Immunogenic composition, inactivated enterovirus vaccine and preparation method thereof

Abstract

The present invention relates to the field of vaccine technology, and in particular to an immunogenic composition, an inactivated enterovirus vaccine and a preparation method thereof. The immunogenic composition provided by the present invention and the vaccine prepared therefrom have high immunogenicity, and can not only induce animals to produce high levels of neutralizing antibodies, but also the maternal antibodies can effectively protect young animals from lethal viral attacks of corresponding enteroviruses. The inactivated vaccine of the present invention can be quantified by the Lowry method for viral particles, and the method is simple and has a wide range of applications. Moreover, the effectiveness of the product and the consistency between batches can be better guaranteed by controlling the ratio of hollow and solid particles of the vaccine product.

Description

Technical Field

[0001] The present invention relates to the field of vaccine technology, in particular to an immunogenic composition, an inactivated enterovirus vaccine and a preparation method thereof. Background Art

[0002] Hand, foot and mouth disease (HFMD) is a common viral infectious disease that is particularly susceptible to children under 5 years old. It is mainly caused by a variety of enteroviruses.

[0003] The pathogens of hand, foot and mouth disease show significant serotype diversity, mainly involving coxsackievirus ( Coxsackievirus ) Group A (CA4-7, 9, 10, 16) and Group B (CB1-3, 5), echovirus ( Echovirus ) some serotypes and enterovirus 71 ( Enterovirus A71, EV-A71, C4 subtype is the current epidemic strain).

[0004] The potency is a key indicator for measuring the effectiveness and batch consistency of vaccines, which is divided into in vivo and in vitro methods. The current potency evaluation of EV71 vaccines uses a dual indicator system of in vivo and in vitro methods, of which the in vivo method relies on BALB / c mice to detect ED. 50 However, there are defects such as large experimental variation (significant individual differences in animals), long cycles (taking several weeks) and ethical disputes (violating the 3R principle of reducing / replacing / optimizing experimental animals), which prompted the European Center for Validation of Alternative Methods to advocate the use of in vitro methods as a substitute. Although in vitro methods have the advantages of simple operation, compliance with the 3R principle and stable results, their development is limited due to insufficient standardized design and lack of unified guidelines.

[0005] It is worth noting that literature data show (such as patent applications such as CN116615234A and CN115429876A) that enterovirus vaccines are generally formulated using antigen titer. And there are two core limitations in the existing system: First, the existing standard products and evaluation methods are only for EV71 and CV-A16 virus strains, and lack an efficacy evaluation system covering other highly pathogenic enteroviruses (such as CV-A10, CV-A6, etc.) or combination vaccines, resulting in a lack of reliable indicators for the development of multivalent vaccines; second, antigen titer (physical and chemical indicators) and immunogenicity (functional indicators) are not completely equivalent, and in vitro efficacy evaluation has not yet established a strong correlation model with clinical protective efficacy. These defects restrict the vaccine's comprehensive prevention and control capabilities for a variety of enterovirus infections, and it is urgent to build a universal evaluation framework and formulation method through cross-strain antigen commonality research and key influencing factor analysis. Summary of the invention

[0006] The invention provides an immunogenic composition, an inactivated enterovirus vaccine and a preparation method thereof.

[0007] In the process of studying the immunogenicity of inactivated Coxsackievirus and enterovirus 71, the present invention unexpectedly found that the ratio of hollow virus particles to solid particles has an effect on the immunogenicity of inactivated enterovirus and its effect is type-specific. Specifically, the immunogenicity of inactivated vaccines such as EV-A71, CV-A10 and CV-A16 is positively correlated with the proportion of solid particles therein, while the level of neutralizing antibodies induced by the inactivated CV-A6 vaccine is asymmetric with the actual protective effect of its maternal antibodies on suckling mice (i.e., a high level of neutralizing antibodies that can be induced does not mean a higher protective efficacy), which means that even if the same ratio of hollow to solid particles is maintained, the existing vaccine preparation method and evaluation system based on physical and chemical indicators (such as antigen titer) still cannot accurately reflect the protective efficacy of inactivated enterovirus vaccines.

[0008] On this basis, the present invention innovatively proposes an immunogenic composition, which can simultaneously improve the neutralizing antibody titer and the passive immune protection rate of maternal antibodies to suckling mice by controlling the concentration threshold of inactivated viral antigen protein and the ratio of hollow and solid particles to prepare the vaccine preparation.

[0009] Based on the above immunogenic composition, the present invention develops monovalent and multivalent inactivated enterovirus vaccines and provides a method for preparing the inactivated vaccine.

[0010] Specifically, the present invention provides the technical solution described below.

[0011] The present invention provides an immunogenic composition, which comprises an enterovirus, wherein the enterovirus is an inactivated enterovirus; In the immunogenic composition, the content of enterovirus antigen protein is not less than 0.5 μg / mL; The inactivated enterovirus comprises solid particles and hollow particles of the enterovirus, wherein the mass percentage of the solid particles to the total amount of the solid particles and the hollow particles is ≥25%.

[0012] In the present invention, the mass percentage of the solid particles in the total amount of the solid particles and the hollow particles is ≥ 25%, which means that in the mixture of the solid particles and the hollow particles, the mass percentage of the solid particles must reach or exceed 25%. As an immunogenic composition, the mass percentage of the solid particles in the total amount of the solid particles and the hollow particles can be any value in the range of 25% to 100%, for example, 25%, 30%, 40%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%.

[0013] Preferably, the protein content of the solid particles and the hollow particles of the enterovirus is the same or has been adjusted to be the same.

[0014] The present invention proposes a new formulation method based on the above-mentioned immunogenic composition, which is significantly different from the existing formulation method based on antigen titer. The existing inactivated enterovirus vaccines are all formulated based on antigen titer, and the quality standard of inactivated enterovirus vaccines does not mention the control of the ratio of antigen protein content combined with hollow and solid particles, and the antigen titer of hollow and solid particles of the virus in the vaccine is not distinguished. Moreover, the existing literature mostly evaluates the immunogenicity of the vaccine based on the titer of neutralizing antibodies induced by inactivated enterovirus vaccines, and does not realize the asymmetric relationship between the neutralizing antibody level and the actual protective effect of maternal antibodies on suckling mice.

[0015] In the present invention, the enterovirus is selected from the group consisting of Coxsackievirus A ( Coxsackievirus A, CVA), Coxsackie virus group B ( Coxsackievirus B, CVB) and echovirus ( Echovirus ) subgroups and at least one of the novel numbered enterovirus serotypes (e.g., EV-A71 and EV-D68).

[0016] In the present invention, the content of the enterovirus antigen protein is measured based on the Lowry method.

[0017] Preferably, the enterovirus is at least one selected from EV-A71, CV-A6, CV-A10, and CV-A16.

[0018] Preferably, in the immunogenic composition of the present invention, the enterovirus should at least comprise CV-A6.

[0019] Preferably, in the immunogenic composition, the content of enterovirus antigen protein is not less than 1.0 μg / mL.

[0020] Depending on the number of virus serotypes contained, the immunogenic composition described above may be monovalent, divalent, trivalent or quadrivalent.

[0021] In some embodiments of the present invention, the immunogenic composition is monovalent. The enterovirus is one selected from EV-A71, CV-A6, CV-A10, and CV-A16; in the immunogenic composition, the content of enterovirus antigen protein is 0.5-4.0 μg / mL. Preferably, it is 1.0-4.0 μg / mL. Preferably, the enterovirus is CV-A6.

[0022] In some embodiments of the present invention, the immunogenic composition is bivalent. The enterovirus is any two selected from EV-A71, CV-A6, CV-A10, and CV-A16; in the immunogenic composition, the content of enterovirus antigen protein is 1.0-8.0 μg / mL (preferably 2.0-8.0 μg / mL), and the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%. Preferably, the enterovirus is CV-A6 and one selected from EV-A71, CV-A10, and CV-A16.

[0023] In some embodiments of the present invention, the immunogenic composition is trivalent. The enterovirus is any three selected from EV-A71, CV-A6, CV-A10, and CV-A16; in the immunogenic composition, the content of enterovirus antigen protein is 1.5-12.0 μg / mL (preferably 3.0-12.0 μg / mL), and the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%. Preferably, the enterovirus is CV-A6 and two selected from EV-A71, CV-A10, and CV-A16.

[0024] In some embodiments of the present invention, the immunogenic composition is tetravalent. The enterovirus is EV-A71, CV-A6, CV-A10 and CV-A16; in the immunogenic composition, the content of enterovirus antigen protein is 2.0-16.0 μg / mL (preferably 4.0-16.0 μg / mL), and the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%.

[0025] The mass percentage of solid particles of each of the above-mentioned enteroviruses to the total amount of solid particles and hollow particles is ≥25%, which means that all kinds of enteroviruses contained in the immunogenic composition meet the requirement that the mass percentage of their solid particles to the total amount of solid particles and hollow particles is ≥25%. For example, if four enteroviruses, EV-A71, CV-A6, CV-A10 and CV-A16, are contained, it is necessary to ensure that the mass percentage of solid particles of each virus EV-A71, CV-A6, CV-A10 and CV-A16 to the total amount of solid particles and hollow particles is ≥25%.

[0026] In order to meet the requirements for preparing human vaccines, the enterovirus is preferably an African green monkey kidney cell (Vero) cell-adapted strain. The Vero cell-adapted strain has the characteristics of being susceptible to Vero cells and having a high titer.

[0027] In some embodiments of the present invention, the deposit number of the EV-A71 strain is CGMCC No. 46210. The deposit number of the CV-A6 strain is CGMCC No. 20386. The deposit number of the CV-A10 strain is CGMCC No. 20388. The deposit number of the CV-A16 strain is CGMCC No. 20387.

[0028] Among them, the EV-A71 strain is the V05090434 strain, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Committee (CGMCC for short, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on November 12, 2024, and was classified and named Enterovirus 71 virus, with the deposit number CGMCC No.46210.

[0029] The CV-A6 strain with the deposit number CGMCC No. 20386 was deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on July 13, 2021, and was classified and named Coxsackievirus A6. The strain has been disclosed in patent application CN114807060A.

[0030] The CV-A10 strain with the deposit number CGMCC No. 20388 was deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on July 13, 2021, and was classified and named Coxsackievirus A10. The strain has been disclosed in patent application CN114774372A.

[0031] The CV-A16 strain with the deposit number CGMCC No. 20387 was deposited at the General Microbiology Center of China Culture Collection Administration (CGMCC for short, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101) on July 13, 2021, and was classified and named Coxsackievirus A16. The strain has been disclosed in patent application CN113564133A.

[0032] In the immunogenic composition described above, EV-A71, CV-A6, CV-A10, and CV-A16 strains have all been inactivated by an inactivator, which may be formaldehyde, β-propiolactone, or the like.

[0033] Preferably, the immunogenic composition further comprises an adjuvant. There is no particular limitation on the adjuvant in the present invention, and adjuvants commonly used in the vaccine field can be used, including but not limited to aluminum adjuvants.

[0034] In some embodiments of the present invention, the adjuvant is an aluminum adjuvant (preferably aluminum hydroxide) with an aluminum content of 0.3-0.7 mg / mL.

[0035] The solvent of the immune composition can be selected from physiological saline, PB and other solutions.

[0036] The present invention provides the use of the above-mentioned immunogenic composition in the preparation of a drug for preventing and / or treating enterovirus infection or hand, foot and mouth disease caused thereby.

[0037] In the above application, the enterovirus includes one or more of EV-A71, CV-A6, CV-A10, and CV-A16.

[0038] In the above applications, the drugs include vaccines, antibodies, antisera, etc.

[0039] The present invention provides the use of the above-mentioned immunogenic composition in the preparation of inactivated enterovirus vaccine.

[0040] The present invention provides an inactivated enterovirus vaccine, which comprises the immunogenic composition described above.

[0041] The above-mentioned vaccine may be a monovalent vaccine or a multivalent vaccine.

[0042] The above-mentioned vaccines may also be added with excipients permitted in the field of vaccine preparations, but the content of enterovirus antigen protein therein shall be guaranteed to meet the content requirements of enterovirus antigen protein in the immunogenic composition, that is, the content of enterovirus antigen protein shall not be less than 0.5 μg / mL (preferably not less than 1.0 μg / mL). For monovalent and multivalent vaccines, the content of enterovirus antigen protein therein shall refer to the above-mentioned monovalent and multivalent immunogenic compositions.

[0043] In some embodiments of the present invention, a monovalent inactivated enterovirus vaccine is provided, which comprises enterovirus EV-A71, CV-A6, CV-A10 or CV-A16 (preferably CV-A6), and the enterovirus is an inactivated enterovirus; in the monovalent inactivated enterovirus vaccine, the content of enterovirus antigen protein is not less than 0.5 μg / mL (preferably not less than 1.0 μg / mL, preferably 0.5~4.0 μg / mL, and more preferably 1.0~4.0 μg / mL); the inactivated enterovirus comprises solid particles and hollow particles of enterovirus, wherein the mass percentage of solid particles to the total amount of solid particles and hollow particles is ≥25%.

[0044] In some embodiments of the present invention, a bivalent inactivated enterovirus vaccine is provided, which comprises any two selected from enterovirus EV-A71, CV-A6, CV-A10, and CV-A16 (preferably comprising CV-A6 and one selected from EV-A71, CV-A10, and CV-A16), and the enterovirus is an inactivated enterovirus; in the bivalent inactivated enterovirus vaccine, the content of enterovirus antigen protein is not less than 1.0 μg / mL (preferably not less than 2.0 μg / mL, preferably 1.0~8.0 μg / mL, and more preferably 2.0~8.0 μg / mL); the inactivated enterovirus comprises solid particles and hollow particles of enterovirus, wherein the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%. Exemplarily, the enteroviruses are EV-A71 and CV-A16, wherein the mass percentage of solid particles of EV-A71 virus to the total amount of its solid particles and hollow particles is ≥25%; and the mass percentage of solid particles of CV-A16 virus to the total amount of its solid particles and hollow particles is ≥25%.

[0045] In some embodiments of the present invention, a trivalent inactivated enterovirus vaccine is provided, which comprises any three of enterovirus EV-A71, CV-A6, CV-A10, and CV-A16 (preferably CV-A6 and two selected from EV-A71, CV-A10, and CV-A16), and the enterovirus is an inactivated enterovirus; in the trivalent inactivated enterovirus vaccine, the content of enterovirus antigen protein is not less than 1.5 μg / mL (preferably not less than 3.0 μg / mL, preferably 1.5~12.0 μg / mL, and more preferably 3.0~12.0 μg / mL); the inactivated enterovirus comprises solid particles and hollow particles of enterovirus, wherein the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%. Exemplarily, the enterovirus is EV-A71, CV-A6 and CV-A10, wherein the mass percentage of the solid particles of the EV-A71 virus to the total amount of its solid particles and hollow particles is ≥25%, the mass percentage of the solid particles of the CV-A6 virus to the total amount of its solid particles and hollow particles is ≥25%, the mass percentage of the solid particles of the CV-A10 virus to the total amount of its solid particles and hollow particles is ≥25%, and the mass percentage of the solid particles of the CV-A16 virus to the total amount of its solid particles and hollow particles is ≥25%.

[0046] In some embodiments of the present invention, a quadrivalent inactivated enterovirus vaccine is provided, which comprises enterovirus EV-A71, CV-A6, CV-A10 and CV-A16, wherein the enterovirus is an inactivated enterovirus; in the quadrivalent inactivated enterovirus vaccine, the content of enterovirus antigen protein is not less than 2.0 μg / mL (preferably not less than 4.0 μg / mL, preferably 2.0-16.0 μg / mL, and more preferably 4.0-16.0 μg / mL); the inactivated enterovirus comprises solid particles and hollow particles of enterovirus, wherein the solid particles of the EV-A71 virus account for ≥25% by mass of the total amount of its solid particles and hollow particles, the solid particles of the CV-A6 virus account for ≥25% by mass of the total amount of its solid particles and hollow particles, the solid particles of the CV-A10 virus account for ≥25% by mass of the total amount of its solid particles and hollow particles, and the solid particles of the CV-A16 virus account for ≥25% by mass of the total amount of its solid particles and hollow particles.

[0047] The immune and protective effects of the above-mentioned multivalent inactivated vaccine are the same as those of the monovalent vaccine with the corresponding proportion. Both can induce the production of high levels of neutralizing antibodies, and the maternal antibodies can protect suckling mice against lethal virus attacks.

[0048] The present invention also provides a method for preparing the above inactivated enterovirus vaccine, which comprises: culturing the enterovirus and harvesting the virus liquid; inactivating the virus liquid and purifying it to obtain a vaccine stock solution, separating the virus solid particles and hollow particles in the vaccine stock solution, and preparing the vaccine according to the content of the antigen protein and the ratio of the solid particles and the hollow particles.

[0049] In the above method, Vero cells are preferably used to culture the enterovirus.

[0050] In some embodiments of the present invention, the method for preparing the inactivated enterovirus vaccine comprises the following steps: (1) amplifying the enterovirus on Vero cells; (2) Inoculate the virus at a multiplicity of infection (MOI) of 0.01-0.0001, continue culturing, and harvest the virus fluid when the lesions reach +++ to +++; (3) Centrifuge the virus solution and inactivate it by any of the following methods: inactivate with β-propiolactone at a ratio of 1:4000 at 5±3°C for 1-3 days, followed by hydrolysis at 37°C for 1-4 hours, or inactivate with formaldehyde at a ratio of 1:1000-1:5000 for 2-6 days; (4) Purifying the inactivated virus solution to obtain the corresponding type of vaccine stock solution; (5) separating the solid virus particles and hollow virus particles in the vaccine stock solution, and preparing the vaccine according to the content of the antigen protein and the ratio of the solid particles to the hollow particles and adding an adjuvant.

[0051] In the above step (4), the purification is preferably ultrafiltration concentration and chromatography column purification.

[0052] In the above step (5), the solid virus particles and hollow virus particles in the vaccine stock solution are separated by sucrose density gradient centrifugation. Before sucrose density gradient centrifugation, the vaccine stock solution is preferably concentrated. After sucrose density gradient centrifugation, a chromatography column is used to remove sucrose, and the collected target peak is concentrated by ultrafiltration.

[0053] Existing inactivated enterovirus vaccines generally use antigen titer as the basis for formulation. The quality standards have neither established a joint control standard for the antigen protein content and the ratio of hollow and solid particles, nor have they implemented differentiated detection of the antigen titer of hollow and solid particles in the vaccine. At the same time, according to existing literature, the industry mainly uses the vaccine-induced neutralizing antibody titer to characterize immunogenicity, but ignores the asymmetric dose-effect relationship between the neutralizing antibody level and the actual protective effect of maternal antibodies on suckling mice. The present invention discovered this phenomenon for the first time, and then creatively established a two-dimensional quality control system: that is, by integrating antigen protein quantitative analysis with empty / solid particle ratio control to achieve synergistic optimization of the vaccine antigen system, thereby constructing a new vaccine quality control standard for monovalent / polyvalent inactivated enterovirus vaccines.

[0054] The beneficial effects of the present invention include at least: (1) The immunogenic composition and the vaccine prepared therefrom provided by the present invention achieve a high immunogenicity by jointly controlling the inactivated virus antigen protein concentration threshold and the solid particle ratio threshold. It can not only induce animals to produce high levels of neutralizing antibodies, but also the maternal antibodies can effectively protect young animals against lethal viral attacks of the corresponding enterovirus, effectively solving the problem of inaccurate evaluation of the efficacy of inactivated enterovirus vaccines by the existing evaluation system.

[0055] (2) Currently, the preparation of inactivated enterovirus vaccines mainly uses the ELISA method for antigen quantification, which is highly dependent on antibodies. The quality of antibodies directly affects the accuracy of the results. Since different manufacturers use different antibodies, the versatility, stability and reliability of this method are limited. The present invention separates the stock solution into hollow and solid particles and uses the Lowry method for quantification. The method is simple and has a wide range of applications, which technically solves the above limitations.

[0056] (3) The inactivated vaccine of the present invention can better ensure the effectiveness of the product and the consistency between batches by controlling the ratio of hollow and solid particles in each batch of products. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0058] Figure 1 These are the immunogenicity test results of different doses of EV-A71\CV-A6\CV-A10\CV-A16 vaccines in Example 2 of the present invention.

[0059] Figure 2 These are electron microscope photos of the hollow and solid particles in Example 3 of the present invention; from left to right, from top to bottom, they are EV-A71 hollow particles, EV-A71 solid particles, CV-A6 hollow particles, CV-A6 solid particles, CV-A10 hollow particles, CV-A10 solid particles, CV-A16 hollow particles, and CV-A16 solid particles.

[0060] Figure 3 These are the results of the EV-A71\CV-A6\CV-A10\CV-A16 mother-suckling mouse protection test in Example 4 of the present invention.

[0061] Figure 4 These are the results of the protective test on mother mice and suckling mice of the bivalent inactivated enterovirus vaccine (Vero cells) composition in Example 5 of the present invention.

[0062] Figure 5 These are the results of the protective test on mother mice and suckling mice of the quadrivalent inactivated enterovirus vaccine (Vero cells) composition in Example 6 of the present invention. DETAILED DESCRIPTION

[0063] The terms used in the present invention are explained as follows.

[0064] In the present invention, hollow particles (EP) refer to particles in the enterovirus particle structure that lack part or all of the viral nucleic acid. Compared with solid particles, hollow particles do not have complete genetic material and may play a specific role in viral infection, immune response or other biological processes.

[0065] In the present invention, full particles (FP) generally refer to particles in the enterovirus structure that contain complete viral nucleic acids. They have complete genetic material and are an important structural basis for the virus to perform functions such as infection and replication.

[0066] In the present invention, the multiplicity of infection (MOI) refers to the ratio of cells infected by the virus. When a suitable MOI value is selected, the higher the infection efficiency of the virus and the expression level of the target protein, the lower the relative cytotoxicity.

[0067] In the present invention, cytopathic effect refers to the phenomenon that, after a certain period of time, cells are cultured and inoculated with cytocidal viruses in an in vitro experiment, cells can be observed to be rounded, necrotic, and fall off the bottle wall under a microscope. This phenomenon is called cytopathic effect, abbreviated as CPE. The degree of CPE is usually expressed by "+" to "++++": "+" means less than 25%; "++" means 25%-50%; "+++" means 50%-75%; "++++" means 75%-100%.

[0068] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0069] The following examples are related to the EV-A71 strain with a biological deposit number of CGMCC No.46210, the CV-A6 strain with a biological deposit number of CGMCC No.20386, the CV-A10 strain with a biological deposit number of CGMCC No.20388, and the CV-A16 strain with a biological deposit number of CGMCC No.20387. It should be clear that the above-mentioned strain examples exist only as typical and schematic examples, and their purpose is to provide intuitive and specific references for subsequent research and explanations, rather than to constitute strict limitations or constraints on the overall research scope and application scenarios. In the actual research and application process, based on different experimental conditions, research purposes and specific application requirements, it may involve the research and application of more types of strains, and there are also diverse choices and adjustment spaces in research methods and application strategies. Therefore, these specific strain examples are only part of many possibilities, intended to help better understand and explain the relevant research content, but should not be regarded as the only or absolute definition of the technical solution and scope of application of the present invention.

[0070] Example 1 Preparation of monovalent inactivated vaccine stock solution

[0071] Put 20-60g / L of flake carrier into a 30L reactor at a density of 2-5×10 5 Vero cells were inoculated with 100 cells / mL of cells, the pH was controlled between 7.0-7.5, the dissolved oxygen was maintained at 50%±30%, the culture temperature was set to 36.5±1°C, and the culture time was 3-7 days. The cell mass was calculated based on the glucose consumption, and the virus was inoculated into the bioreactor at a ratio of MOI=0.0001-0.01. The relevant parameters were set as follows: stirring speed 150±30rpm, temperature 35℃±1℃, pH 6.80~7.40, DO 50.0±30%, and the automatic control of these parameters was turned on. The culture was continued for 4-7 days, and the virus liquid was finally harvested. The harvested virus liquid was treated by centrifugation or filtration and then inactivated. The inactivation method can be to use β-propiolactone at a volume ratio of 1:4000 at 5±3℃ for 1~3 days, followed by hydrolysis at 37℃ for 1~4 hours; or formaldehyde at a volume ratio of 1:1000 to 1:5000 for 2~6 days. The inactivated solution is subjected to ultrafiltration concentration and chromatography column purification, and the purified solution after sterile filtration is the EV-A71, CV-A6, CV-A10, and CV-A16 vaccine stock solution. For the vaccine stock solution, the inspection results of sterility, bacterial endotoxins, free formaldehyde, bovine serum albumin residue, protein purity, etc. all meet the relevant requirements.

[0072] Example 2 Evaluation of different doses of monovalent inactivated vaccine

[0073] Based on the vaccine stock solution prepared in Example 1, four types of inactivated vaccines with three different doses of low, medium and high unit prices (hereinafter also referred to as compositions) were prepared. The stock solution was mixed with aluminum hydroxide adjuvant in an appropriate proportion, and 0.9% saline, PB and other solutions were added at the same time. The adsorption was carried out at room temperature for 15-30 minutes to obtain different doses of various types of monovalent aluminum adsorption products. Among them, the vaccine protein concentration was 0.25-4.0 μg / mL (determined by Lowry method), and the final concentration of aluminum was 0.5 mg / mL. The prepared vaccine was used for mouse immunization. The immunization method is as follows: The mice were randomly divided into 13 groups, 10 in each group, and intraperitoneal immunization was performed according to the immunization schedule on days 0 and 21, 0.5 mL per mouse, and blood was collected on the 35th day, and the immune adjuvant was used as a control.

[0074] The collected serum was tested for neutralizing antibodies. The specific operation method was as follows: the serum was inactivated in a 56°C water bath for 30 minutes, diluted from 1:8, added to a 96-well plate, 2 wells were set for each sample, 100 μL per well, and after 2-fold dilution, 32-320 CCID 50 / 0.05mL virus solution, incubate at 36±1℃, 5% CO2 for 1-2 hours. Add RD cell suspension (1-2×10 5 The cells were cultured in 5% CO2 for 7 days and the results were determined. When the neutralization titer was <8, it was considered negative, and when it was ≥8, it was considered positive.

[0075] The neutralizing antibody titer and positive conversion rate were statistically analyzed (Table 1). After taking the neutralizing antibody titer as Log2, the variance analysis (Tukey method) was used for statistical analysis. The results showed that 14 days after the second immunization, the serum antibody titer of all groups could achieve 100% positive conversion. The neutralizing antibody level of the four-type monovalent vaccine showed a dose-effect relationship. The neutralizing antibody levels of the medium and high doses were significantly higher than those of the low dose (p < 0.05). However, when the doses of each type reached the medium dose level, the neutralizing antibody level was basically stable, and there was no statistically significant difference between the medium dose and the high dose (p > 0.05) ( Figure 1 ). Detailed information of each group is shown in Table 1.

[0076] Table 1. Positive conversion rate and neutralizing antibody titer of each group

[0077] Example 3 Separation of hollow and solid particles of monovalent inactivated virus vaccine stock solution

[0078] The stock solution obtained in Example 1 is separated into hollow and solid particles. The specific operation process is as follows: First, the EV-A71, CV-A6, CV-A10, and CV-A16 virus vaccine stock solutions were concentrated using 30KD ultrafiltration concentrators. Then, a density gradient centrifugation preparation instrument was used to prepare a 15%-45% (w / v) sucrose solution. The stock solution concentrate was aspirated and added dropwise along the tube wall to the top of the sucrose density gradient liquid surface, followed by ultracentrifugation at a speed of 29,000 rpm and centrifugation at 4°C for 5 hours. After centrifugation, the samples were collected from the liquid surface in sections from top to bottom into centrifuge tubes, 1.5 mL per tube, and marked in turn. SDS-PAGE electrophoresis was performed on the samples after ultracentrifugation. The electrophoretic bands of the hollow particles mainly covered VP1, VP0, and VP3, and the electrophoretic bands of the solid particles mainly included VP1, VP2, VP3, and VP4. The samples were merged according to the test results, and the hollow particles were marked as merged solution 1; the solid particles were marked as merged solution 2. The combined solution 1 and the combined solution 2 were desalted by chromatography column to remove sucrose, and the collected target peaks were concentrated by ultrafiltration, and then filtered through a 0.2 μm filter to obtain four types of hollow and solid particles. The hollow and solid particle samples were selected for SDS-PAGE and negative staining transmission electron microscopy observation ( Figure 2) and protein content detection. Full Particle (FP) appears as a dense round particle, the interior of which cannot be negatively stained by phosphotungstic acid (appears as white particles), while the interior of the empty particle (EP) can be negatively stained by phosphotungstic acid to black.

[0079] Example 4 Evaluation of monovalent inactivated vaccines with different hollow and solid particle ratios

[0080] The hollow and solid particle samples separated in the above Example 3 were adjusted to the same protein content using PBS according to the protein content test results. Based on the results of Example 1, a final protein concentration of 1 μg / mL was selected for the preparation of a monovalent vaccine, and the hollow and solid particle ratios shown in Table 2 were used for preparation, and the specific preparation method was the same as in Example 1.

[0081] Table 2. Hollow and solid particle ratio parameters

[0082] (1) Immunogenicity test The vaccine prepared above was used for mouse immunization. The specific operation methods of the immunization method and the neutralizing antibody detection of serum were the same as those in Example 2. The results of the neutralizing antibody titer and positive conversion rate are shown in Table 3. After taking the neutralizing antibody titer as Log2, the variance analysis (Tukey method) was used for ≥3 groups, and the independent sample T test was used for 2 groups for statistical analysis. The results are shown in Tables 4, 5, 6, and 7.

[0083] The results showed that 14 days after the second immunization, the serum antibody titers of all groups could achieve 100% positive conversion. The neutralizing antibody levels of EV-A71, CV-A6, CV-A10, and CV-A16 increased with the increase in the proportion of solid particles in the composition. The neutralizing antibody titers of EV-A71, CV-A10, and CV-A16 solid particles were significantly higher than those of the hollow particle group (p < 0.05), and the neutralizing antibody level of CV-A6 was not significantly correlated with the proportion of hollow and solid particles. There was no significant difference in immunogenicity among the groups with a solid particle proportion of ≥25% (hollow particle proportion ≤75%) for EV-A71, CV-A6, CV-A10, and CV-A16.

[0084] Table 3. Positive conversion rate and neutralizing antibody titer of each group

[0085] Table 4. Statistical analysis of neutralizing antibody titers in each group of EV-A71

[0086] Table 5. Statistical analysis of neutralizing antibody titers in each group of CV-A6

[0087] Table 6. Statistical analysis of neutralizing antibody titers in each group of CV-A10

[0088] Table 7. Statistical analysis of neutralizing antibody titers in each group of CV-A16

[0089] (2) Mother-baby protective examination The vaccine sample prepared above was diluted 100 times and intraperitoneally immunized into female Balb / c mice of 8-12 weeks old. Mating was performed 7 days after the first immunization, and booster injections were given to pregnant mice in the same way 14 days after the first immunization. After mating and giving birth, one-day-old suckling mice were intraperitoneally challenged with a lethal dose of the corresponding type of virus, 50 μL per mouse, 6 mice per group, and a control group was set up at the same time. After the virus attack, the suckling mice were observed for 21 consecutive days, and the health, morbidity and mortality of the suckling mice were recorded. The survival rate of each group of suckling mice at the end of the observation period was statistically analyzed. The data are shown in Table 8, Figure 3 .

[0090] The results showed that when the proportion of solid particles of EV-A71, CV-A10, CV-A6, and CV-A16 was ≥25% (the proportion of hollow particles was ≤75%), the survival rate of suckling mice was 100%.

[0091] Table 8. Survival rate of suckling mice in each group

[0092] Example 5 Evaluation of a bivalent inactivated enterovirus vaccine (Vero cells) (1) Immunogenicity test

[0093] According to the protective results in Example 4, the lowest solid particle ratio group, the all-hollow particle group and the all-solid particle group corresponding to 100% survival rate of suckling mice in EV-A71 and CV-A16 types were selected (Table 9), and a bivalent enterovirus inactivated vaccine (Vero cell) composition was prepared (wherein the antigen protein content of each enterovirus was 1.0 μg / mL, and the antigen protein content was determined by the Lowry method), and the immunogenicity test of mice was carried out, and the specific operation method was the same as that in Example 2. The results of neutralizing antibody titer (GMT) are shown in Table 10. There was no statistical difference in the immunogenicity of the bivalent enterovirus inactivated vaccine (Vero cell) composition with the corresponding monovalent vaccine composition of the same composition (P>0.05) (Table 11). There was a statistical difference in immunogenicity between the bivalent inactivated enterovirus vaccine (Vero cell) compositions with a solid percentage of 0% and that with a solid percentage of 100% (P < 0.05), and there was also a statistical difference in immunogenicity between the bivalent vaccine compositions with a solid percentage of 25% and that with a solid percentage of 0% (P < 0.05) (Table 12).

[0094] Table 9. Composition of bivalent inactivated enterovirus vaccine (Vero cells)

[0095] Table 10. Composition of bivalent inactivated enterovirus vaccine (Vero cells)

[0096] Table 11. Statistical analysis of neutralizing antibody titers between bivalent vaccines and corresponding monovalent vaccines

[0097] Table 12. Statistical analysis of neutralizing antibody titers among the bivalent vaccine groups

[0098] (2) Mother-baby protective examination The above-mentioned bivalent enterovirus inactivated vaccine (Vero cell) composition was diluted 100 times, and the protective test of mother mice and suckling mice was carried out. The specific method was the same as that of Example 4. The survival rate of mice is shown in Table 13. Figure 4 The results showed that under the attack of two types of lethal doses of strains, the pups born to mother mice immunized with the bivalent composition containing solid particles could survive 100%, while the pups born to the bivalent composition without solid particles had a survival rate of no more than 50%.

[0099] Table 13. Survival of suckling mice after immunization of mother mice with bivalent inactivated enterovirus vaccine (Vero cells) composition

[0100] Example 6 Evaluation of the Quadrivalent Inactivated Enterovirus Vaccine (Vero Cell) Composition (1) Immunogenicity test According to the protective results in Example 4, the lowest solid particle ratio group, the full hollow particle group and the full solid particle group corresponding to 100% survival rate of suckling mice in the four types of EV-A71, CV-A6, CV-A10 and CV-A16 were selected (Table 14), and a quadrivalent enterovirus inactivated vaccine (Vero cell) composition was prepared (wherein the antigen protein content of each enterovirus was 1.0 μg / mL, and the antigen protein content was determined by the Lowry method), and the immunogenicity test of mice was carried out. The specific operation method was the same as that in Example 2. The results of neutralizing antibody titer are shown in Table 15. There is no statistical difference in the immunogenicity of the quadrivalent enterovirus inactivated vaccine (Vero cell) composition with the corresponding monovalent vaccine composition of the same composition (P>0.05) (Table 16). The immunogenicity of the quadrivalent vaccine composition with a solid ratio of 0% and a solid ratio of 100% of the quadrivalent enterovirus inactivated vaccine (Vero cell) composition, except for CV-A6, there is no statistical difference, and the other three types are statistically different (P<0.05) (Table 17).

[0101] Table 14. Composition of quadrivalent inactivated enterovirus vaccine (Vero cells)

[0102] Table 15. Composition of quadrivalent inactivated enterovirus vaccine (Vero cells)

[0103] Table 16. Statistical analysis of neutralizing antibody titers between quadrivalent vaccines and corresponding monovalent vaccines

[0104] Table 17. Statistical analysis of neutralizing antibody titers among the four-valent vaccine groups

[0105] (2) Mother-baby protective examination The above-mentioned quadrivalent enterovirus inactivated vaccine (Vero cell) composition was diluted 100 times, and the protective test of mother mice and suckling mice was carried out. The specific method was the same as that of Example 4. The survival rate of mice is shown in Table 18. Figure 5 The results showed that under the attack of lethal doses of four strains, 100% of the pups born to mother mice immunized with the quadrivalent vaccine composition containing solid particles survived, while the survival rate of the pups born to the quadrivalent vaccine composition without solid particles was no more than 50%.

[0106] Table 18. Survival of mice with quadrivalent inactivated enterovirus vaccine (Vero cells) composition

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An immunogenic composition, characterized in that It contains enterovirus, which is an inactivated enterovirus; In the immunogenic composition, the content of enterovirus antigen protein is not less than 0.5 μg / mL; The inactivated enterovirus comprises solid particles and hollow particles of the enterovirus, wherein the mass percentage of the solid particles to the total amount of the solid particles and the hollow particles is ≥25%.

2. The immunogenic composition according to claim 1, characterized in that The enterovirus is selected from at least one of the coxsackievirus group A, coxsackievirus group B, echovirus subgroup and a newly numbered enterovirus serotype.

3. The immunogenic composition according to claim 1, characterized in that The content of the enterovirus antigen protein is determined based on the Lowry method.

4. The immunogenic composition according to any one of claims 1 to 3, characterized in that The enterovirus is one selected from EV-A71, CV-A6, CV-A10, and CV-A16; and the content of the enterovirus antigen protein in the immunogenic composition is 0.5-4.0 μg / mL.

5. The immunogenic composition according to claim 4, characterized in that The enterovirus is CV-A6.

6. The immunogenic composition according to any one of claims 1 to 3, characterized in that The immunogenic composition is any one of the following (1)-(3): (1) The enterovirus is any two selected from EV-A71, CV-A6, CV-A10, and CV-A16; the content of enterovirus antigen protein in the immunogenic composition is 1.0-8.0 μg / mL; and the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%; (2) The enterovirus is any three selected from EV-A71, CV-A6, CV-A10, and CV-A16; the content of enterovirus antigen protein in the immunogenic composition is 1.5-12.0 μg / mL; and the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%; (3) The enterovirus is EV-A71, CV-A6, CV-A10 and CV-A16; the content of enterovirus antigen protein in the immunogenic composition is 2-16.0 μg / mL; and the mass percentage of solid particles of each enterovirus to the total amount of solid particles and hollow particles is ≥25%.

7. The immunogenic composition according to claim 6, characterized in that In (1), the enterovirus is CV-A6 and one selected from EV-A71, CV-A10, and CV-A16; And / or, in (2), the enterovirus is CV-A6 and two selected from EV-A71, CV-A10, and CV-A16.

8. The immunogenic composition according to any one of claims 1 to 3, 5 and 7, characterized in that: The enterovirus is a Vero cell adapted strain; And / or, the immunogenic composition further comprises an adjuvant.

9. Use of the immunogenic composition according to any one of claims 1 to 8 in the preparation of a medicament for preventing and / or treating enterovirus infection or hand, foot and mouth disease caused thereby.

10. Use of the immunogenic composition according to any one of claims 1 to 8 in the preparation of an inactivated enterovirus vaccine.

11. An inactivated enterovirus vaccine, characterized in that: The vaccine comprises the immunogenic composition according to any one of claims 1 to 8.

12. The inactivated enterovirus vaccine according to claim 11, characterized in that The vaccine is a monovalent vaccine or a multivalent vaccine.

13. The method for preparing the inactivated enterovirus vaccine according to claim 11 or 12, characterized in that: The method comprises: culturing the enterovirus and harvesting the virus liquid; inactivating the virus liquid and purifying it to obtain a vaccine stock liquid, separating the virus solid particles and hollow particles in the vaccine stock liquid, and preparing a vaccine according to the content of the antigen protein and the ratio of the solid particles to the hollow particles.

Images