A new coronavirus influenza combined vaccine and preparation method thereof

Abstract

The present invention discloses a novel coronavirus combined vaccine and its preparation method. The combined vaccine comprises the following antigenic components: a recombinant novel coronavirus RBD protein, hemagglutinin from an H1N1 influenza virus strain, hemagglutinin from an H3N2 influenza virus strain, hemagglutinin from a BY influenza virus strain, and hemagglutinin from a BV influenza virus strain. The novel coronavirus combined vaccine provided by the present invention has good compatibility of antigenic components, good immunogenicity, a low aluminum adjuvant content, low safety risks, is amenable to large-scale production, is highly compatible with viral mutations, and has promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of vaccine technology, and in particular relates to a new coronavirus influenza combined vaccine and a preparation method thereof. Background Art

[0002] Influenza is an acute respiratory illness caused by influenza A, B, and C viruses, respectively, and is classified as a Category C infectious disease. It is more common in winter and spring. Flu viruses are prone to mutation, are highly contagious, and are susceptible to infection, resulting in high morbidity and a serious threat to global public health. According to the WHO, of the 3 million to 5 million influenza cases worldwide each year, 290,000 to 650,000 deaths result. Annual influenza vaccination is the most direct and effective way to prevent the flu. Seasonal influenza vaccines are time-sensitive, and the WHO publishes circulating strains annually to ensure that flu vaccines have a high protection rate.

[0003] Both the novel coronavirus and influenza, caused by the flu virus, are respiratory infections. With the emergence of new seasonal flu strains, their co-circulation could pose a persistent threat. Furthermore, novel coronavirus and influenza strains could periodically emerge, potentially causing new pandemics. Therefore, developing a combined vaccine to reduce the number of injections, increase vaccination rates, and conserve medical and public health resources would improve the efficiency of the healthcare system and achieve a high level of protection against both the novel coronavirus and influenza through a single program.

[0004] Although companies such as Sinopharm and Sinovac, which have both COVID-19 and quadrivalent influenza vaccines, are simultaneously conducting safety and efficacy studies on the simultaneous administration of both vaccines (non-combination vaccines, where one dose of COVID-19 vaccine and one dose of quadrivalent influenza vaccine are administered separately), the only companies currently found conducting research on combined influenza and COVID-19 vaccines are Novavax and Moderna, using technologies such as recombinant protein vaccines and mRNA vaccines. Novavax's combined vaccine has entered the clinical research stage, while Moderna is also simultaneously developing a "three-in-one" combination vaccine for COVID-19, influenza, and RSV. Details are as follows:

[0005] Table 1 Global influenza and COVID-19 combined vaccine research status

[0006]

[0007]

[0008] However, how to prepare a combination vaccine with good compatibility of antigen components, good immunogenicity, low aluminum adjuvant content, low safety risk, simple preparation, low production conditions, and conducive to large-scale production and quality control, and good compatibility with virus mutations, still poses a great challenge and has become a technical problem that needs to be solved urgently. Summary of the Invention

[0009] In view of the shortcomings of the existing technology, the present invention provides a new coronavirus influenza combination vaccine and its preparation method, which has good compatibility of antigen components, good immunogenicity, low aluminum adjuvant content, low safety risk, easy industrialization, and good compatibility with virus mutations.

[0010] The present invention provides a new coronavirus influenza combination vaccine, comprising the following antigen components: recombinant novel coronavirus RBD protein, H1N1 influenza virus strain hemagglutinin, H3N2 influenza virus strain hemagglutinin, BY influenza virus strain hemagglutinin and BV influenza virus strain hemagglutinin.

[0011] According to a specific embodiment of the present invention, each 1 mL of the combination vaccine contains the following antigen components: 16-33 μg of recombinant novel coronavirus RBD protein, 10-20 μg of H1N1 influenza virus strain hemagglutinin, 10-20 μg of H3N2 influenza virus strain hemagglutinin, 10-20 μg of BY influenza virus strain hemagglutinin, and 10-20 μg of BV influenza virus strain hemagglutinin.

[0012] According to a specific embodiment of the present invention, it further contains 0.17 to 0.33 mg of aluminum ions.

[0013] According to a specific embodiment of the present invention, each 1 mL of the combined vaccine contains the following components: 25 μg of recombinant novel coronavirus RBD protein, 15 μg of H1N1 influenza virus strain hemagglutinin, 15 μg of H3N2 influenza virus strain hemagglutinin, 15 μg of BY influenza virus strain hemagglutinin, 15 μg of BV influenza virus strain hemagglutinin, and 0.25 mg of aluminum ions.

[0014] The second aspect of the present invention provides a method for preparing the aforementioned new coronavirus and influenza combination vaccine, comprising: diluting the recombinant new coronavirus RBD protein stock solution and the aluminum adjuvant solution separately, mixing them for adsorption, and obtaining a recombinant new coronavirus vaccine; mixing the monovalent stock solutions of H1N1, H3N2, BY, and BV viruses, and diluting them to obtain a quadrivalent influenza virus split vaccine; and mixing the recombinant new coronavirus vaccine with the quadrivalent influenza virus split vaccine to obtain a new coronavirus and influenza combination vaccine.

[0015] According to a specific embodiment of the present invention, in the recombinant novel coronavirus vaccine, the concentration of the recombinant novel coronavirus RBD protein is 50 μg / ml; in the quadrivalent influenza virus split vaccine, the concentrations of the H1N1 influenza virus strain hemagglutinin, the H3N2 influenza virus strain hemagglutinin, the BY influenza virus strain hemagglutinin, and the BV influenza virus strain hemagglutinin are all 30 μg / ml; the volume ratio of the recombinant novel coronavirus vaccine to the quadrivalent influenza virus split vaccine is 1:0.5~2.

[0016] According to a specific embodiment of the present invention, the recombinant novel coronavirus RBD protein stock solution is diluted with a histidine solution containing sodium chloride.

[0017] According to a specific embodiment of the present invention, the aluminum adjuvant solution is diluted with phosphate buffer.

[0018] According to a specific embodiment of the present invention, the adsorption time is 1 to 2 hours.

[0019] The third aspect of the present invention provides the use of the aforementioned new crown influenza combination vaccine in the preparation of a medicament for preventing or treating diseases caused by the new coronavirus and / or influenza virus. Specifically, the quadrivalent influenza virus split vaccine and the recombinant new coronavirus vaccine can be prepared and stored in a prefilled syringe and a vial, respectively. Before use, the quadrivalent influenza virus split vaccine in the prefilled syringe is injected into the vial of the recombinant new coronavirus vaccine for mixing, and then 0.25 to 1.0 mL is extracted using the prefilled syringe of the quadrivalent influenza virus split vaccine, and 0.25 to 1.0 mL is injected intramuscularly at a dose of 0.25 to 1.0 mL per person each time.

[0020] The beneficial effects of the present invention are:

[0021] The novel coronavirus influenza combination vaccine provided by the present invention has the following characteristics: 1) the antigen components have good compatibility and good immunogenicity: 1a) the immunogenicity of the novel coronavirus influenza combination vaccine as a booster immunization is not lower than that of the recombinant novel coronavirus vaccine and the quadrivalent influenza virus split vaccine, and the quadrivalent influenza virus split vaccine in the combination vaccine will not have a significant effect on the immunogenicity of the recombinant novel coronavirus vaccine. Similarly, the recombinant novel coronavirus vaccine in the combination vaccine will not have a significant effect on the immunogenicity of the quadrivalent influenza virus split vaccine; 1b) the aluminum adjuvant in the recombinant novel coronavirus vaccine can enhance the immunogenicity of the quadrivalent influenza virus split vaccine; 1c) after the components of the quadrivalent influenza virus split vaccine and the components of the recombinant novel coronavirus vaccine are mixed, the overall quality attributes of the vaccine do not undergo abnormal changes, and the two vaccines have good compatibility. 2) the aluminum ion content (0.18-0.33 mg / ml) is significantly lower than the aluminum ion content (0.6-1.0 mg / ml) in the existing combination vaccine, which reduces the side effects and safety risks of the vaccine. 3) the number of injections can be reduced and the vaccination rate can be increased.

[0022] The preparation method of the new coronavirus and influenza combination vaccine provided by the present invention is to first prepare influenza vaccine and new coronavirus vaccine separately and store them separately, and then mix them into one dose before use, which has the following advantages: 1) The two vaccines can be produced in different workshops according to this preparation method, reducing the redesign and transformation of the production workshop and achieving higher output; 2) Quality control is to conduct quality inspections on the two vaccines separately, which can better control quality; 3) Both influenza and new coronavirus themselves are very easy to mutate. When vaccines with single antigen components undergo immune escape and their protective effects decrease, the latest new coronavirus vaccine and influenza vaccine can be used for product iteration, greatly improving the protective effects against the currently prevalent new coronavirus and influenza virus strains. DETAILED DESCRIPTION

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] In the following examples, the required reagents are conventional experimental reagents purchased from commercial channels. Among them, the recombinant novel coronavirus RBD protein stock solution and the H1N1, H3N2, BY and BV virus monovalent stock solutions involved in the present invention are all provided by Anhui Zhifei Longcom Biopharmaceutical Co., Ltd.; the experimental methods not mentioned are conventional experimental methods.

[0025] Example 1

[0026] A new coronavirus influenza combined vaccine, each 1 mL contains the following components: 33.3 μg of recombinant novel coronavirus RBD protein, 10 μg of H1N1 influenza virus strain hemagglutinin, 10 μg of H3N2 influenza virus strain hemagglutinin, 10 μg of BY influenza virus strain hemagglutinin, 10 μg of BV influenza virus strain hemagglutinin, and 0.33 mg of aluminum ions.

[0027] The preparation method comprises the following steps:

[0028] 1. Preparation of recombinant novel coronavirus vaccine:

[0029] The recombinant novel coronavirus RBD protein stock solution was diluted with a histidine solution containing sodium chloride to obtain solution I; an aluminum hydroxide solution with an aluminum ion concentration of 1.5 mg / mL was diluted with a phosphate buffer to obtain solution II; solutions I and II were mixed and adsorbed for 1.5 hours to prepare a recombinant novel coronavirus vaccine with an RBD protein concentration of 50 μg / ml and an aluminum ion concentration of 0.5 mg / mL, which was stored in a vial;

[0030] 2. Preparation of quadrivalent influenza virus split vaccine:

[0031] The monovalent stock solutions of H1N1, H3N2, BY, and BV viruses were mixed and then diluted with phosphate buffer to a concentration of 30 μg / ml for each of the H1N1 influenza virus hemagglutinin, the H3N2 influenza virus hemagglutinin, the BY influenza virus hemagglutinin, and the BV influenza virus hemagglutinin to obtain a quadrivalent influenza virus split vaccine, which was stored in a prefilled syringe.

[0032] 3. Preparation of COVID-19 and influenza combined vaccine:

[0033] Before use, inject the quadrivalent influenza virus split vaccine in the prefilled syringe into the vial of recombinant novel coronavirus vaccine, and mix the recombinant novel coronavirus vaccine and quadrivalent influenza virus split vaccine in a volume ratio of 1:0.5 to make 1.0 mL to obtain the new coronavirus influenza combined vaccine.

[0034] Example 2

[0035] A novel coronavirus and influenza combination vaccine, containing the following components per 1 mL: 25 μg of recombinant novel coronavirus RBD protein, 15 μg of hemagglutinin from an H1N1 influenza virus strain, 15 μg of hemagglutinin from an H3N2 influenza virus strain, 15 μg of hemagglutinin from a BY influenza virus strain, 15 μg of hemagglutinin from a BV influenza virus strain, and 0.25 mg of aluminum ion. The preparation method differs from that in Example 1, except that in step 3, the volume ratio of the recombinant novel coronavirus vaccine to the quadrivalent influenza virus split vaccine is 1:1.

[0036] Example 3

[0037] A novel coronavirus and influenza combination vaccine, containing the following components per 1 mL: 16.67 μg of recombinant novel coronavirus RBD protein, 20 μg of hemagglutinin from an H1N1 influenza virus strain, 20 μg of hemagglutinin from an H3N2 influenza virus strain, 20 μg of hemagglutinin from a BY influenza virus strain, 20 μg of hemagglutinin from a BV influenza virus strain, and 0.17 mg of aluminum ion. The preparation method differs from that in Example 1, except that in step 3, the volume ratio of the recombinant novel coronavirus vaccine to the quadrivalent influenza virus split vaccine is 1:2.

[0038] 1. Immunogenicity Trial Study of the COVID-19 and Influenza Combined Vaccine

[0039] (1) 30 female SD rats aged 6 to 10 weeks at SFP level were selected and randomly divided into 5 groups, namely, group 1 of the new crown influenza combined vaccine embodiment, group 2 of the new crown influenza combined vaccine embodiment, group 3 of the new crown influenza combined vaccine embodiment, the recombinant new coronavirus vaccine group, and the quadrivalent influenza virus split vaccine group. Each group was first immunized with one injection of the recombinant new coronavirus vaccine as the basic immunization by intramuscular injection. After 28 days, the booster immunizations were performed with the combination vaccine of embodiment 1, the combination vaccine of embodiment 2, the combination vaccine of embodiment 3, the recombinant new coronavirus vaccine, and the quadrivalent influenza virus split vaccine, respectively. Blood was collected 14 days after the completion of the full immunization to separate serum, and the level of new coronavirus antibodies in the serum of SD rats was detected by IgG binding antibody titer, and the level of influenza antibodies in the serum of SD rats was detected by influenza hemagglutination inhibition test. The results are shown in Tables 2 and 3.

[0040] Table 2 Detection results of SARS-CoV-2-specific IgG binding antibody levels in SD rat serum

[0041]

[0042] Table 3 SD rat serum influenza hemagglutination inhibition results

[0043]

[0044]

[0045] As shown in Table 2, the recombinant novel coronavirus vaccine and the quadrivalent influenza virus split vaccine were mixed into 1.0 mL by volume before use, and the new crown influenza combination vaccine induced the production of new crown antibodies in SD rats at a level not lower than that of the recombinant novel coronavirus vaccine single vaccine group (although the value of the combined vaccine group in Example 3 was lower than that of the recombinant novel coronavirus vaccine single vaccine group, the value was within 2 times the detection error and there was no statistical difference (P>0.05)). As shown in Table 3, the recombinant novel coronavirus vaccine and the quadrivalent influenza virus split vaccine were mixed into 1.0 mL by volume before use, and the H1N1, H3N2, BV, and BY influenza antibodies induced in SD rats were not lower than those of the quadrivalent influenza virus split vaccine single vaccine group. In summary, the mixing ratio of recombinant new coronavirus vaccine: quadrivalent influenza virus split vaccine is 1:0.5 to 1:2, and then 1.0 mL is drawn out using a prefilled syringe of quadrivalent influenza virus split vaccine. The dose is 1.0 mL per person each time, and intramuscular injection has good immunogenicity.

[0046] (2) 30 female SD rats aged 6 to 10 weeks at SFP level were selected and randomly divided into 5 groups, namely the new crown influenza combined vaccine 0.25mL group, the new crown influenza combined vaccine 0.5mL group, the new crown influenza combined vaccine 1.0mL group, the recombinant new coronavirus vaccine group, and the quadrivalent influenza virus split vaccine group. Each group was first immunized with one shot of recombinant new coronavirus vaccine as basic immunity by intramuscular injection. After 28 days, booster immunizations were performed with 0.25mL of Example 2 combined vaccine, 0.5mL of Example 2 combined vaccine, 1.0mL of Example 2 combined vaccine, 0.5_mL of recombinant new coronavirus vaccine, and 0.5_mL of quadrivalent influenza virus split vaccine. Blood was collected 14 days after the full immunization to separate serum, and the new crown antibody level in SD rat serum was detected by IgG binding antibody titer, and the influenza antibody level in SD rat serum was detected by influenza hemagglutination inhibition test. The results are shown in Tables 4 and 5.

[0047] Table 4 Detection results of SARS-CoV-2-specific IgG binding antibody levels in SD rat serum

[0048]

[0049] Table 5 SD rat serum influenza hemagglutination inhibition results

[0050]

[0051] As shown in Table 4, the quadrivalent influenza virus split vaccine (prefilled syringe) was injected into the recombinant novel coronavirus vaccine (vial) for mixing, and then 0.25mL, 0.5mL, and 1.0mL were drawn out using a syringe to obtain the new coronavirus influenza combination vaccine. The level of new coronavirus antibodies induced in SD rats was no lower than that of the recombinant novel coronavirus vaccine single vaccine group (although the value of the 0.25mL combination vaccine group was lower than that of the recombinant novel coronavirus vaccine single vaccine group, the gap was within 2 times). As shown in Table 5, the quadrivalent influenza virus split vaccine (prefilled syringe) was injected into the recombinant novel coronavirus vaccine (vial) for mixing before use, and then 0.25mL, 0.5mL, and 1.0mL were drawn out using a syringe to obtain the new coronavirus influenza combination vaccine. The level of H1N1, H3N2, BV, and BY influenza antibodies induced in SD rats was no lower than that of the quadrivalent influenza virus split vaccine single vaccine group. In summary, the quadrivalent influenza virus split vaccine (prefilled syringe) is injected into the recombinant new coronavirus vaccine (vial) for mixing, and then 0.25mL to 1.0mL is extracted using the prefilled syringe of the quadrivalent influenza virus split vaccine. Intramuscular injection has good immunogenicity.

[0052] (III) 39 female BALB / c mice aged 6 to 10 weeks at SFP level were selected and randomly divided into 3 groups, namely the new crown influenza combined vaccine group of Example 2, the recombinant new coronavirus vaccine group, and the quadrivalent influenza virus split vaccine group. Each group was first immunized with three injections of recombinant new coronavirus vaccine as basic immunization by intramuscular injection. After 28 days, booster immunizations were performed on Example 2 combined vaccine, recombinant new coronavirus vaccine, and quadrivalent influenza virus split vaccine, respectively. Blood was collected 14 days after the full immunization was completed to separate serum, and the new crown antibody level in the mouse serum was detected by IgG binding antibody titer, pseudovirus neutralizing antibody detection, and true virus neutralizing antibody detection. The results are shown in Tables 6 to 8.

[0053] Table 6 Results of mouse specific IgG binding antibody (RBD) level detection

[0054] Booster group GMT Example 2 Combined vaccine group 3033843 Recombinant novel coronavirus vaccine group 3375251 Quadrivalent influenza virus split vaccine group 1161938

[0055] Table 7 Mouse pseudovirus (prototype strain) neutralizing antibody detection results

[0056] Booster group Neutralizing antibody level of prototype novel coronavirus pseudovirus (GMT) Example 2 Combined vaccine group 16732 Recombinant novel coronavirus vaccine group 13369 Quadrivalent influenza virus split vaccine group 5643

[0057] Table 8 Results of neutralizing antibody detection of mouse true virus (prototype strain, Omicron-BA.5 strain)

[0058] Booster group GMT value of neutralizing antibody level of prototype strain GMT value of neutralizing antibody level of Omicron-BA.5 strain Example 2 Combined vaccine group 3500 322 Quadrivalent influenza virus split vaccine group 723 70 Recombinant novel coronavirus vaccine group 2235 97

[0059] (1) As can be seen from Tables 6 and 7, 14 days after the full course of immunization, ① when the mice that had been immunized with three doses of the recombinant novel coronavirus vaccine were immunized again with the combined vaccine of Example 2, the levels of IgG binding antibodies (RBD) and pseudovirus (prototype strain) neutralizing antibodies were as high as 3033843 and 16732, which were comparable to the recombinant novel coronavirus vaccine group, with no statistical difference. This indicates that the immunogenicity of the combined vaccine against the novel coronavirus is not lower than that of the recombinant novel coronavirus vaccine group, and the quadrivalent influenza virus split vaccine in the combined vaccine does not have a significant effect on the immunogenicity of the recombinant novel coronavirus vaccine. ② Compared with the quadrivalent influenza virus split vaccine group, the levels of IgG binding antibodies (RBD) and pseudovirus (prototype strain) neutralizing antibodies in the combined vaccine group of Example 2 were 2.6 times and 3.0 times higher, respectively, indicating that the quadrivalent influenza and recombinant novel coronavirus combined vaccine has good immunogenicity (new coronavirus antigen) when used as a booster shot in mice that had been immunized with three doses of the recombinant novel coronavirus vaccine.

[0060] (2) As shown in Table 8, after 3 doses of recombinant novel coronavirus vaccine as basic immunization and 1 dose of vaccine as booster, the combined vaccine group induced BALB / c mice to produce prototype strain real virus neutralizing antibodies at a level of up to 3500, which was 4.8 times (**P < 0.01) and 1.6 times that of the quadrivalent influenza virus split vaccine group and the recombinant novel coronavirus vaccine group, respectively. This indicates that the combined vaccine can significantly induce BALB / c mice to produce prototype strain real virus neutralizing antibodies, and its immunogenicity against the prototype strain is not lower than that of the recombinant novel coronavirus vaccine group (no significant difference). The combined vaccine group also showed good immunogenicity against the current epidemic strain (Omicron-BA.5), with an EC50 value of 322, which was 4.6 times and 3.3 times that of the quadrivalent influenza virus split vaccine group and the recombinant novel coronavirus vaccine group, respectively. This indicates that the combined vaccine can induce BALB / c mice to produce Omicron-BA.5 strain real virus neutralizing antibodies, and its immunogenicity against Omicron-BA.5 strain is not lower than that of the recombinant novel coronavirus vaccine group.

[0061] (IV) Eighteen SFP-grade SD rats weighing 180 g to 220 g were selected and randomly divided into two groups: the recombinant novel coronavirus vaccine + the combined vaccine of Example 2 group and the normal saline + quadrivalent influenza virus split vaccine group. Each group was first immunized with the "+" pre-test substance by intramuscular injection. 28 days later, booster immunizations were performed with the combined vaccine of Example 2 and the quadrivalent influenza virus split vaccine, respectively. Blood was collected 7, 14, 21, and 28 days after the full course of immunization to separate serum, and the influenza antibody levels in the mouse serum were measured by hemagglutination inhibition method. The results are shown in Tables 9 to 12.

[0062] Table 9 SD rat serum hemagglutination inhibition results after 7 days

[0063]

[0064] Table 10 SD rat serum hemagglutination inhibition results after 14 days

[0065]

[0066] Table 11 SD rat serum hemagglutination inhibition results after 21 days

[0067]

[0068] Table 12 SD rat serum hemagglutination inhibition results after 28 days

[0069]

[0070] (1) As shown in Tables 9 to 12, 14 days after the full course of immunization, the combined vaccine group was able to stimulate SD rats to produce higher levels of H1N1, H3N2, BV, and BY antibodies, which were 6.3 times, 36.3 times, 7.2 times, and 5.0 times higher than those in the quadrivalent influenza virus split vaccine group, respectively. This indicates that the immunogenicity of the combined vaccine against influenza virus is better than that of the quadrivalent influenza virus split vaccine group. This may be because the presence of aluminum adjuvant enhances the immunogenicity of influenza antigens. In addition, the new coronavirus antibodies stimulated by rats immunized with three doses of recombinant new coronavirus vaccine will not affect the immunogenicity of sequential injections of quadrivalent influenza virus split vaccine, indicating that the combined vaccine has good immunogenicity (influenza antigen) when used as a booster shot in mice that have already been immunized with three doses of recombinant new coronavirus vaccine.

[0071] (2) As shown in Tables 9 to 12, after SD rats were immunized with one dose of prototype vaccine and then with the combined vaccine, they were able to induce the production of H1N1, H3N2, BV, and BY influenza antibodies in their bodies. Seven days after the second immunization, the combined vaccine group was 1.8 times, 2.0 times, 3.1 times, and 2.2 times the level of the quadrivalent influenza virus split vaccine group, respectively. Fourteen days after the second immunization, the combined vaccine group was 6.3 times, 36.3 times, 7.2 times, and 5.0 times the level of the saline + quadrivalent influenza virus split vaccine group, respectively. Twenty-one days after the second immunization, the combined vaccine group was 6.4 times, 71.3 times, 5.6 times, and 4.0 times the level of the quadrivalent influenza virus split vaccine group, respectively. Twenty-eight days after the second immunization, the combined vaccine group was 6.4 times, 81.4 times, 10.1 times, and 10.1 times the level of the quadrivalent influenza virus split vaccine group, respectively. The results showed that the combined vaccine as a booster shot after one basic immunization can promote the production of H1N1, H3N2, BV, and BY antibodies in rats, and is better than the quadrivalent influenza virus split vaccine single vaccine group. This may be because the presence of adjuvant enhances the immunogenicity of the rat body to influenza antigens, and the rate of inducing influenza antibody production is not lower than that of the quadrivalent influenza virus split vaccine single vaccine group.

[0072] (3) As shown in Tables 9 to 12, after SD rats were immunized with one dose of prototype vaccine and then with the combination vaccine, the titers of H1N1, H3N2, BV, and BY influenza antibodies produced by the SD rats increased over time. The H1N1 and H3N2 influenza antibodies reached their peak 21 days after the second dose of immunization, while there was no significant change at each time point in the quadrivalent influenza virus split vaccine group. This further proves that the presence of aluminum hydroxide adjuvant in the combination vaccine enhanced the immunogenicity of the rat body to influenza antigens, which may be due to the role of the adjuvant in sustained antigen release and non-specific immune stimulation.

[0073] 2. Safety Trial Study of COVID-19 and Influenza Combined Vaccine

[0074] In accordance with GLP requirements, a single-dose toxicity test in rats, a 4-week repeated-dose toxicity test in rats (accompanied by a rat immunogenicity test), and a rabbit intramuscular injection stimulation test were conducted to scientifically and objectively evaluate the safety of the novel coronavirus influenza combination vaccine (Example 2) of the present invention. The results are as follows:

[0075] (1) The results of the single-dose toxicity test showed that rats were intramuscularly injected with 2 doses / rat of the new crown influenza combined vaccine of the present invention and 1.0 mL / rat of placebo (containing aluminum) (adjuvant control substance). After administration, rats developed nodules at the administration site, but no other abnormal reactions were observed, and no rats died; there was no significant effect on the weight gain of rats. The results of pathological examination showed that all rats in the administration group and the adjuvant control group had obvious inflammatory reactions and adjuvant deposition foci at the injection site, lymph node hyperplasia and lymphatic sinus phagocytosis reaction at the administration site. The pathological changes in the two groups of rats were basically the same, which was considered to be a typical reaction of the injection of aluminum-containing adjuvants at the administration site. No abnormal lesions were found in other tissues and organs, and no toxic target organs were obviously displayed. Therefore, the maximum dosage of a single intramuscular injection of the new crown influenza combined vaccine of the present invention in rats is 2 doses / rat, and the maximum tolerated dose is > 2 doses / rat.

[0076] (2) The results of the 4-week repeated administration toxicity test showed that: under the experimental dose conditions, rats were intramuscularly injected with 1.0 mL (1 dose) of the quadrivalent influenza virus and recombinant novel coronavirus combined vaccine (combined vaccine group in Example 2) and 0.5 mL of the recombinant novel coronavirus protein vaccine (CHO cell) placebo (containing aluminum) (adjuvant control group) for 4 consecutive weeks (a total of 3 doses, 1 time / 2 weeks). The main manifestations were: similar nodules, inflammatory reactions and adjuvant deposition foci at the administration site, lymph node hyperplasia and lymphatic sinus phagocytosis at the administration site, and Fbg ↓ in the combined vaccine group after administration, which were similar to those in the adjuvant control group. The above abnormalities were considered to be common reactions to intramuscular injection of aluminum-containing adjuvant vaccines; some immune-related indicators were affected to a certain extent (neutrophil ↑, A / G ↓, IgG ↑, C3 ↑, CD 3+CD4+↑), which is considered to be related to the immune response caused by vaccination. The results of immunogenicity test showed that the combined vaccine of the present invention can stimulate SD rats to produce new coronavirus IgG antibodies (RBD) and higher levels of influenza virus H1N1, H3N2, BV, and BY antibodies after three injections, but the level of neutralizing antibodies (prototype strains) of the new coronavirus pseudovirus is low. Under the conditions of this experimental dose, rats were injected intramuscularly for 4 consecutive weeks (3 times in total) with the combined vaccine of the present invention. There was no obvious systemic toxicity effect, and the toxic target organs were not obviously shown. Except for the immune response and adjuvant-related local reactions to administration, the no adverse reaction dose (NOAEL) was 1.0mL (1 dose, each dose of combined vaccine contains 15μg of H1N1, H3N2, BV, and BY influenza virus strains hemagglutinin + 25μg of RBD protein) / rat.

[0077] (3) The results of the rabbit muscle stimulation test showed that: under the conditions of the test dose, according to the average score of the muscle stimulation reaction and the "Stimulation Level Judgment Standard", the combined vaccine of the present invention can cause mild muscle stimulation reaction in rabbits, which is basically consistent with the muscle stimulation reaction caused by the recombinant novel coronavirus vaccine and the recombinant novel coronavirus vaccine placebo (containing aluminum) in rabbits, and is considered to be a typical reaction of the injection of aluminum-containing adjuvant at the administration site; after a 14-day recovery period, the lesions showed recovery characteristics and trends (mild hyperplasia of interstitial connective tissue and regeneration of muscle fibers were visible), but were not fully repaired. The quadrivalent influenza virus split vaccine can cause mild muscle stimulation reaction in rabbits, and after a 14-day recovery period (the day after the autopsy 48 hours after drug withdrawal is defined as the first day of the recovery period), the reaction basically disappears.

[0078] In summary, the safety and effectiveness of the quadrivalent influenza virus and recombinant novel coronavirus combined vaccine of the present invention are in line with expectations.

[0079] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A new coronavirus influenza combined vaccine, characterized in that: Each 1 mL of the combined vaccine contains the following antigenic components: 25 μg of recombinant novel coronavirus RBD protein, 15 μg of hemagglutinin from H1N1 influenza virus strain, 15 μg of hemagglutinin from H3N2 influenza virus strain, 15 μg of hemagglutinin from BY influenza virus strain, 15 μg of hemagglutinin from BV influenza virus strain, and 0.25 mg of aluminum ion; The preparation method of the novel coronavirus influenza combined vaccine comprises: The recombinant novel coronavirus RBD protein stock solution and the aluminum adjuvant solution are diluted separately and then mixed for adsorption to obtain a recombinant novel coronavirus vaccine; The virus monovalent stock solutions of H1N1, H3N2, BY and BV are mixed and diluted to obtain a quadrivalent influenza virus split vaccine; Mix the recombinant new coronavirus vaccine and the quadrivalent influenza virus split vaccine in a volume ratio of 1:1 to obtain the new coronavirus and influenza combination vaccine.

2. A method for preparing the COVID-19 combined vaccine according to claim 1, characterized in that: The preparation method comprises: The recombinant novel coronavirus RBD protein stock solution and the aluminum adjuvant solution were diluted separately and then mixed for adsorption to obtain a recombinant novel coronavirus vaccine with a recombinant novel coronavirus RBD protein concentration of 50 μg / ml and an aluminum ion concentration of 0.5 mg / mL; The monovalent stock solutions of H1N1, H3N2, BY, and BV viruses were mixed and diluted to obtain a quadrivalent influenza virus split vaccine with a concentration of 30 μg / ml for each of the H1N1 influenza virus strain hemagglutinin, the H3N2 influenza virus strain hemagglutinin, the BY influenza virus strain hemagglutinin, and the BV influenza virus strain hemagglutinin; Mix the recombinant new coronavirus vaccine and the quadrivalent influenza virus split vaccine in a volume ratio of 1:1 to obtain the new coronavirus and influenza combination vaccine.

3. The preparation method according to claim 2, characterized in that Dilute the recombinant novel coronavirus RBD protein stock solution with histidine solution containing sodium chloride.

4. The preparation method according to claim 2, characterized in that The aluminum adjuvant solution was diluted with phosphate buffer.

5. The preparation method according to claim 2, characterized in that The adsorption time is 1 to 2 h.

6. Use of the COVID-19 and influenza combination vaccine according to claim 1 in the preparation of drugs for preventing or treating diseases caused by the new coronavirus and / or influenza virus.

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