A method for evaluating the immunogenicity of polysaccharide conjugate vaccines based on a weaned rabbit model

Abstract

The present invention provides a method for evaluating the immunogenicity of a polysaccharide conjugate vaccine based on a weaned rabbit model, and belongs to the field of vaccines. The present invention uses weaned rabbits as experimental subjects, and uses a method of increasing the inoculation dose to inject the vaccine multiple times for immunization, and evaluates the immunogenicity of the vaccine by serological detection of the antibody titer produced. The method can be used for preclinical immunogenicity evaluation of polysaccharide conjugate vaccines (such as pneumococcal polysaccharide conjugate vaccines, etc.), and has the advantages of sensitivity and high efficiency, and is particularly suitable for preclinical immunogenicity evaluation of candidate vaccines intended for infants and young children.

Description

Technical Field

[0001] The present invention belongs to the field of vaccines, and in particular relates to a method for evaluating the immunogenicity of polysaccharide conjugate vaccines based on animal experiments. Background Art

[0002] For vaccines that use bacterial polysaccharides as antigens (polysaccharide vaccines), such as pneumococcal polysaccharide vaccines, since polysaccharides are T cell-independent antigens, they directly stimulate B cells to differentiate into plasma cells to produce antibodies by cross-linking B cell receptors (BCRs), which can induce short-term immune effects (lack of memory effects) in children and adults. However, this mechanism is not mature in infants and young children, so polysaccharide vaccines can only cause weak reactions or no reactions in infants and young children. To solve this problem, one approach is to covalently link polysaccharide antigens to carrier proteins (chemical conjugation) to construct polysaccharide conjugate vaccines (conjugates, conjugates), making them T cell-dependent antigens: the carrier protein can be specifically recognized by T helper cells, and the polysaccharide components can activate T helper cells after binding to B cells, thereby amplifying the production of polysaccharide antibodies. Therefore, in theory, polysaccharide conjugate vaccines can effectively stimulate the immune system of infants and young children and produce sufficient protective antibodies.

[0003] Before a candidate vaccine is approved for human use, preclinical studies (animal experiments) and human clinical trials are usually required to demonstrate its safety and immunogenicity. Although polysaccharide conjugate vaccines can theoretically provide effective immune protection for infants and young children, clinical trials are still required to evaluate the safety and immunogenicity of candidate vaccines before they are marketed. Before clinical trials, it is necessary to conduct preliminary studies on the safety and immunogenicity of candidate vaccines using animal models, taking into account factors such as the safety of the subjects, the time and economic costs of the trials. However, due to differences in physiological structure, function, age, etc. between animals, especially non-primates, and humans, the sensitivity and relevance of vaccine evaluation methods based on animal experiments (consistency between animal and human clinical trial results) is an issue worth considering.

[0004] At present, for most vaccines, especially those intended for infants and young children, there are no official regulations or guidelines on how to conduct animal experiments in the preclinical research stage to improve the predictability of human results. Although there are methods for evaluating the immunogenicity of specific vaccines based on animal experiments disclosed in the prior art, most of them are animal vaccines, and the immunization methods for animals are mostly single vaccinations (such as CN106771186B, CN103091484A), or multiple equal vaccinations (such as CN105866424B, CN103830748B), and they have not fully studied the animal experimental methods themselves, their rationality, and immunogenicity.

[0005] For polysaccharide conjugate vaccines intended for infants and young children, there is a lack of animal models that can fully simulate the immune response of infants and young children and corresponding vaccine immunogenicity evaluation methods in the preclinical research stage: (1) The Chinese Pharmacopoeia has not yet clearly stipulated the immunogenicity or potency evaluation methods of such vaccines, and there is no patent or non-patent prior art to study this; (2) The industry generally uses adult model animals as experimental subjects (Ivette Caro-Aguilar et al., Vaccine. 2017 Feb7;35(6):865-872; Chulmin Park et al., Hum Vaccin Immunother. 2017 May; 13(5): 1169–1176). However, the immune response of adult animals may not fully reflect the immune response of infants and young children to polysaccharide conjugate vaccines, and the correlation between the two is poor (Xie, Jinfu et al., The Pediatric Infectious Disease Journal 39(1): p 70-77, January 2020.), which may have an adverse impact on the research and development process of infant vaccines. Therefore, a more sensitive and efficient evaluation method is needed during the animal experiment stage for the immunogenicity assessment of polysaccharide conjugate vaccines, especially those used in infants and young children. Summary of the invention

[0006] To solve the above problems, the present invention provides a method for evaluating the immunogenicity of a candidate polysaccharide conjugate vaccine based on animal experiments, characterized in that weaned rabbits are used as experimental subjects, the vaccine is injected multiple times in a manner of increasing vaccination doses for immunization, and the immunogenicity of the vaccine is evaluated by serological detection of the antibodies produced. Preferably, the user group of the vaccine includes infants and young children (e.g., people aged 0 to 6 years old), and more preferably, the vaccine is a vaccine for infants and young children.

[0007] Furthermore, the weaned rabbits are 30-40 day old rabbits, preferably 30-40 day old New Zealand rabbits.

[0008] Furthermore, the method of gradually increasing the vaccination dose means that, based on the total amount of polysaccharides in the vaccine, except for the first vaccination, each vaccination dose is 1.5 to 2.5 times the previous vaccination dose (that is, the dose of the N+1th vaccination is 1.5 to 2.5 times that of the Nth vaccination, where N is a positive integer), among which 2 times is preferred.

[0009] Furthermore, the multiple injections are 2, 3 or 4 injections; preferably, the multiple injections are 3 injections; further preferably, the 3 injections are performed on the weaned rabbits on days 0, 14 and 28 of the experiment, respectively.

[0010] Furthermore, the serological test refers to blood sampling for neutralizing antibody titer determination; preferably, the specific operation of the serological test is: on the 14th day after the last immunization, blood is collected for neutralizing antibody titer determination.

[0011] The polysaccharide conjugate vaccine referred to in the present invention refers to a vaccine formed by covalently linking a polysaccharide antigen with a non-sugar unit (protein or short peptide, lipid), mainly a polysaccharide protein conjugate vaccine (a conjugate formed by covalently linking a polysaccharide antigen with a carrier protein via a chemical bond, also known as a conjugate or a conjugate, with the English name being conjugate). The polysaccharide may be derived from a bacterial capsular polysaccharide antigen. Therefore, further, the polysaccharide conjugate vaccine may be a bacterial polysaccharide conjugate vaccine, such as a meningococcal polysaccharide conjugate vaccine, a pneumococcal polysaccharide conjugate vaccine, and the like. With respect to pneumococcal polysaccharide conjugate vaccines, 13-valent or higher pneumococcal polysaccharide conjugate vaccines are further preferred, including PCV13 (including 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F capsular polysaccharides), PCV15 (including 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, 33F capsular polysaccharides), PCV20 (including 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14, 15BC, 18C, 19A, 19F, 22F, 23F, 33F capsular polysaccharides), and PCV21 (including 1, 3, 4, 5, 6A, 6B, 7F, 8, 9V, 10A, 11A, 12F, 14 9F, 22F, 23F, 33F capsular polysaccharides), PCV24 (including 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, 33F capsular polysaccharides), etc. Optionally, it can be a monovalent conjugate vaccine (a carrier protein combined with one type of polysaccharide) or a multivalent conjugate vaccine (a carrier protein combined with two or more types of polysaccharides) in the form of a carrier protein used to bind the polysaccharide, and the carrier protein used to bind the polysaccharide can be selected from common carrier proteins such as TT, CRM197, DT, etc. Further preferably, the polysaccharide conjugate vaccine is PCV13, and the vaccination dose is increased in a specific manner as follows: based on the total amount of polysaccharides in the PCV13 vaccine, the first injection is 3±1 μg, the second injection is 6±1 μg, and optionally, the third injection is 9±1 μg; further preferably, the first injection is 3 μg, the second injection is 6 μg; optionally, the third injection is 9 μg.

[0012] Beneficial effects of the present invention:

[0013] In response to the problem of evaluating the immunogenicity of polysaccharide conjugate vaccines, the present invention innovatively proposes using weaned rabbits as animal models, immunizing the experimental animals multiple times with an ascending dose method, and then judging the immunogenicity of the candidate vaccine by serological detection of antibody titers. This method is different from the traditional practice of using adult animals as animal models and vaccinating with a constant dose. It has the advantages of being sensitive and efficient, and being more similar to the trend of clinical trials for infants and young children. It is particularly suitable for preclinical research on candidate vaccines for infants and young children. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Comparison of the immune effects of three different immunization doses in adult rabbits under a constant dose immunization program.

[0015] Figure 2 :Comparison of the first immunization effect of the same dose in adult rabbits and weaned rabbits under a constant dose immunization program.

[0016] Figure 3 :Comparison of the second immunization effect of the same dose in adult rabbits and weaned rabbits under a constant dose immunization program.

[0017] Figure 4 :Comparison of the immunization effects of the third injection of the same dose in adult rabbits and weaned rabbits under a constant dose immunization program.

[0018] Figure 5 : Antibody titers produced by weaned rabbits after the third immunization with constant and increasing doses.

[0019] Figure 6 : Comparison of the immunization trends in clinical trials of weaned rabbits with increasing doses, adult rabbits with constant doses, and infants. DETAILED DESCRIPTION

[0020] Example 1 Animal Model Immunization Dose Selection

[0021] 1.1 Experimental design:

[0022] This example studies the effects of different immunization doses on the immune effect of adult experimental animals. The experimental scheme is shown in Table 1, and is described in detail as follows:

[0023] Experimental materials: The 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13) was used, specifically the PCV13 vaccine produced by Pfizer and already on the market (trade name: Prevnar 13, code: FA1609CN1).

[0024] Experimental animals: Ordinary New Zealand rabbits were used as experimental animals, and adult rabbits (64-74 days old) were selected as immunized subjects, 10 rabbits per group.

[0025] Immunization dose: Three different single-injection doses are designed for adult New Zealand rabbits, namely 1.2μg, 6μg, and 15μg (calculated based on the total amount of each type of pneumococcal polysaccharide in PCV13). A constant dose is used, with a total of three injections.

[0026] Immunization procedure: Immunization injections were carried out on days 0, 14, and 28 of the experiment (Note: the day of the first immunization was defined as day 0), and the drug was administered into the hind leg muscle.

[0027] Serum collection: Blood was collected on the 42nd day (i.e., the 14th day after the third injection), with a blood volume of at least 3 mL per animal, for the purpose of detecting the antibody titer produced after the third injection.

[0028] Table 1: Constant dose immunization conditions for adult rabbits

[0029]

[0030] Note: In the above table, "dose" refers to the ratio of a single-injection dose to the single-injection immunization dose for adults, the same below.

[0031] 1.2 Serological testing:

[0032] Serum treatment: let the blood stand at 2-8℃ for 1 hour, centrifuge at 2500rpm for 10 minutes to separate the serum (repeat the centrifugation if necessary).

[0033] Serum test: Serum test adopts indirect ELISA test method, and various types of 13-valent pneumococcal polysaccharides (Pn1, Pn3, Pn4, Pn5, Pn6A, Pn6B, Pn7F, Pn9V, Pn14, Pn18C, Pn19A, Pn19F, Pn23F) are used as antigens to be coated on different ELISA plates, and then stored at 2-8°C overnight and returned to room temperature the next day for use. The pre-immunization serum, quality control serum, and test serum of each group were diluted 100 times, 50 times, and 50 times with absorption liquid, and incubated in a constant temperature incubator at 37°C for 1 hour for non-specific antibody absorption; after absorption, the quality control serum was diluted into two different multiples of high and low with diluent, and the test serum was diluted 2 times into different dilution multiples, and the diluted serum was added to the cleaned ELISA plate, and incubated in a constant temperature incubator at 37°C for 1.5 hours for antigen-antibody specific reaction. After the reaction, the ELISA plate was washed, goat anti-rabbit IgG antibody was added, and the plate was incubated in a constant temperature incubator at 37°C for 1 hour for enzyme-labeled antibody reaction. After the reaction, the plate was washed, substrate buffer was added, and the plate was reacted in the dark for 30 minutes at room temperature for color development, and then the stop solution was added. The plate was read at wavelengths of 405 nm and 690 nm on the ELISA reader, and the results were judged and calculated based on the OD value.

[0034] 1.3 Experimental results:

[0035] For the serum after three injections (i.e., serum collected on the 42nd day), the serum test results (antibody titers) corresponding to each type of capsular polysaccharide are shown in Table 2, and the corresponding result comparison chart is shown in Figure 1 .

[0036] Table 2: Serum test results of adult rabbits at different immunization doses

[0037]

[0038] The results showed that at different immunization doses in adult rabbits, significant immune responses were produced. However, the antibody titer corresponding to the 6μg / injection × 3 dose group was generally higher than that of the 1.2μg / injection × 3 dose group and the 15μg / injection × 3 dose group, indicating that the immunization dose of 6μg / injection × 3 was sufficient to effectively stimulate the immune system and produce higher serum antibodies. Therefore, 6μg / injection was used as the main immunization dose in subsequent studies.

[0039] Example 2 Animal Model Age Selection

[0040] Based on the results of Example 1, a constant dose administration program was adopted with 6 μg / injection as the immunization dose. In this study, weaned rabbits and adult rabbits were selected as different animal models to conduct research on the immune effects of different animal models.

[0041] 2.1 Experimental Design

[0042] This example studies two different animal models, adult rabbits and weaned rabbits, and the different immune effects under the same immune program and immune dose. The details of the scheme are shown in Table 3, which are described in detail as follows:

[0043] Experimental materials: The 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13) was used, specifically the PCV13 vaccine produced by Pfizer and already on the market (trade name: Prevnar 13, code: FA1609CN1).

[0044] Experimental animals: Ordinary New Zealand rabbits, adult rabbits (64-74 days old), weaned rabbits (30-40 days old), 10 rabbits / group.

[0045] Immunization dose: Three injections in total, the single injection dose is 6μg, and the total dose of three injections is 18μg.

[0046] Immunization procedure: Immunization injections were carried out on days 0, 14, and 28 of the experiment (Note: the day of the first immunization was defined as day 0), and the drug was administered into the hind leg muscles.

[0047] Serum collection: Blood was collected on the 14th and 28th days before immunization (i.e., the 14th day after the 1st and 2nd injections and before the next injection), and on the 42nd day (i.e., the 14th day after the 3rd injection). The blood volume was at least 3 mL per animal each time, which was used to detect the antibody titers produced after the 1st, 2nd, and 3rd injections, respectively.

[0048] Table 3: Constant dose immunization conditions for adult rabbits and weaned rabbits

[0049]

[0050] 2.2 Serum detection: The method and process are the same as in Example 1.

[0051] 2.3 Experimental results:

[0052] Table 4 shows the serum test results of adult rabbits and weaned rabbits on the 14th day after the first, second and third injections. Figure 2~Figure 4 .

[0053] Table 4: Serum test results of adult rabbits and weaned rabbits at the same immunization dose and different immunization times

[0054]

[0055] Figure 2 and Figure 3 It shows that under the same dosage and constant administration immunization program, the immunization effect of the first and second injections of weaned rabbits is not as good as that of adult rabbits, but Figure 4 The results showed that after the third injection, the immune effect of weaned rabbits was significantly improved, and the results of most serotypes were basically the same as or higher than those of adult rabbits. Only 4 serotypes had lower immune effects than adult rabbits. This shows that the young animal model can better reflect the differences between different immunization injections. As the immune system of young animals matures, they can produce a stronger immune response to immune stimulation. This further shows that a constant dose administration method is not necessarily the best immunization procedure for all age groups. Therefore, young animals will be selected as immunization subjects in subsequent studies to increase research on immunization plans.

[0056] Example 3 Constant-dose and increasing-dose immunization regimens

[0057] Based on the results of Example 1 and Example 2, weaned rabbits were used as the animal model. Considering that the immunization dose in the previous study was 6 μg / injection × 3, 18 μg was used as the total dose for three injections in this study to conduct a comparative study of constant dose and increasing dose immunization schemes.

[0058] 3.1 Experimental Design

[0059] This example studies the immune effects of two different immunization programs, a constant dose and an increasing dose, on weaned rabbits. The experimental scheme is detailed in Table 5 and is described in detail as follows:

[0060] Experimental materials: 13-valent pneumococcal polysaccharide conjugate vaccine (PCV13) was used, including the PCV13 vaccine produced by Pfizer (trade name: Prevnar 13, code: FA1609CN1), and the 13-valent pneumococcal conjugate vaccine prepared by the applicant (batch number: 202205003).

[0061] Experimental animals: Ordinary New Zealand rabbits, weaned rabbits (30-40 days old), 10 rabbits / group.

[0062] Immunization dose: three injections in total, with a total injection dose of 18μg, among which: the single injection of the constant dose group is constant at 6μg, and the 1st, 2nd and 3rd injections of the increasing dose group are 3, 6 and 9μg respectively.

[0063] Immunization procedure: Immunization injections were carried out on days 0, 14, and 28 of the experiment (Note: the day of the first immunization was defined as day 0), and the drug was administered into the hind leg muscle.

[0064] Serum collection: Blood was collected on the 42nd day (i.e., the 14th day after the third injection), with a blood volume of at least 3 mL per animal, for the purpose of detecting the antibody titer produced after the third injection.

[0065] Table 5: Immunization conditions of weaned rabbits in constant dose group and increasing dose group

[0066]

[0067] 3.2 Serum detection: The method and process are the same as in Example 1.

[0068] 3.3 Experimental results:

[0069] Table 6 shows the serum test results after the third injection in the constant dose group and the increasing dose group, and the corresponding comparison chart is shown in Figure 5 .

[0070] Table 6: Serological test results of weaned rabbits using constant or increasing immunization schedule

[0071]

[0072] Figure 5It shows that whether it is the applicant's sample group (202205003) or the Pfizer control group (FA1609CN1), the use of increasing doses of pneumococcal polysaccharide conjugate vaccine compared to constant doses generally stimulates more antibodies of various serotypes. Therefore, the immune effects of the sample group and the control group of the 13-valent pneumococcal conjugate vaccine in the form of increasing immunization doses are higher than those of the constant dose group, showing better immune effects, indicating that increasing the immunization dose as the immune system matures is more conducive to stimulating the immune system, making the immune system more sensitive to antigen stimulation, and more conducive to the evaluation of the immunogenicity of vaccine products.

[0073] Example 4 Analysis of trend consistency between animal experiments and clinical trials

[0074] Taking PCV13 as an example, this embodiment aims to compare the titer trends of various types of polysaccharide antibodies produced by the incremental dose immunization method of weaned rabbits proposed in the present invention and the traditional constant dose immunization method of adult rabbits, and the consistency with the clinical trial performance in infants and young children. Therefore, the GMC results of the clinical test data of PCV13 vaccine (Prevnar 13) disclosed by Pfizer for infants (6 months old) (Source: Pediatr Infect Dis J. 2016 Sep;35(9):999-1010., DOI: 10.1097 / INF.00000000000001248, Table 3 GMC data corresponding to Infant Series PCV13 [2,4,6 + 12-mo]) were compared with the antibody titer test results of adult rabbits immunized with Pfizer PCV13 vaccine at a constant dose of 6 μg for 3 times in Example 2 (the last column of data in Table 4), and the antibody titer test results of weaned rabbits immunized with Pfizer PCV13 vaccine at increasing doses of 3, 6, and 9 μg for 3 times in Example 3 (the last column of data in Table 6), and the distribution trend analysis was performed. The results are shown in Table 6. Figure 6 . Figure 6 The results showed that in terms of the distribution trend of serotypes Pn1, Pn3, Pn4, Pn5, and Pn6A, both adult rabbits and weaned rabbits were consistent with the serotype distribution trend of clinical infants and young children. However, compared with the adult rabbit animal model, the weaned rabbit animal model had two more serotypes that were consistent with the serotype distribution trend of clinical infants and young children, including Pn6B and Pn23F. This shows that the ascending dose immunization method of the weaned rabbit animal model can better reflect the antibody data trend of clinical trials of infant vaccines than the constant dose immunization method of the adult rabbit animal model. This further shows that the juvenile animal model combined with the ascending dose immunization method is more suitable for conducting preclinical immunogenicity research trials on vaccines for infants and young children.

[0075] The in-depth analysis and detailed description of the embodiments of the present invention are not intended to limit the scope of application of the present invention, but are only used as representatives to illustrate the process, results and application of the present invention. According to the embodiments of the invention described this time, all other implementation methods that can be derived or obtained by equivalent substitution by specific technicians in the same field without creative efforts are included in the scope of protection of the present invention.

Claims

1. A method for evaluating the immunogenicity of a pneumococcal polysaccharide conjugate vaccine for infants and young children based on animal experiments, characterized in that: Weaned rabbits were used as experimental subjects, and the vaccine was injected multiple times in a manner of increasing inoculation doses for immunization, and the immunogenicity of the vaccine was evaluated by serological detection of the antibodies produced; the weaned rabbits were 30-40 day old New Zealand rabbits.

2. The method according to claim 1, characterized in that The method of increasing the vaccination dose gradually means that, except for the first vaccination, each vaccination dose is 1.5 to 2.5 times the previous vaccination dose, calculated based on the total amount of polysaccharides in the vaccine.

3. The method according to claim 2, characterized in that The method of increasing the vaccination dose gradually means that, except for the first vaccination, each vaccination dose is twice the previous dose, calculated based on the total amount of polysaccharides in the vaccine.

4. The method according to claim 1, characterized in that: The multiple injections are 2, 3 or 4 injections.

5. The method according to claim 4, characterized in that The multiple injections are 3 injections.

6. The method according to claim 5, characterized in that The three injections are: immunizing the weaned rabbits on the 0th, 14th and 28th days of the experiment respectively.

7. The method according to claim 1, characterized in that The serological test refers to blood sampling for determination of neutralizing antibody titer.

8. The method according to claim 7, characterized in that The specific operation of the serological test is: on the 14th day after the last immunization, blood is collected to measure the neutralizing antibody titer.

9. The method according to claim 1, characterized in that: The pneumococcal polysaccharide conjugate vaccine is a pneumococcal polysaccharide conjugate vaccine with 13 valencies or more.

10. The method according to claim 9, characterized in that For the 13-valent pneumococcal polysaccharide conjugate vaccine, the vaccination dose is gradually increased as follows: based on the total amount of polysaccharide in the vaccine, the first injection is 3±1 μg, the second injection is 6±1 μg, and optionally, the third injection is 9±1 μg.

11. The method according to claim 10, characterized in that The specific method of increasing the vaccination dose is as follows: based on the total amount of polysaccharides in the vaccine, the first injection is 3 μg, the second injection is 6 μg, and the third injection is 9 μg.

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