A Universal Mosaic Recombinant Antigen for Seasonal Influenza A and Its Vaccine and Applications

Abstract

The present invention relates to the field of biopharmaceutical technology, and particularly relates to a seasonal influenza A universal Mosaic recombinant antigen, its vaccine and application. The present invention analyzes the amino acid sequences of HA and NA of all human-derived H1N1 and H3N2 from 2009 to 2021 that are known, and uses the Mosaic design strategy to obtain influenza virus HA and NA Mosaic recombinant antigen sequences with the maximum diversity of potential T cell epitopes that can cover natural sequences; the influenza virus HA and NA Mosaic recombinant antigens designed by the present invention are assembled from short peptides of natural sequences. Through antigen epitope coverage analysis, genetic evolution analysis and spatial conformation analysis, they have a high antigen coverage rate, are closely related to vaccine strains and have good structural similarity with natural proteins, and have the potential to be developed into vaccine antigens.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and particularly relates to a seasonal influenza A universal Mosaic recombinant antigen, its vaccine and application. Background Art

[0002] Influenza virus (IV), simply referred to as influenza virus, has numerous subtypes. HA and NA genes from different sources can recombine to form hundreds of different subtypes of viruses. There are also numerous different strains within the same subtype, and it can spread between different hosts. The mutation frequency of the HA antigen of influenza A virus is very high. Under immune pressure, its antigenicity mutates more frequently, and antigenic drift and antigenic shift are extremely likely to occur, enabling the influenza virus to produce immune escape, thus leading to seasonal influenza epidemics and global influenza pandemics.

[0003] Vaccination against influenza is the best intervention measure to prevent influenza, reduce the harm of influenza, and reduce various complications. Due to the high variability of influenza viruses, the World Health Organization (WHO) predicts and recommends the epidemic strains in the Northern and Southern Hemispheres every year. Vaccine manufacturers will use these as vaccine strains to produce influenza vaccines, and the population also needs to be vaccinated against influenza every year to match the strains predicted to be epidemic in that year. If the strains used in the vaccine do not match the epidemic strains, the protective efficacy will be greatly reduced, and the incidence and mortality of influenza will increase. Therefore, the research and development of a relatively broad-spectrum universal vaccine has become a key issue of concern.

[0004] Currently, many universal influenza vaccine strategies have been studied internationally. The viral surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) are the most common targets of vaccines, but their cross-protection effects are limited. Recent research mainly focuses on the HA stem region, which is more conserved than the HA head region. The HA and NA targets rely on inducing antibody responses to provide protection against IAV. However, in the previous development of influenza vaccines, the importance of T cell immune responses has often been overlooked. T cell immune responses are key factors in resisting viral infections and play an important role in controlling influenza viruses and HIV. More and more evidence indicates that T cell immunity may be the key to better vaccine cross-protection and plays an important role in preventing influenza.

[0005] At present, all influenza vaccines on the domestic market are traditional chicken embryo-cultured vaccines, including inactivated and attenuated vaccines, usually trivalent (Influenza A H1N1, Influenza A H3N2, and one type B) or quadrivalent (Influenza A H1N1, Influenza A H3N2, Yamagata lineage of type B, and Victoria lineage of type B) vaccines. On the one hand, these vaccines can only induce systemic humoral responses, and on the other hand, their protection against newly emerging strains is limited. Due to the diversity of influenza viruses and the frequent mutation of antigens, developing a universal vaccine that can resist multiple subtypes of influenza viruses is an ideal choice. Summary of the Invention

[0006] An object of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a universal Mosaic recombinant antigen for seasonal influenza A, its vaccine, and applications.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a universal Mosaic recombinant antigen for seasonal influenza A, and the Mosaic recombinant antigen is:

[0009] (a) an antigen consisting of the amino acid sequence shown in any one of SEQ ID No: 1-4; or,

[0010] (b) an antigen formed by substituting, deleting, or adding one or several amino acids in the amino acid sequence in (a).

[0011] As a preferred embodiment of the universal Mosaic recombinant antigen for seasonal influenza A of the present invention, compared with the Th cell epitopes on the natural HA protein of influenza virus, the Th cell epitope coverage rate of the Mosaic recombinant antigen is greater than 81%; compared with the Th cell epitopes on the natural NA protein of influenza virus, the Th cell epitope coverage rate of the Mosaic recombinant antigen is greater than 84%.

[0012] The Mosaic recombinant antigen of the present application generates a small amount of "mosaic" sequences on the natural HA protein or NA protein sequence, so that it contains the maximum diversity of potential T cell epitopes from the natural protein sequence. Using a genetic algorithm (a computational optimization method) to assemble from fragments of natural proteins, the optimized "mosaic" protein generated has abundant T cell epitopes. After simulating and predicting the three-dimensional structure model of the Mosaic recombinant antigen, the Mosaic recombinant antigen screened by the present invention has a high structural similarity with the natural protein and has the potential to be developed into a vaccine antigen.

[0013] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, the HA protein comprises an H1 protein or an H3 protein, and the NA protein comprises an N1 protein or an N2 protein.

[0014] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, more than 81% of the 12 amino acids of the Th cell epitopes of the Mosaic recombinant antigen are exactly matched with the 12 amino acids of the Th cell epitopes on the natural H1 protein or H3 protein of the influenza virus.

[0015] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, more than 96% of the Th cell epitopes of the Mosaic recombinant antigen with more than 11 amino acids are exactly matched with the 12 amino acids of the Th cell epitopes on the natural H1 protein or H3 protein of the influenza virus.

[0016] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, more than 99% of the 10 amino acids of the Th cell epitopes of the Mosaic recombinant antigen are exactly matched with the 12 amino acids of the Th cell epitopes of the natural H1 protein or H3 protein of the influenza virus.

[0017] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, more than 84% of the 12 amino acids of the Th cell epitopes of the Mosaic recombinant antigen are exactly matched with the 12 amino acids of the Th cell epitopes on the natural N1 protein or N2 protein of the influenza virus.

[0018] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, more than 96% of the Th cell epitopes of the Mosaic recombinant antigen with more than 11 amino acids are exactly matched with the 12 amino acids of the Th cell epitopes on the natural N1 protein or N2 protein of the influenza virus.

[0019] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, more than 98% of the Th cell epitopes of the Mosaic recombinant antigen with more than 10 amino acids are exactly matched with the 12 amino acids of the Th cell epitopes on the natural N1 protein or N2 protein of the influenza virus.

[0020] Verified by experiments on the biological functions of proteins, the Mosaic recombinant antigen with the amino acid sequence shown in SEQ ID No:1 can produce a hemagglutination titer of 2 6 and the Mosaic recombinant antigen with the amino acid sequence shown in SEQ ID No:2 can produce a hemagglutination titer of 2 7 ​

[0021] The Mosaic recombinant antigen of the present invention has the ability to bind to both sialic acid α2,3-galactose receptor and sialic acid α2,6-galactose receptor, and there is a significant difference compared with the PBS group (P≤0.01), and the binding ability to the α2,6-galactose receptor is stronger.

[0022] As a preferred embodiment of the seasonal influenza A universal Mosaic recombinant antigen of the present invention, the Mosaic recombinant antigen is further added with gp67 signal peptide, thrombin cleavage site and 8×His tag, and the original signal peptide of HA or NA is removed; the GCN4pII sequence is added to the HA protein, and the VASP sequence is added to the NA protein;

[0023] The amino acid sequence of the gp67 signal peptide is: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAAD;

[0024] The GCN4pII sequence is MKQIEDKIEEILSKIYHIENEIARIKKLIGEV;

[0025] The VASP sequence is SSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRG.

[0026] The present invention also provides a gene encoding the above-mentioned seasonal influenza A universal Mosaic recombinant antigen.

[0027] The present invention optimizes the gene of the processed Mosaic recombinant antigen, and performs gene modification according to the codon preference of insect cells to obtain the optimized gene.

[0028] As a preferred embodiment of the gene of the present invention, the sequence of the gene is shown in any one of SEQ ID No:5-8.

[0029] The present invention also provides a vector containing the gene.

[0030] As a preferred embodiment of the vector of the present invention, the vector is obtained by ligating the gene into a plasmid. The plasmid is preferably pFastBac-Dual.

[0031] The present invention also provides a cell containing the gene or the vector as described above.

[0032] As a preferred embodiment of the cell of the present invention, the cell is obtained by transferring the gene or the vector into an Escherichia coli host cell.

[0033] In addition, the present invention provides an application of the above-mentioned seasonal influenza A universal Mosaic recombinant antigen in the preparation of a seasonal influenza A universal vaccine.

[0034] The present invention provides a vaccine preparation, which comprises the above-mentioned seasonal influenza A universal Mosaic recombinant antigen, the above-mentioned gene, and the above-mentioned vector.

[0035] As a preferred embodiment of the vaccine preparation of the present invention, the vaccine preparation further comprises an immunologically and pharmaceutically acceptable carrier or adjuvant. The adjuvant includes one or several of aluminum adjuvant, Freund's adjuvant, aluminum phosphate, calcium phosphate, paraffin oil, lanolin, surfactant, calcium alginate, polynucleotide, muramyl peptide, saponin, RIBI adjuvant system, cholera toxin, polymer of acrylic acid or methacrylic acid, oil-in-water emulsion, water-in-oil emulsion.

[0036] The present invention also provides an application of the above-mentioned vaccine preparation in the preparation of a drug for preventing and / or treating seasonal influenza A.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] (1) By analyzing the amino acid sequences of HA and NA of all human-derived H1N1 and H3N2 from 2009 to 2021 known in the prior art, the present invention uses the Mosaic design strategy to obtain influenza virus HA and NA Mosaic recombinant antigen sequences with the maximum diversity of potential T cell epitopes that can cover natural sequences.

[0039] (2) The influenza virus HA and NA Mosaic recombinant antigens designed by the present invention are assembled from short peptides of natural sequences. Through antigen epitope coverage analysis, genetic evolution analysis, and spatial conformation analysis, they have a high antigen coverage rate, a close genetic relationship with vaccine strains, and good structural similarity with natural proteins, and have the potential to be developed into vaccine antigens.

[0040] (3) The influenza virus HA and the constructed Mosaic recombinant protein of the present invention have potential application prospects and values in the development of universal influenza virus vaccines, and the T cell immune response of their antigens is expected to become an important consideration direction in the research and development of influenza vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the average antigen epitope coverage rate of the H1m recombinant sequence for the HA amino acid sequences of all human-derived H1N1 from 2009 to 2021;

[0042] Figure 2Schematic diagram of the average antigenic epitope coverage rate of the H3m recombinant sequence for the HA amino acid sequences of all human H3N2 from 2009 to 2021;

[0043] Figure 3 Schematic diagram of the average antigenic epitope coverage rate of the N1m recombinant sequence for the NA amino acid sequences of all human H1N1 from 2009 to 2021;

[0044] Figure 4 Schematic diagram of the average antigenic epitope coverage rate of the N2m recombinant sequence for the NA amino acid sequences of all human H3N2 from 2009 to 2021;

[0045] Figure 5 Schematic diagram of the antigenic epitope coverage rate of each amino acid of the H1m recombinant sequence;

[0046] Figure 6 Schematic diagram of the antigenic epitope coverage rate of each amino acid of the H3m recombinant sequence;

[0047] Figure 7 Schematic diagram of the antigenic epitope coverage rate of each amino acid of the N1m recombinant sequence;

[0048] Figure 8 Schematic diagram of the antigenic epitope coverage rate of each amino acid of the N2m recombinant sequence;

[0049] Figure 9 Schematic diagram of the antigenic epitope deletion rate of the H1m recombinant sequence;

[0050] Figure 10 Schematic diagram of the antigenic epitope deletion rate of the H3m recombinant sequence;

[0051] Figure 11 Schematic diagram of the antigenic epitope deletion rate of the N1m recombinant sequence;

[0052] Figure 12 Schematic diagram of the antigenic epitope deletion rate of the N2m recombinant sequence;

[0053] Figure 13 Schematic diagram of the genetic evolution analysis of the H1m recombinant sequence;

[0054] Figure 14 Schematic diagram of the genetic evolution analysis of the H3m recombinant sequence;

[0055] Figure 15 Schematic diagram of the genetic evolution analysis of the N1m recombinant sequence;

[0056] Figure 16 Schematic diagram of the genetic evolution analysis of the N2m recombinant sequence;

[0057] Figure 17Schematic diagram of the three-dimensional structure model simulation of the Mosaic recombinant antigen;

[0058] Figure 18 Western Blot result diagram of the Mosaic recombinant protein expressed by the baculovirus system;

[0059] Figure 19 Coomassie brilliant blue staining result diagram of the eluate of the Mosaic recombinant protein;

[0060] Figure 20 Hemagglutination experiment result diagram of the Mosaic recombinant protein;

[0061] Figure 21 Sugar receptor binding experiment result diagram of the Mosaic recombinant protein;

[0062] Figure 22 Neuraminidase activity experiment result diagram of the Mosaic recombinant protein;

[0063] Figure 23 Hemagglutination inhibition animal experiment result diagram of the Mosaic recombinant protein;

[0064] Figure 24 Neuraminidase inhibition animal experiment result diagram of the Mosaic recombinant protein. Detailed implementation manners

[0065] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0066] In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0067] The influenza virus HA and NA proteins play important roles in virus invasion and release, and they have strong immunogenicity and can induce strong specific antibody and T cell responses. The Mosaic vaccine is mainly designed for viruses with variable antigenic epitopes. Its goal is to use natural sequences to generate a small amount of "mosaic" sequences, so that it contains the maximum diversity of potential T cell epitopes from natural sequences. Using genetic algorithms (computational optimization methods) to assemble fragments from natural proteins, the optimized "mosaic" proteins are similar to natural virus proteins and have rich T cell epitopes, so they can be used as antigens for candidate vaccine design.

[0068] Example 1. Construction of a universal Mosaic recombinant antigen for seasonal influenza A

[0069] 1. Design and optimization of the universal Mosaic recombinant antigen sequence:

[0070] 1) Download the amino acid sequences of HA and NA of all human H1N1 and H3N2 from 2009 to 2021 using the GISAID and NCBI databases. After removing duplicate sequences and sequences with poor quality, 7609 H1 amino acid sequences, 9262 H3 amino acid sequences, 8590 N1 amino acid sequences, and 9942 N2 amino acid sequences were obtained.

[0071] 2) Upload the processed amino acid sequences in FAS format to the Mosaic Vaccine Designer program and set the following parameters: Set "Cocktail Size" to "1" to obtain 1 Mosaic sequence for the next step; set the epitope length to "12" to obtain a Mosaic sequence covering more CD4 + Th cell epitopes; set the threshold to "3" to reduce the number of rare epitopes that appear rarely in natural epitopes. After genetic algorithm operation, a series of Mosaic sequences assembled from short peptides composed of 12 amino acids were finally obtained. Subsequently, the genetic algorithm was used to optimize each population in turn, in which new recombinants were generated and their antigen epitope coverage was calculated and tested. Finally, 4 Mosaic recombinant antigen sequences (shown as SEQ ID NO: 1-4) were obtained.

[0072] 2. Screening and identification of universal Mosaic recombinant antigen sequences

[0073] Perform antigen epitope coverage analysis, genetic evolution analysis, and spatial conformation analysis on the obtained Mosaic recombinant antigen sequences.

[0074] 1) Use the Epitope Coverage Assessment Tool (Epicover) to evaluate the antigen epitope coverage of the Mosaic protein. First, add the Mosaic recombinant antigen sequence as the antigen protein to the corresponding position. At the same time, set the complete strain amino acid sequences that were previously downloaded, analyzed, and compared on GISAID and NCBI as the test protein set and add them to the corresponding position. Set the epitope length to 12 and the maximum number of amino acid mismatches to 2. The final result is expressed as the average value of the epitope coverage of the Mosaic recombinant antigen sequence for all background protein sets.

[0075] As shown in Table 1, for the Mosaic recombinant antigen sequences (named H1m or H3m), the 12 amino acids of more than 81% of the Th cell epitopes are completely matched (12 / 12 match) with the 12 amino acids of the Th cell epitopes on the natural H1 protein or H3 protein of influenza A. For more than 96% of the Th cell epitopes on the Mosaic recombinant antigen sequence, more than 11 amino acids are completely matched (11 / 12 match) with the 12 amino acids of the Th cell epitopes on the natural H1 protein or H3 protein of influenza A. For more than 99% of the Th cell epitopes on the Mosaic recombinant antigen sequence, more than 10 amino acids are completely matched (10 / 12 match) with the 12 amino acids of the Th cell epitopes on the natural H1 protein or H3 protein of influenza A.

[0076] For the Mosaic recombinant antigen sequences (named N1m or N2m), the 12 amino acids of more than 84% of the Th cell epitopes of the Mosaic recombinant antigen are completely matched (12 / 12 match) with the 12 amino acids of the Th cell epitopes on the natural N1 protein or N2 protein of the influenza virus. For more than 96% of the Th cell epitopes of the Mosaic recombinant antigen, more than 11 amino acids are completely matched (11 / 12 match) with the 12 amino acids of the Th cell epitopes on the natural N1 protein or N2 protein of the influenza virus. For more than 98% of the Th cell epitopes of the Mosaic recombinant antigen, more than 10 amino acids are completely matched (10 / 12 match) with the 12 amino acids of the Th cell epitopes on the natural N1 protein or N2 protein of the influenza virus.

[0077] The Mosaic recombinant antigen sequences are for the average overall antigen epitope coverage rate as Figures 1-4 shown. The Mosaic recombinant antigen sequences are HAm and NAm.

[0078] Table 1

[0079] Mosaic recombinant antigen Number of natural sequences Off-by-0 Off-by-1 Off-by-2 H1m 7609 88.27％ 98.27％ 99.61％ H3m 9262 81.82％ 96.81％ 99.26％ N1m 8590 84.47％ 96.16％ 98.48％ N2m 9942 84.84％ 97.69％ 99.73％

[0080] 2) Use the Positional Epitope Coverage Assessment Tool (Posicover) to analyze the epitope coverage rate of each amino acid of the Mosaic recombinant antigen. First, add the Mosaic recombinant antigen as the antigen protein to the corresponding position, and at the same time set the amino acid sequences of the strains downloaded and analyzed and compared on GISAID and NCBI before as the test protein set, and also add them to the corresponding position. Set the epitope length to 12, and the final result is represented by the average epitope coverage rate. The schematic diagram of the antigen epitope coverage rate of each amino acid of the Mosaic recombinant antigen sequence is as Figures 5-8 shown; the schematic diagram of the antigen epitope deletion rate of the Mosaic recombinant antigen sequence is as Figures 9-12As shown, the epitope coverage rate of the Mosaic recombinant antigen sequence is relatively high and the overall 12mer deletion rate is relatively low.

[0081] 3) Select the HA and NA genes of seasonal influenza A virus vaccine strains from 2009 to 2022 and HAm and NAm for genetic evolution analysis. After statistical analysis using the maximum likelihood method, phylogenetic trees of the HA and NA genes are drawn. The schematic diagram of the genetic evolution analysis of the Mosaic recombinant antigen sequence is as Figures 13-16 shown. The Mosaic recombinant antigen has a relatively close genetic relationship with multiple vaccine strains, indicating that the designed Mosaic recombinant antigen has the potential to be used as a vaccine antigen.

[0082] 4) Evaluate the innate immune function of the Mosaic recombinant antigen by simulating and predicting its three-dimensional structure model. As Figure 17 shown ( Figure 17 -A is the schematic diagram of the three-dimensional structure model simulation of the H1m recombinant antigen, Figure 17 -B is the schematic diagram of the three-dimensional structure model simulation of the H3m recombinant antigen, Figure 17 -C is the schematic diagram of the three-dimensional structure model simulation of the HA protein of the A / Puerto Rico / 8 / 1934 strain, Figure 17 -D is the schematic diagram of the three-dimensional structure model simulation of the N1m recombinant antigen, Figure 17 -E is the schematic diagram of the three-dimensional structure model simulation of the N2m recombinant antigen, Figure 17 -F is the schematic diagram of the three-dimensional structure model simulation of the NA protein of the A / Aichi / 2 / 1968 strain). The Global Model Quality Estimate (QMQE) scores of the four Mosaic recombinant antigens and their native proteins are as follows: H1m protein: 0.74, H3m protein: 0.78, HA protein: 0.80; N1m protein: 0.81, N2m protein: 0.78, NA protein: 0.79, indicating that the Mosaic recombinant antigen has a high structural similarity with the native protein.

[0083] 3. Gene optimization and synthesis of the general Mosaic recombinant antigen sequence

[0084] For the amino acid sequences of the four Mosaic recombinant antigens H1m, H3m, N1m, and N2m obtained by design, further remove the original signal peptides of HA and NA, add the gp67 signal peptide (MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAAD), add the GCN4pII sequence (MKQIEDKIEEILSKIYHIENEIARIKKLIGEV) to the HA protein sequence; add the VASP sequence (SSSDYSDLQRVKQELLEEVKKELQKVKEEIIEAFVQELRKRG) to the NA protein sequence, and then add the thrombin cleavage site (LVPRGS) and 8×His tag (HHHHHHHH) together. Optimize the coding genes of the processed Mosaic recombinant antigen sequences, and perform gene modification according to the codon preference of insect cells to obtain the optimized gene sequences (as shown in SEQ ID NO.5-8).

[0085] Example 2. Expression of Mosaic recombinant protein

[0086] In this example, the Bac-to-Bac baculovirus expression system of Invitrogen was used to express the required protein.

[0087] 1. Construction of recombinant baculovirus plasmid:

[0088] Perform multiple cloning site analysis on the coding genes of Mosaic recombinant antigens H1m, H3m, N1m, and N2m and the genes of the pFastBac-Dual vector. Select two restriction enzyme sites (EcoRI, HindIII) provided on the pFastBac-Dual vector but not on the target fragment. After amplification, digestion, and recovery of the target fragment, insert the target fragment into the multiple cloning site after the pH promoter of the pFastBac-Dual vector. After transformation with Escherichia coli DH5α competent cells, obtain recombinant plasmids containing the H1m, H3m, N1m, and N2m antigens.

[0089] 2. Extraction of recombinant baculovirus shuttle plasmid (bacmid):

[0090] Transform the recombinant plasmid obtained in step 1 with Escherichia coli DH10Bac competent cells. After blue-white screening, culture overnight in LB containing kanamycin (50 μg / mL), tetracycline (10 μg / mL), and gentamicin (7 μg / mL), and then extract bacmid (purchased from Beyotime, catalog number D0031) to obtain the recombinant baculovirus shuttle plasmid containing the H1m, H3m, N1m, and N2m antigens.

[0091] 3. Bacmid cell transfection and protein expression:

[0092] Transfect the Bacmid described in step 2 and the empty plasmid (as a blank control) into sf9 insect cells respectively, and culture them in a constant temperature shaker at 27°C for 72 h to obtain the P0 generation of recombinant baculovirus. After inoculating sf9 insect cells according to MOI = 3, the P1 generation of recombinant baculovirus is obtained. The expression of the target protein in the cell supernatant is detected by Western Blot (WB), as Figure 18 shown ( Figure 18 -A is the Western Blot result map of the H1m recombinant protein, Figure 18 -B is the Western Blot result map of the H3m recombinant protein, Figure 18 -C is the Western Blot result map of the N1m recombinant protein, Figure 18 -D is the Western Blot result map of the N2m recombinant protein, Figure 18 where 1 represents the cell supernatant of the P1 generation of recombinant baculovirus, 2 represents the cell supernatant of the P2 generation of recombinant baculovirus, 3 represents the cell lysate of the P2 generation of recombinant baculovirus, 4 represents the blank control, and M is the protein ladder).

[0093] 4. Continuous passage and scale-up culture:

[0094] After successful identification of recombinant protein expression, observe the cell mortality rate daily. When the cell mortality rate of the P1 culture is greater than 90%, inoculate sf9 insect cells continuously for two generations according to MOI = 3 to obtain the P3 generation of recombinant baculovirus. When the cell mortality rate of the P3 culture is about 50%, centrifuge at 3000×g for 30 min at 4°C, discard the precipitate, and collect the supernatant.

[0095] 5. Protein concentration and protein purification:

[0096] The supernatant obtained in step 4 was concentrated using a vivaflow 200 membrane package. After three replacements with PBS, 100 mL of concentrated solution was obtained. It was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was collected. The liquid obtained after filtration through a 0.22 μM filter membrane was placed at 4 °C. Using a micro peristaltic pump, the liquid was allowed to flow through a nickel affinity column at a flow rate of 5 mL / min until all the liquid passed through the column. At this time, the protein was bound to the nickel affinity column. Affinity chromatography was performed using an AKTA protein purification system. First, the original column at column position 1 was removed. After plugging the two-line joint where the inlet pipe of the column valve is 1A and the outlet pipe is 1B with a connector, the Buffer in the system was replaced. After the Buffer replacement was completed, the nickel affinity column was connected to column position 1, and continuous gradient elution was started using a phosphate equilibrium buffer containing 5 mM imidazole and a phosphate elution buffer containing 500 mM imidazole, and the eluate was collected.

[0097] According to the ultraviolet absorption peak diagram after elution, the eluate containing the target protein harvested after affinity chromatography was selected and subjected to SDS-PAGE electrophoresis, and then stained with Coomassie Brilliant Blue, as Figure 19 ( Figure 19 -A is the Coomassie Brilliant Blue staining result diagram of the eluate of H1m recombinant protein, Figure 19 -B is the Coomassie Brilliant Blue staining result diagram of the eluate of H3m recombinant protein, Figure 19 -C is the Coomassie Brilliant Blue staining result diagram of the eluate of N1m recombinant protein, Figure 19 -D is the Coomassie Brilliant Blue staining result diagram of the eluate of N2m recombinant protein (the correct band of the target protein expression is within the square). According to the staining result, centrifugal concentration was performed using an ultrafiltration tube until the volume was 0.5 mL. After centrifuging the concentrated solution at 13,000×g for 10 min, the supernatant was taken and aliquoted, quick-frozen in liquid nitrogen, and stored at -80 °C for later use.

[0098] Example 3. Verification experiment on the biological function of Mosaic recombinant protein

[0099] 1. Verification of hemagglutination activity:

[0100] The hemagglutination titers of the purified Mosaic recombinant protein and the cell culture supernatant of the blank control were detected for hemagglutination with 1% guinea pig red blood cells: 50 μL of PBS was added to each well in columns 2-12 of a 96-well hemagglutination plate. 50 μL of the purified Mosaic recombinant protein and the control sample were aspirated and added to column 1 of the 96-well hemagglutination plate respectively. After pipetting and mixing evenly, 50 μL was aspirated and added to column 2, and after pipetting and mixing evenly again, 50 μL was aspirated and added to column 3, and serial dilutions were performed in sequence until 50 μL was discarded from column 11. A new pipette tip was needed each time after pipetting and mixing evenly. 1% guinea pig red blood cells were added to each well, and after shaking and mixing evenly, it was left to stand at room temperature for 25 min and then read. When reading, the well with complete agglutination was used as the hemagglutination titer of the sample.

[0101] The results are as Figure 20 shown, and it can be observed that 10 μg of the H1m recombinant protein can produce a hemagglutination titer of 2 6 , and 10 μg of the H3m recombinant protein can produce a hemagglutination titer of 2 7 . However, no hemagglutination phenomenon was observed in the blank control group (Mock).

[0102] 2. Verification of sugar receptor binding ability:

[0103] Add 50 μL of PBS to the first column of a 96-well plate, and add 50 μL of 3'SLN-PAA-biot and 6'SLN-PAA-biot (purchased from GlycoNZ, catalog numbers GNZ-0036-BP and GNZ-0997-BP) diluted to 500 ng / mL with PBS to the second and third columns. Each protein has two replicates, and incubate overnight at 4°C. Place the plate in a UV crosslinker at a wavelength of 254 nm for about 10 min, discard the liquid in the plate, wash the plate once with PBS for 3 minutes each time, add 100 μL / well of 1 w / v% BSA in PBS for blocking, and incubate overnight at 4°C. Discard the solution in each well, wash 3 times with PBS for 3 min each time, add 50 μL of 2 μg of the HAm recombinant protein to each well, and incubate overnight at 4°C. Wash the plate 6 times with PBST for 3 min each time, add 50 μL of the HA antibody against Influenza A virus H1N1 diluted 1:4000 (purchased from GeneTex, catalog number GTX127357) to each well; the HA antibody against Influenza A virus H3N2 was purchased from GeneTex, catalog number GTX127363), and incubate at room temperature for 2 h. Wash the plate 6 times with PBST for 3 min each time, add 100 μL of HRP-labeled goat anti-rabbit IgG diluted 1:8000 (purchased from Fude Bio, catalog number FDR007) to each well, and incubate at room temperature for 1 h. Wash the plate 6 times with PBST for 3 min each time. Add 100 μL of TMB staining solution to each well and react at room temperature for 30 min; add 50 μL of 2 M H 2 SO 4 solution to terminate the reaction, and immediately measure the absorbance values (OD450nm and OD630nm) at 450 nm and 630 nm using a microplate reader.

[0104] The results are as Figure 21 shown, and the H1m and H3m recombinant proteins have binding ability to both sialic acid α2,3-galactose receptor and sialic acid α2,6-galactose receptor, and there is a significant difference compared with the PBS group (P≤0.01), among which the binding ability to the α2,6-galactose receptor is stronger.

[0105] 3. Neuraminidase Activity Experiment

[0106] This experiment used a neuraminidase detection kit (purchased from Beyotime, catalog number P0306) for detection:

[0107] 1) Preparation for positive and negative control detections: a. Add 70 μL of neuraminidase detection buffer to each well in a 96-well fluorescence microplate. b. Add 10 or 0 μL of neuraminidase to each well respectively. c. Add 10 μL of the solution for dissolving the neuraminidase sample to each well. d. Add 0 or 10 μL of Milli-Q water to each well to make the total volume of each well 90 μL.

[0108] 2) Preparation for sample detections: a. Add 70 μL of neuraminidase detection buffer to each well in a 96-well fluorescence microplate. b. Add 10 μL of the neuraminidase sample to each well. c. Add 10 μL of Milli-Q water to each well to make the total volume of each well 90 μL.

[0109] 3) Detection: a. Vortex and mix well for about 1 minute. b. Add 10 μL of neuraminidase fluorescence substrate to each well. c. Vortex and mix well again for about 1 minute. d. After incubating at 37 °C for 30 minutes, perform fluorescence measurement. The excitation wavelength is 322 nm, and the emission wavelength is 450 nm.

[0110] The results are as Figure 22 shown. Both the N1m and N2m recombinant proteins have relatively high neuraminidase activities, and there are significant differences compared with the PBS group ((P ≤ 0.05 or P ≤ 0.01).

[0111] Example 4. Evaluation of the Immunization Effect of Mosaic Recombinant Proteins

[0112] Using the 4 Mosaic recombinant proteins (H1m, H3m, N1m, and N2m recombinant proteins) expressed and purified in Example 2 as immunogens, BALB / c mice were immunized to investigate the immunization effect of the Mosaic recombinant proteins.

[0113] 1. Immunize mice:

[0114] Use a BCA detection kit (purchased from Beyotime, catalog number P0012) to detect the concentrations of the 4 Mosaic recombinant proteins respectively. Mix the proteins evenly with 70,000 units / mL of IL-2 and 0.1% chitosan to obtain Mosaic recombinant protein vaccines (HAm protein vaccine and NAm protein vaccine).

[0115] Fifteen 6- to 8-week-old female BALB / c mice were randomly divided into 3 groups (blank control group: intramuscular injection of 100 μL PBS; immunized with HAm group: intramuscular injection of 100 μL of 2 HAm protein vaccines containing 60 μg; immunized with NAm group: intramuscular injection of 100 μL of 2 NAm protein vaccines containing 60 μg). At week 0 and week 2, the female BALB / c mice were immunized according to the grouping. At week 0 and week 4 (day 28) after immunization, orbital blood collection was performed on the mice in each group. After standing overnight at 4°C, the serum was obtained by centrifugation at 3000 rpm for 10 min, aliquoted and stored at -80°C for later use.

[0116] 2. Hemagglutination inhibition (HAI) assay:

[0117] 1) Preparation of mouse serum treated with RDE: The receptor-destroying enzyme (RDE, purchased from Seiken, Japan, catalog number 340122) was mixed with the serum of each group of mice at a volume ratio of 3:1 in a test tube and placed in a 37°C water bath for 16 h; the test tube was taken out and placed in a 56°C water bath for 30 min to inactivate RDE; PBS was added to the test tube to dilute the serum to a dilution of 1:10; after cooling to room temperature, chicken red blood cells with 1 / 2 volume of the original serum were added and mixed well, and stored at 4°C for 1 h, during which it was remixed every 15 min; centrifuged at 1200 rpm for 1 min, and the supernatant was aspirated to obtain mouse serum treated with RDE, and left to stand at 4°C for later use.

[0118] 2) Preparation of four-unit standard antigen: The HA titers of A / Victoria / 2570 / 2019 (H1N1 subtype strain, donated by the Chinese Center for Disease Control and Prevention) and A / Cambodia / E0826360 / 2020 (H3N2 subtype strain, donated by the Chinese Center for Disease Control and Prevention) were detected respectively. Each antigen was diluted to 8 hemagglutination units with PBS, and the HA titer was confirmed again and further diluted to 4 hemagglutination units, that is, the four-unit standard antigen was obtained.

[0119] 3) Hemagglutination inhibition assay: Add 25 μL of PBS to each well in columns 2 - 10 and column 12 of a 96-well plate, and add 50 μL of PBS to each well in column 11. Add 25 μL of mouse serum treated with RDE to each well in columns 1 and 2, and mix well. Pipette 25 μL of the mixed solution from column 2 and add it to column 3, and mix well. Repeat the above operation until column 10, and discard 25 μL of the mixed solution in column 10. Add 25 μL of four-unit standard antigen to columns 1 - 10 and column 12. Among them, column 12 serves as the virus control column, and at the same time, add positive serum (mouse serum obtained in the early stage of the laboratory, which has been verified to have hemagglutination titer against the corresponding virus strain) to column 11 as a standard positive control. After thorough mixing, place the 96-well plate at room temperature and let it stand for 45 min. Add 50 μL of 1% chicken red blood cell suspension to each well, let it stand at room temperature for 25 min, tilt the 96-well plate at 45 °C, and observe whether the red blood cells flow in a teardrop shape.

[0120] The results are as Figure 23 shown. The influenza virus-specific hemagglutination inhibition antibody showed that in the mice immunized with HAm, the mouse sera showed certain cross-protective effects against the two vaccine strains of the 2021 - 2022 seasonal influenza virus at week 4 (D28) after the first immunization, and were significantly higher than the blank control group (P ≤ 0.05).

[0121] 3. Neuraminidase Inhibition (NI) assay

[0122] 1) Determination of the dosage of PNA-HPRO: Add 1% BSA in PBST as a sample diluent to a 96-well plate, 216 μL to each well in columns 1 - 11. Thaw the virus, mix well, and add 24 μL to each well in columns 1 - 11. Take out the 96-well plate coated with fetal protein, wash the plate 6 times with PBST, 3 min each time. Transfer 50 μL of the diluted virus in parallel to the 96-well plate coated with fetal protein, add 50 μL of sample diluent to each well, and add 100 μL of sample diluent to column 12. Gently shake and mix well, then place it in an incubator at 37 °C for 16 hours. After incubation, aspirate the liquid in the plate, wash the plate 6 times with PBST, 3 min each time. Add 100 μL of PNA-HRPO (original concentration 1 mg / mL) diluted at 1:200, 1:400, 1:500, 1:800, 1:1000, 1:1600, 1:2000, 1:3000, 1:4000, 1:5000 to each well in columns 1 - 10 in sequence, and incubate at room temperature for 2 h. Wash the plate 6 times with PBST, 3 min each time. Add 100 μL of TMB staining solution to each well, and react at room temperature for 30 min; add 50 μL of 2M H 2 SO 4The reaction of the solution was terminated, and the absorbance value (OD450nm) at 450 nm was immediately measured using a microplate reader. Determine the dosage of PNA-HPRO according to the results.

[0123] 2) Determination of the dosage of NA: Add 1% BSA in PBST as a sample diluent to a 96-well plate, 120 μL per well in columns 2 - 12, and 216 μL per well in column 1. Thaw the virus, mix well and add 24 μL to column 1, and perform serial 2-fold dilutions up to column 11. Take out the 96-well plate coated with fetal protein and wash it 6 times with PBST, 3 minutes each time. Transfer 50 μL of the diluted virus in parallel to the 96-well plate coated with fetal protein, and add 50 μL of the sample diluent to each well. Gently shake and mix well, then place it in an incubator at 37 °C for 16 hours. After incubation, discard the liquid in the plate, and wash the plate 6 times with PBST, 3 minutes each time. Add 100 μL of the PNA-HPRO with the determined dosage in the above step to each well in columns 1 - 11, and incubate at room temperature for 2 h. Wash the plate 6 times with PBST, 3 minutes each time. Add 100 μL of TMB staining solution to each well, and react at room temperature for 30 min; add 50 μL of 2M H 2 SO 4 The reaction of the solution was terminated, and the absorbance value (OD450nm) at 450 nm was immediately measured using a microplate reader. Determine the dosage of NA according to the results.

[0124] 3) Preparation of mouse serum treated with RDE: Mix the receptor-destroying enzyme (RDE, purchased from Nissui Pharmaceutical Co., Ltd., Japan, product number 340122) with the serum of each group of mice at a volume ratio of 3:1 in a test tube, and place it in a water bath at 37 °C for 16 h; take out the test tube, inactivate RDE in a water bath at 56 °C for 30 min; add PBS to the test tube to make the serum dilution reach 1:10; after cooling to room temperature, add chicken red blood cells with a volume of 1 / 2 of the original serum and mix well, place it at 4 °C for 1 h, and remix every 15 min during this period; centrifuge at 1200 rpm for 1 min, aspirate the supernatant to obtain mouse serum treated with RDE, and let it stand at 4 °C for later use.

[0125] 4) Neuraminidase inhibition assay: Add 1% BSA in PBST as the sample diluent to the 96-well plate, 120 μL per well in columns 3 - 11, and 216 μL per well in column 2. Take the mouse serum after RDE treatment and add 24 μL to column 1, and perform serial two-fold dilutions to column 11. Take out the 96-well plate coated with fetoprotein and wash it 6 times with PBST, 3 min each time. Transfer 50 μL of the diluted serum in parallel to the 96-well plate coated with fetoprotein, and add 50 μL of the virus with the NA dosage determined in the previous step to each well in columns 1 - 11. After gently shaking and mixing, place it in an incubator at 37 °C for 16 hours. After incubation, aspirate the liquid in the plate and wash the plate 6 times with PBST, 3 min each time. Add 100 μL of the PNA-HPRO with the dosage determined in the previous step to each well in columns 1 - 11 and incubate at room temperature for 2 h. Wash the plate 6 times with PBST, 3 min each time. Add 100 μL of TMB staining solution to each well and react at room temperature for 30 min; add 50 μL of 2M H 2 SO 4 solution to terminate the reaction, and immediately measure the absorbance value (OD450nm) at 450 nm using a microplate reader.

[0126] The results are as Figure 24 shown. At 4 weeks (day 28) after the first immunization, the mouse sera in the NAm immunization group produced specific neuraminidase inhibitory antibodies against the two vaccine strains A / Victoria / 2570 / 2019 and A / Cambodia / E0826360 / 2020, and were significantly higher than those in the blank control group (P ≤ 0.05 or P ≤ 0.01), indicating that the Mosaic recombinant protein immunization of the present invention had a certain protective effect on mice.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

[0128] SEQ ID NO.1:

[0129] MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTARSWSYIVETSNSDNGTCYPGDFINYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYINDKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYSKKFKPEIATRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFTMERNAGSGIIISDTPVHDCNTTCQTPEGAINTSLPFQNVHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQNAIDKITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLVLLENERTLDYHDSNVKNLYEKVRNQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREKIDGVKLESTRIYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI

[0130] SEQ ID NO.2:

[0131] MKTIIALSYILCLVFAQKIPGNDNSTATLCLGHHAVPNGTIVKTITNDRIEVTNATELVQNSSIGEICDSPHQILDGENCTLIDALLGDPQCDGFQNKKWDLFVERSKAYSNCYPYDVPDYASLRSLVASSGTLEFKNESFNWTGVKQNGTSSACIRGSSSSFFSRLNWLTHLNYTYPALNVTMPNKEQFDKLYIWGVHHPGTDKDQIFLYAQSSGRITVSTKRSQQAVIPNIGSRPRIRDIPSRISIYWTIVKPGDILLINSTGNLIAPRGYFKIRSGKSSIMRSDAPIGKCKSECITPNGSIPNDKPFQNVNRITYGACPRYVKQSTLKLATGMRNVPEKQTRGIFGAIAGFIENGWEGMVDGWYGFRHQNSEGRGQAADLKSTQAAIDQINGKLNRLIGKTNEKFHQIEKEFSEVEGRVQDLEKYVEDTKIDLWSYNAELLVALENQHTIDLTDSEMNKLFEKTKKQLRENAEDMGNGCFKIYHKCDNACIGSIRNETYDHNVYRDEALNNRFQIKGVELKSGYKDWILWISFAISCFLLCVALLGFIMWACQKGNIRCNICI

[0132] SEQ ID NO.3:

[0133] MNPNQKIITIGSICMTIGMANLILQIGNIISIWVSHSIQIGNQSQIETCNQSVITYENNTWVNQTYVNISNTNFAAGQSVVSVKLAGNSSLCPVSGWAIYSKDNSVRIGSKGDVFVIREPFISCSPLECRTFFLTQGALLNDKHSNGTIKDRSPYRTLMSCPIGEVPSPYNSRFESVAWSASACHDGINWLTIGISGPDSGAVAVLKYNGIITDTIKSWRNNILRTQESECACVNGSCFTIMTDGPSDGQASYKIFRIEKGKIIKSVEMKAPNYHYEECSCYPDSSEITCVCRDNWHGSNRPWVSFNQNLEYQMGYICSGVFGDNPRPNDKTGSCGPVSSNGANGVKGFSFKYGNGVWIGRTKSISSRKGFEMIWDPNGWTGTDNKFSIKQDIVGINEWSGYSGSFVQHPELTGLDCIRPCFWVELIRGRPEENTIWTSGSSISFCGVNSDTVGWSWPDGAELPFTIDK

[0134] SEQ ID NO.4:

[0135] MNPNQKIITIGSVSLTISTICFFMQIAILITTVTLHFKQYEFNSPPNNQVMLCEPTIIERNITEIVYLTNTTIEKEICPKPAEYRNWSKPQCGITGFAPFSKDNSIRLSAGGDIWVTREPYVSCDPDKCYQFALGQGTTLNNVHSNNTVRDRTPYRTLLMNELGVPFHLGTKQVCIAWSSSSCHDGKAWLHVCITGDDKNATASFIYNGRLVDSVVSWSKDILRTQESECVCINGTCTVVMTDGNATGKADTKILFIEEGKIVHTSKLSGSAQHVEECSCYPRYPGVRCVCRDNWKGSNRPIVDINIKDHSIVSSYVCSGLVGDTPRKNDSSSSSHCLDPNNEEGGHGVKGWAFDDGNDVWMGRTINETSRLGYETFKVVEGWSNPKSKLQINRQVIVDRGDRSGYSGIFSVEGKSCINRCFYVELIRGRKEETEVLWTSNSIVVFCGTSGTYGTGSWPDGADLNLMHI

[0136] SEQ ID NO.5:

[0137]

[0138] SEQ ID NO.6:

[0139]

[0140] SEQ ID NO.7:

[0141]

[0142] SEQ ID NO.8:

[0143]

Claims

1. A universal Mosaic recombinant antigen for seasonal influenza A, characterized in that, the sequence of the gene encoding the universal Mosaic recombinant antigen for seasonal influenza A is as shown in SEQ ID No: 5; the universal Mosaic recombinant antigen for seasonal influenza A is added with a gp67 signal peptide, a thrombin cleavage site and an 8×His tag, and a GCN4pII sequence is added to the HA protein; the amino acid sequence of the gp67 signal peptide is: MLLVNQSHQGFNKEHTSKMVSAIVLYVLLAAAAHSAFAAD; the GCN4pII sequence is MKQIEDKIEEILSKIYHIENEIARIKKLIGEV; the amino acid sequence of the thrombin cleavage site is LVPRGS.

2. A vector comprising the gene encoding the universal Mosaic recombinant antigen for seasonal influenza A as claimed in claim 1.

3. The vector as claimed in claim 2, characterized in that, the vector is obtained by ligating the gene encoding the universal Mosaic recombinant antigen for seasonal influenza A as claimed in claim 1 into an expression plasmid.

4. A cell comprising the gene encoding the universal Mosaic recombinant antigen for seasonal influenza A as claimed in claim 1 or the vector as claimed in claim 2 or 3.

5. The cell as claimed in claim 4, characterized in that, the cell is obtained by transferring the gene or the vector into an Escherichia coli host cell.

6. Use of a universal Mosaic recombinant antigen for seasonal influenza A as claimed in claim 1 in the preparation of a universal vaccine for seasonal influenza A.

7. A vaccine preparation, characterized in that, the vaccine preparation comprises the gene encoding the universal Mosaic recombinant antigen for seasonal influenza A as claimed in claim 1 or the vector as claimed in claim 2 or 3.

8. The vaccine preparation as claimed in claim 7, characterized in that, it further comprises an immunologically and pharmaceutically acceptable carrier or adjuvant.

9. Use of a vaccine preparation as claimed in claim 7 or 8 in the preparation of a medicament for preventing and / or treating seasonal influenza A.

Images