Pharmaceutical compositions for preventing hepatitis B virus infection and their uses

Abstract

The present invention provides a pharmaceutical composition for preventing hepatitis B virus infection and its use, the pharmaceutical composition comprising: i) a hepatitis B virus surface antigen, an antigenically active fragment thereof, and / or an antigenically active variant thereof; and ii) an immunostimulatory composition comprising an aluminum adjuvant and a CpG oligodeoxynucleotide containing two or more copies of a 5'-TTCGTT-3' motif or a 5'-TCGTCGTCG-3' motif in its sequence. Experimental results have shown that a synergistic effect can be generated between the components of the immunostimulatory composition, thereby achieving the effect of enhancing the immune effect of the hepatitis B virus surface antigen. A good immune effect can be achieved with just two immunizations, and antibody production can be accelerated and high antibody titers can be maintained for a longer period of time than with existing vaccines.

Description

[Technical Field]

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to pharmaceutical compositions and uses thereof for preventing hepatitis B virus infection. [Background technology]

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

[0003] Previously, all commercially available prophylactic hepatitis B vaccines used aluminum hydroxide (Al(OH)3) as an adjuvant, which can enhance Th2 immune responses and humoral immunity, and induce the body to produce and secrete protective antibodies. However, aluminum hydroxide mainly stimulates the production of Th2-related antibodies (including IgE) and is unable to induce the development of ThL cell-mediated immune responses, stimulate ThL activity, or enhance cellular immunity (CTL), blocking the activation of CD8+ CTLs.

[0004] Research has shown that CpG oligodeoxynucleotide (CpG-ODN) adjuvants have immune-enhancing effects and can bind to intracellular Toll-like receptor 9 (TLR9), inducing the generation of Th1 pathway-dependent immune responses, activating B cells to differentiate into plasma cells that secrete antigen-specific antibodies, and stimulating the expression of IFN-γ, thereby promoting cellular immune responses and enhancing the immune effects of non-immune, low-immune, and immunosuppressed individuals. Therefore, when used in combination with aluminum hydroxide adjuvants, such as the combination solution of CpG and aluminum hydroxide described in Patent CN105214083A, better immune effects can be achieved.

[0005] However, research has also found that CpG adjuvants have structural diversity, and different types of CpG adjuvants have significantly different effects in actual use, resulting in high uncertainty regarding the effectiveness of existing products. How to screen for adjuvant compositions with better immune-enhancing effects and improve the preventive effect of hepatitis B vaccines has become an urgent issue in this field. Summary of the Invention [Problem to be solved by the invention]

[0006] In order to overcome the shortcomings of existing technologies, the object of the present invention is to provide a pharmaceutical composition for preventing hepatitis B virus infection, which has a short onset time for the prevention of hepatitis B virus infection and a better immune effect, and its use. [Means for solving the problem]

[0007] The technical solutions to achieve the above objectives of the present invention are as follows:

[0008] A pharmaceutical composition for preventing hepatitis B virus infection, comprising: i) a hepatitis B virus surface antigen, an antigenically active fragment thereof, and / or an antigenically active variant thereof; ii) an immunostimulatory composition comprising an aluminum adjuvant and a CpG oligodeoxynucleotide containing two or more copies of the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif in its sequence.

[0009] In some embodiments of the present invention, the CpG oligodeoxynucleotide may be one or more selected from the CpG oligodeoxynucleotides shown in SEQ ID NOs: 2-4.

[0010] Preferably, the CpG oligodeoxynucleotide is the CpG oligodeoxynucleotide shown in SEQ ID NO:2.

[0011] In some embodiments of the invention, the aluminum adjuvant may be one or more selected from aluminum hydroxide, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum, preferably aluminum hydroxide.

[0012] In some embodiments of the present invention, the CpG oligodeoxynucleotide may comprise phosphorothioate linkages.

[0013] Preferably, the CpG oligodeoxynucleotide is a perthioCpG oligodeoxynucleotide.

[0014] In some embodiments of the present invention, the weight ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 10 to 200:37.5, preferably 60 to 200:37.5, and more preferably 200:37.5.

[0015] According to the present invention, the weight ratio of the CpG oligodeoxynucleotide to 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.

[0016] In some embodiments of the invention, the amino acid sequence of the Hepatitis B virus surface antigen is as set forth in SEQ ID NO:1.

[0017] 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.

[0018] In some embodiments of the present invention, the pharmaceutical composition is a unit dosage form, wherein the content of component i) is ≥ 5 μg / formulation, the content of Al(OH)3 in component ii) is ≥ 187.5 μg / formulation, and the content of CpG is ≥ 300 μg / formulation.

[0019] Preferably, the CpG content is 300 to 1000 μg / preparation.

[0020] Preferably, the CpG content is 1000 μg / preparation.

[0021] According to the present invention, the CpG content may be 300 μg / preparation, 350 μg / preparation, 400 μg / preparation, 450 μg / preparation, 500 μg / preparation, 550 μg / preparation, 600 μg / preparation, 650 μg / preparation, 700 μg / preparation, 750 μg / preparation, 800 μg / preparation, 850 μg / preparation, 900 μg / preparation, 950 μg / preparation or 1000 μg / preparation.

[0022] In some embodiments of the present invention, the pharmaceutical composition may further comprise iii) a pharmaceutically acceptable carrier and / or additive.

[0023] In some embodiments of the present invention, the pharmaceutically acceptable carrier and / or additive may be one or more selected from water, a buffered aqueous solution, an isotonic salt solution, a wetting agent, or an emulsifier. Preferably, the pharmaceutically acceptable carrier and / or additive is one or more selected from phosphate buffer solution (PBS), glucose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, magnesium carbonate, 0.3% v / v aqueous glycerol solution, hyaluronic acid, ethanol, a polyalkylene glycol (e.g., polypropylene glycol), or a triglyceride.

[0024] The type of pharmaceutical carrier used will depend, 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 formulation according to the invention can be administered by any suitable route, for example orally, nasally, intradermal, subcutaneous, intramuscular or intravenous.

[0025] In some embodiments of the present invention, the dosage form of the pharmaceutical composition may be a lyophilized powder injection or an injection solution.

[0026] Another object of the present invention is to provide a vaccine for preventing hepatitis B, which comprises the above pharmaceutical composition.

[0027] Another object of the present invention is to provide a use of said pharmaceutical composition in the preparation of a medicament, such as a vaccine, for the prevention of hepatitis B virus infection and / or hepatitis B virus-induced diseases.

[0028] In some embodiments of the present invention, the hepatitis B virus infection and / or hepatitis B virus-induced disease is one or more selected from hepatitis B, liver cirrhosis, or liver cancer. [Effects of the Invention]

[0029] The experimental results of the present invention have shown that the provided pharmaceutical composition can induce the generation of a significant humoral immune response and can produce an immune response effect earlier and more rapidly than a single adjuvant system, i.e., it can induce a higher level of HBsAb positivity rate earlier after the first immunization (wk02) and produce a higher level of HBsAb antibody after the second immunization.

[0030] The CpG adjuvant in the pharmaceutical composition of the present invention has a good synergistic effect with the aluminum adjuvant, and the combined use of the two adjuvants can improve the protective effect of the hepatitis B vaccine.

[0031] Furthermore, in experiments using cynomolgus monkeys, the two-dose immunization method of the present invention not only achieved the same effect as the three-dose immunization method of commercially available hepatitis B vaccine, but also had a shorter time to onset of immune effect, indicating that the pharmaceutical composition of the present invention can exert preventive and protective effects on humans in a short period of time during actual use. [Brief explanation of the drawings]

[0032] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. [Figure 1] HBsAb antibody levels in mouse serum are shown. [Figure 2] HBsAb antibody levels in cynomolgus monkey serum are shown. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will now be described in more detail with reference to specific embodiments, and the examples given are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0034] Example 1 Preparation of Pharmaceutical Compositions 1. HBsAg protein expression The amino acid sequence of the HBsAg protein is as shown in SEQ ID NO: 1. It is prepared from HBsAg gene-recombinant yeast cells. The yeast cell type can be selected from Hansenula yeast, Saccharomyces cerevisiae, and Pichia pastoris, with Hansenula yeast being preferred. For specific preparation steps, see the disclosure of Chinese Patent Application CN108330145A. HBsAg gene-recombinant Hansen yeast cells were fermented and cultured, and the cells were harvested. Purification was performed by cell disruption, silica gel adsorption, column chromatography, TFF, and other steps. The purified HBsAg protein is used to prepare pharmaceutical compositions.

[0035] 2. Synthesis of CpG oligodeoxynucleotides The CpG oligodeoxynucleotide is a synthetically prepared oligodeoxynucleotide sequence fragment, the sequence of which contains two or more copies of the 5'-TTCGTT-3' motif or the 5'-TCGTCGTCG-3' motif. The CpG oligodeoxynucleotide of the present invention is as shown in any one of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. The preparation steps can be referred 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.

[0036] Example 2 Hepatitis B virus surface antibody (HBsAb) positive rate detection method 1. Experimental steps: Forty female BALB / c mice were used and divided into five groups (8 mice per group), A to E. Group A received HBsAg at 1 μg / mouse, group B received HBsAg at 1 μg / mouse and Al(OH)3 at 37.5 μg / mouse, group C received HBsAg at 1 μg / mouse and CpG at 10 μg / mouse, group D received HBsAg at 1 μg / mouse, Al(OH)3 at 37.5 μg / mouse and CpG at 10 μg / mouse, and group E received a control vaccine (Dalian Hanxin), i.e., HBsAg at 1 μg / mouse and Al(OH)3 at 37.5 μg / mouse. Immunization was performed by intramuscular injection, and the mice were immunized twice at wk (week) 00 and wk04, respectively. After administration, blood samples were taken at wk02, wk04, wk06, wk08, wk12, wk16, and wk20, respectively. The HBsAb positivity rate was detected for wk02 and wk04 sera, and the HBsAb antibody level was detected for wk04, wk06, wk08, wk12, wk16, and wk20 sera.

[0037] A hepatitis B virus surface antibody detection kit (enzyme immunoassay, Shanghai Kehua Bio) was used to qualitatively detect the HBsAb positivity rate of hepatitis B virus surface antibody in mouse serum samples.

[0038] 2. Experimental Results The HBsAb positivity rates in mouse serum at different detection times for each group are shown in Table 1. As can be seen, two weeks after the first immunization (wk02), group D, which contained two adjuvant components, had the highest HBsAb positivity rate, with an antibody positivity rate of 50%, while the antibody positivity rates of the other groups were relatively low, with the control vaccine group, group E, having an antibody positivity rate of only 12.5%. Four weeks after the first immunization (wk04), the HBsAb positivity rates in serum of each group increased, with the antibody positivity rates of groups B, D, and E all reaching 100%.

[0039] The HBsAb positivity rate% of wk02 in group D was much higher than that of groups B and C. This means that the CpG adjuvant of the present invention has a good synergistic effect with the aluminum adjuvant, and the combined use of the two adjuvants can improve the protective effect of the hepatitis B vaccine more quickly.

[0040] [Table 1]

[0041] Example 3 Detection of HBsAb antibody levels in mouse serum 1. Experimental Steps The immunization of animals and collection of serum can both be seen in Example 2. The mouse serum samples were appropriately diluted with sterile PBS (dilution factor n), and the HBsAb levels in the mouse serum samples were quantitatively detected by chemiluminescent microparticle immunoassay (CMIA) using the ARCHITECT Hepatitis B Virus Surface Antibody Assay Kit.

[0042] 2. Experimental Results As shown in Figure 1, the serum HBsAb levels in each group gradually increased over time, reaching a relatively high and stable level by week 16. The induced HBsAb levels among groups A to D tended to be group D > group B > group C > group A. At week 16, the HBsAb levels in groups A to E were 3.27, 4.48, 3.52, 4.78, and 4.57 lg, respectively, demonstrating statistical significance. As shown in Figure 1, *** indicates the data for group D at week 20 compared to group A, p<0.001; ** indicates the data for group D at week 20 compared to group C, p<0.01. Group D had the highest antibody level, and the HBsAb level in the control vaccine group, group E, was consistently lower than that of group D. This indicates that the immune response level and sustained protective effect of the pharmaceutical composition provided by the present invention are superior to commercially available hepatitis B vaccines.

[0043] Example 4 Detection of HBsAb antibody levels in cynomolgus monkey serum 1. Experimental Steps Two to five year old cynomolgus monkeys (Guangdong Chunsheng Biotechnology Development Co., Ltd.), 12 males and 12 females, were divided into four groups, G1 to G4 (6 animals per group, half male and half female).

[0044] [Table 2]

[0045] Groups G1 to G3 were immunized twice by intramuscular injection at wk00 and wk04, respectively, and group G4 was immunized three times by intramuscular injection at wk00, wk04, and wk10, respectively, and the immunization was administered intramuscularly to a single site in the quadriceps or gluteal muscles of the hind legs. After administration, blood samples were taken every two weeks until wk24, and the HBsAb antibody levels were detected in the wk02 to wk24 sera. The detection method was the same as in Example 2.

[0046] 2. Experimental Results As shown in Figure 2, groups G1 to G3 were immunized with pharmaceutical compositions containing different doses of CpG at wk00 and wk04, respectively. The levels of HBsAb antibodies induced and produced thereby all reached a high level at wk06, with the average antibody levels being 22,763, 10,275, and 2,055 mIU / ml, respectively. After this, the antibody levels showed a certain range of fluctuation and tended to stabilize at wk18 to wk24, with the average antibody levels at wk24 being 14,047, 10,871, and 2,641 mIU / ml, respectively. Over the entire experimental period, the trend of HBsAb antibodies induced and produced by groups G1 to G3 was G1>G2>G3, i.e., the antibody levels showed a positive correlation with the CpG dose.

[0047] Group G4 was immunized with the control vaccine on wk00, wk04, and wk10, respectively, and the HBsAb antibody levels induced by this immunization peaked on wk12, with an average antibody level of 27,580 mIU / ml. Thereafter, the antibody levels showed a steady downward trend and tended to stabilize from wk18 to wk24, with an average antibody level of 11,138 mIU / ml on wk24.

[0048] Comparing the HBsAb antibody levels induced by groups G1 to G3 immunized with the double-adjuvant vaccine with those induced by group G4 immunized with the control vaccine over the entire experimental period, between wk06 and wk10 after the second immunization, the immunization groups showed a trend of G1>G2>G4>G3. At wk10, the antibody level in group G4 increased significantly after the third immunization. Between wk12 and wk14, the immunization groups showed a trend of G4>G1>G2>G3, after which the antibody level in group G4 showed a steady decrease. Between wk16 and wk24, the immunization groups showed a trend of G1>G4>G2>G3. At wk24, the antibody level in group G2 was comparable to that of group G4, with no significant difference.

[0049] Compared with the commercially available hepatitis B vaccine, the antibody levels induced by the double adjuvant vaccine containing a high dose of CpG (G1 group) were higher than those induced by the control vaccine (G4 group) during the second immunization, and after the third booster immunization with the control vaccine, the antibody levels induced by it were significantly higher, but even after the antibody levels stabilized, they remained lower than those induced by the double adjuvant vaccine (G1 group).

[0050] To summarize the above, the pharmaceutical composition provided by the present invention has a significant immune effect, and the experimental effects in mice are superior to those of existing technologies, and the experimental effects in cynomolgus monkeys are superior to those of existing three-dose commercially available vaccines, and the hepatitis B vaccine prepared based on this has significant advantages.

[0051] The above examples only represent some embodiments of the present invention, and although the descriptions are specific and detailed, they cannot be understood as limiting the patent scope of the present invention. It should be understood by those skilled in the art that some modifications and improvements can be made without departing from the spirit of the present invention, and these fall within the scope of protection of the present invention. Therefore, the patent scope of the present invention should be governed by the appended claims.

Claims

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

2. The aforementioned CpG oligodeoxynucleotide is one or more selected from the CpG oligodeoxynucleotides shown in Sequence IDs 2 to 4. Preferably, the CpG oligodeoxynucleotide is the CpG oligodeoxynucleotide shown in Sequence ID No.

2. The pharmaceutical composition according to claim 1.

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

4. The aforementioned CpG oligodeoxynucleotide contains a phosphorothioate nucleoside bond, Preferably, the CpG oligodeoxynucleotide is a perthiooligodeoxynucleotide. The pharmaceutical composition according to claim 1.

5. The weight ratio of the CpG oligodeoxynucleotide to the aluminum adjuvant is 10 to 200:37.5, preferably 60 to 200:37.5, and more preferably 200:37.

5. The pharmaceutical composition according to claim 1.

6. The amino acid sequence of the hepatitis B virus surface antigen is as shown in Sequence ID No.

1. The pharmaceutical composition according to claim 1.

7. The weight ratio between components i) and ii) of the pharmaceutical composition is 1:47.5 to 237.5, preferably 1:97.5 to 237.5, and more preferably 1:237.

5. The pharmaceutical composition according to claim 1.

8. The pharmaceutical composition is a unit formulation, where the content of component i) is ≥ 5 μg / formulation, and Al(OH) in component ii) 3 The content of ≥ 187.5 μg / prescription and the content of CpG ≥ 300 μg / prescription. Preferably, the CpG content is 300 to 1000 μg / formulation. Preferably, the CpG content is 1000 μg / formulation. The pharmaceutical composition according to claim 1.

9. The pharmaceutical composition further comprises iii) a pharmaceutically acceptable carrier and / or additive, Preferably, the pharmaceutically acceptable carrier and / or additive is one or more selected from water, buffer, isotonic salt solution, wetting agent, or emulsifier. More preferably, the pharmaceutically acceptable carrier and / or additive is one or more selected from phosphate buffer, 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. A pharmaceutical composition according to any one of claims 1 to 8.

10. The dosage form of the pharmaceutical composition is a lyophilized powder injection or an injection solution. The pharmaceutical composition according to claim 1.

11. A vaccine for the prevention of hepatitis B comprising the pharmaceutical composition described in claim 1.

12. Use of the pharmaceutical composition according to claim 1 in the preparation of a drug for the prevention of hepatitis B virus infection and / or hepatitis B virus-induced disease, such as a vaccine.

13. The aforementioned hepatitis B virus infection and / or hepatitis B virus-induced disease is one or more selected from hepatitis B, cirrhosis, or liver cancer. The use described in claim 12.

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