A novel coronavirus influenza immunogenic composition and its preparation method and application

Abstract

The present invention relates to a novel coronavirus influenza immunogenic composition and a preparation method and application thereof. The novel coronavirus influenza immunogenic composition of the present invention contains a fusion protein comprising the RBD of the novel coronavirus S protein or a functionally active fragment thereof, and the NTD of the novel coronavirus S protein or a functionally active fragment thereof, and a human influenza virus immunogen comprising one or more human influenza virus hemagglutinin (HA) proteins. The immunogenic composition provided by the present invention and the novel coronavirus influenza combination vaccine prepared therefrom have good immunogenicity, can prevent infection by two viruses at the same time with one vaccination, are safe and efficient, have no immune interference, and there is no mutual inhibition between the two immunogenic component vaccines, and can be well compatible.

Description

Technical Field

[0001] The present application belongs to the field of biomedical engineering, and more specifically, relates to a novel coronavirus influenza immunogenic composition and a preparation method and application thereof. Background Art

[0002] The novel coronavirus infection is an acute respiratory infectious disease caused by the novel coronavirus (SARS-CoV-2). After the patient recovers, he or she will basically not be infected again in the short term. However, as the virus continues to mutate and the antibodies in the bodies of recovered patients or vaccinated people gradually decrease or even disappear, there is still a possibility of secondary infection. Therefore, people may need to continuously inject the new coronavirus vaccine or booster shot against a specific strain every year to achieve the purpose of continuous immunity.

[0003] Influenza, also known as flu, is an acute respiratory infection caused by influenza viruses. Annual flu vaccination is the most effective way to prevent influenza, which can significantly reduce the risk of influenza and serious complications.

[0004] The "Technical Guidelines for Influenza Vaccination in China (2022-2023)" points out that, based on the existing research and recommendations, after careful consideration, it is considered acceptable for people aged 18 and above to receive both inactivated influenza vaccines and new crown vaccines at the same time. When vaccinated at the same time, the vaccination sites on both sides of the limbs should be performed separately. The research results of Sinopharm Group China Biologic show that simultaneous vaccination with the new crown inactivated vaccine and the quadrivalent influenza vaccine can increase the chance of immunity in susceptible people, reduce the risk of co-infection and the risk of cross-infection in fever clinics, and improve the vaccination efficiency of vaccination clinics. However, if a new crown influenza combined vaccine can be developed to prevent infection with both viruses at the same time with one vaccination, a lot of manpower, material and financial resources can be saved.

[0005] Novavax, Inc. recently announced clinical data for its COVID-19 infection-influenza combination vaccine (CIC), which combines Novavax's COVID-19 vaccine NVX-CoV2373 (trimeric full-length S protein vaccine) and its quadrivalent nanoparticle influenza vaccine. Both protein-based vaccines use Matrix-M adjuvant. The trial showed that the combination vaccine was feasible, well tolerated and immunogenic. Compared with influenza vaccination alone, there was no difference in influenza antibody titers in the combined vaccination group, but antibody titers against SARS-CoV-2 decreased.

[0006] Therefore, developing a new coronavirus influenza combination vaccine with good immunogenicity, high safety, and no immune interference between immunogens is an urgent problem to be solved by technical personnel in this field. Summary of the invention

[0007] The purpose of the present invention is to provide a novel coronavirus influenza immunogenic composition and a preparation method and application thereof.

[0008] The technical solution of the present invention is as follows:

[0009] The first aspect of the present invention provides an immunogenic composition, which contains a fusion protein and a human influenza virus immunogen, wherein the fusion protein comprises 1) a receptor binding domain (RBD) of a SARS-CoV-2 S protein or a functionally active fragment thereof, and 2) an N-terminal domain (NTD) of a SARS-CoV-2 S protein or a functionally active fragment thereof; and the human influenza virus immunogen comprises one or more human influenza virus hemagglutinin (HA) proteins.

[0010] In some embodiments, the new coronavirus is derived from the prototype strain of the new coronavirus or a mutant thereof.

[0011] In some embodiments, the mutant of the prototype strain has a mutation at one or more amino acid positions among K417, L452, T478, E484, F486, F490, Q493, N501 and Y505.

[0012] In some embodiments, the mutant of the prototype strain comprises one or more amino acid substitutions of K417T / N, L452R / Q, T478K, E484K / Q / A, F486V, F490S, Q493R, N501Y and Y505H.

[0013] In some embodiments, the mutant of the prototype strain is the Alpha variant of the new coronavirus, the NTD contains Δ69H-70V and Δ144 amino acid mutations, and the RBD contains N501Y amino acid mutation.

[0014] In some embodiments, the mutant of the prototype strain is the Beta variant of the new coronavirus, the NTD contains L18F, D80A, D215G, Δ242L-244L and R246I amino acid mutations, and the RBD contains K417N, E484K and N501Y amino acid mutations.

[0015] In some embodiments, the mutant of the prototype strain is the Gamma variant of the new coronavirus, the NTD contains L18F, T20N, P26S, D138Y and R190S amino acid mutations, and the RBD contains K417T, E484K and N501Y amino acid mutations.

[0016] In some embodiments, the mutant of the prototype strain is the Delta variant of the new coronavirus, the NTD contains T19R, G142D, E156G, Δ157F-158R and A222V amino acid mutations, and the RBD contains K417N, L452R and T478K amino acid mutations.

[0017] In some embodiments, the mutant of the prototype strain is the SARS-CoV-2 Kappa variant, the NTD contains G142D and E154K amino acid mutations, and the RBD contains L452R and E484Q amino acid mutations.

[0018] In some embodiments, the mutant of the prototype strain is the SARS-CoV-2 Lambda variant, the NTD contains G75V, T76I and Δ246R-252G amino acid mutations, and the RBD contains L452Q and F490S amino acid mutations.

[0019] In some embodiments, the mutant of the prototype strain is the Mu variant of the new coronavirus, the NTD contains T95I, Y144S and Y145N amino acid mutations, and the RBD contains R346K, E484K and N501Y amino acid mutations.

[0020] In some embodiments, the mutant of the prototype strain is the SARS-CoV-2 Omicron-BA.1 variant, the NTD comprises A67V, Δ69 / 70, T95I, Y145D and L212I amino acid mutations, and the RBD comprises S339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y and Y505H amino acid mutations.

[0021] In some embodiments, the mutant of the prototype strain is the SARS-CoV-2 variant Omicron-BA.2, the NTD comprises T19I, Δ24 / 26, A27S, G142D and V213G amino acid mutations, and the RBD comprises S339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y and Y505H amino acid mutations.

[0022] In some embodiments, the mutant of the prototype strain is the SARS-CoV-2 Omicron-BA.4 / 5 variant, the NTD comprises T19I, Δ24 / 26, A27S, Δ69 / 70, G142D and V213G amino acid mutations, and the RBD comprises S339D, S371F, S373P, S375FT, 376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y and Y505H amino acid mutations.

[0023] In some embodiments, the fusion protein further comprises one or more polypeptides selected from the following groups: P2 or a functionally active fragment thereof, a foldon domain or a functionally active fragment thereof, ferritin or a functionally active fragment thereof, and hepatitis B surface antigen (HBsAg) or a functionally active fragment thereof.

[0024] In some embodiments, the P2 or a functionally active fragment thereof comprises an epitope peptide of tetanus toxin.

[0025] In some embodiments, the foldon domain or its functionally active fragment comprises the amino acid residues at the C-terminus of the phage T4 fiber protein. In some embodiments, the foldon domain in the present application comprises the amino acid sequence shown in SEQ ID NO:14.

[0026] In some embodiments, the ferritin or a functionally active fragment thereof comprises Trichoplusia ferritin or a functionally active fragment thereof.

[0027] In some embodiments, the Trichoplusia ni ferritin or a functionally active fragment thereof comprises the heavy chain or light chain of Trichoplusia ni ferritin.

[0028] In some embodiments, the ferritin or a functionally active fragment thereof comprises Helicobacter pylori ferritin or a functionally active fragment thereof.

[0029] In some embodiments, the amino acid sequence of the fusion protein is, from N-terminus to C-terminus, the amino acid sequence of NTD or its functionally active fragment, the amino acid sequence of RBD or its functionally active fragment, and the amino acid sequence of one or more polypeptides selected from P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen (HBsAg) or its functionally active fragment, which are directly connected or connected through a connecting sequence.

[0030] In some embodiments, the amino acid sequence of the fusion protein is, from N-terminus to C-terminus, the amino acid sequence of NTD or its functionally active fragment directly connected or connected through a connecting sequence, the amino acid sequence of RBD or its functionally active fragment, and the foldon domain.

[0031] In some embodiments, the linker sequence includes but is not limited to GGS or multiple tandem GGS (G and S represent glycine and serine, respectively).

[0032] In some embodiments, the amino acid sequence of the fusion protein is selected from SEQ ID NOs: 1-7.

[0033] In some embodiments, the new coronavirus is the new coronavirus prototype strain WH01, the NTD is positions 14-304 of the prototype strain S protein, the RBD is positions 319-541 of the prototype strain S protein, the fusion protein comprises the amino acid sequence shown in SEQ ID NO:1, and the nucleotide encoding it comprises the sequence shown in SEQ ID NO:12.

[0034] In some embodiments, the new coronavirus is a novel coronavirus Beta variant, and the fusion protein comprises the amino acid sequence shown in SEQ ID NO:2.

[0035] In some embodiments, the new coronavirus is the new coronavirus Gamma variant, and the fusion protein contains the amino acid sequence shown in SEQ ID NO:3.

[0036] In some embodiments, the new coronavirus is the new coronavirus Delta variant, and the fusion protein contains the amino acid sequence shown in SEQ ID NO:4.

[0037] In some embodiments, the new coronavirus is the new coronavirus Omicron-BA.1 strain, and the fusion protein comprises the amino acid sequence shown in SEQ ID NO:5.

[0038] In some embodiments, the new coronavirus is the new coronavirus Omicron-BA.2 strain, and the fusion protein comprises the amino acid sequence shown in SEQ ID NO:6.

[0039] In some embodiments, the new coronavirus is the new coronavirus Omicron-BA.4 / 5 strain, and the fusion protein comprises the amino acid sequence shown in SEQ ID NO:7.

[0040] The beneficial effect of adopting the above technical solution is that the novel coronavirus fusion protein adopted by the present invention is a fusion protein obtained by recombining one or more polypeptides selected from the N-terminal domain, receptor binding domain and P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, and hepatitis B surface antigen (HBsAg) or its functionally active fragment of the novel coronavirus S protein. Compared with the full-length S protein antigen, the fusion protein antigen has more key epitopes, stronger immunogenic conversion potential and higher protein yield. Compared with the RBD subunit vaccine, the fusion protein antigen contains more conservative epitopes and has better cross-protection effect on newly emerging variants.

[0041] In some embodiments, the human influenza virus immunogen comprises hemagglutinin (HA) proteins from H1N1, H3N2, and B human influenza viruses.

[0042] According to the WHO's introduction to seasonal influenza viruses, seasonal influenza viruses are divided into four genera, each with only one species: A (Influenza A viruses, IAV), B (Influenza B viruses, IBV), C (Influenza C viruseses, ICV), and D (Influenza D viruses, IDV). Influenza A virus is the culprit for causing severe influenza symptoms in humans and is also the main pathogen causing the global pandemic. According to the differences in hemagglutinin (HA) protein and neuraminidase (NA) protein, influenza A virus is divided into different subtypes. As of 2019, there are 18 subtypes based on HA protein and 11 subtypes based on NA protein, of which only H1-3 and N1-2 subtypes can be transmitted in humans. Among them, the subtypes currently prevalent in humans are mainly A (H1N1) and A (H3N2). Influenza B virus mainly infects humans and is not divided into subtypes, but into lineages. The lineages currently prevalent in humans are mainly B / Yamagata or B / Victoria. Influenza C virus is rarely detected, and the symptoms it causes are mild, posing less of a threat to public health. Influenza D virus mainly infects cattle, pigs and other animals. The RNA of influenza A and B viruses consists of 8 segments. The 1st, 2nd and 3rd segments encode RNA polymerase, and the 4th segment encodes hemagglutinin; the 5th segment encodes nucleoprotein, and the 6th segment encodes neuraminidase; the 7th segment encodes matrix protein, and the 8th segment encodes a non-structural protein that can splice RNA.

[0043] The human influenza virus immunogen of the present invention can be prepared by chicken embryo culture technology, cell culture technology and recombinant technology.

[0044] In some embodiments, the hemagglutinin (HA) protein is from an influenza virus split vaccine, an influenza virus inactivated vaccine, an influenza live attenuated vaccine, or an influenza virus subunit vaccine.

[0045] The World Health Organization (WHO) will announce the components of the influenza vaccines for the northern and southern hemispheres in the first and second half of each year. For example, the recommended components of the 2022-2023 northern hemisphere seasonal influenza vaccine are: the influenza strains contained in the quadrivalent egg-cultured vaccine are an A / Victoria / 2570 / 2019(H1N1)pdm09-like virus; (EPI_ISL_417210); an A / Darwin / 9 / 2021(H3N2)-like virus; (EPI_ISL_2233240); a B / Austria / 1359417 / 2021(B / Victorialineage)-like virus; (EPI_ISL_2378894) and a B / Phuket / 3073 / 2013(B / Yamagatalineage)-like virus; (EPI_ISL_168822).

[0046] The influenza strains included in the quadrivalent cell culture or recombinant protein vaccine are an A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus; (EPI_ISL_404460); an A / Darwin / 6 / 2021(H3N2)-like virus; (EPI_ISL_2233238); a B / Austria / 1359417 / 2021(B / Victoria lineage)-like virus; (EPI_ISL_2378894) and a B / Phuket / 3073 / 2013(B / Yamagata lineage)-like virus; (EPI_ISL_161843).

[0047] The influenza strains contained in the trivalent egg-grown vaccine are an A / Victoria / 2570 / 2019(H1N1)pdm09-like virus; (EPI_ISL_417210); an A / Darwin / 9 / 2021(H3N2)-like virus; (EPI_ISL_2233240) and a B / Austria / 1359417 / 2021(B / Victoria lineage)-like virus; (EPI_ISL_2378894).

[0048] The influenza strains included in the trivalent cell culture or recombinant protein vaccine are an A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus; (EPI_ISL_404460); an A / Darwin / 6 / 2021(H3N2)-like virus; (EPI_ISL_2233238) and a B / Austria / 1359417 / 2021(B / Victoria lineage)-like virus; (EPI_ISL_2378894).

[0049] Recommended components of the 2021-2022 Northern Hemisphere seasonal influenza vaccine: The influenza strains contained in the trivalent egg-cultured vaccine are an A / Victoria / 2570 / 2019(H1N1)pdm09-like virus; (EPI_ISL_417210); an A / Cambodia / e0826360 / 2020(H3N2)-like virus; (EPI_ISL_806547) and a B / Washington / 02 / 2019(B / Victoria lineage)-like virus; (EPI_ISL_352076).

[0050] The influenza strains included in the trivalent cell culture or recombinant protein vaccine are an A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus; (EPI_ISL_404460); an A / Cambodia / e0826360 / 2020(H3N2)-like virus; (EPI_ISL_944639) and a B / Washington / 02 / 2019(B / Victoria lineage)-like virus; (EPI_ISL_347829).

[0051] In some specific embodiments, the present invention adopts a quadrivalent recombinant influenza vaccine, which refers to the influenza strains that the recombinant quadrivalent vaccine recommended by the WHO for use in the 2021-2022 northern hemisphere influenza season should contain: A / Wisconsin / 588 / 2019(H1N1)pdm09-like virus; (EPI_ISL_404460); A / Cambodia / e0826360 / 2020(H3N2)-like virus; (EPI_ISL_944639); B / Washington / 02 / 2019(B / Victoria lineage)-like virus; (EPI_ISL_347829) and B / Phuket / 3073 / 2013(B / Yamagata lineage)-like virus; (EPI_ISL_161843).

[0052] In some embodiments, the HA proteins of human influenza viruses H1N1, H3N2, B / Victoria and B / Yamagata in the quadrivalent recombinant influenza vaccine comprise the amino acid sequences shown in SEQ ID NOs: 8-11.

[0053] In some specific embodiments, the present invention adopts a commercially available quadrivalent influenza virus split vaccine, which refers to the influenza strains that should be included in the quadrivalent chicken embryo influenza vaccine recommended by the WHO for the 2021-2022 northern hemisphere influenza season: A / Victoria / 2570 / 2019(H1N1)pdm09-like virus; (EPI_ISL_417210); A / Cambodia / e0826360 / 2020(H3N2)-like virus; (EPI_ISL_806547); B / Washington / 02 / 2019(B / Victoria lineage)-like virus; (EPI_ISL_352076) and B / Phuket / 3073 / 2013(B / Yamagata lineage)-like virus; (EPI_ISL_168822).

[0054] The production stage of the quadrivalent influenza virus split vaccine is strictly prepared in accordance with the requirements of the current edition of the "Chinese Pharmacopoeia". After the inspection is qualified, the semi-finished product is prepared. The inspection of the finished product is carried out in accordance with the requirements of the current edition of the "Chinese Pharmacopoeia" to detect whether its hemagglutinin titer and thermal stability test meet the pharmacopoeia standards. The preparation of the quadrivalent influenza virus split vaccine can usually be firstly inoculated with four influenza strains of H1N1, H3N2, B / Victoria and B / Yamagata recommended by WHO and approved by the State Council's drug supervision and administration department. 9-11 days old healthy chicken embryos are obtained after culture and cold embryos. Then it is made after virus inactivation, purification and lysis, which is easily obtained by technicians in this field. Specifically, according to the provisions of the 2020 edition of the "Chinese Pharmacopoeia" Part III-Influenza Virus Split Vaccine, chicken embryos for production: virus seed passage and preparation should come from SPF chickens. Chicken embryos for vaccine production should come from healthy chickens raised in closed houses, and 9-11 days old chicken embryos with no deformities, clear blood vessels and activity should be selected. Name and source of virus strains: The virus strains used in production are influenza A and B virus strains recommended and provided by WHO. Establishment of seed batches: It should comply with the provisions of "Management and quality control of bacteria and virus strains used for production and testing of biological products". Based on the generations of influenza strains recommended and provided by WHO, the master seed batch and working seed batch are established by passage, and the total passage of the finished vaccine virus shall not exceed 5 generations. Verification of seed batches: The master seed batch shall be subjected to the following comprehensive verification, and the working seed batch shall be subjected to at least 2.2.3.1 to 2.2.3.5 verification. Identification of hemagglutinin type: The hemagglutination inhibition test is performed using the specific immune serum of the corresponding (sub) type of influenza virus, and the results should prove that its antigenicity is consistent with the recommended virus strain. The virus titer is checked by the chicken embryo half infectious dose method (EID50), and the virus titer should not be less than 6.5lg EID50 / ml. The hemagglutination titer is detected by hemagglutination method and should not be less than 1:160.

[0055] Preparation of monovalent stock solution: (1) Virus inoculation and culture: Inoculate the working seed batch virus with appropriate dilution into the allantoic cavity of chicken embryos and culture at an appropriate temperature. The working seed batch virus that has not been used up once shall not be refrozen for further use. (2) Virus harvesting: Select the live chicken embryos, cool the embryos at 2-8°C for a certain period of time, and then harvest the allantoic fluid in a container. Take samples from each container for testing of the allantoic harvest fluid. (3) Merge the allantoic harvest fluid: The allantoic fluid containing a single influenza virus that has passed the test in each harvest container can be merged into a monovalent virus pool. (4) Virus inactivation: Add formaldehyde to the monovalent virus pool to inactivate the virus. The specific process parameters, including the protein content of the harvest fluid and the formaldehyde concentration, shall be implemented as approved. After the inactivation period expires, each virus inactivation container should be sampled immediately for virus inactivation verification tests and bacterial endotoxin content determination (it is also possible to add a formaldehyde solution of appropriate concentration to inactivate the virus after or during purification). (5) Virus inactivation verification test: The virus inactivation sample was diluted 10 times in series, and the original, 10-1 and 10-2 times diluted virus liquid was inoculated into the allantoic cavity of chicken embryos in groups. Each group was inoculated with 10 9-11 day old chicken embryos, and 0.2 ml was inoculated for each embryo. The chicken embryos were cultured at 33-35℃ for 72 hours. Those that died within 24 hours were not counted. At least 80% of the chicken embryos in each group must survive. 0.5 ml of allantoic fluid was taken from each surviving chicken embryo, mixed by group, and blindly propagated for one generation. Each group was inoculated with 10 embryos, and 0.2 ml was inoculated for each embryo. After 72 hours of culture at 33-35℃, the allantoic fluid was taken for hemagglutination test. The result should show no hemagglutination reaction. (6) Concentration and purification: 1) Ultrafiltration concentration: After the monovalent virus pool was clarified by centrifugation or other appropriate methods, the virus liquid was concentrated to an appropriate protein content range by ultrafiltration. The concentrated virus liquid should be sampled for bacterial endotoxin content determination. 2) Purification: The monovalent virus pool after ultrafiltration and concentration can be purified by column chromatography or sucrose density gradient centrifugation. When the sucrose density gradient centrifugation is used for purification, ultrafiltration should be used to remove sucrose. After purification, samples are taken for protein content determination. (7) Virus lysis: Virus lysis should be performed within the specified protein content range. Add a lysis agent of appropriate concentration to the purified monovalent virus pool and perform virus lysis under appropriate conditions. (8) Purification after lysis: Column chromatography, sucrose density gradient centrifugation or other appropriate methods are used for re-purification after virus lysis. When the sucrose density gradient centrifugation is used for purification, ultrafiltration should be used to remove sucrose. After ultrafiltration, the virus liquid is sampled for bacterial endotoxin content determination and microbial limit test. The bacterial count in the microbial limit test should be less than 10 CFU / ml. (9) Sterilization filtration: After sterilization filtration, the purified virus lysate can be added with thimerosal of appropriate concentration as an antibacterial agent to obtain the monovalent stock solution. (10) Assay of monovalent stock solution. (11) Storage: Store at 2-8°C.

[0056] Preparation of semi-finished products: According to the hemagglutinin content of each monovalent stock solution, each type of influenza virus is prepared into a semi-finished product with the same hemagglutinin content (the hemagglutinin preparation amount can be in the range of 30-36μg / ml, and each type of influenza virus strain should be prepared with the same hemagglutinin content every year), and thimerosal in an appropriate concentration can be added as an antibacterial agent to obtain a semi-finished product.

[0057] Domestically produced quadrivalent influenza virus split vaccines that have been on the market include those produced by Wuhan Institute of Biological Products Co., Ltd. (National Medicine Approval No. S20200007; S20227010), Beijing Kexing Biological Products Co., Ltd. (National Medicine Approval No. S20200010), Jiangsu Jindik Biotechnology Co., Ltd. (National Medicine Approval No. S20190026; S20227012), Changchun Institute of Biological Products Co., Ltd. (National Medicine Approval No. S20200003), Hualan Biological Vaccine Co., Ltd. (National Medicine Approval No. S20220005; S20180006), Shanghai Institute of Biological Products Co., Ltd. (National Medicine Approval No. S20210007) and Beijing Kexing Biological Products Co., Ltd. (National Medicine Approval No. S20217013), etc.

[0058] The trivalent influenza vaccine can be used to prevent influenza caused by H1N1, H3N2 and B / Victoria influenza viruses. The quadrivalent vaccine has one more type than the trivalent vaccine, namely B / Yamagata influenza virus. The trivalent influenza virus split vaccine and the quadrivalent influenza virus split vaccine are exactly the same in the bulk production stage.

[0059] In some embodiments, the immunogenic composition comprises 20-100 μg of NTD-RBD-foldon fusion protein and 15-80 μg of human influenza virus hemagglutinin (HA) protein.

[0060] In some embodiments, the immunogenic composition comprises 30-60 μg of NTD-RBD-foldon fusion protein and 15-50 μg of human influenza virus hemagglutinin (HA) protein.

[0061] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein and 15 μg of H1N1 influenza virus hemagglutinin (HA) protein.

[0062] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein, 15 μg of H1N1 influenza virus hemagglutinin (HA) protein, and 15 μg of H3N2 influenza virus hemagglutinin (HA) protein.

[0063] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein, and 15 μg of H1N1 influenza virus hemagglutinin (HA) protein, 15 μg of H3N2 influenza virus hemagglutinin (HA) protein, and 15 μg of B / Victoria influenza virus hemagglutinin (HA) protein.

[0064] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein, and 15 μg of H1N1 influenza virus hemagglutinin (HA) protein, 15 μg of H3N2 influenza virus hemagglutinin (HA) protein, 15 μg of B / Victoria influenza virus hemagglutinin (HA) protein, and 15 μg of B / Yamagata influenza virus hemagglutinin (HA) protein.

[0065] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein and 45 μg of H1N1 influenza virus hemagglutinin (HA) protein.

[0066] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein, 45 μg of H1N1 influenza virus hemagglutinin (HA) protein, and 45 μg of H3N2 influenza virus hemagglutinin (HA) protein.

[0067] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein, and 45 μg of H1N1 influenza virus hemagglutinin (HA) protein, 45 μg of H3N2 influenza virus hemagglutinin (HA) protein, and 45 μg of B / Victoria influenza virus hemagglutinin (HA) protein.

[0068] In some embodiments, the immunogenic composition comprises 40 μg of NTD-RBD-foldon fusion protein, and 45 μg of H1N1 influenza virus hemagglutinin (HA) protein, 45 μg of H3N2 influenza virus hemagglutinin (HA) protein, 45 μg of B / Victoria influenza virus hemagglutinin (HA) protein, and 45 μg of B / Yamagata influenza virus hemagglutinin (HA) protein.

[0069] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein and 15 μg of H1N1 influenza virus hemagglutinin (HA) protein.

[0070] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein, 15 μg of H1N1 influenza virus hemagglutinin (HA) protein, and 15 μg of H3N2 influenza virus hemagglutinin (HA) protein.

[0071] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein, and 15 μg of H1N1 influenza virus hemagglutinin (HA) protein, 15 μg of H3N2 influenza virus hemagglutinin (HA) protein, and 15 μg of B / Victoria influenza virus hemagglutinin (HA) protein.

[0072] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein, and 15 μg of H1N1 influenza virus hemagglutinin (HA) protein, 15 μg of H3N2 influenza virus hemagglutinin (HA) protein, 15 μg of B / Victoria influenza virus hemagglutinin (HA) protein, and 15 μg of B / Yamagata influenza virus hemagglutinin (HA) protein.

[0073] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein and 45 μg of H1N1 influenza virus hemagglutinin (HA) protein.

[0074] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein, 45 μg of H1N1 influenza virus hemagglutinin (HA) protein, and 45 μg of H3N2 influenza virus hemagglutinin (HA) protein.

[0075] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein, and 45 μg of H1N1 influenza virus hemagglutinin (HA) protein, 45 μg of H3N2 influenza virus hemagglutinin (HA) protein, and 45 μg of B / Victoria influenza virus hemagglutinin (HA) protein.

[0076] In some embodiments, the immunogenic composition comprises 50 μg of NTD-RBD-foldon fusion protein, and 45 μg of H1N1 influenza virus hemagglutinin (HA) protein, 45 μg of H3N2 influenza virus hemagglutinin (HA) protein, 45 μg of B / Victoria influenza virus hemagglutinin (HA) protein, and 45 μg of B / Yamagata influenza virus hemagglutinin (HA) protein.

[0077] The beneficial effect of using NTD-RBD-foldon fusion protein and HA protein from quadrivalent recombinant influenza vaccine to form an immunogenic composition is that the quadrivalent recombinant influenza vaccine has good tolerability, immunogenicity, protection durability, cross-protection ability and production scalability, while having higher biosafety.

[0078] The beneficial effect of using NTD-RBD-foldon fusion protein and HA protein from quadrivalent influenza virus split vaccine to form an immunogenic composition is that the production process of the quadrivalent influenza virus split vaccine used is mature and easy to obtain, which is conducive to shortening the vaccine production cycle and saving research and development costs when an epidemic breaks out.

[0079] In some embodiments, the immunogenic composition further comprises a lyoprotectant.

[0080] In some embodiments, the lyoprotectant is prepared by mixing 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80 and 10 mM PB buffer (Na 2 HPO 4 , NaH 2 PO 4 ) is prepared according to the formula.

[0081] The second aspect of the present invention provides a new crown-influenza combined protein vaccine, which comprises the immunogenic composition described in the first aspect and an adjuvant, and the adjuvant is selected from one or more of aluminum adjuvant, MF59, MPL, QS-21, GLA, CpG, PolyI:C, AS01, AS02, AS03, and AS04 adjuvants, preferably AS03 or MF59 adjuvant.

[0082] In some embodiments, a dose of AS03 adjuvant for one human is 0.25 ml, and the ingredients include 4.86 mg polysorbate 80 (Tween 80), 10.69 mg squalene and 11.86 mg α-tocopherol.

[0083] In some embodiments, a human dose of MF59 adjuvant is 0.25 ml, and the ingredients include 1% squalene, 0.5% Tween 80 and 0.5% Span 85.

[0084] In some embodiments, the protein vaccine comprises 30-60 μg of NTD-RBD-foldon fusion protein, 15-50 μg of each type of HA protein, and AS03 adjuvant.

[0085] In some embodiments, the protein vaccine comprises 40 μg of NTD-RBD-foldon fusion protein, 15 μg of each type of HA protein, and AS03 adjuvant.

[0086] In some embodiments, the protein vaccine comprises 40 μg of NTD-RBD-foldon fusion protein, 45 μg of each type of HA protein, and AS03 adjuvant.

[0087] In some embodiments, the protein vaccine comprises 50 μg of NTD-RBD-foldon fusion protein, 15 μg of each type of HA protein, and AS03 adjuvant.

[0088] In some embodiments, the protein vaccine comprises 50 μg of NTD-RBD-foldon fusion protein, 45 μg of each type of HA protein, and AS03 adjuvant.

[0089] The third aspect of the present invention provides a method for preparing the immunogenic composition of the first aspect, comprising the following steps:

[0090] S1. Prepare an antigen stock solution 1 containing a fusion protein of the new coronavirus;

[0091] S2, preparing an antigen stock solution 2 containing H1N1, H3N2 and B human influenza virus immunogens;

[0092] S3. Mix the antigen stock solution 1 and the stock solution 2 to obtain the immunogenic composition.

[0093] In some embodiments, S1 specifically comprises: constructing a recombinant expression plasmid using a nucleotide sequence encoding the novel coronavirus fusion protein; transforming the constructed recombinant expression plasmid into a host bacterium and screening the correct recombinant expression plasmid; transfecting mammalian host cells with the screened recombinant expression plasmid for expression; collecting the cell culture supernatant and purifying to obtain the antigen stock solution 1 containing the novel coronavirus fusion protein.

[0094] In some embodiments, when the human influenza virus immunogen in S2 is derived from an influenza virus subunit vaccine, the specific preparation method is: inserting the nucleotide sequence encoding the influenza virus rHA into the pFastBac series vector, and transforming the competent cells containing the baculovirus genome, so that the nucleotide sequence of rHA is inserted into the baculovirus genome through homologous recombination to obtain the recombinant baculovirus genome rBacmid; transfecting the rBacmid into insect cells, culturing and collecting the supernatant by centrifugation to obtain the recombinant baculovirus seed virus; infecting the insect cells with the recombinant baculovirus, culturing and collecting the precipitate by centrifugation; lysing the cells and collecting the lysate, and purifying to obtain the influenza virus antigen stock solution 2 containing various types of rHA proteins.

[0095] In some embodiments, when the human influenza virus immunogen in S2 is derived from an influenza virus split vaccine, those skilled in the art can prepare antigen stock solutions 2 containing various types of human influenza virus immunogens according to the requirements of the current edition of the Chinese Pharmacopoeia. Usually, the H1N1, H3N2 and B influenza strains recommended by the WHO and approved by the State Council's drug regulatory department can be used to inoculate healthy chicken embryos aged 9-11 days, culture and freeze the embryos to obtain virus liquid, and then inactivate the virus, purify and lyse the virus to prepare influenza virus antigen stock solutions 2 containing various types of HA proteins.

[0096] In some embodiments, the antigen stock solution 2 is directly prepared by using a commercially available influenza virus split vaccine. Domestically produced quadrivalent influenza virus split vaccines that have been listed include, for example, Wuhan Institute of Biological Products Co., Ltd. (National Medicine Standard S20200007; S20227010), Beijing Kexing Biological Products Co., Ltd. (National Medicine Standard S20200010), Jiangsu Jindik Biotechnology Co., Ltd. (National Medicine Standard S20190026; S20227012), Changchun Institute of Biological Products Co., Ltd. (National Medicine Standard S20200003), Hualan Bio Vaccine Co., Ltd. (National Medicine Standard S20220005; S20180006), Shanghai Institute of Biological Products Co., Ltd. (National Medicine Standard S20210007) and Beijing Kexing Biological Products Co., Ltd. (National Medicine Standard S20217013).

[0097] There is no specific order relationship between S1 and S2, and S1 and S2 can be performed in any order or simultaneously. In some embodiments, S3 further comprises additional steps such as adding a lyoprotectant and freeze drying.

[0098] The fourth aspect of the present invention provides a combined COVID-19 and influenza DNA vaccine, which comprises a DNA sequence encoding the immunogenic composition described in the first aspect.

[0099] The fifth aspect of the present invention provides a combined COVID-19 and influenza mRNA vaccine, which comprises an mRNA sequence encoding the immunogenic composition described in the first aspect.

[0100] The sixth aspect of the present invention provides a broad-spectrum immunogenic composition, which comprises the immunogenic composition described in the first aspect of the present invention and at least one RSV antigenic polypeptide selected from the following group: RSV glycoprotein G or its immunogenic fragment, glycoprotein F or its immunogenic fragment, glycoprotein SH, nucleocapsid protein N or its immunogenic fragment and matrix (M) protein or its immunogenic fragment.

[0101] In some embodiments, the glycoprotein F comprises at least one modification or mutation that enhances its immunogenicity compared to unmodified RSV glycoprotein F. The modification or mutation comprises substitution, deletion or insertion of one or more amino acids, for example, one or more of deletion of a transmembrane domain, inactivation of an intervening furin cleavage site, engineered disulfide bond mutation, cavity filling mutation and electrostatic mutation.

[0102] The engineered disulfide bond mutations include, but are not limited to, S55C and L188C; S155C and S290C; T103C and I148C; and L142C and N371C.

[0103] The cavity filling mutations include but are not limited to S62I, S190F, T54Y, T219I, G151A, A147L, V164I, V192H, V207L ​​and V300Y.

[0104] The F1 polypeptide and F2 polypeptide of the recombinant RSV soluble F protein into which one or more mutations are introduced can be derived from any wild-type RSV F protein known in the art or discovered in the future, including but not limited to the amino acid sequences of RSV subtype A and subtype B strains (including A2 Ontario and Buenos Aires) or any other subtype F proteins.

[0105] The preparation of RSV glycoprotein F generally comprises the following steps: culturing mammalian host cells to express the RSV glycoprotein F; purifying the RSV glycoprotein F obtained by expression; wherein, additional steps such as construction of an expression vector and freeze-drying of the purified protein are also included. Respiratory syncytial virus (RSV) is a respiratory virus that infects the lungs and respiratory tract. It is the leading cause of severe viral lower respiratory tract disease in infants worldwide and an important cause of respiratory disease in the elderly. However, there is currently no approved vaccine for the prevention of RSV infection.

[0106] The beneficial effect of adopting the above-mentioned technical solution is that it allows the vaccine recipient to obtain immunity against three viruses, namely the new coronavirus, influenza and respiratory syncytial virus, with one dose of vaccine, thereby reducing the probability of serious complications after infection with the three viruses at the same time.

[0107] The seventh aspect of the present invention provides a method for preparing the above-mentioned broad-spectrum immunogenic composition, which comprises the following steps:

[0108] S1. Prepare an antigen stock solution 1 containing a fusion protein of the new coronavirus;

[0109] S2, preparing an antigen stock solution 2 containing H1N1, H3N2 and B human influenza virus immunogens;

[0110] S3, preparing an antigen stock solution 3 containing RSV antigenic polypeptides;

[0111] S4. Mix the antigen stock solution 1, the stock solution 2 and the stock solution 3 to obtain the immunogenic composition.

[0112] The preparation methods of antigen stock solution 1 and antigen stock solution 2 are the same as those described in the third aspect above.

[0113] Among them, S3 is usually culturing host CHO cells to express the modified RSV glycoprotein F; purifying the modified RSV glycoprotein F obtained by expression. In some embodiments, additional steps such as construction of an expression vector and purification of proteins are also included.

[0114] There is no specific order relationship between S1, S2 and S3, and S1, S2 and S3 can be performed in any order or simultaneously. In some embodiments, S3 further comprises additional steps such as adding a lyoprotectant and freeze drying.

[0115] The eighth aspect of the present invention provides a new coronavirus-influenza-respiratory syncytial virus combined protein vaccine, which comprises the broad-spectrum immunogenic composition described in the sixth aspect and an adjuvant, and the adjuvant is selected from one or more of aluminum adjuvant, MF59, MPL, QS-21, GLA, CpG, PolyI:C, AS01, AS02, AS03, and AS04 adjuvants, preferably AS03 or MF59 adjuvant.

[0116] The ninth aspect of the present invention provides a new coronavirus-influenza-respiratory syncytial virus combined DNA vaccine, which contains a DNA sequence encoding the above-mentioned broad-spectrum immunogenic composition.

[0117] The tenth aspect of the present invention provides a new coronavirus-influenza-respiratory syncytial virus combined mRNA vaccine, which contains an mRNA sequence encoding the above-mentioned broad-spectrum immunogenic composition.

[0118] The eleventh aspect of the present invention provides the use of the above-mentioned immunogenic composition in the preparation of a vaccine for the simultaneous prevention and / or treatment of the new coronavirus and influenza virus; preferably, the vaccine is used for single immunization or sequential immunization with other types of new coronavirus vaccines; further preferably, the other types of new coronavirus vaccines include inactivated vaccines.

[0119] The twelfth aspect of the present invention provides the use of the above-mentioned broad-spectrum immunogenic composition in the preparation of a vaccine for the simultaneous prevention and / or treatment of the new coronavirus, influenza virus and respiratory syncytial virus; preferably, the vaccine is used for single immunization or sequential immunization with other types of new coronavirus vaccines; further preferably, the other types of new coronavirus vaccines include inactivated vaccines.

[0120] Compared with the prior art, the present invention has the following beneficial effects:

[0121] The immunogenic composition provided by the present invention and the new crown influenza combined vaccine prepared therefrom have good immunogenicity, can induce the production of high titers of anti-influenza antibodies and new coronavirus neutralizing antibodies, achieve the purpose of preventing two viral infections at the same time with one vaccination, reduce the risk of co-infection and cross infection in fever clinics, and improve the vaccination efficiency of vaccination clinics, saving a lot of manpower, material and financial resources. In addition, the immunogenic composition provided by the present invention and the new crown influenza combined vaccine prepared therefrom are safe and efficient, with less immune interference between the two immunogens, can be well compatible, and can be used as a new crown vaccine booster shot. BRIEF DESCRIPTION OF THE DRAWINGS

[0122] The following drawings are intended only to illustrate and explain the present invention, and are not intended to limit the scope of the present invention.

[0123] Figure 1 This is a diagram of the SDS-PAGE and WB verification results of the NTD (WH01) -RBD (WH01) -foldon purified protein in Example 2 of the present invention.

[0124] Figure 2 This is a diagram of the SDS-PAGE and WB verification results of the NTD(Beta)-RBD(Beta)-foldon purified protein in Example 2 of the present invention.

[0125] Figure 3 This is a diagram of the SDS-PAGE and WB verification results of the NTD(Gamma)-RBD(Gamma)-foldon purified protein in Example 2 of the present invention.

[0126] Figure 4 This is a graph showing the evaluation results of the immune effect of the vaccine against influenza virus in Example 17 of the present invention.

[0127] Figure 5 This is a graph showing the evaluation results of the booster immune effect of the vaccine against the new coronavirus in Example 17 of the present invention. DETAILED DESCRIPTION

[0128] The present invention will be further described below by non-limiting examples, and it is well known to those skilled in the art that many modifications may be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention. The following examples are only intended to illustrate the present invention and should not be considered to limit the scope of the present invention, because the embodiments are necessarily diverse. The terms used in this specification are only intended to illustrate specific embodiments, and not as a limitation, and the scope of the present invention is defined in the appended claims.

[0129] Unless otherwise specified, all technical and scientific terms used in this specification have the same meaning as those generally understood by technicians in the technical field to which this case belongs. The preferred methods and materials of the present invention are described below, but any methods and materials similar to or equivalent to the methods and materials described in this specification can be used to implement or test the present invention. The following experimental methods are all conventional methods or methods described in the product manual unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified. The quadrivalent influenza virus split vaccine stock solution containing H1N1, H3N2, B / Victoria and B / Yamagata types involved in the present invention was purchased from Jiangsu Jindik Biotechnology Co., Ltd. (GDK), and each human dose (1HD) is 0.5 ml, containing 15 μg of hemagglutinin of various types of influenza virus strains.

[0130] Definition of terms

[0131] In the present application, when "one embodiment" is mentioned, it means that the specific parameters, steps, etc. described in the embodiment are at least included in one embodiment of the present invention. Therefore, in the present application, if terms such as "according to one embodiment of the present invention" or "in one embodiment" are used, they are not used to specifically refer to the same embodiment, and if terms such as "in another embodiment", "according to different embodiments of the present invention", or "according to another embodiment of the present invention" are used, they are not used to specifically refer to that the mentioned features can only be included in specific different embodiments. It should be understood by those skilled in the art that the specific parameters, steps, etc. disclosed in one or more embodiments of the present specification can be combined in any suitable manner.

[0132] In the present application, the term "mutant" generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids.

[0133] In this application, the term "fusion protein" generally refers to a protein molecule with biological functional activity obtained by genetic engineering technology. In this application, the fusion protein is a fusion protein composed of NTD or its functionally active fragment, RBD or its functionally active fragment and any of the following fragments: P2 or its functionally active fragment, foldon domain or its functionally active fragment, ferritin or its functionally active fragment, hepatitis B surface antigen HBsAg or its functionally active fragment.

[0134] GISAID uses the full genome sequence (EPI_ISL_402124) of the strain hCoV-19 / Wuhan / WIV04 / 2019 (WIV04) as the official reference sequence. The WIV04 strain is generally defined as a wild strain, an original strain, or a prototype strain. In this application, the term "new coronavirus prototype strain" generally refers to the WIV04 strain.

[0135] In the present application, the term "P2" generally refers to an epitope peptide of tetanus toxin. For example, P2 may be a peptide segment of epitope peptide 830 to 844 of tetanus toxin or a mutant thereof. For example, P2 may be a peptide segment of epitope peptide 830 to 845 of tetanus toxin or a mutant thereof. For example, P2 may be a peptide segment of epitope peptide 829 to 844 of tetanus toxin or a mutant thereof. For example, P2 may be a peptide segment obtained by truncating the peptide segment of epitope peptide 829 to 844 of tetanus toxin or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids at the N-terminus or C-terminus. For example, P2 may be a peptide segment obtained by truncating the peptide segment of epitope peptide 830 to 845 of tetanus toxin or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids at the N-terminus or C-terminus. For example, P2 can be a peptide segment obtained by truncating or adding 1, 2, 3, 4, 5, 6 or 10 amino acids at the N-terminus or C-terminus of the epitope peptide 830 to 844 of tetanus toxin. In the present application, "mutant" generally refers to a sequence that is different from a reference sequence because it contains one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.

[0136] In the present application, the term "foldon domain" generally refers to the residues at the C-terminus of the phage T4 fiber protein. In the present application, the foldon domain can be the 27 residues or mutants at the C-terminus of the phage T4 fiber protein. In the present application, the foldon domain can be a truncation or an extension obtained by truncating the 27 residues at the C-terminus of the phage T4 fiber protein or adding 1, 2, 3, 4, 5, or 6 or 10 amino acids at the N-terminus or C-terminus. In the present application, "mutant" generally refers to a sequence that is different from a reference sequence because it contains one or more differences (mutations). The difference can be a substitution, deletion or insertion of one or more amino acids.

[0137] In the present application, the terms "ferritin", "ferritin" generally refer to Trichoplusia ferritin or Helicobacter pylori ferritin. In the present application, Trichoplusia ferritin has a heavy chain and a light chain (ferritin LC and ferritin HC). In the present application, Helicobacter pylori ferritin has a single-chain structure. In the present application, ferritin may be a mutant of Trichoplusia ferritin or Helicobacter pylori ferritin. In the present application, "mutant" generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids. In the present application, "light chain of Trichoplusia ferritin", "LC" may be used interchangeably with "ferritin LC". In the present application, "heavy chain of Trichoplusia ferritin", "HC" may be used interchangeably with "ferritin HC".

[0138] In the present application, the term "hepatitis B surface antigen (HBsAg)" generally refers to a coat protein in the outermost envelope of the hepatitis B virus. For example, the amino acid sequence of the hepatitis B surface antigen may be a sequence corresponding to the protein sequence accession number AAA45524, ANJ76941, CAA24234 or AAC34729 in NCBI, or may be appropriately truncated or have 1, 2, 3, 4, 5, 6 or 10 amino acids at the N-terminus or C-terminus, or may have a protein mutation, such as deletion, substitution or insertion of one or more amino acids.

[0139] In the present application, the term "functionally active fragment" generally refers to a fragment having similar biological activity to RBD, NTD, P2, foldon domain, ferritin, and hepatitis B surface antigen HBsAg.

[0140] In the present application, the term "engineered disulfide bond mutation" refers to a pair of amino acid residues in the wild-type RSV F protein that mutates into a pair of cysteine ​​residues. The introduced cysteine ​​residues allow for the formation of a disulfide bond between the introduced cysteine ​​residues, which is used to stabilize the configuration or oligomeric state of the protein, such as the pre-fusion configuration. For the pre-fusion configuration of the stable mutant, the residue pair for mutation into cysteine ​​should be very close in the pre-fusion configuration, but far away in the post-fusion configuration.

[0141] In the present application, the term "cavity filling mutation" refers to the amino acid replacement of the amino acid residue in the wild-type RSV F protein by the expected filling of the internal cavity of the mature RSV F protein. In a kind of application, such cavity filling mutation helps to stabilize the pre-fusion configuration of the recombinant RSV soluble F protein. The cavity in the pre-fusion configuration of RSV F protein can be identified by methods known in the art, such as by visual inspection in the crystal structure of the RSV F of the pre-fusion configuration, or by using computational protein design software.

[0142] In this application, the term "electrostatic mutation" refers to an amino acid mutation introduced into a wild-type RSV F protein that reduces ionic repulsion or increases ionic attraction between residues that are close to each other in a folded structure in the protein. Since hydrogen bonding is a special case of ionic attraction, electrostatic mutations can increase hydrogen bonding between such close residues.

[0143] In this application, the term "adjuvant" refers to a substance that has the function of enhancing immune response and can be clinically applied to humans or has the prospect of being applied to humans, including various adjuvants that are currently approved and may be approved in the future, such as but not limited to aluminum adjuvants and various forms of adjuvant compositions.

[0144] In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "for", "consisting of...".

[0145] In the present application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0146] In the present application, the term "host cell" generally refers to a plasmid or vector that may or has contained a nucleic acid molecule described in the present application, or an individual cell, cell line or cell culture that can express an antibody or its antigen-binding fragment described in the present application. The host cell may include progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be completely identical in morphology or genome to the original parent cell, but can express the fusion protein described in the present application. The host cell can be obtained by transfecting cells in vitro using the vector described in the present application. The host cell can be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, such as a COS cell, a Chinese hamster ovary (CHO) cell, a HeLa cell, a HEK293 cell, a COS-1 cell, a NS0 cell or a myeloma cell). In some embodiments, the host cell is a mammalian cell. For example, the mammalian cell can be a CHO cell.

[0147] Example 1. Construction of NTD-RBD-foldon-8*His recombinant expression plasmid

[0148] A signal peptide (MGWSCIILFLVATATGVHS, SEQ ID NO: 13) was added, and the amino acid sequence was codon optimized according to the host CHO cells.

[0149] Cloning of fragments The primers of NTD(WH01)-RBD(WH01)-foldon-8*His were amplified by PCR to obtain a fragment PCR product, which was recombined into the target vector pcDNA3.1(+) (BamHI-XhoI restriction vector) by multi-segment recombination to obtain a recombinant expression plasmid. The reaction system is shown in Table 1, and the connection solution was connected at a constant temperature of 52°C for 30 minutes.

[0150] Table 1. The reaction system of the treated target fragment and the vector ligation

[0151] name volume Vector after enzyme digestion 5μl Purified PCR products 5μl Seamless assembly MIX 10μl Total volume 20μl

[0152] Conversion : Pipette 1-3μl of the recombinant expression plasmid at a concentration of 100ng / μl into 100μl of competent cells, gently shake and rotate to mix, and place on ice for 3 minutes; place in a 42℃ water bath for 90s without shaking; place in an ice bath for about 3 minutes; add 500-800μl of LB medium pre-warmed at 37℃ to each tube, and gently shake at 200rpm at 37℃ for 40 minutes.

[0153] Verification of recombinant expression plasmid : Prepare agar plates containing corresponding resistance; take 100 μl of bacterial solution and spread it on the agar plate containing corresponding resistance, use a sterile glass applicator to gently spread the bacteria on the surface of the plate, and incubate the plate at 37°C for 15 minutes; invert the plate and incubate at 37°C for 12-16 hours until colonies appear; pick bacteria on the plate, shake the bacteria at 37°C, 250r / min for 14 hours, use the bacterial solution for PCR identification, and send the positive clone bacterial solution for sequencing.

[0154] Identification of cloned plasmids : PCR amplification of NTD-RBD-foldon-8*His fragment, the identification primer sequence was synthesized by the company's internal primer department. The expected amplified fragment length was 1761bp. The PCR reaction used a 20μl system: primer 0.5μl, template bacterial solution 2μl, polymerase buffer 0.5μl, buffer 3μl, ddH 2O 14μl. Cycle parameters: 96℃ pre-denaturation for 3min; 95℃15s, 58℃15s, 72℃20s, 23 cycles, 72℃ final extension for 1min. Screen positive clones by bacterial liquid PCR method, obtain positive bacterial liquid 37℃ shake bacterial plasmid, and sequence. Sequencing alignment correct plasmid, double enzyme digestion with BamHI-XhoI, obtain 1761bp and 5372bp two fragments.

[0155] Recombinant expression plasmid extraction : 1% E. coli cells (Stbl3) containing each recombinant expression plasmid were inoculated into 2 ml LB medium and cultured overnight at 37°C with shaking. After treating the cells, 100 μg of plasmid was extracted using a plasmid extraction kit. The correct plasmid was sequenced and double-digested with BamHI-XhoI to obtain two fragments of 1761 bp and 5372 bp.

[0156] The same method was used to construct NTD(Beta)-RBD(Beta)-foldon-8*His and NTD(Gamma)-RBD(Gamma)-foldon-8*His recombinant expression plasmids.

[0157] Example 2. Cell transfection and protein purification

[0158] Cell transfection: The obtained plasmid was introduced into CHO cells by transient transfection. After culture, the protein was collected and purified. CHO cells were subcultured and expanded using ExpiCHO Expression Medium (EXPICHO from Thermo). During all cell manipulations, the host cells were gently rotated to mix; vigorous mixing / pipetting was avoided. Subculture and expand CHO cells until the cell density reached 4×10 6 ~6×10 6 Day -1: Cell expansion, expand the cultured cells to 3×10 6 ~4×10 6 Day 0: Transfect cells and measure viable cell density and viability. The cell density should reach 7 × 10 6 ~10×10 6 / mL, and the survival rate reaches 95-99% for transfection. Fresh cell expression medium is preheated to 37°C and the cells are diluted to a final density of 6×10 6 cells / mL, incubated in a 50 mm shaking incubator at 90 rpm, 37°C, 8% CO 2Use OPti-PRO SFM medium to prepare transfection reagent and recombinant expression plasmid complex (4°C), for example: prepare 40μl OPti-PRO SFM for 1ml cells, add 0.8μg corresponding recombinant expression plasmid, mix and place for 5min; prepare 40μl OPti-PRO SFM, add 3μl Expi Fectamine CHO reagent, mix and place for 5min, at room temperature for 1 to 5 minutes, then slowly transfer the solution to a shake flask and shake it, gently shake the shake flask during the addition process. Place the cells in a 50mm amplitude incubator at 90rpm, 37°C, 8% CO 2 18-22 hours after transfection, add Enhancer and ExpiCHO Feed and perform standard experimental protocol. For example, add 6μl Enhancer and 0.24ml ExpiCHO Feed to 1ml cells and culture the cells in a 50mm amplitude incubator at 90rpm at 37℃ and 8% CO 2 The target purified protein was obtained after HPLC-SEC purification 8 days after transfection. The results are shown in Table 2.

[0159] Table 2 Protein purification results

[0160]

[0161] SDS-PAGE and WB verification results are as follows Figures 1 to 3 shown.

[0162] Example 3. Baculovirus Preparation

[0163] 3.1 Transfection of Sf9 cells and preparation of P1 virus

[0164] After Sf9 cells were counted, 9*10 5cells (one of which is set as a negative control), and cultured with 2 ml of IB905 medium for at least 1 hour to allow the cells to adhere to the wall. Prepare a mixture of recombinant plasmid and cell transfection reagent Cellfectin. While incubating the recombinant plasmid and transfection agent mixture, wash one well of cells to be transfected with 2 ml of Grace's medium without additives and discard the washing solution. Take 0.8 ml of Grace's medium without additives and add it to the mixture of plasmid and transfection reagent. After gently mixing, the total volume is about 1 mL. Add the mixture of plasmid and transfection reagent to the corresponding cell wells after washing in the previous step, and add 1 mL of Grace's medium without additives to the blank well, and continue to culture at 27°C for 5 hours. Discard the plasmid transfection reagent mixture and add 2 ml of IB905 medium to the cells. Incubate at 27°C for 96 hours, collect the culture supernatant containing the virus, centrifuge at 500g for 5 minutes, remove cells and debris, and the supernatant is the P1 generation virus stock solution.

[0165] 3.2 Virus propagation

[0166] Sf9 cells were 2*10 6 cells / mL density was inoculated in a shake flask, and the P1 virus titer was 10 6 pfu / mL, and the virus was inoculated at MOI = 0.1. The shaker temperature was set at 27°C and the speed was set at 120 rpm. The culture was shaken for 72 hours, and the culture supernatant containing the virus was collected and centrifuged at 500g for 5 minutes to remove cells and debris. The supernatant was the P2 virus stock solution. Sf9 cells were cultured at 2*10 6 The cells / mL density was inoculated in a shake flask, and the P2 virus titer was 10 7 pfu / mL, and the virus was inoculated at MOI = 0.1. The remaining P2 generation virus stock solution was divided and frozen at -80°C for later use. The shaker temperature was set to 27°C and the speed was set to 120 rpm. The shaking culture was performed for 72 hours, and the culture supernatant containing the virus was collected and centrifuged at 500g for 5 minutes to remove cells and debris. The supernatant was the P3 generation virus stock solution.

[0167] Example 4. Expression of rHA protein and cell collection

[0168] The full-length HA gene sequences of H1N1, H3N2, B / Victoria and B / Yamagata were amplified by RT-PCR to obtain the full-length HA gene fragments of each type of influenza virus. The full-length HA gene fragments were inserted into the pFastBac1 vector to construct a recombinant expression vector, which was diluted to 200 pg / μl for later use.

[0169] The E. coli DH10Bac competent cells containing the baculovirus genome were transformed by heat shock method. 1 ng (5 μl) of plasmid DNA was added to the DH10Bac competent cells and gently mixed. Incubated on ice for 30 min, then heat shocked at 42°C for 45 s, and immediately placed on ice for 2 min to obtain the recombinant baculovirus genome rBacmid.

[0170] The rBacmid was transfected into insect cells, and the supernatant collected after cultivation by centrifugation was the recombinant baculovirus seed virus.

[0171] Insect cells were cultured at 1.5*10 6 The initial density of cells / ml was inoculated in 40 ml IB905 medium, and the virus was inoculated at a multiplicity of infection (MOI) of 0.01. The shaker temperature was set at 28°C and the speed was set at 120 rpm. After shaking culture for 72 hours, the cells were centrifuged at 4000 rpm, the supernatant was discarded, and the cell pellet was collected.

[0172] Example 5. Lysing cells and collecting lysate

[0173] The cell pellet was resuspended in lysis buffer (10 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (PB), 1% Triton X-100, pH 7.0-7.2) at a resuspension ratio of 1:35, i.e., 1 g of cell pellet was resuspended in 35 mL of lysis buffer, stirred on a magnetic stirrer at 400-800 rpm for 0.5-1.0 h, and centrifuged at 10000 g for 30 min to separate and collect the lysis supernatant, which is the lysate containing rHA of various types of human influenza viruses.

[0174] Example 6. Purification of influenza virus rHA protein

[0175] The rHA lysate was initially purified by anion exchange chromatography (UniGel 80Q) at a flow rate of 4 mL / min, and the chromatography buffer was 10 mM PB, 0 mM / 1000 mM NaCl, 0.5% Tween 20, pH 7.0-7.2. The rHA flowed through the anion column, and some impurities bound to the column. The anion column flow-through (Q-FT) was collected for further purification. Q-FT was subjected to cation exchange chromatography (SP Bestarose HP) at a flow rate of 4 mL / min, and the buffer was 10 mM PB, 0 mM / 1000 mM NaCl, 0.5% Tween 20, pH 7.0-7.2. The rHA in the Q-FT bound to the cation column, and most impurities flowed through. The bound rHA was eluted with buffer 1 (10 mM PB, 200 mM NaCl, 0.5% Tween 20, pH 7.0-7.2), and the eluate (SP-E) was collected; the conductivity of SP-E was adjusted to about 70 mS / cm with an adjustment buffer (10 mM PB, 2000 mM NaCl, 0.5% Tween 20, pH 7.0-7.2), and hydrophobic interaction chromatography (Phenyl Bestarose HP) was performed at a flow rate of 4 mL / min. The buffer was 10 mM PB, 1000 mM / 0 mM NaCl, 0.5% Tween 20, pH 7.0-7.2. rHA bound to the Phenyl Bestarose HP medium, and some impurities flowed through. The bound rHA was eluted with elution buffer 2 (10 mM PB, 0 mM NaCl, 0.5% Tween 20, pH 7.0-7.2), collect the eluate (HIC-E), concentrate HIC-E with a 100 kDa ultrafiltration tube and replace it with storage buffer (10 mM PB, 150 mM NaCl, 0.005% Tween 20, pH 7.0-7.2) to make the final rHA concentration 0.5-1.0 mg / mL.

[0176] Example 7. Preparation of immunogenic compositions

[0177] NTD-RBD-foldon fusion protein 40 μg, H1N1 HA protein 45 μg, H3N2 HA protein 45 μg, B / Victoria HA protein 45 μg, B / Yamagata HA protein 45 μg, 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80 and 10 mM PB buffer (Na2O3) at pH 7.5 2 HPO 4 , NaH 2 PO 4), dispense into 2 mL vials, 0.5 mL per vial, and place in a freeze dryer for freeze drying.

[0178] Example 8. Preparation of immunogenic compositions

[0179] NTD-RBD-foldon fusion protein 40 μg and GDK quadrivalent influenza virus split vaccine (IIV4) (GDK quadrivalent influenza virus split vaccine contains H1N1 HA protein 15 μg, H3N2 HA protein 15 μg, B / Victoria HA protein 15 μg and B / Yamagata HA protein 15 μg), 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80 and 10 mM PB buffer (Na2SO4) at pH 7.5. 2 HPO 4 , NaH 2 PO 4 ), dispense into 2 mL vials, 0.5 mL per vial, and place in a freeze dryer for freeze drying.

[0180] Example 9. Preparation of a broad-spectrum immunogenic composition

[0181] NTD-RBD-foldon fusion protein 40 μg, H1N1 HA protein 45 μg, H3N2 HA protein 45 μg, B / Victoria HA protein 45 μg, B / Yamagata HA protein 45 μg, RSV glycoprotein F 50 μg, 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80 and 10 mM PB buffer (Na2O3) at pH 7.5 2 HPO 4 , NaH 2 PO 4 ), dispense into 2 mL vials, 0.5 mL per vial, and place in a freeze dryer for freeze drying.

[0182] Example 10. Preparation of a broad-spectrum immunogenic composition

[0183] NTD-RBD-foldon fusion protein 40 μg, GDK quadrivalent influenza virus split vaccine (IIV4) (GDK quadrivalent influenza virus split vaccine contains H1N1 HA protein 15 μg, H3N2 HA protein 15 μg, B / Victoria HA protein 15 μg and B / Yamagata HA protein 15 μg) and RSV glycoprotein F 50 μg, 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80 and 10 mM PB buffer (Na2O3) at pH 7.5 2 HPO 4 , NaH 2PO 4 ), dispense into 2 mL vials, 0.5 mL per vial, and place in a freeze dryer for freeze drying.

[0184] Example 11. Preparation of COVID-19-Influenza Combined Vaccine

[0185] Preparation of the novel coronavirus fusion protein-quadrivalent recombinant influenza combined vaccine: The immunogenic composition described in Example 7 further comprises an adjuvant, the adjuvant bottle is 0.25 ml, and the ingredients include 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na 2 HPO 4 , 0.09 mg KH 2 PO 4 and water for injection.

[0186] Example 12. Preparation of COVID-19-Influenza Combined Vaccine

[0187] Preparation of the novel coronavirus fusion protein-quadrivalent influenza split combined vaccine: The immunogenic composition described in Example 8 further comprises an adjuvant, the adjuvant bottle is 0.25 ml, and the ingredients include 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na 2 HPO 4 , 0.09 mg KH 2 PO 4 and water for injection.

[0188] Example 13. Preparation of a COVID-19-Influenza-Respiratory Syncytial Virus Combined Vaccine

[0189] The broad-spectrum immunogenic composition described in Example 9 further comprises an adjuvant, the adjuvant bottle is 0.25 ml, and the ingredients include 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na 2 HPO 4 , 0.09 mg KH 2 PO 4 and water for injection.

[0190] Example 14. Preparation of a COVID-19-Influenza-Respiratory Syncytial Virus Combined Vaccine

[0191] The broad-spectrum immunogenic composition described in Example 10 further comprises an adjuvant, the adjuvant bottle is 0.25 ml, and the ingredients include 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na 2 HPO 4 , 0.09 mg KH 2 PO 4 and water for injection.

[0192] Example 15. Preparation of quadrivalent influenza virus recombinant protein vaccine

[0193] 45 μg of H1N1 HA protein, 45 μg of H3N2 HA protein, 45 μg of B / Victoria HA protein, 45 μg of B / Yamagata HA protein, 1% (w / v) sucrose; 2% (w / v) glycine; 0.02% (w / v) Tween 80 and 10 mM PB buffer (Na2SO4) at pH 7.5 2 HPO 4 , NaH 2 PO 4 ), divided into 2 mL tube bottles, 0.5 mL per tube, and placed in a freeze dryer for freeze drying. It further comprises an adjuvant, the adjuvant bottle is 0.25 ml, and the ingredients include 10.69 mg of squalene, 11.86 mg of α-tocopherol, 4.86 mg of Tween 80, 3.53 mg of NaCl, 0.09 mg of KCl, 0.51 mg of Na 2 HPO 4 , 0.09 mg KH 2 PO 4 and water for injection.

[0194] Example 16. Construction of pseudovirus

[0195] China Science Guobang (Beijing) Inspection and Testing Co., Ltd. was commissioned to construct a pseudovirus of the new coronavirus WH01 strain and conduct corresponding tests in preparation for subsequent in vitro neutralization tests.

[0196] Example 17. Evaluation of vaccine immunogenicity

[0197] 17.1 Evaluation of immune effects against influenza virus

[0198] BALB / c mice (female, 8-10 weeks old, purchased from the Institute of Laboratory Animals, Chinese Academy of Medical Sciences) were used for group immunization and random grouping, and the immunization dose was 1 / 10HD. The mouse grouping and immunization dose are shown in Table 3. Gr1 is the quadrivalent influenza virus recombinant protein vaccine prepared in Example 15, Gr2 is the new crown fusion protein-quadrivalent recombinant influenza combined vaccine prepared in Example 11, Gr3 is the GDK quadrivalent influenza virus split vaccine, Gr4 is the new crown fusion protein-quadrivalent influenza split combined vaccine prepared in Example 12, and Gr5 is a blank control.

[0199] Table 3 Mouse grouping and immunization dose

[0200]

[0201]

[0202] The first immunization was performed on day 0. 21 days after the first immunization, blood was collected to separate serum, and the anti-influenza antibody titer in the mouse serum was determined by the hemagglutination inhibition method. Figure 4 shown.

[0203] The results showed that compared with Gr1, the hemagglutination inhibition antibody titers of H1N1, H3N2 and BY types produced by Gr2-induced mice were not statistically different, and the hemagglutination inhibition antibody titers of BV type decreased slightly; compared with Gr3, the hemagglutination inhibition antibody titers of H1N1 type produced by Gr4-induced mice decreased slightly, and the hemagglutination inhibition antibody titers of other types were not statistically different. This shows that both combined vaccines can basically maintain the same immunogenicity as the corresponding influenza vaccine, and will not cause immune interference due to the addition of NTD (WH01)-RBD (WH01)-foldon.

[0204] 17.2 Evaluation of the effect of booster immunity against the new coronavirus

[0205] BALB / c mice (female, 8-10 weeks old, purchased from the Institute of Laboratory Animals, Chinese Academy of Medical Sciences) were used for group immunization and random grouping. On days 0 and 21, 1 / 10 of the human dose of Sinopharm COVID-19 inactivated vaccine was used for intramuscular immunization. Eight months after two injections of inactivated vaccine immunization, booster immunization was performed with COVID-19 fusion protein-quadrivalent influenza split combined vaccine and COVID-19 fusion protein single vaccine, with 100 μl of immune sample injected intramuscularly each time. Blood was collected 21 days after booster immunization to separate serum and detect the titer of SARS-COV-2 pseudovirus neutralizing antibodies before and after booster. The mouse grouping and immunization scheme are shown in Table 4.

[0206] Table 4 Mouse grouping and immunization scheme

[0207]

[0208] The method for detecting the neutralizing antibody titer of the new coronavirus pseudovirus (hereinafter referred to as pseudovirus) is as follows: In a 96-well plate, the immune mouse serum is diluted in a 2-fold gradient ratio, then mixed with the pseudovirus and incubated at 37°C for 1 hour. The immune serum-pseudovirus mixture is transferred to a 96-well plate filled with Vero cells. After 15 hours, the positive cell value is calculated by CQ1 confocal cell imager (Yokogawa), and then the fitting curve is drawn in GraphPad Prism software to calculate the reciprocal of the serum dilution corresponding to 50% neutralization, which is the 50% pseudovirus neutralization titer.

[0209] The results are as follows Figure 5 As shown, after two doses of the inactivated COVID-19 vaccine, booster vaccination with either the COVID-19 fusion protein single vaccine or the COVID-19-IIV4 combined vaccine can significantly increase the COVID-19 pseudovirus neutralizing antibody titer. Three weeks after the booster, the neutralizing antibody titer of the COVID-19 fusion protein single vaccine was slightly higher than that of the COVID-19-IIV4 combined vaccine, but there was no statistical difference. This shows that when the COVID-19-influenza combined vaccine is used as a COVID-19 booster shot, it can not only achieve the purpose of effective booster immunization, but also the interference of influenza antigens on COVID-19 antigens is small, which can be ignored or eliminated by changing the amount of antigen. In summary, the immunogenic composition provided by the present invention can be used for dual immunization of the new coronavirus and influenza virus, and has the potential for application in single immunization or sequential immunization with other types of new coronavirus vaccines.

[0210] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An immunogenic composition, characterized in that Containing a fusion protein and a human influenza virus immunogen, the fusion protein comprises 1) a receptor binding domain (RBD) of the SARS-CoV-2 S protein or a functionally active fragment thereof, and 2) an N-terminal domain (NTD) of the SARS-CoV-2 S protein or a functionally active fragment thereof, and the amino acid sequence of the fusion protein is shown in SEQ ID NO: 1; the human influenza virus immunogen is composed of human influenza virus hemagglutinin (HA) proteins from H1N1, H3N2 and B types.

2. The immunogenic composition according to claim 1, characterized in that The hemagglutinin (HA) protein is from an influenza virus split vaccine, an influenza virus inactivated vaccine, an influenza live attenuated vaccine or an influenza virus subunit vaccine.

3. The immunogenic composition according to claim 1 or 2, characterized in that The immunogenic composition comprises 20-100 μg of fusion protein and 15-80 μg of human influenza virus hemagglutinin (HA) protein.

4. The immunogenic composition according to claim 3, characterized in that The immunogenic composition further comprises a lyoprotectant.

5. The immunogenic composition according to claim 4, characterized in that The lyoprotectant is prepared according to the formula of 1% (w / v) sucrose, 2% (w / v) glycine, 0.02% (w / v) Tween 80 and 10 mM PB buffer at pH 7.

5.

6. A COVID-19-influenza combined protein vaccine, characterized in that: The protein vaccine comprises the immunogenic composition of any one of claims 1 to 5 and an adjuvant.

7. The COVID-19-influenza combined protein vaccine according to claim 6, characterized in that: The adjuvant is selected from one or more of aluminum adjuvant, MF59, MPL, QS-21, GLA, CpG, PolyI:C, AS01, AS02, AS03, and AS04 adjuvants.

8. The COVID-19-influenza combined protein vaccine according to claim 7, characterized in that: The adjuvant is AS03 or MF59 adjuvant.

9. The COVID-19-influenza combined protein vaccine according to claim 8, characterized in that: The protein vaccine contains 30-60 μg of fusion protein, 15-50 μg of each type of HA protein, and AS03 adjuvant.

10. A method for preparing the immunogenic composition according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1. Prepare an antigen stock solution 1 containing a fusion protein of the new coronavirus; S2, preparing an antigen stock solution 2 containing H1N1, H3N2 and B human influenza virus immunogens; S3. Mix the antigen stock solution 1 and the stock solution 2 to obtain the immunogenic composition.

11. Use of the immunogenic composition according to any one of claims 1 to 5 in the preparation of a vaccine for the simultaneous prevention of novel coronavirus and influenza virus.

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