments 23-120 and 134-136, wherein the subject is a mammal, a human, a canine, a feline, or a ruminant.

138. The method of embodiment 137, wherein the ruminant is selected from a cow, cattle, a bull, a bison, a yak, a buffalo, an antelope, a goat, a sheep, a deer, a giraffe, a caribou, a gazelle, a macropod, a llama, a camel, and an alpaca.

139. The method of any one of embodiments 23-120 and 134-138, wherein the subject is cattle.

140. The method of embodiment 139, wherein the cattle is selected from a pregnant cow, heifer, bull, and steer.

141. The method of any one of embodiments 23-120 and 134-140, wherein the subject is an adult.

142. The method of embodiment 141, wherein the subject is adult cattle selected from:

- (a) a cow that is about 6-8 months to about 2-7 years old;
- (a) a beef adult that is about 1.5 years to about 2.5 years old; and
- (b) a dairy adult that is about 4 years to 7 years old.

143. The method of any one of embodiments 23-120 and 134-140, wherein the subject is a young subject (e.g., before weaning or below 2 years of age).

144. The method of embodiment 143, wherein the subject is young cattle selected from:

- (a) a newborn calf and a pre-weaned calf that is about 0 month to about 3 months old; and
- (b) a weaned cow that is about 3 months to 8 months old.

145. The method of any one of embodiments 23-120 and 134-140, wherein the subject is a pregnant female subject.

146. The method of any one of embodiments 23-120 and 134-140, wherein the subject is an offspring (e.g., calf) of the vaccinated female subject that received the milk comprising an antibody that binds at least one methanogen.

147. The method of any one of embodiments 23-120 and 134-146, wherein the vaccine is administered to a subject as a part of a pre-existing vaccination program to which the subject is subject (e.g., Table 14).

148. The method of any one of embodiments 23-120 and 134-147, wherein the vaccine is administered to a subject when the subject is subject to or receives at least one other vaccine, wherein the at least one other vaccine is against infectious bovine rhinotracheitis (IBR), bovine virus diarrhea (BVD), parainfluenza-3 (PI3), bovine respiratory syncytial virus (BRSV), clostridia, E. Coli mastitis, leptospirosis, Mannheimia hemolytica, Brucella, vibriosis, Campylobacter, trichomonas, trichomoniasis, rotavirus, coronavirus, and/or respiratory disease.

149. The method of any one of embodiments 23-120 and 134-148, wherein the vaccine is administered to a subject when the subject changes in hands and/or a changes in environment.

150. The method of any one of embodiments 23-120 and 134-149, wherein the vaccine reduces methane and/or hydrogen production in the lower intestinal track (lower bowel) or the rumen of the subject.

151. The method of embodiment 150, wherein the method results in at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10, 1%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% reduction in the level of methane and/or hydrogen produced by the subject, optionally wherein the reduction in the level of methane and/or hydrogen is compared to an untreated subject.

152. A kit comprising the vaccine composition of any one of embodiments 1-22.

153. The kit of embodiment 152, wherein the vaccine composition comprises no more than one polypeptide or peptide.

154. The kit of embodiment 152, wherein the vaccine composition comprises:

- (a) at least two polypeptides;
- (b) at least two peptides; or
- (c) at least one polypeptide and at least one peptide.

155. The kit of embodiment 152, wherein the kit comprises at least two vaccine compositions, each comprising different polypeptide(s) and/or peptide(s).

156. The kit of embodiment 155, wherein the at least two vaccine compositions are in separate containers.

157. The kit of any one of embodiments 152-156, wherein the kit comprises about or at least about 1, 2, 3, 4, 5, 10, 20, 50, 100, 250, 500, 1000, 1500, 2000, 2500, or 3000 doses of the vaccine composition.

158. The kit of embodiment 157, wherein

- (a) all doses are in a single container;
- (b) each dose is in an individual container; or
- (c) about or at least about 2, 3, 4, 5, 10, 25, 50, or 100 doses are pooled in a single container.

159. The kit of any one of embodiments 152-158, further comprising at least one adjuvant.

160. The kit of embodiment 159, wherein the vaccine composition and the at least one adjuvant are in separate containers.

161. The kit of embodiment 159 or 160, wherein the kit comprises at least about 1, 2, 3, 4, 5, 10, 20, 50, 100, 250, 500, 1000, 1500, 2000, 2500, or 3000 doses; and

- (a) all doses are in a single container, and the at least one adjuvant for all doses are in a separate single container;
- (b) each dose is in an individual container, and the at least one adjuvant for all doses are in a separate single container;
- (c) each dose is in an individual container, and the at least one adjuvant for each dose is in a separate individual container; or
- (d) about or at least about 2, 3, 4, 5, 10, 25, 50, or 100 doses are pooled in a single container, and the pooled amount of at least one adjuvant for the pooled doses are in a separate single container.

162. The kit of any one of embodiments 159-161, wherein the kit comprises at least two adjuvants that are different.

163. A method of treating a subject afflicted with a disease, the method comprising to the subject:

- (a) the vaccine composition of any one of embodiments 1-22;
- (b) the antibody of any one of embodiments 121-125;
- (c) the milk and/or derivative thereof of any one of embodiments 126-130;
- (d) the animal feed of any one of embodiments 131-133;
- (e) at least one agent that reduces methane and/or hydrogen production in a subject, optionally wherein the at least one agent is selected from the agents in Tables 9-13; or
- (f) any combination of two or more thereof.

164. The method of embodiment 163, wherein the subject is selected from a mammal, ruminant, a canine, a feline, and a human, optionally wherein the ruminant is selected from a cow, cattle, a bull, a bison, a yak, a buffalo, an antelope, a goat, a sheep, a deer, a giraffe, a caribou, a gazelle, a macropod, a llama, a camel, and an alpaca.

165. A method of reducing CH₄emissions in a ruminant comprising administering to the ruminant a vaccine composition comprising at least one polypeptide and/or at least one peptide of at least one cell surface protein or a fragment thereof (e.g., an antigenic fragment, e.g., a fragment comprising an epitope, e.g., a fragment comprising an extracellular domain or a portion thereof) of at least one methanogen, wherein the CH₄emissions are reduced by at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% as compared to an untreated control ruminant.

166. A method of reducing H₂emissions in a ruminant comprising administering to the ruminant a vaccine composition comprising at least one polypeptide and/or at least one peptide of at least one cell surface protein or a fragment thereof (e.g., an antigenic fragment, e.g., a fragment comprising an epitope, e.g., a fragment comprising an extracellular domain or a portion thereof) of at least one methanogen, wherein the H₂emissions are reduced by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% as compared to an untreated control ruminant.

167. A method of increasing the productivity of a ruminant comprising administering to the ruminant a vaccine composition comprising at least one polypeptide and/or at least one peptide of at least one cell surface protein or a fragment thereof (e.g., an antigenic fragment, e.g., a fragment comprising an epitope, e.g., a fragment comprising an extracellular domain or a portion thereof) of at least one methanogen, wherein the productivity is increased by at least about 0.50%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.50%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or 15% as compared to an untreated control ruminant.

168. The method of any one of embodiments 165-167, wherein the vaccine composition comprises the vaccine composition of any one of embodiments 1-22.

169. The method of any one of embodiments 165-168, wherein the ruminant is cattle.

170. An animal injected subcutaneously with the vaccine composition of any one of embodiments 1-22, wherein the vaccine composition comprises about a dosage of between 0.1 mg/kg and 250 mg/kg, or any range in between or any value in between.

171. A method of producing a low carbon animal product, the method comprising:

- (a) administering to an animal (e.g., a mammal, a ruminant) a vaccine composition of any one of embodiments 1-22;
- (b) determining an amount of emissions of at least one greenhouse gas (e.g., CO₂, CH₄, N₂O, and/or H₂) from the animal of (a) following administration until animal product harvesting;
- (c) determining a first carbon intensity as a ratio of a first amount of emissions from the vaccinated animal in (b) and the amount of harvested animal product;
- (d) determining a second carbon intensity as a ratio of a second amount of carbon emissions from an untreated animal and the same amount of harvested animal product; and
- (e) determining the difference between the first carbon intensity and the second carbon intensity.

172. The method of embodiment 171, wherein determining the amount of emissions comprises measuring the emissions using a GreenFeed system.

173. The method of embodiment 171, wherein the animal product is selected from the group consisting of meat, milk, and wool.

174. The method of embodiment 171, further comprising administering to the animal at least one agent that reduces methane and/or hydrogen production.

175. The method of embodiment 171, further comprising certifying the animal product as a low carbon intensity product based on the determined difference between the first carbon intensity and the second carbon intensity.

EXAMPLES
Example 1: Computational Selection of M. gottschalkii Antigens
Dataset Generation

The complete list of 1,800 predicted proteins from the Methanobrevibacter gottschalkii DSM 11977 genome was obtained from NCBI and included both the protein identifiers, amino acid sequence, and native or genomic nucleic acid (DNA) sequence.

Protein Metadata Generation
Localization Predictions

A successful protein antigen would need to be located on the exterior of the methanogen cell in order for it to be accessible to antibodies for binding. Therefore, we were interested in understanding the subcellular localization of all proteins in the M. gottschalkii genome. Several computational tools were used to predict both the overall localization of the entire protein and the residue-specific localization.

Subcellular localization for whole proteins was performed using the pSORTb (World Wide Web at hub.docker.com/r/brinkmanlab/psortb_commandline/) command line interface (CLI) tool. PSORTb is a bioinformatics tool for predicting subcellular localization for a given set of protein sequences. We report the localization with the highest score per protein.

Localization per-residue was predicted using TMbed (World Wide Web at github.com/BernhoferM/TMbed). This tool predicts for every residue whether it is predicted to be inside the cell, outside the cell, or embedded in the plasma membrane. We used the predictions from this tool to select extracellular fragments from proteins that may be only partially exterior to the cell.

Specificity and Conservation

A successful antigen in a methanogen vaccine should be both specific to methanogens and conserved among them; that is, widespread among methanogen species but not among non-methanogen microbial species. To identify proteins that are both specific to and conserved among methanogens, we analyzed the prevalence of two types of annotations within ruminal microbes (both methanogens and non-methanogens). We analyzed Gene Ontology (GO terms), which describe the predicted function and localization of a protein, and Pfam domain annotations, which describe conserved protein sequence features shared among members of a protein family. Both analyses were performed using the same methods.

To perform a comparative genomics analysis of methanogen genomes, we used data from Stewart et al., 2019 (World Wide Web at doi.org/10.1038/s41587-019-0202-3). In this paper, rumen samples from 283 beef cattle were DNA sequenced and 4941 genomes were assembled, of which 126 were archaea and 111 were from the Methanobrevibacter genus.

Stewart et al. annotated genes using DIAMOND to search against several public databases including UniRef; 9 million UniRef100 genes were annotated across the 4,941 genomes. Stewart et al. classified each genome's taxonomy. We defined “methanogens” as archaeal genomes containing methanogenesis genes and identified ˜235,000 genes from those genomes for further analysis.

The UniRef100 genes' Gene Ontology terms were obtained and GO term enrichment analysis was performed on each genome. A hyper-geometric test was used to quantify the degree of enrichment of each GO term in every genome (p-values adjusted with the Benjamini-Hochberg method). Then, for each GO term we used a two-sided t-test to measure the significance of the difference between the means of the adjusted p-values from the methanogen and non-methanogen genomes. GO terms that were enriched (hypergeometric test; adjusted p-value <0.05) in at least 50% of the methanogen genomes (i.e., at least 63 genomes) and were significantly different between methanogens and non-methanogens (two-sided t-test; adjusted p-value <0.05) were considered “preferred GO terms”. See Table A below for a list of preferred GO terms.

TABLE A

go_id
count
go_name
ttest_pval_adj

GO: 0016491
126
oxidoreductase activity
1.112e−164

GO: 0044272
122
sulfur compound biosynthetic process
0

GO: 0015948
117
methanogenesis
0

GO: 0000178
117
exosome (RNase complex)
0

GO: 1901576
116
organic substance biosynthetic process
0

GO: 0016020
114
membrane
8.154e−47

GO: 0019386
112
methanogenesis, from carbon dioxide
0

GO: 0032259
112
methylation
0

GO: 0016151
111
nickel cation binding
0

GO: 0000287
109
magnesium ion binding
1.933e−142

GO: 0006082
108
organic acid metabolic process
0

GO: 0009236
106
cobalamin biosynthetic process
2.213e−183

GO: 0006730
101
one-carbon metabolic process
0

GO: 0006777
100
Mo-molybdopterin cofactor biosynthetic process
5.948e−273

GO: 0003899
96
DNA-directed 5′-3′ RNA polymerase activity
0

GO: 0030677
93
ribonuclease P complex
0

GO: 0000428
93
DNA-directed RNA polymerase complex
0

GO: 0046982
91
protein heterodimerization activity
0

GO: 0050454
84
coenzyme F420 hydrogenase activity
0

GO: 0015252
83
proton channel activity
0

GO: 0008270
81
zinc ion binding
1.057e−173

GO: 0005267
77
potassium channel activity
0

GO: 0030269
74
tetrahydromethanopterin S-methyltransferase activity
0

GO: 0003743
72
translation initiation factor activity
0

GO: 0050524
71
coenzyme-B sulfoethylthiotransferase activity
0

GO: 0008234
71
cysteine-type peptidase activity
5.831e−42

GO: 0003735
71
structural constituent of ribosome
3.140e−14

GO: 0006412
70
translation
4.23le−13

GO: 0008168
66
methyltransferase activity
0

GO: 0052645
66
F420-0 metabolic process
0

GO: 0006824
65
cobalt ion transport
0

GO: 0046872
63
metal ion binding
2.974e−17

Pfam enrichment analysis was performed similarly; however as Stewart et al. did not perform Pfam annotation, we annotated these protein sequences using eggNOG-mapper (World Wide Web eggnog-mapper.embl.de/), a tool that first determines the orthology of a sequence based on pre-computed phylogenies prior to annotation. This approach can be more accurate than homology-based approaches (e.g. BLAST) since orthologs, or genes in a different species that evolved from a common ancestor, tend to retain their function as they evolve. EggNOG-mapper provides annotations from multiple public databases, one of which is the Pfam database composed of protein families, domains, motifs, and repeats. Pfam IDs were obtained for each genome's set of protein sequences, and similar to the GO term enrichment, all combinations of Pfam IDs and genomes were analyzed for enrichment. Pfams passing the same statistical tests were considered “preferred Pfams”. See Table B for a list of preferred Pfams.

TABLE B

Pfams enriched in at least 63 methanogen genomes

and with an adjusted p-value of <0.05

pfam_id
count
ttest_pval_adj

Fer4
126
2.66E−11

DNA_pol_B
124
0

Rad51
118
0

RLI
116
0

TGT_C2
116
0

eRF1_2
115
0

DUF1743
115
0

eRF1_3
115
0

eRF1_1
115
0

TF_Zn_Ribbon
114
0

TFIIB
114
0

ECR1_N
113
0

Big_3_5
113
0

CBFD_NFYB_HMF
113
0

FMN_red
113
6.00E−40

PMBR
112
0

Beta_helix
110
1.38E−78

DUF4013
109
0

Fer4_4
109
4.74E−60

DUF2115
107
0

FTR
107
0

Class_IIIsignal
107
0

FTR_C
107
0

HTH_5
107
3.34E−58

Fer4_17
106
1.46E−24

CBS
106
1.19E−07

DUF3100
103
0

HEAT_2
103
4.49E−166

NosD
102
0

UDG
101
3.42E−08

AAA_17
99
0

TFIIS_C
99
0

RNA_POL_M_15KD
99
0

DUF1724
99
0

LSM
99
0

DUF447
98
0

DUF11
97
1.30E−280

zinc_ribbon_2
97
6.25E−74

Aldo_ket_red
97
9.41E−09

DUF1211
95
0

PIN_6
94
0

DUF2112
94
0

Transglut_core
94
2.68E−36

HycI
93
0

NiFeSe_Hases
93
8.00E−101

S-AdoMet_synt_C
93
0.00127743

S-AdoMet_synt_M
93
0.00131966

S-AdoMet_synt_N
93
0.0018158

FrhB_FdhB_N
92
2.40E−239

Rubredoxin
92
2.13E−29

HIGH_NTasel_ass
91
0

FrhB_FdhB_C
91
9.65E−178

GGGtGRT
91
9.47E−16

HTH_12
87
0

DUF2097
86
0

MMR_HSR1_C
86
0

FlpD
86
0

Peptidase_C1
85
1.60E−31

Regulator_TrmB
84
0

DUF2149
83
0

CbiM
83
5.61E−180

zf-ribbon_3
82
1.32E−247

FdhD-NarQ
81
0

Oxidored_q6
81
1.55E−107

Flavodoxin_4
81
8.47E−29

HTH_45
79
0

Chlam_PMP
79
0

TOBE
79
1.84E−25

Ribosomal_S28e
78
0

CbiZ
78
0

DUF4012
78
0

Ion_trans_2
78
1.34E−166

S_layer_C
77
0

O_anti_polymase
75
0

Glyphos_transf
75
8.67E−76

Abi
75
7.14E−55

MTD
73
0

MtrA
71
0

DUF2085
71
0

MFS_1
71
0.00344293

PKD
70
2.11E−67

Radical_SAM
70
3.62E−10

Fer4_3
69
0

HTH_33
69
2.44E−296

ThiC_Rad_SAM
69
2.30E−92

NAD_binding_4
69
2.16E−71

MCM_OB
68
0

MCM
68
0

OrfB_Zn_ribbon
68
2.49E−53

UDPG_MGDP_dh_C
68
9.87E−06

UDPG_MGDP_dh_N
68
1.03E−05

HxIR
67
2.36E−47

MarR
67
3.57E−13

UDPG_MGDP_dh
67
1.65E−05

CARDB
66
5.57E−172

MCM_N
65
0

DEAD_2
65
1.09E−74

Pilin_N
64
0

DDE_Tnp_IS240
64
7.40E−266

Condensation
64
2.44E−39

PadR
64
3.96E−22

DDE_Tnp_1
64
3.59E−10

DUF3344
63
0

TelA
63
1.69E−36

Gene Expression—Metatranscriptomics and Transcriptomics
Methods

Growth of M. gottschalkii Monocultures

Triplicate M. gottschalkii DSM 11977 cultures were grown anaerobically in 100 mL of BY medium in 500 mL bottles at 38° C. The headspace was regularly exchanged and pressurized to ˜10 psi with 80% H₂/20% CO₂. Growth of the cultures was monitored via methane measurements. While the cultures were in exponential phase, 50 mL samples were anaerobically collected at 4° C. after centrifugation at 5000×g for 6 min. Cell pellets were immediately frozen on dry ice and stored at −80° C. until sent on dry ice to Texas A&M University (TAMU).

Rumen Sampling Via Ruminal Cannula

Rumen samples were provided by the Wickersham Lab at Texas A&M University (TAMU). Samples were previously collected via the ruminal cannula, strained through three layers of cheesecloth, and separated into liquid and solid phases. Individual 50 mL samples of both liquid and solid phases were retained in conical tubes. Sub-samples of the solid phase were immediately flash frozen using liquid nitrogen and transferred to −20° C. storage until further processing and analysis.

Total RNA Extraction, Library Preparation, and Sequencing

Previously frozen biological samples were thawed on wet ice and further processed by the Dass Lab at TAMU. Total RNA extraction was completed using the protocol defined in Wang et al. 2011, and RNA cleanup using the RNeasy mini kit (Qiagen) was performed. All total RNA samples were treated with DNAase to remove any DNA contamination. Total RNA was delivered to Texas A&M AgriLife Research's TxGen for sequencing library preparation and subsequent RNA-sequencing. Total RNA samples were stored at −80° C. before and after transport and were transported to TxGen on dry ice.

Samples were QC'ed according to TxGen's SOP prior to library preparation. The bulk of the ribosomal RNA was removed using the Illumina Ribo-Zero Plus rRNA Microbiome depletion kit. The remaining RNA was processed using the PerkinElmer NextFlex Rapid Directional RNA-Seq kit 2.0 to obtain the final libraries. All libraries were stored at or below −20° C. prior to sequencing. RNA-Seq libraries were sequenced using the Illumina NovaSeq 6000 platform and 2×150 bp paired-end reads.

M. gottschalkii Transcriptomic Analysis

The M. gottschalkii transcriptomic data was processed using the latest release of the standardized RNA-Seq Nextflow pipeline (nf-core/maseq: World Wide Web at nf-co.re/rnaseq/3.13.2). Using various tools, the pipeline trims and filters low quality reads (TrimGalore!: World Wide Web at bioinformatics.babraham.ac.uk/projects/trim_galore/), filters out ribosomal RNA (SortMeRNA: World Wide Web at github.com/sortmema/sortmema), builds a transcriptome assembly (StringTie: World Wide Web at ccb.jhu.edu/software/stringtie/), performs multiple alignment (STAR: World Wide Web at github.com/alexdobin/STAR) and quantification (Salmon: World Wide Web at combine-lab.github.io/salmon/) of reads against the transcriptome and reference genome, and compiles a quality assessment report (MultiQC: World Wide Web at multiqc.info/). No major changes were made to the pipeline. The M. gottschalkii DSM 11977 genome in FASTA and GTF file format was used as a reference (NCBI RefSeq Assembly GCF_003814835.1).

Bovine Rumen Meta-Transcriptomic Analysis: Metatdenovo Nextflow Pipeline Development

In order to analyze the meta-transcriptomic data we utilized the development version of the metatdenovo Nextflow pipeline, published by nf-core (metatdenovo: World Wide Web at nf-co.re/metatdenovo/dev). The metatdenovo pipeline is still under development, so we added a number of improvements to adapt the pipeline to our dataset (FIG. 2). First, we added filtering steps to filter unnecessary input reads: adapter sequences and low-quality reads (Trimmomatic: World Wide Web at usadellab.org/cms/?page=trimmomatic), contamination from bovine sequences (bowtie2: World Wide Web at github.com/BenLangmead/bowtie2), ribosomal RNA (SortMeRNA: World Wide Web at github.com/sortmema/sortmema), and unnecessary bacterial and non-archaeal reads (Kraken2: World Wide Web at ccb.jhu.edu/software/kraken2/). We also developed a novel parallelization strategy in Nextflow to split input reads into smaller file chunks to increase analysis throughput by leveraging cloud resources to run multiple filtering steps simultaneously.

We then combine the filtered reads into concatenated FastQ files, which are merged and deduplicated (BBMap: World Wide Web at jgi.doe.gov/data-and-tools/software-tools/bbtools/bb-tools-user-guide/bbmap-guide/). The deduplicated reads are then assembled (Megahit: World Wide Web at github.com/voutcn/megahit), as in the original nf-core/metatdenovo pipeline. We then use this assembly to: quantify reads (Salmon: World Wide Web at combine-lab.github.io/salmon/about/), annotate contig information (eggNOG-mapper: World Wide Web at github.com/eggnogdb/eggnog-mapper and HMMER: World Wide Web at hmmer.org/), identify archaeal contigs (Kraken2), and report some pipeline and assembly statistics (MultiQC, Transrate: World Wide Web at hibberdlab.com/transrate/, Kraken2).

Generation of Heatmaps

Heatmaps of expressed transcripts were generated in R with Pretty Heatmaps (pheatmap: Pretty Heatmaps: World Wide Web at cran.r-project.org/package=pheatmap). Counts matrices containing read mapping values for both the M. gottschalkii transcriptome data and the rumen meta-transcriptome data were first normalized for library size and variance-scaled transformed. The normalized and transformed counts were used as inputs for the heatmaps.

Nextflow Tower & AWS Batch

While our Nextflow pipeline can be run on standard personal computer machines, the scale of our experiment makes that impractical for the billions of reads sequenced in a meta-transcriptomic experiment. We designed our pipeline to be compatible with Seqera's Nextflow Tower (now known as Segera Platform) to connect our cloud-stored data with computational resources. Seqera Platform utilizes a Fusion file system to access input and reference data without copying data, saving on wall time.

Our pipeline is run on AWS Batch, a batch computing cloud platform that allows for scalable computational jobs. This synergizes with our parallelized pipeline to allow for thousands of pipeline steps to be run at the same time, reducing 30 years worth of compute time to only 24 hours of wall time. We reduced the amount of unnecessary bacterial reads present in the meta-transcriptome, and our meta-assembly was able to complete in less than 2 hours because of the parallelized effort.

Data Storage on AWS S3

After sequencing was completed on the rumen and cultured methanogen samples, raw FastQ files were uploaded to our AWS S3 bucket. Reference databases and files are also stored in S3, which allows us to scale up storage capacity nearly infinitely.

Metatranscriptomic and Transcriptomic Results

From the M. gottschalkii transcriptomic data, 192 genes were selected for comparison of their gene expression values. These genes were selected based on their potential for use as protein antigens in future vaccine products. Three lines of evidence were used to assess their potential: whether they contain a Gene Ontology (GO) term enriched in methanogens, whether they contain a Pfam enriched in methanogens, and their presence in four mass spectrometry samples of M. gottschalkii proteins exposed and reactive to ArkeaBio's Trial 2 sera. Details of the GO and Pfam enrichment analyses are described above. In total 192 genes met at least 1 of these lines of evidence and 32 genes met at least 2. Of the 48 genes whose corresponding protein was found in at least 1 mass spectrometry sample, 15 were present in at least two samples. In addition, several genes of interest are expressed by M. gottschalkii well above baseline, indicating their active use during normal growing conditions (FIG. 3). Common GO term groups include those related to membrane localization, enzymatic activity (hydrolase, endopeptidase, etc.), ion transport and binding, methanogenesis, and energy use or production (ATP-related or FAD-related). Common Pfam domains include those involved in metal ion binding or transport (zinc_ribbon_2, Fer4, Ion_trans 2), glycosyl- or glycerophospho-transferases, methanogenesis (MtrH), and several domains of unknown function (DUF).

The same 192 M. gottschalkii genes were compared using DIAMOND BLASTp against the metatranscriptomic de novo assembly to determine if they were expressed in the bovine rumen. Of the 192 genes, 127 matched to at least one assembly contig, with 1700 different hits overall. To visualize this, a stringent filter (bit score >80 and percent sequence similarity >50%) was used to obtain the best hits, yielding 97 different gene and contig pairings. When combined with the metatranscriptome expression data from the 24 rumen samples, all but 3 contigs had read mapping support for expression in the rumen after low abundance filtering (>1 read in at least one sample; FIG. 4). This indicates that the 192 M. gottschalkii genes of interest not only match sequences sourced from the rumen in terms of homology but are also being actively expressed within it. Both gene rows and sample columns are also clustered by expression values, indicating similarity of expression patterns across different conditions. Table C lists the metatranscriptomic de novo assembly contigs with the highest average expression values across all rumen samples, the unique M. gottschalkii proteins that mapped to them, and a short description of their functional annotation.

Mean expression Z-scores were calculated as follows: the metatranscriptomic and transcriptomic counts per sample were transformed into per-sample Z-scores, then the mean Z-score was calculated per protein.

The Z-score is a statistical measurement that describes a value's relationship to the mean of a group of values. Z-score denotes the number of standard deviations a value is away from the mean. If a Z-score is 0, it indicates that the data point's score is identical to the mean score.

The difference in expression between cultured and rumen samples was calculated by subtracting the cultured Z-score from the rumen Z-score. A large positive value indicates that the gene has higher relative expression in the rumen than in culture, while a large negative value indicates the opposite.

TABLE C

The 10 highest expressed metaT de novo assembly contigs on average across all rumen samples,

the M. gottschalkii proteins that mapped to them, and their functional annotations.

metaT de novo
Mean

M. gottschalkii
assembly contig
normalized

SEQ ID NO
protein ID
ID
expression
functional annotation

71358
RPF51679.1
k141_250835
7.991
MCR beta subunit

71521
RPF52611.1
k141_53717
7.186
MtrA

71178
RPF50774.1
k141_202519
5.732
hypothetical protein

71282
RPF51428.1
k141_294869
4.664
MtrA

71469
RPF51995.1
k141_106620
4.612
cation diffusion facilitator family

transporter

71124
RPF50456.1
k141_88927
2.903
V/A-type H+-transporting ATPase

subunit A

71694
RPF53127.1
k141_262553
1.998
ferrous iron transport protein B

71189
RPF50793.1
k141_92432
1.657
P-type E1-E2/heavy metal translocating

ATPase

71432
RPF51892.1
k141_157370
1.527
multimeric flavodoxin WrbA

71679
RPF53090.1
k141_167452
1.352
FlaA1/EpsC-like NDP-sugar epimerase

Proteomic Analysis of Trial 2 Active Sera

Briefly, sera identified as having binding antibodies in the whole cell methanogen ELISA assay were used as the source of primary antibodies in Western blots to identify bands of interest for proteomic analysis. The rationale is that identified proteins bind to antibodies from vaccinated animals and therefore might represent antigens of interest. A protein is more preferred if it was identified in more than one proteomic sample (maximum of four).

Preferred Antigens

Preferred antigens were selected based on the following criteria.

- Full-length proteins—Set 1
  - Antigens include full-length proteins from the M. gottschalkii genome with a localization of “Cellwall”, “Cytoplasmic Membrane”, “Extracellular”, or “Unknown” by pSORTb predictions, or at least one amino acid residue predicted to be outside the cell by TMbed.
- Full-length proteins—Set 2
  - Antigens include any full-length protein from Set 1 that also was annotated with at least one preferred GO term or at least one preferred Pfam.
- Full-length proteins—Set 3
  - Antigens include any full-length protein from Set 2 with either above average expression in the rumen transcriptome (Z-score >0), or a rumen-cultured difference >2, or was detected in the Western blot proteomics analysis.
- Peptide Fragments—Set 4
  - Antigens include any portion or a fragment of a protein from the M. gottschalkii genome where the amino acid residues are predicted to be outside the cell by TMbed. The peptide fragment contains at least 10 amino acids.
- Peptide Fragments—Set 5
  - Antigens include any peptide fragments from Set 4 that originated from a protein that was annotated with at least one preferred GO term or at least one preferred Pfam.
- Peptide Fragments—Set 6
  - Antigens include any peptide fragments from Set 5 that originated from a protein with either above average expression in the rumen transcriptome (Z-score >0), or a rumen-culture difference >2, or was detected in the Western blot proteomics analysis.

Example 2: Informatic Analysis of Antigen Targets and Vaccination

This example demonstrates informatic selection of 40 methanogen cell surface proteins and generation of nucleic acid vaccines encoding those cell surface proteins

Cell Surface Protein Selection

Briefly, Methanobrevibacter ruminantium and Methanobrevibacter gottschalkii were identified as the two most prevalent ruminal methanogens in the abundance analysis. Each open reading frame (ORF) from their published genomes were curated first database comprising ˜4500 ORFS. Subcellular localization for each ORF was predicted using pSORTdb (Lau (2020) Nucleic Acids Research) and the predicted localization was appended to the first database. The 741 predicted non-intracellular proteins were then ranked based on published gene expression changes in response to lauric acid stress (Zhou (2018) BMC Research Notes) resulting in a second database comprising 45 proteins. The DNA sequences of the proteins were collected from NCBI and were used to search a subset of rumen transcriptomic studies in the Sequence Read Archive (SRA). Proteins were given additional emphasis for inclusion in the final set of candidates based on if there was evidence that either the exact nucleotide sequence or a highly similar sequence was found in transcriptomic data. From examination of the combination of all of these datasets, as well as information based on the predicted functions of the proteins (adhesin or metal binding for example), a final list of sequences was output for additional studies comprising 40 sequences. The protein IDs were used to download the protein sequences from NCBI. As an additional step prior to ordering constructs, the proteins were analyzed using a signal peptide predictor and signal peptides were removed from the final designs. In addition, some of the proteins were too long for synthesis of the template for mRNA production. In this case, the proteins were examined and truncated versions of the protein were chosen based on predicted domain boundaries. This final list of sequences (Table D, ARK016-055) encompasses a list of proteins from two methanogens commonly found in the rumen of cattle. These proteins are predicted to be outside of the cells and there is evidence indicating that they are expressed.

Table D. Selected Methanogen Cell-Surface Proteins

See SEQ ID NO: 16689 to SEQ ID NO: 16728 for the native nucleic acid sequences of selected methanogen cell-surface proteins (ARK016-ARK055, respectively). See SEQ ID NO: 16729 to SEQ ID NO: 16768 for the Bos Taurus codon-optimized and uridine-depleted nucleic acid sequences of selected methanogen cell-surface proteins (ARK016-ARK055, respectively). See SEQ ID NO: 16769 to SEQ ID NO: 16808 for the amino acid sequences of selected methanogen cell-surface proteins (ARK016-ARK055, respectively).

Example 3: Ruminal Metagenomic Mining of Methanogen Cell Surface Proteins

This example demonstrates informatic selection of candidate methanogen cell surface proteins, generation of protein and/or peptide vaccines encoding those cell surface proteins or a fragment thereof.

To identify candidate methanogen cell surface proteins, a comparative genomics analysis of ruminal methanogen genomes from rumen samples from 283 beef cattle was used (Stewart (2019) Nature Biotechnology, which is incorporated herein by reference). In total, the analysis comprised 4941 genomes were assembled, of which 126 were archaea and 111 were Methanobrevibacter. Genes were annotated in the 4941 genomes using DIAMOND, resulting in first database comprising 9,712,545 total open reading frames. Methanogen genes were identified by narrowing the total 4941 genomes to those that were archaeal genomes comprising one or more methanogenesis metabolic pathway genes. From those, a second database comprising 235,935 methanogen open reading frames was generated. UniRef100 gene's Gene Onotology terms were obtained and GO term enrichment analysis was performed on each of the ORFs in the second database. The p-values for the enrichment analysis were used as input to a machine learning classification model to classify genomes as methanogen or non-methanogen. This resulted in a third database comprising 10,640 methanogen-specific genes. GO terms most specific to methanogens were used to select candidate genes for localization prediction. pSORTb and TMbed was used to predict subcellular localization and selected whole genes and domains predicted to be accessible to antibodies (e.g., localized to the cell membrane or extracellularly). Based on the subcellular localization, 225 whole proteins and ˜7500 peptide fragments were selected for vaccine production. The bioinformatic selection is shown in FIG. 11A (left). FIG. 11A (right) further shows a network map of the enriched cell surface protein functions, with key methanogen enriched cell surface proteins having functions related to adhesin-like proteins, metal binding, ATP processing, proteolysis, and transport. Many of the adhesin-like proteins comprise cysteine protease domains that are important for cell health, survival, and membrane remodeling.

Table E. Select Non-Methanobrevibacter Genes

See SEQ ID NO: 16809 to SEQ ID NO: 16832 for the native nucleic acid sequences of select non-Methanobrevibacter genes. See SEQ ID NO: 16833 to SEQ ID NO: 16856 for the bovine codon-optimized nucleic acid sequences of select non-Methanobrevibacter genes. See SEQ ID NO: 16857 to SEQ ID NO: 16880 for the amino acid sequences of select non-Methanobrevibacter genes.

Table F. Select Protein Fragment Sequences

These fragments are isolated functional domains based on predicted computation folding. For example, a hypothetical protein in FIG. 11B comprising 3 folded domains would yield 3 fragments, each of which is a stable domain on its own and can be used as an antigen for vaccine.

See SEQ ID NO: 16999 to SEQ ID NO: 35021 for the native nucleic acid sequences encoding the select protein fragments. See SEQ ID NO: 35022 to SEQ ID NO: 53044, and SEQ ID NO: 71068 for the bovine codon-optimized and uracil-depleted nucleic acid sequences encoding the select protein fragments. See SEQ ID NO: 53045 to SEQ ID NO: 71067 for the amino acid sequences of the select protein fragments.

Example 4: Vaccination of Animals with a Protein Vaccine Composition

This example demonstrates a method of preparing and administering a vaccine composition to an animal (e.g., cattle). The vaccine composition used in this study contains a full-length protein of Methanobrevibacter gottschalkii, which upon vaccination to animals resulted in reduction in the animal emitted methane, hydrogen, and/or carbon dioxide normalize methane; and/or increase emitted carbon dioxide.

Preparation of a Vaccine Composition

A full-length protein of Methanobrevibacter gottschalkii is expressed in bacteria (e.g., E. Coli) or yeast (e.g., S. cerevisiae or P. pastoris). The purified protein is combined with an equal volume of Freund's complete adjuvant (ThermoFisher, Waltham, MA) for primary injections. The purified protein is combined with an equal volume of Freund's incomplete adjuvant (ThermoFisher, Waltham, MA) for booster injections.

Vaccination and Sample Collection

Healthy, weaned, Angus cross steers are weaned for a minimum of sixty days and received pre-weaned vaccinations a minimum of sixty days prior to initiation of the study. Animals are transferred to the study site fourteen days prior to vaccination to allow for acclimation to the study diet and environment. Each vaccine formulation is administered by intramuscular (IM) injection to three animals. In total, 120 animals are vaccinated with the protein vaccine comprising the full-length protein of Methanobrevibacter gottschalkii and 3 animals are vaccinated with adjuvant alone. Intramuscular injections are delivered into the cow's right neck muscle by veterinary-trained staff using an 18-gauge 1-1.5″ needle. Animals are boosted with a second vaccine administration on day 21. Total blood samples (˜100 mL) are collected from the jugular vein by veterinary-trained staff and processed by ultracentrifugation within 24 hours of sample collection. Isolated serum samples are aliquoted, labeled and stored in cryogenic tubes at −20° C. until use.

Example 5: Vaccination of Animals with a Peptide-Fragment Vaccine Composition

This example demonstrates a method of preparing and administering a vaccine composition to an animal (e.g., cattle). The vaccine composition used in this study contains a peptide fragment of Methanobrevibacter gottschalkii, which upon vaccination to animals resulted in reduction in the animal emitted methane, hydrogen, and/or carbon dioxide normalize methane; and/or increase emitted carbon dioxide.

Preparation of a Vaccine Composition

A peptide fragment of Methanobrevibacter gottschalkii is synthesized in vitro using methods known in the art via a vendor (GenScript, Piscataway, NJ). The purified peptide fragment is combined with an equal volume of Freund's complete adjuvant (ThermoFisher, Waltham, MA) for primary injections. The purified protein is combined with an equal volume of Freund's incomplete adjuvant (ThermoFisher, Waltham, MA) for booster injections.

Vaccination and Sample Collection

Healthy, weaned, Angus cross steers are weaned for a minimum of sixty days and received pre-weaned vaccinations a minimum of sixty days prior to initiation of the study. Animals are transferred to the study site fourteen days prior to vaccination to allow for acclimation to the study diet and environment. Each vaccine formulation is administered by intramuscular (IM) injection to three animals. In total, 120 animals are vaccinated with the protein vaccine comprising the peptide fragment of Methanobrevibacter gottschalkii and 3 animals are vaccinated with adjuvant alone. Intramuscular injections are delivered into the cow's right neck muscle by veterinary-trained staff using an 18-gauge 1-1.5″ needle. Animals are boosted with a second vaccine administration on day 21. Total blood samples (˜100 mL) are collected from the jugular vein by veterinary-trained staff and processed by ultracentrifugation within 24 hours of sample collection. Isolated serum samples are aliquoted, labeled and stored in cryogenic tubes at −20° C. until use.

Example 6: Ruminal Microbiome Measurement

˜50 mL of fresh rumen samples are collected by esophageal tubing and retained in conical vials. Samples are strained through three layers of cheesecloth, with the liquid phase aliquoted into 50 mL conical vials. All samples re snap-frozen in liquid N₂and stored at ˜20° C. for subsequent processing. Samples are processed within twenty-four hours of collection. Sample aliquots are labeled with animal number, collection method, collection date and time.

Isolation of DNA from the rumen follows the methods defined in Henderson (Henderson, G. et al. Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities. PLOS One 8, e74787 (2013), which is incorporated herein by reference), with preference given to methods involving both phenol-chloroform and mechanical lysis steps (PCQI, PCBB, PCSA). PCR amplification of the hypervariable V6-V8 regions of the 16S rRNA gene is performed using the archaea-specific Ar915aF/Ar1386R primer set with Illumina adapters as defined in Table 1 of Kittelmann 2015 (Kittelmann et al. Buccal swabbing as a noninvasive method to determine bacterial, archaeal, and eukaryotic microbial community structures in the rumen. Appl Environ Microbiol 81:7470-7483 (2015), which is incorporated herein by reference). and PCR cycle conditions as defined in Kittelmann 2013 (Kittelmann et al. Simultaneous Amplicon Sequencing to Explore CoOccurrence Patterns of Bacterial, Archaeal and Eukaryotic Microorganisms in Rumen Microbial Communities. PLOS One 8(2): e47879 (2013), which is incorporated herein by reference). PCR products are stored at the appropriate conditions until subsequent use. The PCR products are then purified, quality checked, prepared into sequencing libraries, and analyzed for 16S rRNA sequencing within three weeks of the final rumen sample collection (Day 90). Libraries are generated using the PerkinElmer NextFlex DNA-Seq kit. The 16S rRNA amplicon libraries are sequenced on the Illumina MiSeq v3 600-cycle (2×300 bp) platform. Following the completion of 16S rRNA sequencing, the resulting data is analyzed for methanogen abundance. Methanogen abundance is reduced by ˜10-80% after treatment with vaccines encoding methanogen cell surface proteins as compared to pre-vaccination.

Example 7: Methane Production Measurements

Cow enteric methane production is monitored using GreenFeed Systems (C-Lock, Inc., Rapid City, SD). Briefly, feed intake is recorded daily throughout the study period using GrowSafe Systems. Animals are fed once daily in the morning and have access to a water source at all times. Body weight is recorded periodically, at the time of total blood draws. Enteric methane, hydrogen, and carbon dioxide emissions are measured daily throughout the study period using GreenFeed Systems (C-Lock, Inc., Rapid City, SD). Animals have free access to the GreenFeed System throughout the study period; animals are coerced to use the system. Methane yield and intensity is calculated using dry matter intake for each measurement period separately. Methane production is reduced by ˜10-80% when treated with nucleic acid vaccines encoding methanogen cell surface proteins as compared to pre-vaccination.

This example demonstrates informatic selection of methanogen cell surface proteins for formulation into protein vaccines, vaccination into subjects, and subsequent reductions in methane in vivo.

Example 8: Analysis of Antibody Binding
Sera Collection and Confirmation of Sera Antibody Binding to Methanogens

Total blood samples are collected under the supervision of veterinary-trained staff. Samples are collected via jugular vein needle puncture into blood tubes. Collected blood samples are then clarified via ultracentrifugation within 24 hours of collection in order to harvest serum. Clarified serum samples are aliquoted, labeled and stored at ≤−20° C.

Specifically, jugular blood draws are accomplished by first restraining the animal in a squeeze chute with a head gate. The neck area was then cleaned by wiping the area with isopropyl alcohol, e.g., rubbing alcohol, and gauze to remove any superficial dirt and debris. The jugular vein is occluded by applying pressure at the base of the jugular groove in order to visualize the raised vein. After a sufficient volume of blood is collected, the needle is removed and disposed of in an approved sharps container.

Confirmation of Sera Antibody Binding to Methanogens Using a Methanogen ELISA.

In order to determine a humoral immune response to the vaccine in the immunized animals, a methanogen ELISA is developed and utilized to assess sera antibody binding to M. gottschalkii.

Specifically, each well of a 96-well high binding ELISA plate, e.g., plate, is coated with 50 μL of 0.001% polylysine. The plate is then stored at 4° C. for at least 10 hours, and up to 2 weeks. Polypeptide and/or peptide samples are diluted in DPBS to an empirically determined dilution for each plate preparation (typically 1:10). Next, the volume of 0.001% polylysine is removed from the plate, and 50 μL of diluted, the polypeptide and/or peptide sample is added to each well. The plate is then centrifuged for 5 minutes at 900×g to facilitate binding of the polypeptide and/or peptide sample. Next, 50 μL of 0.1% glutaraldehyde is added to each well, mixed well, and incubated for 20 minutes at room temperature. The liquid from each well is removed, and 200 μL of 5% goat serum in PBST (blocking solution) is added to each well. The plate is then incubated for 1 hour at room temperature to prevent non-specific binding of the detection antibodies in subsequent steps, also referred to as ‘blocking’.

The clarified sera collected from the animals enrolled in this study as described above are diluted in 5% goat serum in PBST, e.g., blocking solution. Next, the blocked plates are washed twice by adding 200 μL of PBST to each well, and then removing the liquid. 50 μL of PBST-diluted sera is added to each well and incubated for 1 hour at room temperature. The plate is then washed three times with 200 μL of PBST per well. 50 μL of 1:1,000 rabbit anti-bovine-HRP conjugate is added to each well and incubated for 1 hour at room temperature. The plate is then washed three times with 200 μL of PBST per well. Next, 50 μL of TMB solution is added to each well, incubated for 20 minutes at room temperature, to develop. Finally, 50 μL of 2 M H2SO4 is added to each well to stop the reaction. OD450 is measured for each well using a Synergy plate reader (BioTek, Winooski, VT).

Confirmation of Sera Antibody Binding to Methanogens Using FACS.

In order to confirm the development of a humoral immune response to the vaccine in animals immunized with a vaccine composition comprising a protein or a fragment thereof of M. gottschalkii, fluorescence-activated cell sorting (FACS) is utilized to observe sera antibody binding to M. gottschalkii.

Specifically, 5 μL of fixed M. gottschalkii cells are added to 50 μL of PBS-T in an Eppendorf tube. Then, where applicable, 1 μL of clarified sera collected from the animals enrolled in this study as described above, is added to the relevant Eppendorf tube and mixed well. A detection solution is prepared by adding 150 μL of PBST to 1.5 μL of rabbit anti-bovine IgG biotin and 1.5 μL of SA-PECy7 and mixing well. Once prepared, 50 μL of detection solution is added to sample in Eppendorf tube(s) and mixed well. Sample in Eppendorf tubes, including detection solution, is incubated for 30 minutes at room temperature. Finally, 5 μL of each previously incubated sample is added to 1 mL of DPBS immediately before running on SONY SH800 (FSC gain: 5; threshold: 0.04%; all other settings: default).

Confirmation of Sera Antibonding Binding to Methanogens Using Western Blot.

In order to confirm the development of a humoral immune response to the vaccine in animals immunized with the vaccine composition, a methanogen Western blot is utilized to observe the sera antibody binding to the polypeptide and/or peptide to which the animals are vaccinated. The Western blot analysis detects the interaction of (a) the sera antibodies previously collected from the animals enrolled in this study as described above, and (b) methanogen protein antigens resolved on an SDS-PAGE gel.

Specifically, a polypeptide and/or peptide sample is mixed with 100 to 200 μL of 1×SDS sample buffer. Next, the polypeptide and/or peptide sample is boiled for 5 minutes at 100° C.

Between 5 and 10 μL of the boiled sample is loaded into individual wells of a 4-15% SDS-PAGE gel. Next, the SDS-PAGE gel is run for 10 minutes at 100V, and then 30 minutes at 200V. The proteins are then transferred using wet transfer technique for 1 hour at 100V onto a 0.22 μm PVDF membrane. Next, the 0.22 μm PVDF membrane is incubated in SuperBlock SuperBlock T20 TBS buffer solution (blocking solution) for 1 hour at room temperature to prevent non-specific binding of the detection antibodies in subsequent steps. The blocked 0.22 μm PVDF membrane is then exposed to various dilutions of clarified sera samples, previously collected from the animals enrolled in this study as described above, in blocking solution for 1 hour at room temperature. Next, the 0.22 μm PVDF membrane is washed three times (5 minutes for each wash) in TBS-T20 to remove any unbound sera and/or antibodies. The 0.22 μm PVDF membrane is then exposed to HRP-conjugated anti-bovine IgG secondary antibody in SuperBlock T20 TBS buffer solution for 1 hour at room temperature to detect any bound bovine sera antibodies. Next, the 0.22 μm PVDF membrane is washed three times (10 minutes for each wash) in TBS-T20 to remove any unbound HRP-conjugated anti-bovine IgG antibodies. Finally, the 0.22 μm PVDF membrane is developed using chromogenic TMB blotting solution, or an appropriate chemiluminescence substrate.

Example 9: Sample Collection and Additional Analyses
Saliva Collection

Saliva samples are collected using a suction tube in the oral cavity, into a conical flask or directly into a sterile Eppendorf tube. Equipment contacting saliva, e.g., collection flask or plastic tubing, is changed out after each animal's collection; either single-use or autoclavable materials are used. Sample are immediately put on wet ice, and then transported to the lab for storage at ≤−80° C.

Rumen Fluid Collection

First, the ruminant, e.g., steer, is restrained in a squeeze chute with a head gate and nose tucked down toward the chest. Then, a long flexible plastic tube is inserted into the animal's nostril and down into the stomach. A hand-operated rumen fluid pump, attached to the flexible plastic tube, is then used to harvest 20-50 mL of rumen fluid per collection. Harvested rumen fluid samples are stored in conical vials and transferred to the lab. Finally, within 24 hours of collection, rumen fluid samples are strained through three layers of cheesecloth, and the liquid phase is aliquoted into 50 mL conical vials and snap-frozen in liquid nitrogen for cryostorage at ≤−20° C.

Recording and Analysis of Dry Matter Intake

This example describes a method to record and analyze daily dry matter intake of vaccinated animals in order to determine whether the administration of a vaccine has an adverse effect on individual daily feed intake. Furthermore, such a record may be used to normalize inter-animal, e.g., ruminant, for example, cattle, comparisons of daily emitted methane.

Animals are fed once daily in the morning and had access to a water source at all times for the duration of the study. Feed intake is recorded continuously using GrowSafe systems (GrowSafe Systems, Calgary, AB, CA). Briefly, an individual animal's electronic identification tag, e.g., EID, which is typically affixed to the ear, is read by the GrowSafe feed bunk when the animal's head is in sufficiently close proximity to the bunk, e.g., when the animal is feeding from the bunk. During recorded feed events, the animal's EID is associated with the amount of feed that animal consumes from the bunk, which is automatically measured using scales equipped within the bunk. A comparable method for tracking individual dry matter intake may be used where GrowSafe systems are unavailable.

Recording and Analysis of Animal Body Weight

This example demonstrates a method of recording individual animal body weight (lb) in order to confirm that administration of a vaccine composition does not have an adverse effect on the rate of individual animal weight gain.

Animal body weight is recorded periodically, at the time of total blood draws, throughout the study. Body weight is recorded using a livestock scale. Specifically, animals to be weighed are walked through a pipe alley from their housing area to the holding pens. After being weighed, they are released to return to their holding pen with other animals from the same pen. At the end of the weighing, animals are then walked back to their assigned pen.

Measurement of Animal Emitted Methane, Hydrogen and Carbon Dioxide

The amount of animal-emitted methane, hydrogen, and carbon dioxide is measured using GreenFeed systems (C-Lock, Inc., Rapid City, SD). Briefly, the GreenFeed system uses a pelletized feed to incentivize animals to visit multiple times per day. When an animal visits a GreenFeed system to consume the dispensed feed pellets, its individual animal's electronic identification tag, e.g., EID, which is typically affixed to the ear, is read by GreenFeed and a measurement period associated with that animal is recorded. During the measurement period, GreenFeed records the amount of animal-emitted methane, hydrogen, and carbon dioxide.

Example 10: Pregnant and Postpartum Animal Vaccination

A vaccine is used to vaccinate a pregnant animal (e.g., ruminant). The unborn fetus is exposed to the antibodies against methanogen generated by the mother. After birth, the offspring receives milk and/or colostrum laden with the antibodies against the at least one methanogen surface antigen. The offspring is vaccinated with at least one vaccine of the present disclosure prior to weaning. The offspring may be vaccinated with a polypeptide and/or peptide vaccine that is the same or different from the vaccine used to vaccinate the mother.

Example 11: Combinatorial Therapies

A subject is treated with a combinatory therapy, which comprises any two or more selected from a vaccine, antibodies, milk and/or derivative thereof, animal feed, an agent (e.g., an agent that reduces methane production in a subject, a probiotic bacterial strain, a small molecule inhibitor, etc.), and other composition of the present disclosure (e.g., those reducing methane production in a subject). Any one of the combinatory therapy may be given in any order, i.e., before, concurrently with, or after any other combinatory therapy. Here, a reduction in methane production that is greater than the use of a single therapy alone is achieved.

Example 12: Synthesis of 3NOP

Adapted from “Structure-Based Design, Synthesis, and Biological Evaluation of Indomethacin Derivatives as Cyclooxygenase-2 Inhibiting Nitric Oxide Donors” (J. Med. Chem. 2007, 50, 6367-6382).

All reagents purchased from Sigma-Aldrich and Fisher Scientific and used as received.

3-Bromopropanol (78.0 g, 0.56 mol) in acetonitrile (300 mL) was added dropwise within minutes to a solution of silver nitrate (145.9 g, 0.86 mol) in acetonitrile (600 mL) and stirred at room temperature for 24 hr. The solution was protected from light by being covered with aluminum foil. After 24 hr, 5:1 excess of brine was added to the reaction mixture and stirred for 1 hr. Silver halide was filtered through Celite and filtrate was extracted with diethyl ether (300 mL×3). The organic layer was washed with brine (300 mL×3). Dried over sodium sulfate, and concentrated (59 g, 86% yield). Product confirmed via NMR (>97% purity based on HPLC).

3NOP pH and Temperature Stability: Confirmed via HPLC over a 1-month time period at temperature between 4-30 C and pH 4-10.

3NOP Volatility: 500 mg of 3NOP in a 5 dram vial was left open to air at ˜18-24 C at 15-30% RH over the course of 6 days. Average mass loss is ˜0.25% per day.

Compaction Study

The viability of different binders for densification were examined. 0.5 g of powder was tableted under 20 kN in a 13 mm pellet die. Potential binders are summarized in Table 16.

TABLE 16

Potential binders ranked based on densification.

3: powdery
2: compacts,

break,
no strength

4: clean
retains
1: compacts,

5: Unbreakable
braking,
moderate
disintegrates

tablet by hand
no dust
strength
0: no compaction

Xanthan gum
Lignin
Pea protein
Chitosan

k-carrageenan
Inulin
Soy protein
Hydroxyethyl

cellulose

Sodium alginate
Wood flour
Gelatin
Talc

Cellulose acetate
Corn Starch
Casein
Sodium starch

glycolate

Ethyl cellulose
Amylopectin

Pentaerythritol

Hydroxypropyl
Hemp protein

Calcium

cellulose

phosphate

Cellulose acetate

Diatomaceous

phthalate

earth (Celite)

Maltodextrin

Biochar

Dextran

Tablets for Encapsulation of 3NOP

A multilayer encapsulation system was employed for extended-release formulations consisting of 3 main parts:

- 1) Adsorbent—inert, non-toxic, solid-support for 3NOP (ie. silica, biochar, activated charcoal, talc, arginine, lysine, calcium carbonate, carbon black, cyclodextrin, calcium phosphate, celite (diatomaceous earth), glutamine, betaine, bismuth phosphate, bismuth citrate, iron phosphate, etc)
- 2) Binder/matrix—allow for granulation of adsorbent to form solid core (ie, starches, flours, sugars, proteins, gums, gelatin, cellulose and derivatives, etc)
- 3) Coating—single to multilayer coatings to decrease water penetration and modify release kinetics

All materials purchased from Sigma-Aldrich and Fisher Scientific and used as received unless otherwise specified.

Tablet Preparation

3NOP was adsorbed onto the corresponding adsorbent at a given weight percent (noted in formulations) by dropwise addition to adsorbent stirring on a hot plate. The mixture was left to stir for more than 30 minutes until a free-flowing powder was obtained. The resulting mixture was speed mixed with the corresponding binder for 1 minute followed by compaction of in a carver press using an 8-13 mm pellet die under various forces of 10-60 kN.

Coated Tablet Preparation

Selected coating materials were dissolved in various solvents at 5-20 wt %. Tablets were then dip coated multiple times allowing for drying in between to give the desired coating weight.

3NOP Release Quantification and Corresponding Tablet Formulations

13 mm, 0.5 g tablets were placed in a sealed 5 dram vial with 10 mL of distilled water and incubated at 70 F until full dissolution of 3NOP. 1 mL aliquots were taken every few days and refreshed with 1 mL of distilled water. Aliquots were filtered through a 0.22 um PVDF filter before being quantified via HPLC.

Uncoated Tablets

All formulation percentages are based on weight. 2 replicates of 13 mm disc-shaped, 0.5 g tablets were compacted under 20 kN. The uncoated formulations had the following compositions:

- AH-01-7.5% 3NOP, 12.5% propylene glycol, 60% Cargill hemp protein
- AH-02-8.57% 3NOP, 22.86% Celite, 68.57% hemp protein
- AH-03-8.57% 3NOP, 22.86% Wakefield Biochar, 68.57% Cargill hemp protein
- AH-04-8.57% 3NOP, 22.86% K10 montmorillonite, 68.57% Cargill hemp protein
- AH-05-8.57% 3NOP, 22.86% activated charcoal, 68.57% Cargill hemp protein

The 3NOP release of the uncoated tablets

after 12 hours is shown in Table 17.

Total Average

Concentration of 3NOP
Standard deviation

Formula
(mM)
(mM)

AH-01
24.56
2.09

AH-02
24.50
1.76

AH-03
19.61
0.17

AH-04
33.10
0.10

AH-05
18.58
0.07

Coated Tablets

Tablets for coating experiments consist of 10% 3NOP, 20% Wakefield biochar, and 70% cellulose acetate (Mn: 50,000). Tablets were then coated with ˜0.2 wt % of the corresponding coating material, as shown in the table in Table 18. The release profile over periods of 4, 6 and 11 days for each formula is also shown.

TABLE 18

3NOP release profile of coated tablets over 4 days.

Total Average
Standard

Time
Concentration
deviation

(days)
(mM)
(mM)

Cellulose Acetate Phthalate
4
34.21
2.23

(CAP)

CAP
6
30.65
0.04

CAP
11
32.38
0.24

Ethyl cellulose (EC)
4
29.92
0.06

EC
6
31.07
0.01

EC
11
31.50
0.26

Hydroxy propyl cellulose (HPC)
4
31.70
0.05

HPC
6
30.35
0.01

HPC
11
32.53
0.4

Polycaprolactone (CAPA 6800)
4
32.08
0.29

CAPA 6800
6
31.00
0.14

CAPA 6800
11
31.99
0.23

Alignate (layer 1) Chitosan
4
30.30
0.17

(layer 2)

Alignate (layer 1) Chitosan
6
30.22
0.17

(layer 2)

Alignate (layer 1) Chitosan
11
31.90
0.10

(layer 2)

Polycaprolactone Mn: 2000
4
33.68
0.01

Polycaprolactone Mn: 2000
6
32.25
0.18

Polycaprolactone Mn: 2000
11
33.23
0.09

PEG Mn: 1500
4
32.74
0.22

PEG Mn: 1500
6
31.88
1.30

PEG Mn: 1500
11
33.48
0.29

Further formulations were prepared and tested. 3 replicates of 13 mm disc-shaped, 0.5 g tablets were compacted under 6 kN. All formulas consist of 23.8% 3NOP, 28.6% silica, and 47.6% ethyl cellulose. Tablets were then coated with 60-80 wt % of the corresponding coating material. Control tablet (CTRL) is uncoated. “R” denotes replicate number. CAP coating solution was 18 wt % in 70% ethanol/water. Cellulose acetate (CA) coating was 6 wt % in 70% ethanol/ethyl acetate with an additional 5 wt % triethyl citrate, 5 wt % propylene glycol, and 5 wt % N-methyl-2-pyrollidone. The release profiles were analyzed at 1, 4, 8, 11, and 15 days, as summarized in Table 19.

TABLE 19

3NOP release profile of coated tablets over 15 days.

Total concentration of 3NOP is listed in mM.

CAP R1
25.74
47.79
59.39
61.05
59.68

CAP R2
18.72
43.71
52.56
53.74
53.37

CAP R3
23.12
50.66
57.23
59.23
58.75

AVG
22.53
47.39
56.39
58.01
57.26

STD
3.54
3.49
3.49
3.81
3.41

CA R1
50.90
75.98
73.80
66.97
61.48

CA R2
71.29
78.41
73.11
66.66
60.18

CA R3
63.86
69.38
65.56
61.28
57.00

AVG
62.01
74.59
70.82
64.97
59.55

STD
10.32
4.67
4.57
3.20
2.31

CTRL R1
45.80
80.64
80.33
73.64

CTRL R2
45.80
84.41
83.39
76.22
69.25

CTRL R3
45.80
77.15
75.92
70.11
62.74

AVG
45.80
80.73
79.88
73.32
66.00

STD
0.00
3.63
3.76
3.07
4.60

Coated Tablets

All formulation percentages are based on weight. 3 replicates of 13 mm disc-shaped, 0.5 g tablets were compacted under 60 kN. All formulas consist of 23% 3NOP, 30% silica, and 47% ethyl cellulose. Tablets were then coated with 60-80 wt % of the corresponding coating material. Control tablet (CTRL) is uncoated. V1 coating solution was 20 wt % cellulose acetate Mn: 50000 in 70% acetone/ethyl acetate with BYK coating additives. V2 coating solution was 20 wt % in 70% acetone/ethyl acetate cellulose acetate Mn: 50000 with BYK coating additives. Ethyl cellulose coating solution was 20 wt % in 70% ethanol/water with 15 wt % triacetin. CAP coating solution was 18 wt % in 70% ethanol/water with 10 wt % propylene glycol. The 3NOP release profile of the coated tablets over 18 days is shown in Table 20. PGP-49;

TABLE 20

3NOP release profile of coated tablets over 18 days.

Total concentration of 3NOP is listed in mM.

Time
1
5
8
12
18

V1 R1
33.04
70.32
70.57
66.04
60.88

V1 R2
57.54
77.54
77.42
72.65
67.44

V1 R3
61.42
71.21
70.08
65.71
61.19

AVG
50.67
73.02
72.69
68.13
63.17

STD
15.39
3.93
4.10
3.91
3.70

V2 R1
31.15
66.47
65.62
61.03
56.78

V2 R2
32.55
66.81
67.61
63.67
59.55

V2 R3
53.59
69.37
71.28
67.53
63.01

AVG
39.10
67.55
68.17
64.08
59.78

STD
12.57
1.59
2.87
3.27
3.12

EC R1
17.99
39.67
45.39
47.12
50.00

EC R2
15.84
38.15
43.53
45.83
49.27

EC R3
21.21
52.28
58.64
60.42
63.56

AVG
18.35
43.37
49.19
51.13
54.28

STD
2.70
7.75
8.24
8.08
8.05

CAP R1
24.07
57.62
63.10
69.28
68.61

CAP R2
23.24
56.30
61.85
60.67
59.42

CAP R3
37.58
63.57
71.80
69.89
70.48

AVG
28.30
59.17
65.58
66.61
66.17

STD
8.05
3.87
5.42
5.15
5.92

CTRL R1
61.62
97.29
89.52
81.26
74.20

CTRL R2
45.40
82.69
79.42
73.16
67.07

CTRL R3
53.75
83.30
76.77
70.18
64.12

AVG
53.59
87.76
81.90
74.87
68.47

STD
8.11
8.26
6.73
5.74
5.18

Bilayer Coated Tablets

All formulation percentages are based on weight. 3 replicates of 13 mm disc-shaped, 0.5 g tablets were compacted under 60 kN. All formulas consist of 15% 3NOP, 20% silica, and 65% ethyl cellulose. Bilayer coatings were applied with ˜10 wt % of the first coating material and ˜20 wt % of the second coating material. CAP/EC denoted first and second coating material. The 3NOP release profile of bilayer coated tablets over 27 days is shown in Table 21.

TABLE 21

3NOP release profile of bilayer coated tablets over

27 days. Total concentration of 3NOP is listed in mM.

Time (days)

1.25
2.5
5.5
8
10
14
17
21
27

CAP/EC
6.27
8.80
15.97
18.49
20.41
24.15
24.59
24.46
26.21

R1

CAP/EC
4.39
7.77
15.50
17.78
19.54
22.90
23.44
23.74
25.94

R2

CAP/EC
5.63
8.43
15.44
17.84
19.29
20.36
20.94
21.18
22.65

R3

AVG
5.43
8.33
15.64
18.04
19.75
22.47
22.99
23.13
24.93

STD
0.96
0.52
0.29
0.39
0.59
1.93
1.87
1.73
1.98

EC/CAP
9.75
14.58
22.35
23.09
23.76
26.40
26.04
26.12
26.82

R1

EC/CAP
10.72
17.13
24.89
26.10
26.78
29.51
28.89
28.52
29.31

R2

EC/CAP
9.84
15.34
22.33
24.08
25.21
28.03
27.91
27.56
28.17

R3

AVG
10.10
15.68
23.19
24.42
25.25
27.98
27.61
27.40
28.10

STD
0.54
1.31
1.47
1.54
1.51
1.56
1.45
1.21
1.25

CTRL
26.11
28.76
36.26
37.49
36.75
35.77
33.69
31.26
32.66

R1

CTRL
26.12
28.43
38.45
39.57
38.58
37.45
34.91
32.16
33.30

R2

CTRL
25.23
29.94
40.65
41.23
45.02
42.91
39.10
27.92
−2.04

R3

AVG
25.82
29.05
38.45
39.43
40.12
38.71
35.90
30.45
21.31

STD
0.51
0.79
2.19
1.87
4.34
3.73
2.84
2.24
20.22

Adsorbent Study

Various adsorbents were assessed for potential binding affinity and adsorptive capacity of 3NOP. Activated carbon/charcoal and silica were identified as the most suitable adsorbents based on their high binding affinity and adsorptive capacity.

Briefly, 0.2 g of adsorbent was added to 4 mL of 2.5% 3NOP/H2O stock solution or 10 mL of 1% 3NOP/H2O stock solution. The solution was then placed in an ultrasonic bath for ˜4 hr and then allowed to equilibrate over 24 h. All materials were purchased from Sigma-Aldrich and used as received. After 24 h, adsorbents were tested for their binding affinity and adsorptive capacity by looking at the reduction of 3NOP concentration via HPLC at 230 nm.

The following adsorbents were tested and integrated areas were converted to mM concentrations of 3NOP based on calibration standards of known 3NOP concentration in distilled water, as seen in Table 22. FIG. 14 is graph showing the concentration of 3NOP (mM) v. adsorbent (20 mM stock solution).

TABLE 22

Change in concentration v. control of 0.2

g of adsorbent in 4 mL of 2.5% 3NOP/H2O.

Adsorbent
3NOP concentration (mM)

Control
20.649

Calcium Carbonate
20.649

Talc
20.463

Silica Gel (40-60 um)
20.043

Silica (5-20 nm)
19.920

Arginine
19.782

Lysine
18.583

Carbon Black
17.422

Activated Charcoal
11.063

Table 23 shows the change in concentration v. control of 0.2 g of adsorbent in 10 mL of 1% 3NOP/H2O, and FIG. 15 shows 3NOP concentration (mM) v. adsorbent (˜8 mM stock solution).

TABLE 23

Change in concentration v. control of 0.2

g of adsorbent in 10 mL of 1% 3NOP/H2O.

Adsorbent
3NOP concentration (mM)

Control
8.258

Lignin (Alkaline)
8.326

Bismuth Sulfate
8.079

Bismuth Citrate
8.072

Lignin (Dealkaline)
8.056

Diatomaceous Earth
7.895

Copper Sulfate
7.814

Molecular Sieve (4 Å)
7.771

Cyclodextrin
7.676

Glutamine
7.588

Iron Phosphate
7.503

Betaine
7.202

Activated Charcoal
1.358

Activated carbon shows strong binding affinity for 3NOP filtering ˜45 wt % in concentrated (20 mM) conditions with ˜80 wt % in more dilute concentrations (8 mM). It appears that the adsorbent pore size should be greater than 4 Å for adsorption of 3NOP. Mesoporous adsorbents with high surface area such as silica and activated carbon show high adsorptive capacity with no visible welting seen until ˜75 wt %. Layer-by-Layer

Polyelectrolytes Coatings

Formulated tablets containing 3NOP, an adsorbent, and a binder can be coated with sequential layers of oppositely charged poly electrolytes. While not being bound by theory, it is believed that ionic crosslinking between the layers can result in a controlled release of 3NOP out of the tablets. Examples of polyelectrolyte pairs that can be used include polyglutamic acid and polylysine, polyallylamine hydrochloride and polyacrylic acid, and polyallylamine hydrochloride and polystyrene sulfonate. An exemplary formulated tablet includes an adsorbent of silica, arginine, lysine, and activated charcoal (30-45%) and a binder of ethyl cellulose and hydroxypropyl cellulose (40-55%).

TABLE 24

Formulation examples of 3NOP tablets.

Tablet weight varied from 0.2-0.5 g.

Tablet Formulation

Part per

Part per

formulation

formulation

Adsorbent
(wt %)
Binder
(wt %)

Silica, Arginine,
30-45
Ethyl Cellulose,
40-55

Lysine, Activated

Hydroxypropyl

Charcoal

Cellulose

The release data of 3NOP tablets coated with multilayers of some polyelectrolytes is shown in Table 25.

TABLE 25

Release (%) of 3NOP out of tablets formulated with

15% 3NOP, 30% silica, and 55% ethyl cellulose.

Release Compared to Control Tablets (%)

PE Pair
Day 1
Day 3
Day 6
Day 8
Day 14
Day 16
Day 21
Day 30

PLK20 +
58.1
56.6
61.3
62.6
64.7
63.6
63.8
63.2

PRE20

PAH +
61.5
62.5
67.1
70.2
70.4
71.4
71.7
71.6

PAA

PAH +
61
66.5
74.7
76
77.5
76.6
76.7
74.8

PSS

Weight of tablets ~0.5 g. Calculations are based on a comparison to control tablets with no coating.

3NOP to Adsorbent Ratio Study

Optimal loading of 3NOP onto adsorbents was determined by varying ratios of 3NOP to adsorbent. 3NOP and adsorbent were speed mixed at 2000 rpm for 1 min at the following ratios by weight: 1:1.5, 1:2, 1:2.5. The adsorbed 3NOP powder mixture was then placed in distilled water at 1% 3NOP loading. The solution was allowed to equilibrate for 24 hr and then an aliquot was taken for HPLC to determine the reduction in 3NOP concentration. Table 26 shows the average integrates are of 3NOP in solution at various adsorbent ratios.

TABLE 26

Average integrated area of 3NOP in solution at varying 3NOP:adsorbent ratios

3NOP:Adsorbent

1:1.5
1:2
1:2.5

Average

Average

Average

Integrated
Standard
Integrated
Standard
Integrated
Standard

Area
Deviation
Area
Deviation
Area
Deviation

3NOP
2566.4
51.3
2566.4
51.3
2566.4
51.3

Silica
2499.4
293.2
2321.9
99.3
2340.9
30.3

Activated
1751.2
29.6
1581.9
8.5
1403.7
14.5

charcoal

Lysine
2336.2
61.7
2308.9
23.9
2266.6
56.1

Arginine
2406.0
113.2
2372.8
61.2
2435.2
230.2

Glutamic
2445.9
149.9
2452.5
78.9
2446.3
85.0

acid

Higher ratios of activated carbon to 3NOP result in higher adsorptive capacity. At ratio of 1:2.5 an adsorption capacity of ˜45% is observed and at a ratio of 1:1.5 an adsorption capacity of ˜30% is observed.

Activated Carbon Tablets

Based on the 3NOP:adsorbent ratio study, the same ratios were tested for tablet integrity, friability, porosity, etc to determine the optimal 3NOP loading. 500 mg, 13 mm tablets were compacted under 60 kN.

High ratios of activated carbon (1:2.5 3NOP:adsorbent) resulted in tablets that absorb more water and coating solutions. This water/solvent absorption of non-wetted charcoal causes difficulty in producing uniform, defect free coatings. High loadings of activated charcoal >25% in tablets result in poor tablet integrity characterized by loss in hardness, increased porosity, tablet spilling, and poor shape retention. The preferred loading for tablets with poor disintegration was 1:1.5 3NOP:adsorbent.

FIG. 16 shows a multilayer tablet formulation above consisting of 10 wt % 3NOP, 15 wt % activated carbon, and 75 wt % ethyl cellulose. The first day release of coated tablets is ˜40% less than uncoated tablets and after 30 days, ˜30% 3NOP remains adsorbed on activated carbon.

FIG. 17 shows a multilayer tablet formulation consisting of 10 wt % 3NOP, 25 wt % activated carbon, and 75 wt % ethyl cellulose. After 30 days, ˜50% 3NOP remains adsorbed on activated carbon. These adsorptive capacities are in-line with what was observed during the 3NOP:adsorbent ratio study.

FIG. 18 shows a multilayer formulation consisting of 10 wt % 3NOP, 15 wt % activated carbon, 5 wt % sodium lignosulfonate, and 70 wt % ethyl cellulose. The addition of 5 wt % sodium lignosulfonate improves coating adhesion to the tablet resulting in ˜30% reduction in the first day 3NOP release. Over time, the release rate remains similar to previous formulations with a residual 30% 3NOP remaining adsorbed to activated carbon.

FIG. 19 shows a multilayer formulation consisting of 10 wt % 3NOP, 15 wt % activated carbon, 5 wt % hydroxypropyl cellulose, and 70 wt % ethyl cellulose. The addition of 5 wt % hydroxypropyl cellulose, a hydrophilic water-soluble binder, results in increased release between day 4 and day 10. After 30 days, ˜30% 3NOP remains adsorbed to activated carbon.

Silica v. Activated Carbon Controls to Assess Binding Affinity of 3NOP

Various control experiments were conducted to confirm the adsorbed 3NOP to activated carbon after 30 days. Activated carbon was compared to silica as preferred adsorbents. 500 mg of 1:1.5 3NOP:adsorbent were speed mixed at 2000 rpm for 1 min. The resulting powder mixture was placed in 10 mL of distilled water and 10 mL of acetonitrile (ACN) separately. The solutions were placed in a sonic bath for ˜5 hr, removed, and allowed to equilibrate for 24 hr.

Aliquots were filtered through a 0.22 um PTFE filter, and taken for HPLC to measure the 3NOP concentration in solution.

The same trend was observed in the adsorbent study. However, 3NOP is recoverable by washing the activated carbon with acetonitrile or a similar organic solvent with high 3NOP solubility (ie. ethanol, acetone, ethyl acetate, tetrahydrofuran, chloroform).

TABLE 27

Silica powder release v. activated carbon release

in distilled water and acetonitrile.

24 hr in H2O
24 hr in ACN

Integrated Area
Integrated Area

Si-1
8415.7
8847

Si-2
8507.3
8822.2

Average
8461.5
8834.6

AC-1
4940.8
8103.9

AC-2
5577.5
8082.8

Average
5259.2
8093.4

After 24 hr in water, less than 5 wt % 3NOP remained adsorbed to silica compared to the powder mix placed in acetonitrile. After 24 hr in water, ˜35 wt % 3NOP remained adsorbed to activated carbon compared to the powder mix placed in acetonitrile. By washing the activated carbon in acetonitrile, the adsorbed 3NOP can be recovered in solution proving that the remaining 30-50% 3NOP that is seen in FIG. 11-FIG. 14 remains adsorbed. Further studies need to be conducted to determine the driving forces for 3NOP desorption from activated carbon under different environmental conditions to utilize adsorptive capacity of activated carbon. This study also confirms that from previous tablet formulations, diffusion is the primary driver for 3NOP release of unbound 3NOP. This can be seen by very similar release profiles between silica-based adsorbent tablets and activated carbon-based adsorbent tablets. FIG. 20 compares a silica-based tablet and activated carbon-based tablet over 8 days. Small differences in release profiles could be due to differences in porosity between tablets using silica or activated carbon.

Compounding of 3NOP

3NOP was compounded with a variety of matrix materials using a DSM Xplore MC15 conical twin-screw extruder. The extrudate was pelletized and used as is or further compression molded into tablets as a suitable 3NOP extended-release form.

3NOP was adsorbed onto the corresponding adsorbent and speed mixed with binder prior to compounding is a DSM Xplore MC15 conical twin-screw extruder at 100 C and 50 rpm. The strand was then pelletized, heated, and molded into a 13 mm disc-shaped tablet using a carver press under 40 kN of compaction force. Formula consists of 8.3% 3NOP, 25.0% arginine, 66.7% CAPA 6800.

TABLE 28

3NOP release profile of compounded tablets over 24 days.

Time
1
3
7
10
14
20
24

Arg R1
8.97
16.37
22.55
23.42
24.11
23.07
24.56

Arg R2
12.49
15.85
20.43
21.45
21.60
19.33
19.97

Arg R3
9.90
17.78
24.52
24.50
24.36
22.60
22.33

AVG
10.45
16.67
22.50
23.13
23.36
21.67
22.29

STD
1.82
1.00
2.04
1.55
1.52
2.04
2.29

Total concentration of 3NOP is listed in mM.

Formulas consists of 11.1% 3NOP, 13.9% silica, 75.0% CAPA 6800 denoted “11PCL” and 19.4% 3NOP, 24.2% silica, 56.5% CAPA 6800 denoted “19PCL.”

TABLE 29

3NOP release profile of compounded tablets over 24

days. Total concentration of 3NOP is listed in mM.

Time
1
3
7
13
17

11PCL R1
9.79
18.77
24.31
29.15
27.93

11PCL R2
10.34
19.81
24.99
30.02
29.49

11PCL R3

19.05
22.51
27.14
26.95

AVG
10.06
19.21
23.94
28.77
28.12

STD
0.39
0.54
1.28
1.48
1.28

19PCL R1
23.55
40.56
49.99
55.00
3.48

19PCL R2
24.19
40.59
51.03
53.46
51.64

19PCL R3
25.05
42.76
52.21
57.93
55.77

AVG
24.26
41.30
51.08
55.47
36.96

STD
0.75
1.26
1.11
2.27
29.07

Compounded Polyelectrolyte Complexes for Extended Release of 3NOP

Solid polyelectrolyte complexes can be processible when plasticized with aqueous electrolytes to act as a matrix for 3NOP molecules. An example of a polyelectrolyte pair for compounding is polydiallyldimethylammonium chloride (PDADMAC), as a cationic polyelectrolyte, and polystyrene sulfonate (PSS), as an anionic polyelectrolyte. At no salt conditions, the complex is brittle and not ideal for processing and the addition of salt, such as sodium chloride, can help with the plasticization and further processing. A trial formulation used for compounding includes mixing of equal charge stoichiometry of PDADMAC and PSS solutions with a concentration of 100 mM, with respect to each polymer, and a total volume of 200 mL (as per preparation method described in ACS Appl. Mater. Interfaces 2015, 7, 895-901). The polyelectrolyte complex can be separated, removed from the supernatant phase, and soaked in salt water with a concentration of 1M for 24 h. The polyelectrolyte complex should be decanted and ready for compounding. A typical formulation used for compounding is summarized in Table 30.

TABLE 30

Compounding formulation with polyelectrolyte complexes for extended

release of 3NOP. Total materials used 20 g. 3NOP was mixed with

arginine (as in solid powder) overnight at 30 C.

PEC Compounding Formulation

Part per

Part per

formulation

formulation

3NOP Complex
(wt %)
PEC Complex
(wt %)

3NOP: Arginine
25
PDADMAC: PSS
75

(ratio 1:3, wt %)

(Equimolar with

respect to the

monomer)

Table 31 shows release (%) of 3NOP out of compounded PEC formulated as per Table 21. The compounded PECs were cut into 5-10 mm pellets and soaked in water (10 mL) for the release study.

TABLE 31

Release percentages

Release (%)

Day 1
Day 4
Day 6
Day 12

22.4
34.4
35
35.7

Compounded Pellets

3NOP was adsorbed onto the corresponding adsorbent and speed mixed with binder prior to compounding is a DSM Xplore MC15 conical twin-screw extruder at temperatures of 110-130 C and 100 rpm. The extrudate was then pelletized forming ˜2 mm pellets. Material suppliers are listed in Table 30. Polybutylene succinate grades FZ71, FZ91, FD92 were supplied by Mitsubishi Chemical. Polycaprolactone-based formulations are summarized in the Table in FIG. 21. FIG. 22 shows 3NOP release in mM for polycaprolactone-based formulations.

TABLE 32

Polybutylene succinate-based formulations

Code
65FZ71
65FZ91
65FD92a
65FD92b

Material

Polybutylene succinate
65.0%
65.0%
65.0%
65.0%

Activated charcoal
20.0%
20.0%
20.0%
20.0%

3NOP
15.0%
15.0%
15.0%
15.0%

On total solids

Glycerol monooleate
0.5%
0.5%
0.5%
0.0%

Compounding Temperature
120 C.
120 C.
130 C.
110 C.

FIG. 24 is chart showing the normalized 3NOP concentration v. Release time in days of polybutylene succinate-based formulations. Lower 3NOP loadings (<10%) have a less pronounced burst release over the first 7 days of monitored release. Glycerol monooleate was identified at a potent processing aid at loadings <1%. High levels <25 wt % activated carbon can easily be processed unlike silica which leads to high viscosity and screw torque. Cross-linked systems show slower release in comparison to un-crosslinked systems. High shear conditions of compounding appear to destroy activated carbon structure and adsorption capacity is lost.

Microcapsules for the Encapsulation of 3NOP

Polyelectrolyte complexes (PECs) are used to form microcapsules and encapsulate 3NOP. PECs are formed through attractive forces such as ionic interactions, hydrogen bonding, hydrophobic, and pi-interactions between the polyelectrolytes. Polyelectrolytes are polymers with ionic groups bonded to counter ions and can dissociate in a solution to make positively or negatively charged polymers. Herein, PEC microcapsules refer to interconnecting networks of polyelectrolytes formed upon the interaction between oppositely charged polymers.

Examples of polyelectrolytes used in this invention include synthetic polyelectrolytes such as polystyrene sulfonate (PSS), polyacrylic acid (PAA), and polyallylamine hydrochloride (PAH). Further examples include naturally occurring polyelectrolytes such as chitosan and alginate or ionic biopolymers such as proteins, enzymes, and charged polypeptides.

Examples of Polyelectrolytes (charged polymers with positively or negatively charged repeating units) Used:

Synthetic Polyelectrolytes

- Polystyrene sulfonate (PSS), molecular weight ˜200,000 Da, Sigma Aldrich
- Sodium Lignosulfonate (SLS), molecular weight ˜52000 Da, Sigma Aldrich
- Polyethyleneimine (PEI), molecular weight ˜2000 Da, Polysciences
- Polyallylamine hydrochloride (PAH), molecular weight ˜17,500 Da, Sigma Aldrich

Commercially Available Peptide Sequences

- Poly (L-lysine) (PLK10), degree of polymerization: 10, molecular weight: 1600 Da, Alamanda Polymers
- Poly (L-lysine) (PLK20), degree of polymerization: 20, molecular weight: 3300 Da, Alamanda Polymers
- Poly (L-arginine) (PLR10), degree of polymerization: 10, molecular weight: 1900 Da, Alamanda Polymers
- Examples of Crosslinker Used:
- Glutaraldehyde, 25% solution in water, Sigma Aldrich
- EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), Thermo Fisher.

PEC Microcapsules Preparation

Equal stoichiometry of oppositely charged polyelectrolytes is sequentially added to water or water-3NOP solutions followed by vortexing for 10 s, after the addition of each component, to form PEC microcapsules. Some samples are chemically crosslinked (glutaraldehyde is an example of the crosslinker used). The PEC microcapsules may also be formed with non-stoichiometric ratios of polyelectrolytes. While stoichiometric ratios of polyelectrolytes provide almost a neutral microcapsule, microcapsules prepared with non-stoichiometric ratios are positively or negatively charged. All polyelectrolyte solutions are prepared in water and pH adjusted. The concentration of solutions is based on the monomer charge. The stock solution of 3NOP is prepared in water with a concentration of 100 mM and pH adjusted to 8.

High-performance liquid chromatography (HPLC) is used to determine the concentration of 3NOP in PEC microcapsules and calculate the release of 3NOP from the microcapsules. Samples are centrifuged to separate the supernatant phase from the complex phase. The supernatant phase is then removed carefully by using a micropipette and transferred to a 2 mL glass vial for further analysis. The 3NOP release of some PEC microcapsules prepared at neutral pH conditions and after 96 h of preparation is summarized in Table 31.

TABLE 33

3NOP release (%) from the PEC microcapsules. Samples

are prepared with a 3NOP concentration of 100 μM. Final

pH solution ~7. K, L, F, and E refer to lysine, leucine,

phenylalanine, and glutamic acid, respectively. PSS

refers to polystyrene sulfonate. Table 33 discloses

(KKLF)3 as SEQ ID NO: 79312, (EELF)3 as SEQ ID NO:

79313 and (kKlF)3 as SEQ ID NO: 79314.

3NOP Concentration

Release (%) at
in Microcapsules

Polyelectrolyte Pair
96 h timepoint
(μM)

(KKLF)3 + (EELF)3
63
37

(KKLF)3 + PSS
64
36

(kKlF)3 + PSS
66
34

(kKlF)3 + (EELF)3(crosslinked)
70
30

(kKlF)3 + PSS (crosslinked)
72
28

FIG. 24 is a graph showing 3NOP release (%) from the PEC microcapsules. Samples are prepared with a 3NOP concentration of 100 μM. Final pH solution ˜7. K, L, F, and E refer to lysine, leucine, phenylalanine, and glutamic acid, respectively. PSS refers to polystyrene sulfonate.

Examples of release profiles of some microcapsules made of positively charged polypeptides with either polystyrene sulfonate (PSS) or sodium lignosulfonate (SLS) are shown in FIG. 25 (3NOP release (%) from the PEC microcapsules. Samples are prepared with a 3NOP concentration of 100 μM. Final pH solution ˜7. PLR refers to poly (L-arginine), and PLK refers to poly (L-lysine). SLS and PSS refer to sodium lignosulfonate and polystyrene sulfonate).

The encapsulation efficiency of some PEC microcapsules prepared at neutral pH conditions is summarized in Table 34. Table 35 shows the encapsulation efficiency of samples prepared at pH-8.

TABLE 34

Encapsulation efficiency of the PEC microcapsules. Samples are prepared

with a 3NOP concentration of 100 μM. Final pH solution ~7.

Upper and lower cases in (kKlF)3 sequences represent L and D-chirality,

respectively. Table 34 discloses (KKLF)3 as SEQ ID NO: 79312, (EELF)3

as SEQ ID NO: 79313 and (kKlF)3 as SEQ ID NO: 79314.

Total Concentration
Encapsulation
3NOP Concentration

(monomer charge basis)
Efficiency
in Microcapsules

Polyelectrolyte Pair
(mM)
(%)
(μM)

PLK20 + PRE20
5
7.4
7.4

PLR10 + PRE20
5
0
0

(KKLF)3 + (EELF)3
5
100
100

(kKlF)3 + (EELF)3
5
22.2
22.2

(KKLF)3 + PSS
5
37
37

(kKlF)3 + PSS
5
100
100

PAH + PSS
5
30.9
30.9

TABLE 35

Encapsulation efficiency of the PEC microcapsules. Samples are prepared with

a 3NOP concentration of 2.5 mM. Final pH solution ~8. Upper and lower cases

in (kKlF)3 (SEQ ID NO: 79314) sequences represent L and D-chirality of the amino

acid residue, respectively. Table 35 discloses (kKlF)3 as SEQ ID NO: 79314.

Total Concentration
Encapsulation
3NOP

(monomer charge
Efficiency
Concentration in

Polyelectrolyte Pair
basis) (mM)
(%)
Microcapsules (mM)

PLR10 + PRE20
5
50
1.25

PLK20 + PRE20
5
53.8
1.34

(kKlF)3 + PRE20
5
50.2
1.26

Physical Cross/Inking of 3NOP with Small Molecules

3NOP molecules can form hydrogen bonds with small molecules such as amino acids to make a larger size conjugate resulting in enhanced encapsulation efficiency. For example, an equal stoichiometry of 3NOP and two selective amino acids, arginine and lysine, were reacted in aqueous solution for 24 h, and the solution was used for encapsulation studies. The results of such encapsulation with some PEC microcapsules are shown in Table 36.

TABLE 36

Encapsulation efficiency of the PEC microcapsules. Samples are prepared

with a 3NOP concentration of 125 μM. Final pH solution ~7. Table

36 discloses (KKLF)3 as SEQ ID NO: 79312 and (EELF)3 as SEQ ID NO: 79313.

Encapsulation
3NOP Concentration in

Polyelectrolyte
Efficiency (%)
Microcapsules (μM)

Pair
Arginine-3NOP
Lysine-3NOP
Arginine-3NOP
Lysine-3NOP

PLR10 + PRE20
4.6
19.1
5.75
23.9

PLK20 + PRE20
34.3
28.4
42.9
35.5

(KKLF)3 + (EELF)3
27.4
34.8
34.25
43.5

PAH + PAA
30.3
47.1
37.9
58.9

PAH + PSS
67.4
67.6
84.3
84.5

Example 13: Vaccination of Cattle Population

It is demonstrated herein that vaccination of exemplary vaccines of the present disclosure resulted in generation of antibodies which were effective in neutralizing ruminal methanogens.

Treatment Groups

TABLE 37

Treatment descriptions

Treatment ID
Count
Treatment Description

9A
(n = 20)
Untreated

9G
(n = 5)
1 mL of RPF51698.1 (SEQ ID NO: 11032) +

ADC46800.1 (SEQ ID NO: 5382)

1 mL of Enable C1

9H
(n = 4)
0.5 mL of RPF51698.1 (SEQ ID NO: 11032)

0.5 mL of Enable C1

9I
(n = 2)
0.5 mL of ADC46800.1 (SEQ ID NO: 5382)

0.5 mL of Enable C1

1. Conclusions
1.1 Vaccine Formulations

Treatment with protein antigen formulations (9G+9H+91) did not adversely affect daily feed intake nor average daily gain. Any trends in enteric emissions changes reported herein are present when CH₄production (g/d) is normalized for DMI (i.e., CH₄yield) or ADG (i.e., CH₄intensity). Furthermore, no adverse events were reported for animals treated with protein antigen formulations.

Antigen-specific ELISA assay of sera from animals vaccinated with treatments 9G and 9H demonstrated elevated IgG's against RPF51698.1 protein. IgG response peaked in the d42 sample. Antigen-specific ELISA assay of sera from animas vaccinated with treatments 9G, 9H, and 9I demonstrated elevated IgG's reactive to ADC46800.1 protein. This further demonstrates that vaccines comprising RPF51698.1 and/or ADC46800.1 protein antigen induce an immune response generating antibodies capable of binding ADC46800.1. This observation suggests one or more epitopes on ADC46800.1 raise cross reactive IgG response to both protein homologs.

Treatment 9G and 91 formulations appeared to be efficacious following primary vaccination (d0), while treatment 9H formulations appeared to be efficacious following secondary vaccination. Efficacy for protein antigen formulations was determined using a covariate analysis (i.e., AA) using the pre-prime period (p0) as the baseline for emissions.

When compared to the untreated control animals, treatment with protein antigen formulations resulted in mitigation of ˜46 kg of CO_2eover 10 weeks (p1, p2+p3). Specifically, treatment resulted in a reduction in CH₄intensity of ˜10%, ˜15 and ˜17% in p1, p2 and p3, respectively, as compared to p0.

2. Protein Antigen Vaccine Formulation
2.1. Preparation of Recombinant Protein

Recombinant proteins were expressed using an E. coli T7 based bacterial expression system. Briefly, genes encoding the proteins of interest were amplified from genomic DNA using PCR primers designed to facilitate Gibson assembly into a linearized pRSET expression plasmid. Sanger or nanopore sequencing was used to confirm the sequence of the target gene and its intended subcloning into the expression vector. Subcloning included the addition of an N-terminal histidine affinity tag to facilitate purification and confirmation of expression. Expression plasmids were used to transform a lon-expression strain of E. coli such as BL21(DE3) pLysS, BL21(DE3), or clear coli (a DE3 lysogen strain engineered for reduced levels of endotoxin). Shake flask expression conditions were determined empirically and used either LB medium with IPTG induction or commercial autoinduction medium (Magic Medium). Cells were lysed with BugBuster reagent.

Purifications were conducted using HiTrap HP Nickel columns using either an FPLC to apply a linear imidazole elution gradient or manually driven syringes to create a step gradient. Fractions containing target protein were pooled and concentrated using Centricon concentrators. Concentrated purified protein was dialyzed against DPBS using slide-alyzer cassettes. If needed, endotoxin levels of preparations were reduced using polymixin-B resin followed by another round of concentration and dialysis. To reduce the likelihood of cross contamination of the proteins, purification media was used for a single target protein only. Preparations were sterilized by passing through 0.22 um syringe filters. Endotoxin levels were monitored either out-of-house by Charles River Laboratory or in-house using a Charles River NextGen-PTS instrument. Protein concentrations were routinely monitored by use of OD280 and final concentrations were determined relative to BSA standards using Bradford Assay.

Purified proteins were diluted to a concentration of 200 μg per 500 uL in DPBS for single protein treatment group doses or 200 ug each protein/1 mL for the dual protein treatment group. Purity of final material was confirmed by SDS-PAGE with coomassie brilliant blue staining. Purity of at least 85% was confirmed using densiometry of gel images with ImageJ. Endotoxin levels were confirmed to be less than 2000 EU per dose. Table 38 describes the final protein preparations.

TABLE 38

Protein preparations summary

Protein
% Purity
EU per dose
Group

RPF51698.1
87
780
9H

ADC46800.1
85
1,100
9I

RPF51698.1 + ADC46800.1
86 (avg)
1,900
9G

3. Vaccine Preparation+Administration
3.1. Treatment Assignments

Prior to the study's initiation, steers were transferred to the study site, McGregor Research Center (McGregor, TX 76657) for a GreenFeed training period lasting approximately 8 weeks to learn behaviors associated with GreenFeed systems, as well as to allow for adaptation to a grower's diet (i.e., 40% rolled corn, 25% dried distillers grains, 2.5% mineral premix, 7.5% molasses and 25% sudan hay).

Only healthy, weaned Black Angus cross steers were utilized for this Project. All animals were weaned and received pre-weaned vaccinations a minimum of sixty days (60 d) prior to initiation of the study. In addition, animals demonstrated no clinical signs of health concerns (e.g., dull or sunken eye, depression, signs of scours, listlessness, weakness, or raspy breathing) at the time of enrollment. All animals selected were within approximately one hundred (100) lbs of each other at the time of enrollment.

3.2. Vaccine Preparation

Vaccines were prepared in their final formulation, i.e., antigen mixed with adjuvant, onsite on the following dates: Jun. 10, 2024 and Jul. 1, 2024.

Briefly, vaccines were prepared by mixing antigen prepared as described above, and the appropriate adjuvant at a 1:1 volumetric ratio via inversion less than 24 hours prior to injection. Exact mixing volumes are defined in Table 37.

Specifically, frozen protein antigens were shipped on dry ice overnight to the Research Center, with storage at −80° C. before and after shipment. Protein antigens were packaged as single aliquots, e.g., 1 mL each. Prior to injection, antigen aliquots were thawed on wet ice and mixed with equal volume of veterinary adjuvant (1:1 mix ratio) using gentle inversion to prepare a final vaccine volume of 0.4-2 mL. Vaccines were kept at 4° C. and/or on wet ice until injection, less than 24 hours.

3.3. Adjuvant Selection

ENABL C1 (Huvepharma, cat. #7010201) was chosen due to its known performance profile and USDA-approved 21 d withdrawal period.

3.4. Vaccine Administration

Animals were assigned to one of nine treatment groups, detailed below in Appendix A. Vaccines were administered subcutaneously by veterinary-trained staff. Just prior to injection, animals were restrained using a cattle silencer, and injections were administered into the animal's neck region using an 18-gauge 0.5-0.75″ needle 4. Dry matter intake, body weight, and daily gain 4.1. Dry matter intake (DMI) To ensure that vaccination did not adversely affect intake, dry matter intake was recorded daily using GrowSafe feed bunks. DMI for Individual animals was recorded by GrowSafe using known RFIDs.

Throughout the entirety of the study period, animals were fed once daily in the morning and had access to a water source at all times.

Individual daily feed intake (kg/d) was used to normalize enteric methane emissions, as well as to confirm that vaccination did not have a deleterious effect on intake. Intake was recorded from May 21, 2024 through Aug. 13, 2024 using GrowSafe feed bunks. Animals also consumed alfalfa pellets from the GreenFeed systems.

Total daily feed intake was determined by summing the feed intake recorded using the GrowSafe feed bunks and the estimated pellet consumption from the GreenFeeds, using the reported good visit duration. Daily dry matter intake was then calculated by correcting the total feed intake for the dry matter content of the feed type; average dry matter content of the feed consumed from the GrowSafe bunks and the pellets consumed from the GreenFeed System.

Dry matter content of the growing diet and GreenFeed pellets were determined.

5. Results
5.1. Body Weight

To ensure that vaccination did not adversely affect animal weight gain, body weight (kg) was recorded weekly throughout the study period. Body weight was recorded using a livestock scale. Specifically, animals to be weighed were walked through a pipe alley from their housing area to the holding pens. After being weighed, they were released to return to their holding pen with other animals from the same pen. At the end of the weighing, animals were walked back to their assigned pen in the barn.

Bodyweight measurements were used to determine average daily gain (ADG) throughout the study period (Jun. 4, 2024-Aug. 13, 2024). ADG was determined by taking the linear regression of weekly body weight measurements collected over 11 weeks.

ADG=SLOPE(body weight_{period X-Y},measurement date_{period X-Y})

Except for Group 9I, ADG over the entire study period was not significantly different from the untreated control (Group 9 A+9B). Group 9I was significantly greater (P=0.0004) than the untreated control, suggesting that there was no deleterious effect following any of the treatments; the observed difference between Group 9I and the control (Group 9 A+9B) may be attributable to the number of biological replicates in Group 9I (n=2), as compared to other treatment groups. When all protein antigen vaccines were considered together (Group 9G+9H+9I), there was no significant difference in ADG as compared to the untreated animals (Group 9 A+9B).

TABLE 39

ADG (kg per day) averaged per treatment group

Treatment Group
ADG (kg/d)
P value

Untreated control
1.12
N/A

9 A + 9B

All gen2
1.20 (↑)
0.187

9G + 9H + 9I

9G
1.16 (↑)
0.368

9H
1.17 (↑)
0.373

9I
1.33 (↑)
0.0004

ADG (kg/per day) for individual animals is shown in FIG. 26 and Appendix B.

6.1. Enteric Emissions Measurement

Enteric methane, hydrogen and carbon dioxide emissions were measured daily throughout the study period using GreenFeed skid systems and GrowSafe feed bunks. Animals had free access to the GreenFeed systems throughout the study period; animals were not physically coerced to use the system. Methane yield and intensity was normalized for dry matter intake for each measurement period separately. Measurement periods were binned into 21-day intervals to align with the prime-boost vaccination schedule, as well as allow for sufficient GreenFeed Spot visits; ≥20 spot visits per measurement period are recommended for analysis.

TABLE 40

GreenFeed measurement periods

Avg. GreenFeed

Period
Start Date
End Date
Spot Visits

0
May 21, 2024
Jun. 10, 2024
35

1
Jun. 11, 2024
Jul. 1, 2024
38

2
Jul. 2, 2024
Jul. 22, 2024
37

3
Jul. 23, 2024
Aug. 19, 2024*
37

*_#Extended + 7 d beyond the planned period end date (28 d total). Drop in wk 7 GreenFeed visitation would have resulted in 15 animals failing GreenFeed visitation criteria (>=20 visits) if p3 were not extended.

Period 3 (p3) was extended +7 d to from Aug. 12, 2024 to Aug. 19, 2024 in order to meet the minimum visitation threshold (>=20 spot visits) for the majority of animals. GreenFeed visitation greatly decreased in the first week of p3 (i.e., wk 7). If p3 had not been extended, 15 of 71 animals would have been excluded from the final emissions analysis for poor visitation; between Jul. 23, 2024 and Aug. 12, 2024, the average GreenFeed visitation was 27. The decrease in GreenFeed visits during wk 7 may be attributable to a drop in ambient air temperature, as no equipment malfunctions or loss of data was reported. The GreenFeed units are located beneath a shade structure, and, with the corresponding drop in temperature, it's possible that animals spent more time in the sun, away from the GreenFeed unit and therefore did not visit as often (FIG. 28).

7.1. Methane Emissions

A covariate analysis was used to interpret average daily methane production, yield and intensity. The rate of change between measurement periods in treatment groups was compared against the rate of change between measurement periods in the untreated control (Group 9A+9B) in a AA analysis (FIG. 29). The following equations were used to perform this analysis Definitions:

C
⁢

H
4

⁢

production

=

grams
⁢

of
⁢

CH4
⁢

per
⁢

day

CH
4

⁢

yield

=

grams
⁢

of
⁢

CH4
⁢

per
⁢

day

kg
⁢

of
⁢

DMI
⁢

per
⁢

day

CH
4

⁢

intensity

=

grams
⁢

of
⁢

CH4
⁢

per
⁢

day

kg
⁢

of
⁢

bodyweight
⁢

gained
⁢

per
⁢

day
⁢

(
ADG
)

Equations:

A
⁢
vg

⁢

⁢

CH
4

⁢

production

=

AVERAGE
(

CH4
⁢

massflow
⁢

values

period
⁢

X

)

Avg
⁢

DMI

=

[

[
DEFINE
]

]

CH
4

⁢

yield

=

[

[
DEFINE
]

]

CH
4

⁢

intensity

=

(

Avg
⁢

CH4
⁢

production

)

period
⁢

X

ADG

Δ
⁢
C

⁢

H
4

⁢

production

=

(

Avg
⁢

CH4
⁢

production

)

period
⁢

X

-

(

Avg
⁢

CH4
⁢

production

)

period
⁢

Y

ΔΔ
⁢

C
⁢

H
4

⁢

production

=

Δ
⁢

CH
4

⁢

production

group
⁢

X

;

period
⁢

X

-
Y

-

Δ
⁢
Avg
⁢

CH4
⁢

production

control
;

period
⁢

X

-
Y

Efficacy for protein antigen formulations was determined using a covariate analysis (i.e., AA) using the pre-prime period (p0) as the baseline for emissions.

Treatment with protein antigen formulations (Group 9G+9H+91) resulted in an average mitigation of ˜46.5 kg of CO_2eper animal over 10 weeks (p1, p²+p3).

TABLE 41

ΔΔ CH₄production (g/d) for protein antigen formulations

Avg ΔΔ CH₄Production

Treatment Groups
Period
(g/d), per animal*
P value

9G + 9H + 9I
p1 - p0
−16.01 (↓)
0.006

(n = 10)
p2 - p0
−23.71 (↓)
0.002

p3 - p0
−25.89 (↓)
0.001

9G + 9I
p1 - p0
−21.97 (↓)
0.0003

(n = 7)
p2 - p0
−24.64 (↓)
0.00001

p3 - p0
−27.43 (↓)
0.001

*_#Δ CH₄production relative to untreated animals (Groups A + B) over the same periods of time.

CH₄intensity was significantly reduced following treatment protein antigen formulations, as compared to changes in CH₄intensity in the control animals over the same measurement periods (Table 42). Treatment with protein antigen formulations resulted in an average reduction in CH₄intensity of ˜0.10%, ˜15% and ˜17% per animal in p1, p2 and p3, respectively, as compared to p0.

TABLE 42

ΔΔ CH₄intensity (g/kg ADG) for protein antigen formulations

Avg ΔΔ CH₄Intensity

Treatment Groups
Period
(g/kg ADG), per animal*
P value

9G + 9H + 9I
p1 - p0
−15.27 (↓)
0.003

(n = 10)
p2 - p0
−23.73 (↓)
0.0002

p3 - p0
−25.67 (↓)
0.0002

9G + 9I
p1 - p0
−19.36 (↓)
0.001

(n = 7)
p2 - p0
−23.44 (↓)
0.00001

p3 - p0
−26.26 (↓)
0.0004

*_#Δ CH₄intensity relative to untreated animals (Groups A + B) over the same periods of time.

8.1. Sample collection+analysis

Total blood samples were collected periodically throughout the duration of the immune monitoring period for subsequent processing. For a detailed sample collection, see Table 43 below.

TABLE 43

Sample collection schedule

Date

Vaccination
Total blood

Jun. 11, 2024
d 0
X
X

Jul. 2, 2024
d 21
X

Jul. 9, 2024
d 28

X

Jul. 23, 2024
d 42

X

Aug. 13, 2024
d 63

X

No adverse events were observed.

8.1.1. Sera
8.1.1.1. Sample Collection

Generally, total blood samples were collected under the supervision of veterinary-trained staff. Samples were collected via jugular vein needle puncture into blood tubes. Collected blood samples were then clarified, via ultracentrifugation, to collect serum within twenty-four hours of collection. Clarified serum samples were aliquoted, labeled and stored at or under −20° C.

8.1.1.2. Sera Sample Receipt+Processing

Frozen sera samples returned from the field were thawed at 4° C. overnight or rapidly in an ambient temperature water bath. Thawed samples were mixed by inversion at least 3 times, aliquoted to 4×1 mL in 1.5 mL Eppendorf tubes. Additionally, a 5 mL pooled sera sample was made for each treatment group, which was also aliquoted. A working aliquot of each sample was held at 4° C., and the remaining volumes refrozen and held at −80° C.

8.1.1.3. Determination of IgG Response Using WC or Protein ELISA

Protein ELISA plates were prepared from recombinant methanogen protein preparations. Briefly, ˜0.2 micrograms of protein in carbonate buffer was applied to wells of a high binding plate and allowed to coat overnight at 4° C. Coated plates were washed with PBS-T and then blocked with SuperBlock T20 (ThermoFisher) for 3 hrs at 4° C. After blocking, plates were emptied, dried at room temperature overnight, vacuum sealed with a desiccant pack, and held at 4° C. until use.

Sera samples diluted to 1:25,000 were applied to ELISA plates and binding IgG's were detected after washing using a commercially available anti-bovine IgG H & L chain secondary antibody. The WC ELISA data shown below represents the average of technical triplicates (i.e., the same sera dilution run in multiple ELISA plates).

APPENDIX A

Treatment assignments

Visual ID
RFID
Treatment ID

112L
000000000840003242071747
9A

1165L
000000000840003234349454
9A

674L
000000000840003242071873
9A

459L
000000000840003234349414
9A

687L
000000000840003242071828
9A

598L
000000000840003234349578
9A

587L
000000000840003242071497
9A

1025L
000000000840003234349264
9A

135L
000000000840003242071667
9A

078L
000000000840003242071732
9A

1186L
000000000840003234349477
9A