Panax and are active ingredients in ginseng. In some embodiments, the ginsenoside is preferably a monomer such as ginsenoside Rgl, ginsenoside Rg3, ginsenoside Rbl, ginsenoside Re, or a mixture of two or more of these saponin monomers. The platycodin is preferably platycodin D, platycodin D2 or a mixture thereof. The astragaloside is preferably a monomer such as astragalin A (astragaloside IV), astragaloside I, astragaloside II, and the like, or a mixture of two or more of these saponin monomers. The notoginsenoside is preferably notoginsenoside R1, or the like. The ophiopogonin is preferably ophiopogonin D, or the like. The saikosaponin is preferably saikosaponin a, saikosaponin d, or a mixture thereof. The cortex albiziae saponin is preferably cortex albiziae total saponins or the like. The glycyrrhizin is preferably total glycyrrhizins or the like. The Panax japonicus saponin is preferably Panax japonicus total saponins or the like.

As used herein, “Iscom adjuvant” is an immunostimulatory complex adjuvant, specifically ISCOM MATRIX that does not comprise an antigen, which is an adjuvant composed of a phospholipid, a saponin, and cholesterol with a cage-like structure.

As used herein, “a therapeutically effective amount” or “an effective amount” refers to a dosage sufficient to show its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, would depend on the own conditions and severity of the subject being treated. A prescription of treatment (e.g. determination of dosage, etc.) is ultimately the responsibility of, and determined by, general practitioners and other physicians, often taking into account the disease to be treated, the conditions of the individual patient, the site of delivery, the method of administration, and other factors known to physicians.

As used herein, the term “mammal” refers to a human, and may also be other animals, such as wild animals (e.g. herons, storks, cranes, etc.), domestic animals (e.g. ducks, geese, etc.) or laboratory animals (e.g. chimpanzees, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

In other embodiments, the composition of the present invention may further comprise an additional additive, such as a pharmaceutically acceptable carrier or additive, particularly when presented as a pharmaceutical formulation form.

The preferred pharmaceutical carrier is especially water, buffered aqueous solutions, preferably isotonic saline solutions such as PBS (phosphate buffer), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides, etc. The types of pharmaceutical carrier used depend inter alia on whether the composition according to the present invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the present invention may comprise a wetting agent, an emulsifying agent or buffer substance as an additive.

The pharmaceutical composition, vaccine or pharmaceutical formulation according to the present invention may be administered by any suitable route, for example, oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration.

The present invention is further illustrated by the following description of specific embodiments in conjunction with the accompanying drawings, which are not to be construed as limitation of the present invention, and various modifications or improvements can be made by those skilled in the art in light of the basic concepts of the present invention, which are all within the scope of the present invention, as long as they do not deviate from the basic concepts of the present invention.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention is illustrated below with reference to the specific examples. Those skilled in the art will appreciate that these examples are merely illustrative of the present invention and not intended to limit the scope of the present invention in any way.

The experimental methods in the following examples are conventional, unless otherwise specified. The raw materials, reagent materials and the like used in the following examples are commercially available products, unless otherwise specified.

Example 1 Preparation of Screening Experiment on Immunostimulatory Compositions

1. HBsAg stock solution: the amino acid sequence of the HBsAg protein is shown by SEQ ID NO: 1.

The HBsAg protein was prepared from recombinant yeast cells of the HBsAg gene, and the types of yeast cells include Hansenula, Saccharomyces cerevisiae and Pichia, preferably Hansenula. For the specific preparation steps, reference was made to Chinese patent application CN108330145A. The recombinant Hansenula cells of the HBsAg gene were cultured by fermentation and the mycelia were harvested. The mycelia were subjected to disruption treatment and purified by the steps of silica gel adsorption, column chromatography and TFF, etc.

2. HBcAg stock solution: the amino acid sequence of the HBcAg protein is shown by SEQ ID NO: 2.

The HBcAg protein was prepared from recombinant yeast cells of the HBcAg gene, and the types of yeast cells include Hansenula, Saccharomyces cerevisiae and Pichia, preferably Hansenula. For the specific preparation steps, reference was made to Chinese patent application CN108047316A. The recombinant Hansenula cells of the HBcAg gene were cultured by fermentation and the mycelia were harvested. The mycelia were subjected to disruption treatment and purified by the steps of ammonium sulfate treatment, column chromatography and TFF, etc. to prepare the HBcAg stock solution.

3. QS-21 was purchased from BRENNTAG, CAS. NO. A010-023.

4. Preparation method of CPG oligodeoxynucleotide raw materials:

Oligodeoxynucleotides are synthetically prepared fragments of oligodeoxynucleotide sequence containing one or more CpG motifs. The CPG sequences used in this example are shown in Table 1:

TABLE 1

Specific sequences of CPG oligodeoxynucleotide

Typing of CPG
CPG

Oligodeoxynucleotide
Oligodeoxynucleotide
Sequence

Type B
CpG 1018
TGACTGTGAACGTTCGAGATGA (SEQ ID NO: 3)

CpG 7909
TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 4)

CpG 1826
TCCATGACGTTCCTGACGTT (SEQ ID NO: 5)

CpG T1
TCGTTCGTTCGTTCGTTCGTT (SEQ ID NO: 6)

CpG T2
TCG TTC GTT CGT TCG TTC GTT CGT T (SEQ ID

NO: 7)

CpG T3
TCG TCG TCG TCG TCG TCG TCG (SEQ ID NO: 8)

CpG 684
TCGACGTTCGTCGTTCGTCGTTC (SEQ ID NO: 9)

CpG 1668
TCC ATG ACG TTC CTG ATGCT (SEQ ID NO: 10)

CpG D2
TGTCGTCGTCGTTTGTCGTTTGTCGTT (SEQ ID

NO: 11)

Type A
CpG 2216
GGGGGACGATCGTCGGGGGG (SEQ ID NO: 12)

ODN 2336
GGGGACGACGTCGTGGGGGGG (SEQ ID NO: 13)

Type C
ODN 2395
TCGTCGTTTCGCGCGCGCCG (SEQ ID NO: 14)

ODN M362
TCGTCGTTCGTTCGTCGAACGACGTTTGAT

(SEQ ID NO: 15)

The specific preparation method: a conventional solid phase phosphoramidite-phosphotriester chemical synthesis method was used for the preparation, starting from the 3′ end, i.e., 1) Deprotection: first removing the protecting group DMT (dimethoxytrityl) of the nucleotide connected to CpG with trichloroacetic acid to obtain free 5′ hydroxyl for the next step of condensation reaction; 2) Activation: mixing a phosphoramidite-protected nucleotide monomer and a tetrazole activator into a synthesis column to form a phosphoramidite tetrazole active intermediate, which undergoes a condensation reaction with a deprotected nucleotide on CpG; 3) Connection: when the phosphoramidite tetrazole reactive intermediate encounters the deprotected nucleotide on CpG, it will undergo an affinity reaction with its 5′ hydroxyl, condense and remove the tetrazole, upon which the oligonucleotide chain is extended forward by one base; 4) Oxidation: during the condensation reaction, the nucleotide monomer is connected to the oligonucleotide connected to CpG via a phosphite bond, while the phosphite bond is unstable and prone to be hydrolyzed by an acid or a base, upon which the phosphoramidite is oxidized into a phosphotriester with a sulphur-phosphorus double bond using a thio-substitution reagent, thereby obtaining a stable oligonucleotide; and 5) Blocking: in order to prevent the unreacted 5′ hydroxyl connected to CpG from being extended in the subsequent circular reaction after the condensation reaction, this terminal hydroxyl is often blocked by acetylation. After the above five steps, one deoxynucleotide is connected to the nucleotide of CpG. The above deprotection, activation, connection, oxidation and blocking processes are repeated to obtain a crude DNA fragment. Finally, it is subjected to post-synthesis treatments, such as cleavage, deprotection, purification and quantification, etc.

5. The HbsAg stock solution and the HbcAg stock solution were diluted to 200 μg/ml and 100 μg/ml respectively, using a PBS solution (purchased from Hyclone). All the CPG raw materials were separately dissolved and diluted to 100 μg/ml using the PBS solution, for the next step.

Example 2 Screening Experiment of CPG Oligodeoxynucleotides

1. Experimental animals: C57BL/6(N) mice, male, 4 weeks old, 135 mice, Shanghai Lingchang Laboratory Animal Technology Co. Ltd.

2. Experimental grouping: see Table 2. The dosage for each injection was 100 μL/mouse, and Group A was the negative control (the PBS solution, 100 μL/mouse).

TABLE 2

Grouping of experimental animals

Component (μg/mouse)

Number
HBs
HBc
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CpG
CPG
CpG
ODN
ODN
ODN

Group
(animals)
Ag
Ag
T1
T2
T3
1018
7909
1826
684
1668
D2
2216
2336
2395
M362

Control
9

Antigen
9
20
10

T1
9
20
10
10

T2
9
20
10

10

T3
9
20
10

10

1018
9
20
10

10

7909
9
20
10

10

1826
9
20
10

10

684
9
20
10

10

1668
9
20
10

10

D2
9
20
10

10

2216
9
20
10

10

2336
9
20
10

10

2395
9
20
10

10

M362
9
20
10

10

3. Experimental steps: on Day 7 after the immunization of mice, the spleen lymphocytes were prepared according to a conventional method, and the details were as follows: the spleens were taken aseptically by being cut with sterile forceps and scissors, and placed in a 70 μm cell strainer, which was placed in a plate containing 2 ml of pre-chilled 2% FBS (purchased from GIBCO)-PBS; the spleens were ground using a grinding rod, and the spleen cells entered the plate through the meshes to obtain a cell suspension, and then the suspension was filtered by a 40 μm cell strainer (purchased from BD) and put into a 50 ml sterile centrifuge tube by using a Pasteur pipet; it was centrifuged at 500×g at 4° C. for min; the supernatant was discarded, and then 2 ml of 1× erythrocyte disruption agent (purchased from BD) was added to re-suspend the cells, and the resultant was allowed to stand for 5 min at 4° C., protected from light to disrupt the red blood cells; 10 ml of 2% FBS-PBS was added to terminate the erythrocyte disruption reaction; the resultant was centrifuged at 500×g at 4° C. for 5 min, the supernatant was discarded, and then 5 ml of 2% FBS-PBS was added to re-suspend the cells for later use. The spleen cells were stimulated with the stimulators, HBsAg-specific peptide library PS4 and HBcAg-specific peptide library PCP, respectively. An ELISPOT kit (BD) was used to detect the secretion levels of HBsAg and HBcAg antigen-specific IFN-γ according to the kit instructions. The spot number measured by the ELISPOT kit were read using ImmunoSPOT Series 3 Elispot analyzer (refer to Example 7 of Chinese patent CN104043120B for the specific operation steps).

The sequences of HBsAg-specific peptide library refer to Example 7 of Chinese patent CN104043120B, and the sequences of HBcAg-specific peptide library are shown by SEQ ID NO: 16-30.

4. Experimental results: the results of ELISPOT spot are shown in FIG. 1 and FIG. 2. The results show that the CpG adjuvants of type B with different sequences had different immune effects. Among them, CpG T1-T3, CpG 1018, CpG 7909, CpG 1826 and CpG 684 as a whole were superior to the CpG adjuvants of type A and the CpG adjuvants of type C, while CpG 1618 and CPG D2 had poorer immune effects, and the induced production levels of HBsAg- and HBcAg-specific IFN-γ were all lower than those induced by the CpG adjuvants of type A and the CpG adjuvants of type C.

Example 3 Screening Experiment of Immunostimulatory Compositions

1. Experimental animals: C57BL/6(N) mice, male, 4 weeks old, 81 mice, Shanghai Lingchang Laboratory Animal Technology Co. Ltd.

2. Experimental grouping: see Table 3. The dosage for each injection was 100 μL/mouse, and Group A was the negative control (the PBS solution, 100 μL/mouse).

TABLE 3

Grouping of experimental animals

Component (μg/mouse)

Number

CpG
CpG
CpG
CpG
CpG
CpG
CpG

Group
(animals)
HBsAg
HBcAg
T1
T2
T3
1018
7909
1826
684
QS21

Control
9

Antigen
9
20
10

T1
9
20
10
10

5

T2
9
20
10

10

5

T3
9
20
10

10

5

1018
9
20
10

10

5

7909
9
20
10

10

5

1826
9
20
10

10

5

684
9
20
10

10
5

3. Experimental steps: following Example 2.

4. Experimental results: the results of ELISPOT spot are shown in FIG. 3 and FIG. 4. The results show that use of CpG T1-T3 in combination with QS21 resulted in a high-efficiency synergistic effect, and the induced production levels of HBsAg- and HBcAg-specific IFN-γ were significantly higher than those induced by other CpG adjuvants, such as CpG 1018, CpG 7909, etc., with unexpected immune effect.

Example 4 Effects of Different Amounts of Adjuvant on Immune Effect of Pharmaceutical Composition

1. Experimental animals: C57BL/6(N) mice, male, 4 weeks old, 60 mice, Shanghai Lingchang Laboratory Animal Technology Co. Ltd.

2. Reagents and materials:

- 1) The HBsAg protein, HBcAg protein and CpG T1 were obtained from Example 1.
- 2) QS-21 (CAS. NO. A010-023, purchased from BRENNTAG).
- 3) The HBsAg stock solution and HBcAg stock solution were diluted to 200 μg/ml and 100 μg/ml respectively, using a PBS solution (purchased from Hyclone); QS21 was diluted to 5 μg/ml, 50 μg/ml and 100 μg/ml respectively; CpG T1 was dissolved and diluted to 50 μg/ml, 100 μg/ml and 2 mg/ml respectively, using the PBS solution; and CPG 7909 was dissolved and diluted to 100 μg/ml using the PBS solution, for the next step.

3. Experimental grouping: see Table 4. The dosage for each injection was 100 μL/mouse, and Group A was the negative control (the PBS solution, 100 μL/mouse).

4. Experimental steps: following Example 2.

5. Experimental results: the results of ELISPOT spot are shown in FIG. 5 and FIG. 6. The results show that the dosage changes of CpG T1 and QS21 had significant effects on the vaccine composition, and the immunostimulatory compositions having a dosage higher than Dosage 5 induced the production levels of HBsAg- and HBcAg-specific IFN-γ which were significantly higher than that of the CPG 7909 group. However, due to the species difference, a further increase of adjuvant dosage did not induce a significant increase of the effect, presumably because the mice could not accurately reflect the immune intensity of adjuvant.

Dosages 1, 2 and 4 were equivalent to CPG 7909 group in terms of immunostimulatory effect, but the adjuvant dosages used were lower than that of the equivalent CPG 7909 group, thus they also had a certain advantage.

TABLE 4

Grouping of experimental animals

Component (μg/mouse)

Number

CpG

Group
(animals)
HBsAg
HBcAg
T1
QS21
CPG7909

Control
5

Antigen
5
20
10

Dosage 1
5
20
10
5
0.5

Dosage 2
5
20
10
5
5

Dosage 3
5
20
10
5
10

Dosage 4
5
20
10
10
0.5

Dosage 5
5
20
10
10
5

Dosage 6
5
20
10
10
10

Dosage 7
5
20
10
200
0.5

Dosage 8
5
20
10
200
5

Dosage 9
5
20
10
200
10

CPG7909
5
20
10

5
10

Example 5 Experimental Group Setting and Immunization Process for Hepatitis B Vaccines

1. Experimental animals and model establishment:

C57BL/6(N) mice, male, 4 weeks old, 81 mice, Shanghai Lingchang Laboratory Animal Technology Co. Ltd.; rAAV 8-HBV adenovirus, purchased from Beijing FivePlus Molecular Medicine Institute Co. Ltd. A C57BL/6(N) mouse model infected persistently with rAAV 8-HBV was established by intravenously injecting rAAV 8-HBV adenovirus into the upper tail vein of C57BL/6 (N) mice.

2. Reagents and materials:

1) HBsAg protein: obtained from Example 1.

2) HBcAg protein: obtained from Example 1.

3) The HBsAg stock solution, HBcAg stock solution and QS-21 were diluted to 200 μg/ml, 100 μg/ml and 50 μg/ml respectively, using a PBS solution (purchased from Hyclone), and CpG was dissolved and diluted to 100 μg/ml using the PBS solution, for the next step.

3. Experimental grouping: see Table 5. The dosage for each injection was 100 μL/mouse, and Group A was the negative control and injected with the PBS solution at 100 μL/mouse).

TABLE 5

Grouping of experimental animals and injection amounts for each group

Number
Component (μg/mouse)

Group
(animals)
HBsAg
HBcAg
CpG T1
CpG 7909
QS-21

A
9

B
9

10

C
9

5

D
9

10

5

E
9
20
10

F
9
20
10
10

G
9
20
10

5

H
9
20
10
10

5

I
9
20
10

10
5

4. Animal immunization: all the groups were administrated by intramuscular injection once every 2 weeks and the inoculation site was at the right rear thigh, with a total of 6 administrations at 4th, 6th, 8th, 10th, 12th and 14th week respectively, after the tail vein intravenous injection of rAAV 8-HBV virus. The blood was collected once every 2 weeks after the start of administration, i.e., at 4th, 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th and 22th week, respectively. All the mice were sacrificed at 22th week.

Example 6 Effects of Hepatitis B Vaccines on HBsAg Level in Serum

1. Detection steps for serum HBsAg: Nanjing Drum Tower Hospital was entrusted for detection.

Using a two-step immunoassay, the binding between the sample to be detected and the paramagnetic particles coated with the hepatitis B surface antibody was firstly detected; after washing, an acridinium ester-labeled hepatitis B surface antibody conjugate was added; after washing again, a pre-excitation solution and an excitation solution were added to the reaction mixture, and the relative luminescence units (RLU) of the sample to be detected was determined; there was a positive correlation between the content of HBsAg in the sample and RLU, and the concentration of HBsAg in the mouse serum sample was determined via a generated ARCHTITECT HBsAg standard curve; finally, the concentration of HBsAg in the mouse serum sample was 50 to 200 times of the determined value.

2. Analysis of results (FIG. 7): Group H vaccine containing the immunostimulator according to the present invention showed a significant downtrend in the corresponding HBsAg level, and maintained a stable long-lasting immune effect after the end of the immunization process (from Week 14), with a significant advantage compared to the CpG group alone (Group F) and the QS-21 group alone (Group G). The HBsAg level in Group H decreased from >6350 IU/ml at the onset to about 50 IU/ml. In this group, the HBsAg level decreased by more than 30% after the second immunization (Week 6) and decreased by more than 70% after the third immunization (Week 8), and the average decrease rate was maintained at about 92% after the end of immunization at Week 14. A superior immune effect was found. Compared with the dual adjuvant control (Group I), Group H still maintained a stable immune effect after the end of immunization at Week 14, and the immune level was significantly better than that of Group I.

Example 7 Evaluation of Humoral Immune Effects of Hepatitis B Vaccines

1. Detection steps for serum HBsAb: Nanjing Drum Tower Hospital was entrusted for detection.

Using a two-step immunoassay, the sample to be detected was first mixed with the paramagnetic particles coated with the recombinant HBsAg (rHBsAg); after washing, an acridinium ester-labeled rHBsAg conjugate was added; after washing again, a pre-excitation solution and an excitation solution were added to the reaction mixture, and the relative luminescence unit (RLU) of the sample to be detected was determined; there was a positive correlation between the content of HBsAb in the sample and RLU, and the concentration of HBsAg in the mouse serum sample was determined via a generated ARCHTITECT HBsAb standard curve; finally, the concentration of HBsAb in the mouse serum sample was 50 to 200 times of the determined value.

2. Analysis of results (FIG. 8): Group H vaccine containing the immunostimulator began to generate HBsAb (>10 mIU/ml) after the second immunization (Week 6), and the level of HBsAb showed a trend of continuous increase as the number of immunizations increased, and the trend of increase was significantly superior to those of the CpG group alone (Group F) and the QS-21 group alone (Group G). Two weeks after the end of immunization (Week 16), the HBsAb level was close to saturation, reaching an HBsAb level of 4.0 logs, i.e. about 10000 mIU/ml. The antibody level generated was also significantly superior to that of the dual adjuvant control (Group I).