WO2018219266A1

WO2018219266A1 - Composition for preparing hepatitis b vaccine, preparation method therefor, and application thereof

Info

Publication number: WO2018219266A1
Application number: PCT/CN2018/088817
Authority: WO
Inventors: 靳广毅; 王竹林; 朱征宇; 唐黎; 胡云龙
Original assignee: 深圳大学; 深圳康泰生物制品股份有限公司
Priority date: 2017-05-31
Filing date: 2018-05-29
Publication date: 2018-12-06
Also published as: CN107281484A

Abstract

A composition for preparing hepatitis B vaccine, a preparation method therefor, and an application thereof. The composition comprises immune agonist activated ester, HBsAg and an aluminum adjuvant. On the basis of maintaining the hepatitis B virus specific antibody of an existing prophylactic vaccine in animal and human body, the composition can perform inactivation and clearance on hepatitis B virus inflected cells, thus having a dual effect of preventing and treating hepatitis B.

Description

Composition for preparing hepatitis B vaccine and preparation method and application thereof

Technical field

The invention relates to the technical field of prevention and treatment of hepatitis B vaccine, and more particularly to a composition for preparing hepatitis B vaccine, a preparation method and application thereof.

Background technique

Hepatitis B vaccine is a drug used to prevent hepatitis B. After vaccination, it can stimulate the immune system to produce protective antibodies, so that the human body has the immunity to prevent hepatitis B and achieve the purpose of preventing hepatitis B infection. However, there are 90 million people infected with hepatitis B virus (HBV) in China, including 20 million patients with chronic hepatitis B. For patients with chronic hepatitis B infection, preventive hepatitis B vaccine is basically ineffective. Therefore, there is a great need for a "therapeutic hepatitis B vaccine" that both prevents and eliminates chronic hepatitis B virus infection.

Summary of the invention

The object of the present invention is to provide a composition for preparing hepatitis B vaccine, a preparation method and application thereof, and solve the problem that the hepatitis B vaccine in the prior art cannot simultaneously prevent and eliminate chronic infection of hepatitis B virus.

The technical solution adopted by the present invention to solve the technical problem is: a composition for preparing hepatitis B vaccine, comprising: an immunoagonist activated ester, HBsAg and an aluminum adjuvant, wherein the composition ratio is: an immunoagonist activated ester :HBsAg:Al=n:m:q, HBsAg stands for recombinant hepatitis B surface antigen, Al stands for aluminum in aluminum adjuvant; n, m, q represent the molar ratio or weight ratio of activated agonist activated ester, HBsAg, Al, respectively. And the representative molar ratio is n:m:q=4:1:18181; wherein the immunoagonist activated ester (T7) has the following structural formula:

Wherein R is OH or SH.

In the composition of the present invention, the amino acid sequence of HBsAg includes 200-400 amino acids, and the molecular weight of HBsAg is 20,000 Da-44000 Da.

In the composition of the present invention, the amino acid sequence of HBsAg includes 200-250 amino acids; and the molecular weight of HBsAg is 20,000 Da-25000 Da.

In the compositions of the invention, the amino acid sequence of HBsAg is as follows:

In the composition of the present invention, the aluminum adjuvant is an aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum.

In the composition of the present invention, the preparation method of the immunoagonist activated ester comprises: dissolving the reaction starting material in anhydrous DMF, adding EDC and NHS in portions, reacting at room temperature, and pouring the reaction solution into ice water. Filtering, washing with water, drying to obtain an immunoagonist activated ester in the form of a white solid; the mass ratio of the reaction starting material to anhydrous DMF is from 0.075 g/ml to 0.1 g/ml; the amount of EDC added is The mass ratio of the reaction starting material is 0.45-0.6; the mass ratio of the NHS added to the reaction starting material is 0.25-0.4;

Wherein, the structural formula of the reaction starting material is:

In the composition of the present invention, the reaction time at room temperature is from 1.5 h to 4 h.

In the compositions of the present invention, the use of the immunoagonist activated ester in the preparation of a therapeutic hepatitis B vaccine, and in the preparation of human and animal immunomodulatory drugs.

The invention also provides a preparation method of the above composition, comprising: mixing an immunoagonist activated ester with a parent liquid of HBsAg in a solvent of DMSO or physiological saline, shaking at 4 ° C - 8 ° C overnight, and then 9 times the volume. The aluminum adjuvant solution is mixed to obtain a composition for preparing a prophylactic and therapeutic hepatitis B vaccine.

The present invention also provides the use of the above composition for the preparation of a medicament for increasing antibody titer and effector T cells against hepatitis B surface antigen HBsAg, and for the preparation of vaccines and medicaments for the prevention and treatment of hepatitis B.

The composition for preparing hepatitis B vaccine of the present invention and the preparation method and application thereof have the following beneficial effects: the present invention provides a composition for preparing hepatitis B vaccine for chronic hepatitis B infection, and a preparation method and application thereof, It can inactivate and eliminate cells infected with hepatitis B virus in animal and human body based on the hepatitis B virus-specific antibody which maintains the original prophylactic vaccine, and has the dual effects of prevention and treatment; the present invention activates the ester and the immunoconjugate The original antigen HBsAg binds to form a new high-activity antigen, and achieves a stronger immunostimulatory effect than the original HBsAg antigen in synergy with the aluminum adjuvant. The benefit is enhanced by enhanced immune cell factor induction and higher antibody production, especially The high level of gamma-interferon and IgG2a antibodies marks Th1 type immune production and is significantly innovative and practical for the removal of hepatitis B virus.

DRAWINGS

Figure 1 is a schematic diagram of the synthesis of an immunoagonist-activated ester (T7) and a schematic diagram of binding of an immunoagonist-activated ester (T7) to the original antigen HBsAg;

Figure 2 is a mass spectrum of an immunoagonist activated ester (T7);

Figure 3 shows the effect of (HBsAg)-(T7) mixture on IL-6 in different molar ratios (1:1, 1:4, 1:40, 1:100);

Figure 4 shows the effect of INF-γ on the (HBsAg)-(T7) mixture with aluminum adjuvant and no aluminum adjuvant in different molar ratios (1:1, 1:4, 1:40, 1:100);

Figure 5 is a 1:4 molar ratio of (HBsAg)-(T7) mixture plus aluminum adjuvant for IL-6 induction;

Figure 6A is a comparison diagram of a humoral-specific IgG1 humoral immune response induced by a solution and a control group for preparing a composition for preventing and treating hepatitis B vaccine, respectively;

Figure 6B is a comparison diagram of a humoral-specific IgG2a humoral immune response induced by a solution and a control group for preparing a composition for preventing and treating hepatitis B vaccine, respectively;

Figure 7A is a photograph of a solution for the preparation of a composition for preventing and treating hepatitis B vaccine and a control group inducing an antigen-specific CD4+ reaction, respectively;

Figure 7B is a comparative diagram showing the expression rate of IFNγ which induces an antigen-specific CD4+ response in a solution for preparing a composition for preventing and treating hepatitis B vaccine and a control group;

Figure 7C is a photograph of a solution for the preparation of a composition for preventing and treating hepatitis B vaccine and a control group inducing an antigen-specific CD8+ reaction, respectively;

Fig. 7D is a comparative diagram of the expression rate of IFNγ which induces an antigen-specific CD8+ response in a solution for preparing a composition for preventing and treating hepatitis B vaccine and a control group, respectively.

detailed description

The immunoagonist activated esters and compositions of the present invention, and methods for their preparation and use are further described below in conjunction with the accompanying drawings and examples:

The advantage of using a small molecule immunoagonist as a vaccine adjuvant is that it is structurally stable and easy to synthesize, purify and manufacture. However, the shortcomings of small molecule immunoadjuvants are that the body absorbs quickly and the metabolism is fast, so it cannot be metabolized with the antigenic protein in a good synergistic distribution. The small molecule immunoagonist activated ester and the recombinant hepatitis B surface antigen HBsAg used in the invention are covalently coupled to form a new whole antigen, and the distribution and action in vivo are consistent. These advantages of the hepatitis B vaccine adjuvant in the present invention greatly enhance the immunological synergy between the adjuvant and the antigen, and thus are also significantly innovative in terms of mechanism.

The immunoagonist activates the ester (T7) and has the structural formula:

Wherein R is OH or SH.

The present invention also provides a composition for preparing a prophylactic and therapeutic hepatitis B vaccine comprising the above-mentioned immunoagonist activated ester, HBsAg and aluminum adjuvant, wherein the composition ratio is: an immunoagonist activated ester: HBsAg: Al = n: m:q, HBsAg stands for recombinant hepatitis B surface antigen, Al stands for aluminum content in aluminum adjuvant; n, m, q represent the molar ratio or weight ratio of activated agonist activated ester, HBsAg, Al, respectively, and representative moles The ratio is n:m:q=4:1:18181.

Preferably, the amino acid sequence of HBsAg comprises 200-400 amino acids and the molecular weight of HBsAg is 20000Da-44000Da. More preferably, the amino acid sequence of HBsAg comprises 200-250 amino acids; the molecular weight of HBsAg is 20000Da-25000Da. The typical amino acid sequence of HBsAg is as follows (containing 226 amino acids) (Chinese Journal of Bioengineering 1995, 15(2): 43-45):

The aluminum adjuvant is an aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum.

The principle of the invention uses the immunoagonist activated ester to combine with the original antigen HBsAg to form a new high active antigen, and achieves a stronger immune activation effect than the original HBsAg antigen in synergy with the aluminum adjuvant, and the benefit is enhanced by the enhanced immune cytokines. Induction, higher antibody production effects, especially high levels of gamma-interferon and IgG2a antibodies, Th1 type immune production, has significant innovation and utility for the removal of hepatitis B virus.

The details will be described below by way of specific examples.

Example 1: Synthesis of an immunoagonist activated ester (T7)

As shown in Figure 1, 1 g of compound 1 (2.25 mmol) was dissolved in 12 ml of anhydrous DMF, and EDC (518 mg, 2.7 mmol) and NHS (337 mg, 2.93 mmol) were added in portions, and reacted at room temperature for 2 h. After the completion of the reaction, the reaction mixture was poured into 100 ml of ice water, filtered, washed with water and dried to give a white solid. As shown in Figure 2, ESI-MS: m/z = 542.10 [M + H] +.

It should be noted that EDC is a water-soluble carbodiimide having a chemical formula of C ₈ H ₁₇ N ₃ .HCl, specifically 1-(3-dimethylaminopropyl)-3-ethyl carbon. Diimine hydrochloride; NHS is N-hydroxysuccinimide; DMF dimethylformamide.

Example 2:

1 g of Compound 1 (2.25 mmol) was dissolved in 13.4 ml of anhydrous DMF, and EDC 600 mg and NHS 400 mg were added in portions, and reacted at room temperature for 4 h. The reaction was detected by LC-MS. After the reaction was completed, the reaction solution was poured into 100 ml of ice water and suction filtered. It was washed with water and dried to give a white solid. ESI-MS: m/z = 542.10 [M+H]+.

Example 3:

1 g of Compound 1 (2.25 mmol) was dissolved in 10 ml of anhydrous DMF, and EDC 450 mg and NHS 250 mg were added in portions, and reacted at room temperature for 1.5 h. The reaction was detected by LC-MS. After the reaction was completed, the reaction solution was poured into 100 ml of ice water and suction filtered. It was washed with water and dried to give a white solid. ESI-MS: m/z = 542.10 [M+H]+.

Example 4: Preparation method of composition for preparing hepatitis B vaccine

More specifically, the method refers to a method for preparing a solution for preparing a composition for preventing and treating hepatitis B vaccine.

As shown in Figure 1, the immunoagonist-activated ester T7 (2 mg/3.7 μM) was mixed with 100 mL of a 220 μg/mL HBsAg mother liquor (0.92 μM; average molecular weight 24 KD; supplied by Shenzhen Kangtai Biological Products Co., Ltd.). Shake overnight at -8 ° C, and then mix with 900 mL of aluminum adjuvant solution (containing Al(OH) 3 concentration of 1.44 mg / mL; aluminum concentration of 0.5 mg / mL) to obtain representative for the preparation and prevention of hepatitis B vaccine A solution of the composition is coupled to a vaccine solution. Wherein the weight ratio (T7): (HBsAg): (Al): = n: m: q = 1: 11: 225; molar ratio n: m: q = 4: 1:18181.

Regardless of which aluminum adjuvant is used in the above ratio, the aluminum content is calculated by the molar ratio in the examples.

The solvent for the mother broth of the immuno agonist is DMSO or physiological saline; the other aluminum adjuvant is replaced by the effective content of aluminum (containing 0.5 mg/mL of aluminum). The form of HBsAg may be a monomer or a multipolymer having a mother liquor concentration ranging from 220 μg/mL to 250 μg/mL.

The biological activity test is specifically described below.

Example 5: In vitro cytokine stimulation

Spleen lymphocytes derived from Balb/c mice were used as a test system. (HBsAg): (T7) was cultured in a mixture of different molar ratios with 1×106/mL of spleen lymphocytes for 24 hours. After collecting the supernatant, the concentration of IL-6 and INF-γ in the supernatant was detected by Elisa method. Add as a negative control. The results are shown in Figures 3-5. The (HBsAg)-(T7) mixture and the aluminum-added adjuvant have significant stimulatory effects on the immune cytokines IL-6 and IFN-γ.

Example 6: Solution for preparing a composition for preventing and treating hepatitis B vaccine (conjugated hepatitis B vaccine mixture) to induce antigen-specific humoral immune response

Balb/c mice (5 per group) were immunized with (HBsAg): (T7) in a mixture of different molar ratios in the case of aluminum plus adjuvant and no aluminum adjuvant at 0, 14 and 28 days. Mouse sera were isolated on day 35 and HBsAg-specific IgG1 and IgG2a antibody titers were measured by Elisa assay. Commercial hepatitis B vaccine (HBsAg + Al) and PBS injection were used as a control group. The results are shown in Figures 6A and 6B, and the effect of the solution on antigen-specific humoral immune response was evident.

Example 7: Solution for preparing a composition for preventing and treating hepatitis B vaccine (conjugated hepatitis B vaccine mixture) induces antigen-specific CD4+ and CD8+ T cell responses

Balb/c mice (5 per group) were immunized with (HBsAg):(T7) in the presence of a mixture of 1:4 molar ratios on days 0, 14 and 28 in the presence of an aluminum adjuvant. Mouse spleen lymphocytes were isolated on day 35, and the ratio of HBsAg-specific CD4+ and CD8+ T cells was identified by intracellular IFNγ staining and flow cytometry. Commercial hepatitis B vaccine (HBsAg + Al) was used as a control group. Referring to Figures 7A, 7B, 7C and 7D, the results show that the solution induces an effect of antigen-specific CD4 ⁺ and CD8 ⁺ T cell responses.

It is to be understood that those skilled in the art can make modifications and changes in the above-described description, all of which are within the scope of the appended claims.

Claims

A composition for preparing a hepatitis B vaccine, comprising an immunoagonist activated ester, HBsAg and an aluminum adjuvant, wherein the composition ratio is: an immunoagonist activated ester: HBsAg: Al = n: m: q, HBsAg Represents recombinant hepatitis B surface antigen, Al represents aluminum in aluminum adjuvant; n, m, q represent the molar ratio or weight ratio of activated agonist activated ester, HBsAg, Al, respectively, and the representative molar ratio is n: m: q=4:1:18181; wherein the immunoagonist-activated ester (T7) has the following structural formula:

Wherein R is OH or SH.
The composition of claim 1 wherein the amino acid sequence of HBsAg comprises from 200 to 400 amino acids and the molecular weight of HBsAg is from 20,000 Da to 44000 Da.
The composition according to claim 2, wherein the amino acid sequence of HBsAg comprises from 200 to 250 amino acids; and the molecular weight of HBsAg is from 20,000 Da to 25000 Da.
The composition according to claim 3, wherein the amino acid sequence of HBsAg is as follows:
The composition of claim 1 wherein the aluminum adjuvant is an aluminum hydroxide gel, aluminum phosphate, aluminum sulfate, ammonium alum or potassium alum.
The composition according to claim 1, wherein the preparation method of the immunoagonist activated ester comprises: dissolving the reaction starting material in anhydrous DMF, adding EDC and NHS in portions, reacting at room temperature, and reacting the reaction. The liquid is poured into ice water, filtered with suction, washed with water, and dried to obtain an immunoagonist activated ester in the form of a white solid; the mass ratio of the reaction starting material to anhydrous DMF is from 0.075 g/ml to 0.1 g/ml; The mass ratio of the amount of EDC added to the reaction starting material is 0.45-0.6; the mass ratio of the amount of the NHS added to the reaction starting material is 0.25-0.4;

Wherein, the structural formula of the reaction starting material is:
The composition according to claim 6, wherein the reaction at room temperature is from 1.5 h to 4 h.
The composition according to claim 1, wherein the immunoagonist activated ester is used in the preparation of a therapeutic hepatitis B vaccine, and in the preparation of a human and animal immunomodulatory drug.
The method for preparing a composition according to any one of claims 1 to 8, comprising: mixing an immunoagonist activated ester with a parent aqueous solution of HBsAg in a solvent of DMSO or physiological saline at 4 ° C to 8 ° C The composition for preventing and treating hepatitis B vaccine was obtained by shaking overnight and mixing with 9 volumes of aluminum adjuvant solution.
Use of the composition according to any one of claims 1 to 8 for the preparation of a medicament for increasing antibody titer and effector T cells against hepatitis B surface antigen HBsAg, and for preparing vaccines and medicaments for preventing and treating hepatitis B Application in .