Pharmaceutical composition for preventing hepatitis b virus infection and use thereof

Abstract

The present invention provides a pharmaceutical composition for preventing hepatitis B virus infection and use thereof. The pharmaceutical composition comprises i) hepatitis B virus surface antigen, a fragment thereof with antigenic activity, and / or a variant thereof with antigenic activity; and ii) an immunostimulatory composition, which comprises an aluminum adjuvant and a CpG oligodeoxynucleotide, wherein the sequence of the CpG oligodeoxynucleotide comprises two or more copied 5'-TTCGTT-3' motifs or 5'-TCGTCGTCG-3' motifs. Experimental results show that the components contained in the immunostimulatory composition can have a synergistic effect, thereby greatly enhancing the immune effect on hepatitis B virus surface antigen; a mere two doses of the composition are enough to produce a good immune effect, and the composition causes antibody production earlier and maintains high-titer antibody levels for a longer time than existing vaccines.

Description

A pharmaceutical composition for preventing hepatitis B virus infection and its application Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to a pharmaceutical composition for preventing hepatitis B virus infection and an application thereof. Background Art

[0002] Hepatitis B virus (HBV) infection is a serious public health problem worldwide. HBV infection is a major cause of chronic hepatitis B, cirrhosis, and hepatocellular carcinoma (Fattovich GJ Hepatol. 2008;48:335-352). Currently, widely used hepatitis B protein vaccines achieve preventive goals by inducing humoral immunity and producing protective neutralizing antibodies.

[0003] Currently, all preventive hepatitis B vaccines on the market use aluminum hydroxide (Al(OH)3) as an adjuvant, which can enhance Th2 immune responses and humoral immunity, inducing the body to produce and secrete protective antibodies. However, aluminum hydroxide mainly stimulates the production of Th2-related antibodies (including IgE), and cannot induce Th1 cellular immune responses, stimulate Th1 activity, enhance cell-mediated immunity (CTL), or block the activation of CD8+ CTL.

[0004] Studies have found that CpG oligodeoxynucleotide (CpG-ODN) adjuvants have an immunopotentiating effect. They can bind to TLR9 (Toll-like receptor 9) in cells to induce an immune response that relies on the Th1 pathway, activate B cells to differentiate into plasma cells that secrete antigen-specific antibodies, and stimulate IFN-γ expression, thereby promoting cellular immune responses and increasing the immune effect of people with no immunity, low immune response, and immunosuppression. Therefore, combined use with aluminum hydroxide adjuvants can achieve better immune effects, such as the combination of CpG and aluminum hydroxide mentioned in patent CN105214083A.

[0005] However, research has also revealed that CpG adjuvants exhibit structural diversity, resulting in significant differences in the effectiveness of different types of CpG adjuvants in practical applications and a high degree of uncertainty regarding their efficacy. Screening for adjuvant compositions with superior immune-enhancing properties to enhance the preventive efficacy of hepatitis B vaccines remains an urgent challenge in this field.

[0006] Summary of the Invention

[0007] In order to address the deficiencies of the prior art, the present invention aims to provide a pharmaceutical composition for preventing hepatitis B virus infection and its use, which has a short onset time for preventing hepatitis B virus infection and a better immune effect.

[0008] The technical solutions of the present invention for achieving the above-mentioned purpose are as follows:

[0009] A pharmaceutical composition for preventing hepatitis B virus infection, comprising:

[0010] i) hepatitis B virus surface antigen, antigenically active fragments thereof and / or antigenically active variants thereof; and

[0011] ii) An immunostimulatory composition comprising an aluminum adjuvant and a CpG oligodeoxynucleotide, wherein the sequence of the CpG oligodeoxynucleotide comprises two or more copies of a 5'-TTCGTT-3' motif or a 5'-TCGTCGTCG-3' motif.

[0012] In some embodiments of the present invention, the CpG oligodeoxynucleotide can be selected from one or more of the CpG oligodeoxynucleotides shown in SEQ ID NOs: 2 to 4.

[0013] Preferably, the CpG oligodeoxynucleotide is the CpG oligodeoxynucleotide shown in SEQ ID NO: 2.

[0014] In some embodiments of the present invention, the aluminum adjuvant can be selected from one or more of aluminum hydroxide, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum, preferably aluminum hydroxide.

[0015] In some embodiments of the invention, the CpG oligodeoxynucleotide may comprise a phosphorothioate bond.

[0016] Preferably, the CpG oligodeoxynucleotide is an all-phosphorylation CpG oligodeoxynucleotide.

[0017] In some embodiments of the present invention, the weight ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 10-200:37.5, preferably 60-200:37.5, and more preferably 200:37.5.

[0018] According to the present invention, the weight ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is selected from 10:37.5, 20:37.5, 30:37.5, 40:37.5, 50:37.5, 60:37.5, 70:37.5, 80:37.5, 90:37.5, 100:37.5, 110:37.5, 120:37.5, 130:37.5, 140:37.5, 150:37.5, 160:37.5, 170:37.5, 180:37.5, 190:37.5 or 200:37.5.

[0019] In some embodiments of the present invention, the amino acid sequence of the hepatitis B virus surface antigen is shown in SEQ ID NO: 1.

[0020] In some embodiments of the present invention, the weight ratio between components i) and ii) of the pharmaceutical composition is 1:47.5-237.5, preferably 1:97.5-237.5, and more preferably 1:237.5.

[0021] In some embodiments of the present invention, the pharmaceutical composition is a unit preparation, wherein the content of component i) is ≥5 μg / dose, the Al(OH)3 content in component ii) is ≥187.5 μg / dose, and the CpG content is ≥300 μg / dose.

[0022] Preferably, the CpG content is 300-1000 μg / dose.

[0023] Preferably, the CpG content is 1000 μg / dose.

[0024] According to the present invention, the CpG content can be 300 μg / dose, 350 μg / dose, 400 μg / dose, 450 μg / dose, 500 μg / dose, 550 μg / dose, 600 μg / dose, 650 μg / dose, 700 μg / dose, 750 μg / dose, 800 μg / dose, 850 μg / dose, 900 μg / dose, 950 μg / dose or 1000 μg / dose.

[0025] In some embodiments of the present invention, the pharmaceutical composition may further comprise: iii) a pharmaceutically acceptable carrier and / or excipient.

[0026] In some embodiments of the present invention, the pharmaceutically acceptable carrier and / or excipient may be selected from one or more of water, a buffered aqueous solution, an isotonic saline solution, a wetting agent, or an emulsifier. Preferably, the pharmaceutically acceptable carrier and / or excipient is selected from one or more of phosphate buffered saline (PBS), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% v / v glycerol aqueous solution, hyaluronic acid, ethanol, polyalkylene glycol (e.g., polypropylene glycol), or triglycerides.

[0027] The type of carrier used for the pharmaceutical composition depends inter alia on whether the pharmaceutical composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The pharmaceutical composition, vaccine or pharmaceutical preparation according to the invention can be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

[0028] In some embodiments of the present invention, the pharmaceutical composition may be in the form of a lyophilized powder injection or an injection solution.

[0029] Another object of the present invention is to provide a preventive vaccine for hepatitis B, which comprises the above-mentioned pharmaceutical composition.

[0030] Another object of the present invention is to provide use of the pharmaceutical composition in the preparation of a medicament, such as a vaccine, for preventing hepatitis B virus infection and / or hepatitis B virus-mediated diseases.

[0031] In some embodiments of the present invention, the hepatitis B virus infection and / or hepatitis B virus-mediated disease is selected from one or more of hepatitis B, cirrhosis or liver cancer.

[0032] The experimental results of the present invention show that the provided pharmaceutical composition can induce a significant humoral immune response, and can produce an immune response effect earlier and faster than a single adjuvant system: a higher level of HBsAb seroconversion rate can be induced earlier after a single immunization (wk02), and a higher level of HBsAb antibodies can be produced after a second immunization.

[0033] The CpG adjuvant and the aluminum adjuvant in the pharmaceutical composition of the present invention have a good synergistic effect, and the combination of the two adjuvants can improve the protective effect of the hepatitis B preventive vaccine.

[0034] In addition, in experiments on crab-eating macaques, compared with the three-dose immunization regimen of the commercially available hepatitis B vaccine, the two-dose immunization regimen of the present invention can achieve the same effect, and the immune onset time is shorter, indicating that in actual application, the pharmaceutical composition of the present invention can play a preventive and protective role on the human body in a shorter period of time.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0037] Figure 1 shows the HBsAb antibody levels in mouse serum;

[0038] FIG2 shows the HBsAb antibody levels in cynomolgus monkey serum.

[0039] Modes for Carrying Out the Invention

[0040] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0041] Example 1 Preparation of pharmaceutical composition

[0042] 1. HBsAg protein expression

[0043] The amino acid sequence of the HBsAg protein is shown in SEQ ID NO: 1. It is prepared from yeast cells recombinant to the HBsAg gene. The yeast cell species can be selected from Hansenula, Saccharomyces cerevisiae, and Pichia pastoris, preferably Hansenula. For detailed preparation steps, refer to the disclosure of Chinese patent application CN108330145A. The recombinant Hansenula cells are fermented and harvested. The cells are then purified by disrupting the bacteria and silica gel adsorption, column chromatography, and TFF. The purified HBsAg protein is used to prepare a pharmaceutical composition.

[0044] 2. Synthesis of CpG oligodeoxynucleotides

[0045] CpG oligodeoxynucleotides are synthetically prepared oligodeoxynucleotide sequence fragments having two or more copies of a 5'-TTCGTT-3' motif or a 5'-TCGTCGTCG-3' motif in the sequence. The CpG oligodeoxynucleotides of the present invention are shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. The preparation steps are described with reference to Chinese Patent CN104043120B. The following examples use the CpG oligodeoxynucleotide shown in SEQ ID NO: 2 as an example to illustrate the effects of the immunostimulatory composition of the present invention.

[0046] Example 2 Hepatitis B virus surface antibody (HBsAb) positive conversion rate detection method

[0047] 1. Experimental steps:

[0048] Forty female BALB / c mice were used and divided into 5 groups (AE, 8 mice / group); group A was given HBsAg 1 μg / mouse; group B was given HBsAg 1 μg / mouse and Al(OH)3 37.5 μg / mouse; group C was given HBsAg 1 μg / mouse and CpG 10 μg / mouse; group D was given HBsAg 1 μg / mouse, Al(OH)3 37.5 μg, and CpG 10 μg / mouse; group E was given a control vaccine (Dalian Hanxin), HBsAg 1 μg / mouse and Al(OH)3 37.5 μg / mouse. Immunization was performed by intramuscular injection twice, at wk00 and wk04, respectively. Blood samples were collected at wk02, wk04, wk06, wk08, wk12, wk16, and wk20 after administration. The HBsAb positive conversion rate was tested on the sera of wk02 and wk04, and the HBsAb antibody level was tested on the sera of wk04, wk06, wk08, wk12, wk16, and wk20.

[0049] The hepatitis B virus surface antibody detection kit (enzyme-linked immunosorbent assay, Shanghai Kehua Biotechnology) was used to qualitatively detect the HBsAb positive conversion rate of hepatitis B virus surface antibodies in mouse serum samples.

[0050] 2. Experimental results:

[0051] The HBsAb positive conversion rate in the mouse serum of each group at different detection times is shown in Table 1. The results show that two weeks after the first immunization (wk02), the HBsAb positive conversion rate of group D containing dual adjuvant components was the highest, with an antibody positive conversion rate of 50%, while the antibody positive conversion rates of other groups were relatively low. The antibody positive conversion rate of the control vaccine group E was only 12.5%; four weeks after the first immunization (wk04), the HBsAb positive conversion rate of the serum of each group increased, and the antibody positive conversion rates of groups B, D and E all reached 100%.

[0052] The experimental results show that the wk02HBsAb positive conversion rate (%) in group D is much higher than that in groups B and C, which means that the CpG adjuvant of the present invention and the aluminum adjuvant have a good synergistic effect, and the combination of the two adjuvants can improve the protective effect of the hepatitis B vaccine earlier.

[0053] Table 1 HBsAb positive conversion rate in mouse serum

[0054] Example 3 Detection of HBsAb Antibody Levels in Mouse Serum

[0055] 1. Experimental steps:

[0056] Animal immunization and serum collection are as described in Example 2. Mouse serum samples were diluted appropriately with sterile PBS (dilution factor n), and the HBsAb levels in the mouse serum samples were quantitatively determined using the ARCHITECT Hepatitis B Virus Surface Antibody Assay Kit and chemiluminescent microparticle immunoassay (CMIA).

[0057] 2. Experimental results:

[0058] As shown in Figure 1, with the extension of time, the HBsAb antibody level in the serum of each group gradually increased, and at wk16, the HBsAb antibody reached a relatively high level and tended to stabilize; the trend of the induced HBsAb antibody level among the AD groups was group D>group B>group C>group A, and at wk16, the HBsAb antibody levels of the AE groups were 3.27, 4.48, 3.52, 4.78 and 4.57 Lg, respectively, showing statistical differences: as shown in Figure 1, *** refers to the data of group D relative to group A at week 20, p < 0.001, ** refers to the data of group D relative to group C at week 20, p < 0.01, the antibody level in group D was the highest, and the HBsAb antibody level in the control vaccine group E was always lower than that in group D, indicating that the immune response level and sustained protection effect of the pharmaceutical composition provided by the present invention are superior to those of commercially available hepatitis B vaccines.

[0059] Example 4 Detection of HBsAb Antibody Levels in Cynomolgus Monkey Serum

[0060] 1. Experimental steps:

[0061] Twelve male and 12 female cynomolgus monkeys (Guangdong Chunsheng Biotechnology Development Co., Ltd.) aged 2-5 years were divided into 4 groups (G1-G4) (6 monkeys / group, half male and half female).

[0062] Table 2 Experimental animal groups

[0063] Groups G1-G3 were immunized intramuscularly on week 00 and week 04, for a total of two immunizations; group G4 was immunized intramuscularly on week 00, week 04, and week 10, for a total of three immunizations. The drug was administered intramuscularly via a single site in the quadriceps or gluteal muscles of the hind limbs. Blood samples were collected every two weeks after administration and continued until week 24. Serum levels of HBsAb were measured from week 02 to week 24 using the same method as in Example 2.

[0064] 2. Experimental results:

[0065] As shown in Figure 2, groups G1-G3 were immunized with pharmaceutical compositions containing different doses of CpG at wk00 and wk04, respectively. The HBsAb antibody levels induced by them reached a high level at wk06, with average antibody levels of 22763, 10275 and 2055 mIU / ml, respectively. Thereafter, they showed a certain degree of variation and tended to stabilize at wk18-wk24, with average antibody levels of 14047, 10871 and 2641 mIU / ml, respectively. During the entire experimental period, the trend of HBsAb antibody induced by groups G1-G3 was: G1>G2>G3, that is, there was a positive correlation between antibody level and CpG dose.

[0066] The G4 group was immunized with the control vaccine at wk00, wk04 and wk10, and the induced HBsAb antibody level reached a peak at wk12, with an average antibody level of 27580mIU / ml. Thereafter, it showed a certain downward trend and stabilized at wk18-wk24, with an average antibody level of 11138mIU / ml at wk24.

[0067] During the entire experimental period, the HBsAb antibody levels induced by the G1-G3 groups immunized with the double-adjuvant vaccine and the G4 group immunized with the control vaccine were compared. During wk06-wk10 after the second immunization, the immune groups showed a trend of G1>G2>G4>G3; at wk10, after the third immunization of the G4 group, its antibody level increased significantly, and during wk12-wk14, the immune groups showed a trend of G4>G1>G2>G3; thereafter, the antibody level of the G4 group showed a certain downward trend, and during wk16-wk24, the immune groups showed a trend of G1>G4>G2>G3. Among them, at wk24, the antibody levels between the G2 group and the G4 group were comparable, with no significant difference.

[0068] Compared with commercially available hepatitis B vaccines, during the second immunization period, the antibody level induced by the double-adjuvant vaccine containing a higher CpG dose (G1 group) was higher than that of the control vaccine (G4 group); after the third booster immunization of the control vaccine, the antibody level induced by it increased significantly, but after the antibody level reached a relative stability, its antibody level was still lower than that of the double-adjuvant vaccine in the G1 group.

[0069] In summary, the pharmaceutical composition provided by the present invention has a significant immune effect. The experimental results conducted on mice are better than the existing technology, and the experimental results conducted on crab-eating macaques are better than the existing three-dose commercially available vaccine. The hepatitis B vaccine prepared based on this has significant advantages.

[0070] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A pharmaceutical composition for preventing hepatitis B virus infection, comprising: i) hepatitis B virus surface antigen, a fragment thereof having antigenic activity and / or a variant thereof having antigenic activity; and ii) An immunostimulatory composition comprising an aluminum adjuvant and a CpG oligodeoxynucleotide, wherein the sequence of the CpG oligodeoxynucleotide comprises two or more copies of a 5'-TTCGTT-3' motif or a 5'-TCGTCGTCG-3' motif.

2. The pharmaceutical composition according to claim 1, wherein the CpG oligodeoxynucleotide is selected from one or more of the CpG oligodeoxynucleotides shown in SEQ ID NOs: 2 to 4; Preferably, the CpG oligodeoxynucleotide is the CpG oligodeoxynucleotide shown in SEQ ID NO:

2.

3. The pharmaceutical composition according to claim 1 or 2, wherein the aluminum adjuvant is selected from one or more of aluminum hydroxide, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum, preferably aluminum hydroxide.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the CpG oligodeoxynucleotide comprises a phosphorothioate internucleoside bond; Preferably, the CpG oligodeoxynucleotide is an all-thiophosphate oligodeoxynucleotide. 5 . The pharmaceutical composition according to claim 1 , wherein the weight ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 10-200:37.5, preferably 60-200:37.5, and more preferably 200:37.

5.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the amino acid sequence of the hepatitis B virus surface antigen is as shown in SEQ ID NO:

1.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the weight ratio between components i) and ii) of the pharmaceutical composition is 1:47.5-237.5, preferably 1:97.5-237.5, and further preferably 1:237.

5.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the pharmaceutical composition is a unit preparation, wherein the content of component i) is ≥5 μg / dose, the content of Al(OH)3 in component ii) is ≥187.5 μg / dose, and the content of CpG is ≥300 μg / dose; Preferably, the CpG content is 300 to 1000 μg / dose; Preferably, the CpG content is 1000 μg / dose.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition further comprises: iii) a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutically acceptable carrier and / or excipient is selected from one or more of water, buffer, isotonic saline solution, wetting agent or emulsifier; Further preferably, the pharmaceutically acceptable carrier and / or excipient is selected from one or more of phosphate buffer, glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% v / v aqueous glycerol solution, hyaluronic acid, ethanol, polyalkylene glycol (e.g., polypropylene glycol) or triglycerides.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the dosage form of the pharmaceutical composition is a lyophilized powder injection or an injection solution.

11. A hepatitis B preventive vaccine comprising the pharmaceutical composition according to any one of claims 1 to 10.

12. Use of the pharmaceutical composition according to any one of claims 1 to 10 in the preparation of a medicament, such as a vaccine, for preventing hepatitis B virus infection and / or hepatitis B virus-mediated diseases.

13. The use according to claim 12, wherein the hepatitis B virus infection and / or hepatitis B virus-mediated disease is selected from one or more of hepatitis B, liver cirrhosis or liver cancer.

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