Influenza B virus HA protein mutant and application thereof in self-replicating RNA vaccine

Abstract

The invention provides a mutant of influenza B virus HA protein and application of the mutant in a self-replicating RNA (Ribonucleic Acid) vaccine. The mutant comprises four mutation sites of S469D, S462F, S426P and D430P; according to the present invention, three mutation sites, i.e., G268C, T35C and E464C, and seven mutation sites, i.e., S469D, S462F, S426P, D430P, G268C, T35C and E464C, are provided; wherein the S469D is added with a lysine (K406) salt bridge; the hydrophobicity of the L414 / I466 space is increased by the S462F; the stability of the antigen epitope of the HA protein is improved by the S426P and the D430P; and a new disulfide bond is generated by G268C, T35C and E464C, so that the conformation stability before HA protein fusion is enhanced. According to the present invention, the specific amino acid site of the HA protein is modified, such that the self-replicating RNA (self-amplifying RNA) vaccine based on the mutant continuously expresses the HA protein more stable than the natural structure in vivo, and the generation of the antibody having the neutralizing activity is induced so as to enhance the anti-virus efficacy of the vaccine;

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an influenza B virus HA protein mutant and an application thereof in a self-replicating vaccine. Background Art

[0002] Influenza virus belongs to the Orthomyxoviridae family of enveloped viruses. Its genome consists of eight negative-strand RNA fragments, encoding a total of 11 proteins. According to the difference in the antigenicity of nucleoprotein and matrix protein, influenza virus is divided into four categories: A, B, C, and D. Among them, there are two glycoproteins on the surface of type A and type B viruses, hemagglutinin (HA) and neuraminidase (NA), while type C and type D viruses only have hemagglutinin-esterase fusion (HEF). Type A influenza virus can be divided into different subtypes according to the combination of HA and NA glycoproteins on the surface of the virus. The current main popular combinations are A / H1N1 and A / H3N2 subtype viruses. Since 1983, type B influenza virus has gradually evolved into Yamagata and Victoria strains.

[0003] Influenza A and B viruses include many different strains, and different strains cause regular seasonal outbreaks of influenza, among which influenza A and influenza B viruses alternate to form global transmission. The Chinese Center for Disease Control and Prevention conducted a nationwide prospective surveillance of patients of all ages with acute respiratory infections from 2009 to 2019. Influenza virus is the most important viral pathogen, with a much higher infection rate than respiratory syncytial virus and rhinovirus, at 28.5%, 16.8%, and 16.7%, respectively.

[0004] Vaccination is the best way to prevent influenza. WHO and other partners continuously monitor the activities of influenza viruses around the world through the Global Influenza Program and the Global Influenza Surveillance and Response System. They recommend seasonal influenza vaccine ingredients twice a year for the northern and southern hemisphere influenza seasons and guide countries in tropical and subtropical regions on which vaccine formula to use. Influenza vaccines are mainly trivalent or quadrivalent attenuated live vaccines or inactivated vaccines, which are produced by breeding viruses in chicken embryos or mammalian cells, which takes a long time to produce and has many adverse reactions.

[0005] There is no mRNA vaccine for influenza on the market yet. Moderna, a US company, has conducted clinical trials of the influenza mRNA vaccine mRNA-1010, which has demonstrated ideal safety. However, in the Phase III clinical trial conducted during the 2022 southern hemisphere influenza epidemic season, interim results showed that the seroconversion rates (i.e., neutralizing antibodies reaching the positive standard) for influenza A H1N1 and H3N2 were superior to existing vaccines. In the comparison of the geometric mean titer (GMT, Geometric Mean Titer), the anti-H3N2 antibody level achieved superiority (i.e., the test drug response was better than the control drug (positive or placebo control)), and the anti-H1N1 antibody level was non-inferior (i.e., the test drug response was clinically not inferior to the comparison preparation (positive or placebo control)). However, mRNA-1010 did not achieve non-inferiority in both indicators for the influenza B virus strains Yamagata and Victoria.

[0006] The viruses that cause seasonal influenza epidemics are mainly the H1N1 and H3N2 subtypes of influenza A virus and the Victoria strain of influenza B virus. Therefore, improving the efficacy of the B-Victory strain mRNA vaccine is crucial to preventing the spread of influenza B virus. Summary of the invention

[0007] In order to solve the problem of limited efficacy of B-Victory strain mRNA vaccine, the present invention provides a mutant of hemagglutinin (HA) protein of B-Victoria strain of influenza B virus and its application in self-replicating vaccine, wherein the mutant includes four mutation sites of S469D, S462F, S426P and D430P; three mutation sites of G268C, T35C and E464C and three mutation modes of seven mutation sites of S469D, S462F, S426P, D430P, G268C, T35C and E464C. The present invention, through the transformation of specific amino acid sites of HA protein, the self-replicating RNA (self-amplifying RNA, saRNA) vaccine based on the mutant continuously expresses HA protein that is more stable than the natural structure in vivo, induces the production of antibodies with more neutralizing activity, thereby enhancing the antiviral efficacy of the vaccine.

[0008] On the one hand, the present invention provides a mutant of influenza B virus HA protein, which is based on the sequence of wild-type influenza B virus HA protein and has mutation sites, including a combination of any one or more mutation sites of S469D, S462F, S426P, D430P, G268C, T35C, and E464C.

[0009] The present invention is designed and studied for HA protein, hoping to form salt bridges, hydrophobic bonds or disulfide bonds in the HA protein through mutation sites, thereby increasing the stability of the HA protein, so that it can be continuously expressed and have high stability. When used for preparing vaccines, it can also enhance the body's ability to recognize antigens and achieve long-term immunity of the body, thereby improving the immune effect of the vaccine.

[0010] It is understandable that mutation of any one or more of the seven mutation sites of HA protein, S469D, S462F, S426P, D430P, G268C, T35C, and E464C, has the effect of improving the conformational stability of HA protein.

[0011] Furthermore, the mutant includes a combination of any three, four or seven mutation sites of S469D, S462F, S426P, D430P, G268C, T35C and E464C.

[0012] In addition, there are many other combinations, such as selecting any 1 to 7 of the seven mutation sites for combination, or combining with other mutation sites that are expected to improve the stability and expression of HA protein, or combining with any carrier used to prepare vaccines (such as combining with the replicase with mutation sites described in the domestic patent CN2024114401133 applied for by this research team), all of which have certain beneficial effects.

[0013] Furthermore, the wild-type influenza B virus is a Victoria strain, and the amino acid sequence of its HA protein is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2.

[0014] In some embodiments, the mutant of the wild-type influenza B virus HA protein includes a mutation site S469D, and the amino acid sequence of the HA protein mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4.

[0015] In some embodiments, the mutant of the wild-type influenza B virus HA protein includes a mutation site S462F, and the amino acid sequence of the HA protein mutant is shown in SEQ ID NO.5, and the nucleotide sequence is shown in SEQ ID NO.6.

[0016] In some embodiments, the mutant of the wild-type influenza B virus HA protein includes a mutation site S426P, and the amino acid sequence of the HA protein mutant is shown in SEQ ID NO.7, and the nucleotide sequence is shown in SEQ ID NO.8.

[0017] In some embodiments, the mutant of the wild-type influenza B virus HA protein includes a mutation site D430P, and the amino acid sequence of the HA protein mutant is shown in SEQ ID NO.9, and the nucleotide sequence is shown in SEQ ID NO.10.

[0018] In some embodiments, the mutant of the wild-type influenza B virus HA protein includes a mutation site G268C, and the amino acid sequence of the HA protein mutant is shown in SEQ ID NO.11, and the nucleotide sequence is shown in SEQ ID NO.12.

[0019] In some embodiments, the mutant of the wild-type influenza B virus HA protein includes a mutation site T35C, and the amino acid sequence of the HA protein mutant is shown in SEQ ID NO.13, and the nucleotide sequence is shown in SEQ ID NO.14.

[0020] In some embodiments, the mutant of the wild-type influenza B virus HA protein includes a mutation site E464C, and the amino acid sequence of the HA protein mutant is shown in SEQ ID NO.15, and the nucleotide sequence is shown in SEQ ID NO.16.

[0021] In some embodiments, the mutant includes four mutation sites: S469D, S462F, S426P, and D430P, and has an amino acid sequence as shown in SEQ ID NO.17.

[0022] Among them, S469D increases the K406 salt bridge, S462F increases the hydrophobicity of the L414 and I466 spaces, and S426P and D430P increase the stability of the HA protein antigenic epitope.

[0023] In the present invention, the mutant is named BV-HA 01, and the DNA sequence corresponding to the amino acid sequence of BV-HA 01 is shown in SEQ ID NO.18.

[0024] In some embodiments, the mutant includes three mutation sites, G268C, T35C, and E464C, and has an amino acid sequence as shown in SEQ ID NO. 19. The mutant is named BV-HA 02, and the DNA sequence corresponding to the amino acid sequence of BV-HA 02 is shown in SEQ ID NO. 20.

[0025] The three mutation sites G268C, T35C, and E464C generate new disulfide bonds, enhancing the stability of the HA protein.

[0026] In some embodiments, seven mutation sites, including S469D, S462F, S426P, D430P, G268C, T35C, and E464C, have an amino acid sequence as shown in SEQ ID NO. 21. The mutant is named BV-HA 03, and the DNA sequence corresponding to the amino acid sequence of BV-HA 03 is shown in SEQ ID NO. 22.

[0027] The protein expressed in cells by the mRNA after the above-mentioned modification is more stable than the HA protein with the natural sequence, and can thus induce the production of more efficient neutralizing antibodies in mice, thereby enhancing the immune efficacy against the virus.

[0028] In addition to the HA protein coding region described above (carrying several point mutations), the synthetic product precursor also contains other functional elements: a cap structure and a 5'UTR sequence at the 5' end; non-structural proteins nsP1, nsP2, nsP3, and nsP4 from alphavirus; a subgenomic promoter, a 3'UTR sequence; and elements such as a PolyA tail structure (such as Figure 1 ).

[0029] In another aspect, the present invention provides an influenza vaccine comprising the mutant as described above.

[0030] Furthermore, the present invention adds three mutation sites G268C, T35C, and E464C on the basis of the above-mentioned four mutation sites S469D, S462F, S426P, and D430P. The neutralizing antibody titer produced by the vaccine designed based on this is further improved, thereby enhancing the immune efficacy against influenza virus.

[0031] Furthermore, the vaccine includes one or more of a traditional single-stranded mRNA vaccine, a self-replicating RNA vaccine, a circular RNA vaccine, a DNA vaccine or a protein subunit vaccine.

[0032] It is understandable that the above-mentioned HA protein mutants with higher stability can be used to prepare any form of vaccine and can effectively improve the immune effect of the vaccine.

[0033] In some embodiments, the vaccine comprises a self-replicating RNA vaccine.

[0034] In another aspect, the present invention provides a use of a mutant for preparing a vaccine with improved efficacy against influenza B virus, wherein the mutant is based on the sequence of wild-type influenza B virus HA protein and has a mutation site, wherein the mutation site includes any one or more of S469D, S462F, S426P, D430P, G268C, T35C, and E464C.

[0035] Furthermore, the mutant includes a combination of any three, four or seven mutation sites of S469D, S462F, S426P, D430P, G268C, T35C and E464C.

[0036] In another aspect, the present invention provides a use of a mutant for preparing a reagent for improving the stability of influenza B virus HA protein, wherein the mutant is based on the sequence of wild-type influenza B virus HA protein and has a mutation site, and the mutation site includes any one or more of S469D, S462F, S426P, D430P, G268C, T35C, and E464C.

[0037] Compared with the prior art, the present invention has the following technical effects or advantages:

[0038] 1. The 10 mutants of HA protein provided by the present invention can all improve the stability of HA protein;

[0039] 2. The antigens expressed in mice by the saRNA vaccine based on the modified HA protein mutant can induce the production of neutralizing antibodies more efficiently and lastingly, thereby enhancing the immune efficacy against the virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0041] Figure 1 Schematic diagram of the saRNA encoding the influenza B-Victory strain HA protein mutant in the present invention.

[0042] Figure 2 is the expression level of saRNA encoding HA protein mutants in HEK293 cells;

[0043] Figure 3 The left figure shows the HA expression level of saRNA vaccine in HEK293 cells, and the right figure shows the average fluorescence intensity result.

[0044] Figure 4 These are the results of a study on the persistence of HA expressed by saRNA vaccines in HEK293 cells.

[0045] Figure 5 This is the neutralizing antibody level after mice were immunized with saRNA vaccine. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0047] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0048] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0049] The evaluation results show that the neutralizing antibody titers of BV-HA 01, BV-HA 02, and BV-HA 03 vaccines based on the mutants of the present invention after immunization of mice are significantly higher than those of vaccines designed based on wild-type HA proteins. This indicates that the HA protein expressed in vivo by the saRNA vaccine corresponding to the mutant modified by the present application is more stable than the HA protein of the natural sequence, and can induce more efficient neutralizing antibody production, thereby enhancing the immune efficacy against the virus.

[0050] The saRNA vaccine based on the wild-type HA protein mutant of the present application will be described in detail below in combination with the examples and experimental data.

[0051] Example 1: Screening of HA protein mutation sites

[0052] Based on the amino acid sequence of B / Austria / 1359417 / 2021 (B / Victoria lineage)-like virus HA, the experience of amino acid modification reported in the literature (Science, 2020, PMID: 32703906; Trends Microbiol. 2018, PMID: 29681430) and the classic design principles of structural biology (including salt bridges, proline mutations, additional disulfide bonds, hydrogen bonds and hydrophobic pockets, etc.), we tried to obtain a more stable prefusion conformation by changing amino acids. We first predicted the B / Austria / 1359417 / 2021 (B / Victorialineage)-like virus HA protein structure through Alfafold3 and found that it has 5 pairs of disulfide bonds, namely C503-C507, C19-C496, C307-C333, C72-C75, and C109-C158. So we redesigned new disulfide bond pairs based on the spatial position of amino acids. We searched for amino acids that may pair to form disulfide bonds in the 3A range of C193, and mutated G at position 268 to C (i.e., G268C) to form a disulfide bond with C193; and after the T at position 35 was transformed into C and the E at position 464 was transformed into C, the formation of the T35C-E464C disulfide bond promoted the connection between the LOOP region and the Helix region, improving the structural stability). On the other hand, in order to maintain the prefusion conformation of HA and avoid changes to the postfusion conformation, we modified the sites S426 and D430 that can affect conformational changes in the conformationally variable loop region, and enhanced the conformational stability of prefusion by amino acid modification S426P / D430P, which is more conducive to maintaining the antigenic epitope; in addition, based on the hydrophobic environment generated by L414 and I466 in the helix region, S462 in the helixA domain was modified to F462, thereby enhancing the interaction between the two helix structures. In addition, the modification of S469D in the helix-A fragment can form a salt bridge with K406, thereby stabilizing the prefusion conformation and reducing the conversion to the postfusion conformation. Finally, we designed a total of seven mutation sites: S469D, S462F, S426P, D430P, G268C, T35C, and E464C. Mutation of any one or more of these seven mutation sites in the HA protein can effectively improve the conformational stability of the HA protein.

[0053] Table 1 Information on 7 mutation sites in HA protein

[0054]

[0055] Example 2: Effect of mutation on HA protein stability

[0056] In this example, according to the 7 mutation sites provided in Example 1, HA protein mutants were constructed based on the wild-type influenza B virus HA protein, including 10 types: mutant 1: mutation S469D; mutant 2: mutation S462F; mutant 3: mutation S426P; mutant 4: mutation D430P; mutant 5: G268C; mutant 6: mutation T35C; mutant 7: mutation E464C; BV-HA01: mutations S469D, S462F, S426P and D430P; BV-HA02: mutations G268C, T35C and E464C; BV-HA 03: mutations S469D, S462F, S426P, D430P and G268C, T35C, E464C, and 10 HA protein mutants were obtained by in vitro expression.

[0057] The stability of the mutants was evaluated by measuring the thermal stability Tm value of the mutants using circular dichroism (CD) spectroscopy, and compared with the wild-type viral HA protein. The test results are shown in Table 2. Tm represents the midpoint temperature of thermal denaturation of the protein, that is, the temperature at which the protein is 50% unfolded, reflecting the trend of protein conformation change during the temperature change process. The higher the Tm, the better the stability.

[0058] Table 2 Stability of HA protein mutants

[0059]

[0060]

[0061] According to Table 2, compared with the wild-type influenza B virus HA protein, the thermal stability of the mutants has been significantly improved. Among the mutants with a single mutation site, the stability of mutant 3 has been improved most significantly, with a Tm of 58°C, followed by mutant 5, with a Tm of 57°C. The thermal stability of the mutants after combined mutations is higher than that of the single mutation site (the improvement of BV-HA 02 is not as obvious as that of BV-HA 01 and BV-HA 03), and BV-HA 03 has the highest thermal stability.

[0062] The higher the thermal stability, the more stable the spatial conformation of the protein, and the more stable the prepared HA protein mutant. At the same time, the subsequent embodiments of the present invention also found that mutants with higher thermal stability can express antigens more stably and persistently when used in vaccine antigen design, thereby improving the body's ability to produce neutralizing antibodies.

[0063] Example 3. Preparation of mRNA of influenza B HA protein mutants provided by the present invention

[0064] The DNA sequence encoding the influenza B HA protein was commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. Among them, the RNA of the HA protein, based on the wild-type B-Victory strain HA protein (WT), has 10 mutants: saRNA 1: mutation S469D; saRNA 2: mutation S462F; saRNA 3: mutation S426P; saRNA 4: mutation D430P; saRNA 5: G268C; saRNA 6: mutation T35C; saRNA 7: mutation E464C; BV-HA 01: mutations S469D, S462F, S426P and D430P; BV-HA 02: mutations G268C, T35C and E464C; BV-HA 03: mutations S469D, S462F, S426P, D430P and G268C, T35C, E464C. The isolate ID of the B-Victory strain in the GISAID EpiFlu database is EPI_ISL_1519459, from which the amino acid sequence and nucleotide sequence of the HA protein were obtained, the corresponding amino acid sequence is SEQ ID No.1, and the DNA sequence is SEQ ID No.2; the amino acid sequence of saRNA 1 is SEQ ID No.3, and the DNA sequence is SEQ ID No.4; the amino acid sequence of saRNA 2 is SEQ ID No.5, and the DNA sequence is SEQ ID No.6; the amino acid sequence of saRNA 3 is SEQ ID No.7, and the DNA sequence is SEQ ID No.8; the amino acid sequence of saRNA 4 is SEQ ID No.9, and the DNA sequence is SEQ ID No.10; the amino acid sequence of saRNA 5 is SEQ ID No.11, and the DNA sequence is SEQ ID No.12; the amino acid sequence of saRNA 6 is SEQ ID No.13, and the DNA sequence is SEQ ID No.14; the amino acid sequence of saRNA 7 is SEQ ID No.15, and the DNA sequence is SEQ ID No.16; the amino acid sequence of BV-HA 01 is SEQ ID No.17, and its DNA sequence is SEQ ID NO.18; the amino acid sequence of BV-HA 02 is SEQ ID No.19, and its DNA sequence is SEQ ID NO.20; the amino acid sequence of BV-HA 03 is SEQ ID No.21, and its DNA sequence is SEQ ID NO.22.

[0065] The synthesized DNA sequence was inserted between the subgenomic promoter and 3'UTR of the self-replicating RNA vector to construct a self-replicating RNA vaccine sequence template plasmid, wherein the self-replicating RNA backbone sequence was referenced to the published literature (McKay, PF, Hu, K., Blakney, AK et al. Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. Nat Commun 11, 3523 (2020)), and the SARS-CoV-2 antigen sequence was replaced with a synthesized HA antigen sequence, transferred into E. Coli and positive clones with stable presence of PolyA were selected. After the shake flask was expanded and cultured, the bacteria were collected, and after alkaline lysis, neutralization, clarification, concentration and liquid replacement, purification, linearization and enzyme digestion, and template purification, a high-quality template was obtained for in vitro transcription (IVT). The template was subjected to an IVT reaction in vitro through optimized T7 RNA polymerase and transcription conditions to obtain the saRNA of the above 10 mutants.

[0066] Example 4. Expression of saRNA encoding HA protein mutants in HEK293 cells

[0067] The different saRNAs synthesized in Example 3 were transfected in HEK293T cells, and the groups were set as follows: PBS control group, saRNA-WT, saRNA 1, saRNA 2, saRNA 3, saRNA 4, saRNA 5, saRNA 6, saRNA 7, BV-HA01, BV-HA 02, BV-HA 03. The experimental method was as follows: HEK293T cells were inoculated in a culture dish, and when the cells grew to 80%, 11 kinds of saRNAs (including blank control) were added at 5 μg / mL, and the cells were collected after 24 hours. After fixing the cells with 4% paraformaldehyde for 15 minutes at room temperature, the cells were permeabilized with 0.2% Saponin for 10 minutes, and then the HA protein in the cells was labeled with anti-B-Victory HA antibody (NIBSC 22 / 228). After incubation at room temperature for 1 hour, the fluorescent secondary antibody was added and continued to be incubated at room temperature for 1 hour. After washing the cells with PBS, the fluorescence intensity was detected by flow cytometry.

[0068] The results are as follows Figure 2As shown, compared with the wild-type HA protein, both the single mutation saRNA1-7 and the combined mutation BV-HA 01-03 have increased MFI values, indicating that the level of expressed HA protein is increased, among which the expression of HA protein of the combined mutation BV-HA01-03 is even higher than that of the single mutation saRNA1-7.

[0069] Example 5. Preparation of saRNA vaccine

[0070] (I) Purification of saRNA

[0071] The saRNA synthesized in Example 3 was purified by oligo dT affinity, the buffer was replaced to sodium acetate by lateral flow filtration, and sterile filtration was performed. RNA integrity was measured by microfluidic parallel capillary electrophoresis (Fragment Analyzersystem 5200, Agilent), and the concentration, pH value, residual DNA, protein and double-stranded RNA impurities of the solution were also measured. Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the 5' end capping efficiency and 3'-polyadenosine (poly A) tail of mRNA.

[0072] (II) Encapsulation of saRNA

[0073] 1. Dissolve SM102, DSPC, cholesterol and DMG-PEG2000 in ethanol solution at a concentration of 10 mg / ml. According to the ratio of SM102: DSPC: cholesterol: DMG-PEG2000 of 50:10:38.5:1.5 (molar ratio), mix the four lipids and add anhydrous ethanol to control the total lipid concentration to 10 mM.

[0074] 2. Dilute different antigen RNAs in a citrate buffer (pH 4.0) at a mass ratio of 1:1, where the input amount of RNA and lipid must satisfy the N / P of 6. The prepared organic phase (lipid) and aqueous phase (RNA) are used to prepare RNA-encapsulated LNP vaccine particles through a microfluidic instrument, where the flow rate of the aqueous phase is 12 mL / min and the flow rate of the organic phase is 4 mL / min.

[0075] 3. Immediately after obtaining the LNP vaccine particles, they were diluted with sterile ultrapure water, and the dilution volume was 10 times. After dilution, the vaccine particles were ultrafiltered using a 100kDa ultrafiltration tube, and the centrifugation conditions were 3000rpm and 4°C. When the liquid was centrifuged to 1mL, sterile ultrapure water was added to 15mL, and centrifugation was continued for 3 times to reduce the ethanol content in the vaccine particles.

[0076] 4. During the final centrifugation, the vaccine particles are concentrated to 1 mL to ensure that the required immune dose is met.

[0077] saRNA is encapsulated into lipid nanoparticles (LNPs) by ethanol lipid mixtures (ionic cationic lipids, phosphatidylcholine, cholesterol and polyethylene glycol lipids) (Maier et al, Mol Ther, 2013 21 (8): 1570-8). Before immunization, the concentrated vaccine particles are adjusted to isotonicity using 10×PBS. The product has been analyzed and characterized, including the determination of particle size and polydispersity, encapsulation rate, pH value, endotoxin and bioburden, and is suitable for in vivo and in vitro scientific research in the preclinical stage. Among them, the vaccine based on wild-type HA is named saRNA-LNP-WT, the vaccine based on mutant BV-HA01 is named saRNA-LNP-01, the vaccine based on mutant BV-HA02 is named saRNA-LNP-02, and the vaccine based on mutant BV-HA-03 is named saRNA-LNP-03.

[0078] Example 6. Expression of saRNA vaccine in HEK293 cells

[0079] HEK293T cells were inoculated in a culture dish. When the cells grew to 80%, 4 kinds of saRNAs 5μg / mL (including BV-HA 01, BV-HA 02, BV-HA 03 and blank control) were added respectively, and the cells were collected after 24 hours. After fixing the cells with 4% paraformaldehyde for 15 minutes at room temperature, the cells were permeabilized with 0.2% Saponin for 10 minutes, and then the HA protein in the cells was labeled with anti-B-Victory HA antibody (NIBSC 22 / 228). After incubation at room temperature for 1 hour, fluorescent secondary antibody was added, and incubation continued at room temperature for 1 hour. After washing the cells with PBS, the fluorescence intensity was detected by flow cytometry.

[0080] The results are as follows Figure 3 As shown, the four saRNAs were expressed in more than 80% of HEK293T cells, and the mean fluorescence intensity (MFI) of cells transfected with saRNA-LNP-01, saRNA-LNP-02, and saRNA-LNP-03 were 5923.4, 9360.2, and 28578.2, respectively, which were significantly higher than the wild type (saRNA-LNP-WT). Among them, saRNA-LNP-03 had the highest MFI, indicating that the saRNA encoding BV-HA03 expressed the most HA protein in the cell.

[0081] Example 7: Stable expression of saRNA vaccine in cells

[0082] As described in Example 3, the four saRNAs were transfected into HEK293 cells for 24, 48, 96, and 144 hours, and the cells were collected. After the cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, the cells were permeabilized with 0.2% Saponin for 10 minutes, and then the HA protein in the cells was labeled with anti-B-Victory HA antibody (NIBSC 22 / 228). After incubation at room temperature for 1 hour, fluorescent secondary antibody was added, and the cells were incubated for another 1 hour at room temperature. After washing the cells with PBS, the fluorescence intensity was detected by flow cytometry.

[0083] The results are as follows Figure 4 As shown, the four saRNAs were expressed in more than 80% of HEK293T cells, and the mean fluorescence intensity (MFI) of cells transfected with saRNA-LNP-01, saRNA-LNP-02, and saRNA-LNP-03 was more stable at different time points and significantly higher than that of the wild type (saRNA-LNP-WT). Among them, the MFI of saRNA-LNP-03 was the highest, indicating that the saRNA encoding BV-HA 03 expressed the most HA protein in the cell.

[0084] Example 8. Antibody levels produced by saRNA vaccines in mice

[0085] 8-week-old C57BL / 6 mice were randomly divided into 8 groups, each group was set as: blank control group, saRNA-LNP-WT, saRNA-LNP-01, saRNA-LNP-02, saRNA-LNP-03. The saRNA vaccine was injected into the mice by intramuscular injection on the outside of the right hind leg, 5μg / mouse, injection volume was 50μL / mouse. The blank control group was injected with only the same volume of normal saline.

[0086] On the 21st day and the 49th day, 100 μL of blood was collected from each group of mice, and the serum was separated. The chicken hemagglutination inhibition experiment was used to detect the anti-HA antibody titer in the serum. The specific experimental method is as follows:

[0087] Preparation of 1.1% chicken red blood cell suspension: Centrifuge the Aldrich solution containing red blood cells at 1500rpm for 5min, discard the supernatant, add at least twice the volume of physiological saline to gently resuspend the cells, centrifuge at 1500rpm for 5min again, wash three times, and centrifuge at 1500rpm for 10min for the last time, discard the supernatant, and the deposited red blood cells are concentrated chicken red blood cells. According to the volume of the configured solution, suck out 1 / 100 volume of chicken red blood cells, repeatedly blow with physiological saline for more than 10 times, that is, prepare a 1% chicken red blood cell suspension, and place it at 4°C for use.

[0088] 2. Treatment of serum samples to be tested: prepare a 30-fold dilution of Vibrio cholerae filtrate, mix the serum with the dilution of Vibrio cholerae filtrate at a ratio of 1:4, and place at 37°C overnight for 16 to 18 hours to remove non-specific inhibitors. Inactivate Vibrio cholerae by placing the treated serum in a 56°C water bath for 50 minutes, then add 25 μL of packed chicken blood red blood cells to the inactivated serum, mix thoroughly and incubate at 2 to 8°C, re-mix every 15 minutes, centrifuge at 2000rpm for 5 minutes after 1 hour, aspirate the serum supernatant, remove non-specific agglutinins, and prepare for testing. 3. Determination of hemagglutination titer of standard antigen and whole virus stock solution: add 50 μL of normal saline to the first well of the hemagglutination plate, and add 25 μL of normal saline to each well from the 2nd to the 12th well. Add 10 μL of standard antigen or whole virus stock solution to the first well, pipette repeatedly 8-10 times to mix, aspirate 25 μL to the second well, dilute to the 12th well in turn, aspirate 25 μL from the 12th well and discard. That is, the dilution of the first well of the hemagglutination plate is 1:5, the second well is 1:10, the third well is 1:20, and so on, the 12th well is 1:10240. Set up 2 more wells, add 25 μL of physiological saline to each well as a negative control. Add 25 μL of 1% chicken blood red blood cell suspension to each well, let stand at room temperature for 45 minutes, observe and record the red blood cell agglutination results, and the highest dilution at which "++" hemagglutination appears is the hemagglutination titer of the antigen.

[0089] 4. Prepare 4 hemagglutination unit antigen solution: According to the hemagglutination titer of the standard antigen or whole virus stock solution, prepare 4 hemagglutination unit antigen solution (for example, if the hemagglutination titer is 1:320, 1 unit is used, and 4 units are diluted 1:80).

[0090] Re-measure 4 hemagglutination units antigen solution: Add 50μL of 4 hemagglutination units antigen solution to the first well of the microhemagglutination plate. Add 25μL of physiological saline to each of the 2nd to 6th wells, aspirate 25μL from the 1st well to the 2nd well, and dilute the same volume from the 2nd well to the 6th well in the same manner, and aspirate 25μL from the 6th well and discard. Then the hemagglutination units of the 1st well are 4, the hemagglutination units of the 2nd well are 2, the hemagglutination units of the 3rd well are 1, the hemagglutination units of the 4th well are 1 / 2, the hemagglutination units of the 5th well are 1 / 4, and the hemagglutination units of the 6th well are 1 / 8. Add 25μL of 1% chicken red blood cell suspension to each well, flick the hemagglutination plate to fully mix the red blood cells and viruses, let it stand at room temperature for 45min, tilt the hemagglutination plate 60° and leave it for 30 seconds. If the 1st to 3rd wells are hemagglutination positive and the 4th well is hemagglutination negative, then the four hemagglutination units are correct and the test is valid.

[0091] Table 3 Results of the re-determination of 4 hemagglutination units antigen solution

[0092] Hole No. 1 2 3 4 5 6 Hemagglutination unit 4 2 1 1 / 2 1 / 4 1 / 8 result ++++ ++ + － － －

[0093] 5. Test serum hemagglutination inhibition test: add 25 μL of physiological saline to the 1st to 12th wells of the hemagglutination plate, add 25 μL of the serum to be tested to the 1st and 12th wells, pipette and mix well in the 1st well, and then pipette 25 μL to the 2nd well, and dilute the same volume ratio from the 2nd well to the 11th well by analogy, and pipette 25 μL from the 11th well and discard; add 25 μL of 4 hemagglutination unit antigen solution to each well of 1-11 wells, mix well, and let stand at room temperature for 45 minutes. Add 25 μL of 1% chicken blood red blood cell suspension to each well, let stand at room temperature for 45 minutes, tilt the hemagglutination plate 60° to observe and record the hemagglutination results of red blood cells. The highest dilution of serum that shows no agglutination is the hemagglutination inhibition (HI) titer of the serum.

[0094] The results are as follows Figure 5 It shows that saRNA-LNP-01, saRNA-LNP-02, and saRNA-LNP-03 can significantly increase the neutralizing antibody titer level in mouse serum, and the neutralizing antibody titer produced by saRNA-LNP-03 is the highest and most durable. According to the results, the average geometric titers of the blank control group, saRNA-LNP-WT, saRNA-LNP-01, saRNA-LNP-02, and saRNA-LNP-03 were calculated to be 6.06, 776.05, 2352.53, 5404.70, and 12416.75 after immunization on the 21st day, and 6.06, 675.59, 2048.00, 5404.70, and 10809.41 after immunization on the 49th day, indicating that saRNA-LNP-03 can provide a better level of immune protection. Among them, saRNA-LNP-03 produced the highest and most durable antibody titer, and the hemagglutination inhibition titer (HI titre) was maintained at more than 10 times that of the wild type on both days 21 and 49. The above results suggest that the vaccine saRNA-LNP-03 prepared from the mutant BV-HA-03 is significantly better than the vaccine prepared based on the wild-type sequence in preventing influenza virus B-Victory strain.

[0095] In summary, the mutants of the wild-type HA protein provided by the present invention and their applications, through the modification of the amino acid sequence of the expressed HA protein, the modified protein expressed by the mutant saRNA in vivo can maintain a more stable conformation than the natural protein, induce the production of more and more effective neutralizing antibodies, thereby enhancing the antiviral efficacy.

[0096] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0097] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0098] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

[0099] Sequence Listing

[0100] SEQ ID NO.1:

[0101] Amino acid sequence of wild-type influenza B virus HA protein:

[0102] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0103] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0104] TNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0105] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0106] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0107] QEAINKITKN LNSLSELEVK NLQRLSGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLSNEGIINSE DEHLLALERK

[0108] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0109] LMIAIFVVYM VSRDNVSCSI CL

[0110] SEQ ID NO.2:

[0111] DNA sequence of wild-type influenza B virus HA protein:

[0112] ATGAAGGCAA TAATTGTACT ACTCATGGTA GTAACATCCA ATGCAGATCG AATCTGCACTGGGATAACAT CGTCAAACTC ACCACATGTC GTCAAAACTG CTACTCAAGG GGAGGTCAAT GTGACTGGTGTAATACCACT GACACTAAAGA CCCAATCATTGCAAAT CCAGGGGGAAACTATGCCCA AAATGCCTAA ACTGCACAGA TCTGGATGTA GCCTTGGGCA GACAAATG CACAGGAAAATACCCTCTG CAAGGGTTTC AATACTCCAT GAAGTCAGAC CTGTTACATC TGGGTGCTAGTT ACCGTAGATAACTCTCAAGATA AGGATACGAA CATGTCAGGT TATCAACTCACAACGTTATC AATACAGAAG ATGCACCAGG AGGACCCTAC GAAATTGGAA CCTCAGGGTC TTGCCTCAACTACCAATG GAAAAGGATT CTTCGCAACA ATGGCTTGGG ACCAACCATCACACAATCAAACAACAA TACATTTGTA CAGAAAGA AGACCAAATT ACCGTTTGGG GGTTCCACTGACGACGAG ACCCAAATGG CAAGGCTCTA TGGGGATTCA AAGCCCCAGA AGTTCACCTC ATCTGCAACGGAGTGACCA CACACTACGT CTCACAGATT GGTGCAGAGCATGA AATTGTTGTT GATTACATGG TGCAAAAATC TGGAAAAACA GGAACAATTA CCTATCAAAGGTATTT TTGCCTCAAA AGGTGTGGTG CGCAAGTGGC AAGAGCAAGG

[0113] TAATAAAAGG ATCCTTGCCC TTAATTGGAG AAGCAGATTG CCTCCATGAA AAATACGGTGGATTAAACAA AAGCAAGCCT

[0114] TACTACACAG GGGAACATGC AAAGGCCATA GGAAATTGCC CAATATGGGT GAAAACACCCTTGAAGCTGG CCAATGGAAC CAAATATAGA CCTCCTGCAA AACTATTAAA GGAAAGAGGT TTCTTCGGAGCCATTGCTGG TTTCTGAGAG GGAGGGATTTGGGG CATCCCATGGGGCACATGGA GTAGCGGTGG CAGCTGACCT TAAGAGCACT CAGGAGGCCA TAAACAAGAT AACAAAAAATCTCAACTCTT TGAGTGAGCT GGAAGTAAG AATCTTCAAA GACTAAGCGG TGCCATGAT GAACTCAG GAGAACGATCAATACT AGCTGATACA ATAAGCTCAC AGATAGAACTCGCAGTCCTG CTTTCCAATG AAGGAATAAT AAACAGTGAA GATGAACATC TCTTGGCGCT TGAAAGAAAGCTGAAAAA TGCTGGGCCC CTCTGCTGTA GAGATAGGAA ATGGACTTCAAGCTT CAAGCACAACCA ATAGCTGCTG GTACCTTTGA TGCAGGAGAA TTTTCTCTCC CCACCTTTGATTCACTGAAT ATTACTGCTG CATCTTTAAA TGACGATGGA TTGGACAATC ATACTATACT GCTTTACTACTCAACTGCTG CCTCCAGTTT GGCTGTTATTAGTAG T CTGATGATTAG GTCTCCAGACAATGTTTC TTGCTCCATT TGTCTATAA

[0115] SEQ ID NO.3:

[0116] Amino acid sequence of saRNA 1 mutant:

[0117] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0118] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0119] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0120] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0121] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0122] QEAINKITKN LNSLSELEVK NLQRLSGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLSNEGIINDE DEHLLALERK

[0123] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0124] LMIAIFVVYM VSRDNVSCSI CL

[0125] SEQ ID NO.4:

[0126] DNA sequence of saRNA 1 mutant:

[0127] ATGAAGGCCA TAATCGTTCT TCTCATGGTT GTCACCTCTA ATGCCGATAG AATTTGCACTGGTATAACCT CTTCCAACAG TCCTCACGTA GTGAAAACTG CCACTCAAGG CGAGGTCAAC GTCACTGGAGTGATCCCGCT GACTACGACC CCCACTAAGA

[0128] GCCACTTCGC TAATTTGAAA GGGACTGAGA CACGTGGTAA GTTATGCCCC AAATGCCTGAACTGCACTGA CCTCGACGTT

[0129] GCATTAGGCA GGCCTAAATG CACGGGCAAG ATACCGAGTG CTAGAGTCTC TATCTTGCACGAAGTTCGAC CGGTGACTTC GGGGTGTTTC CCAATCATGC ACGATCGCAC AAAGATCCGT CAGCTACCTAACCTTTTGCG AGGATGAG CATCGTAGTGACAGCAA TCGAT ATGCCCCTGGAGGCCCATAT GAGATAGGCA CGAGCGGCTC GTGTCTAAAT ATAACCAATG GAAAGGGGTT CTTCGCGACTATGGCATGGG CTGTACCTAA GAATAAAACA GCTACAAATC CCTTGACGAT TGAAGTACCT TATATGAGTAGTACATTAGAGAT TGACACGAA ACACAGATGG CGAGATTATATGGCGATTCA AAACCGCAGA AGTTTACCTC CAGCGCTAAC GGCGTTACAA CTCACTATGT ATCACAAATAGGTGGATTTC CTAATCAGAC GGAGGACGGC GGCCTACCGC AGAGCGGTAG GATAAGAGTGAATGGTGCCACTAG CGTATCAGCG AGGAATCTCAAA CTGCCTCAAA AGGTTTGGTGCGTCTGGC AAGAGCAAAG TAATTAAAGG AAGTTTACCC TTAATTGGCG AGGCGGATTG TCTGCATGAAAAATGGGGG GTTTGAACAA ATCTAAACTT TATGAGCCAGATGGAGGAGGGA AAAAAACGCCA CTCAAGCTCG CCAACGGGAC AAAATACAGG CCACCTGCCA AACTCCTCAAGGAGCGAGGA TTCTTTGGCG CGATAGCCGG ATTTTGAA GGTGGCTGGG AAGGCATGAT AGCCGGTTGGCATGGATATA CGTCCCATGGGGCGCATGGC GTCGCGGTTG CAGCAGACCT CAAGTCTACA CAGGAGGCCATCAACAAGAT AACCAAAAAC CTCAACTCGC TCAGTGAGTT AGAAGTAAAA AATTTACAGC GGTTGTCAGGAGCTATGGAT GAGTTGCATA ATGAGATACT CGAACTGGAT GAAAAGGTAG ACGATCTACG CGCTGATACTATATCATCCC AAATAGAGCT GGCTGTGCTT CTCTCCAACG AAGGCATAAT AAATGATGAA GACGAGCATCTCCTGGCGTT GGAAAGAAAG CTAAAAAAAA TGTTGGGGCC GAGTGCTGTC GAGATTGGTA ACGGATGTTTCGAAACGAAG CATAAATGCA ATCAGACGTG TCTCGACCGC ATCGCTGCAG GGACCTTCGA CGCTGGTGAATTCTCACTGC CGACGTTCGA CTCATTAAAC ATCACAGCCG CCTCACTGAA TGACGACGGT CTTGATAATCATACTATTTT ACTGTATTAC AGTACGGCCG CTTCTTCTTT AGCAGTTACT CTTATGATAG CTATTTTCGTAGTCTATATG GTTTCGCGTG ATAATGTCAG CTGTTCTATT TGCCTA

[0130] SEQ ID NO.5:

[0131] Amino acid sequence of saRNA 2 mutant:

[0132] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0133] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0134] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0135] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0136] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0137] QEAINKITKN LNSLSELEVK NLQRLSGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLFNEGIINSE DEHLLALERK

[0138] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0139] LMIAIFVVYM VSRDNVSCSI CL

[0140] SEQ ID NO.6:

[0141] DNA sequence of the saRNA 2 mutant:

[0142] ATGAAAGCCA TTATAGTACT ATTAATGGTC GTTACCTCTA ACGCGGATCG TATTTGTACCGGAATAACTA GTTCTAATTC ACCGCACGTG GTGAAGACTG CAACACAAGG AGAGGTAAAT GTAACAGGGGTGATACCTTT GACTACGACA CCGACTAAAA GGTCATTGATTGATTGATTGATT CCAGGGGCAAGCTATGTCCG AAATGTCTGA ACTGTACAGA TTTGGACGTA GCGCTAGGTC GACCGAAATG CACGGGTAAAATACCGTCCG CTAGGGTAAG TATTTTGCAC GAAGTGCGTC CCGTTACTTC TGGATGTTTT CCGATAATGCATGACCAC CATAGGACCATTCTAGCTGTC GGGGTACGAA CATGTGCGTC TTTCTACGCAATGTTATA AATACGGAAG ACGCACCAGG AGGCCCATAC GAAATAGGGA CATCAGGTTC GTGCCTAAATATCACCAATG GGAAGGGGTT TTTTGCTACC ATGGCTTGGG CCGTCCCTAA GAACAAAACCTACCTAGGACCATTA TACATCTGCA CAGAAGAAGA AGACCAGATC ACAGTGTGGG GTTTTCATTCCGATGATGAA ACGCAAATGG CGCGGCTCTA CGGAGATAGT AAACCACAGA AGTTCACCAG CAGTGCTAATGGGGTAACGA CTCATTATGT ATCACAAATA GGGGGTTGATC GGAAGGATTC GGCTTACCACAGTCGGGAAG AATTGTTGTA GATTACATGG TTCAAAAGTC CGGAAAAACA GGCACCATTA CATACCAACGTGGAATACTT CTGCCTCAGA AGGTTTGGTG CGCGTCGGGA AAGTCGAAGG TAATTAAGGG ATCGCTACCATTAAATTGGGATTG ATCGCTACCATTATTTGCATGAA AAGTATGGTG GGCTGAATAA GAGTAAACCC TACTACACAGGGGAACATGC CAAGGCTATA GGCAATTGTC CCATATGGGT TAAAACTCCT CTCAAATTAG CCAACGGGACCAAGTATAGG CCACCGGCGA AACTTCTAAA GGAACCGGA GCTTTGCCGGTTGCCG GTTCCTTGAAGGCGGCTGGG AGGGCATGAT TGCAGGATGG CATGGCTACA CTAGTCACGG AGCCCATGGC GTTGCAGTGGCGGCGGATCT AAAATCTACT CAGGAGGCTA TCAACAAGAT TACAAAGAAC CTGAATTCTT TGAGTGAGTTAGAACTCATCACTCACCAGGAGGAG CGCAATGGAT GAATTACACA ACGAAATCCT TGAGTTAGATGAAAAAGTGG ATGACTTACG AGCCGATACT ATAAGCTCGC AAATAGAATT GGCTGTTCTA CTTTTCAACGAGGGGATTAT AAAATTCAGAG GACGAGCACC TTCTGGCACT TGAACGTAAAA CCTGATTGATTGACCGTTGATTGATT GAGATTGGTA ACGGTTGCTT TGAGACCAAG CACAAATGCA ACCAAACTTG CCTTGATCGCATTGCGGCCG GCACGTTTGA TGCAGGGGAA TTTAGTCTAC CGACGTTCGA TTCGCTGAAT ATAACCGCTGCGAGCCTCAA CGACGACGGC TTGGACAACCCATACCATACTCACTGACTGCACT ATTCG CCTCGTCGTTAGCGGTCACG CTTATGATTG CTATATTCGT AGTCTATATG GTCTCTAGGG ATAACGTGTC TTGTCCAATATGTCTC

[0143] SEQ ID NO.7:

[0144] saRNA 3 Report Ad:

[0145] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0146] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0147] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0148] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0149] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0150] QEAINKITKN LNSLSELEVK NLQRLPGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLSNEGIINSE DEHLLALERK

[0151] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0152] LMIAIFVVYM VSRDNVSCSI CL

[0153] SEQ ID NO.8:

[0154] DNA sequence of saRNA 3 mutant:

[0155] ATGAAGGCTA TCATTGTCTT GCTAATGGTG GTCACCTCGA ACGCCGATCG AATATGCACAGGAATTACTA GCTCAAACTC CCCTCATGTC GTGAAGACAG CCACGCAGGG GGAAGTTAAT GTAACAGGTGTTATACCTACT AACGACGACG CCACTGATTGAAAT GTACAACCATA CCCGCGGTAAACTATGCCCT AAGTGCCTCA ATTGTACCGA TCTAGATGTC GCTTTAGGCA GACCAAAATG CACGGGGAATTTCCCAGTG CTCGAGTGTC AATTTTGCAC GAGGTGCGCC CAGTCACATC GGGGTGTTTC CCCATAATGACCACGAACGATTATCATTCACAAGAC CATGTCAGAT TAAGCACCCACAATGTCATT AACACAGAAG ATGCCCCCATAC GAGATCGGCA CGAGTGGTTC CTGTTTAAATATCACTAATG GTAAAGGATT CTTCGCAACC ATGGCGTGTGG CCGTACCCAA GAACAACTATCCATCCCTCGATCGAA CGGAAAGA GGATCAAATA ACAGTGTGGG GCTTTCACTCTGACATGAA ACTCAAATGG CAAGACTATA TGGCGACTCG AAGCCTCAAA AATTCCACGTC TTCTGCCAATGGAGTCACCA CTCACTATGT TAGCCAGATC GGAGGTTAGGGACCAGGATCCGGAC GGAG AATAGTTGTC GACTACATGG TTCAGAAGTC TGGCAAGACG GGGACTATAA CATACCAACGCGGCATCCTA TTGCCTCAAA AAGTGTGGTG CGCCAGTGGT AAGAGTAAGG TCATAAAAGG TTCACTCCCGCATCGGAG AAGCAGATTGTTTACACGAG AAGTACGGCG GGCTGAACAA GTCTAAGCCC TATTACACTGGAGAGCACGC AAAAGCTATA GGGAATTGCC CGATATGGGT CAAAACCCCC CTTAAACTCG CCAACGGGACTAAGTACAGA CCGCCTGCGA AACTACTTAA AGAGCGAGGT TTGATTGGGGGGG TTTTCTAGAGGGAGGCTGGG AGGGCATGAT AGCGGGTTGG CACGGGTATA CCTCGCACGG GGCGCACGGT GTAGCTGTCGCAGCTGACCT AAAGTCTACT CAAGAAGCTA TTAACAAGAT CACTAAAAAC CTGAACTCCT TATCAGAACTAGAAGCTGAAG ACCATCACCATGCGAG GCCGAG GAATTACACA ATGAAATACT GGAGTTGGACGAAAAGGTCG ATGACTTACG TGCGGATACC ATTTCAAGTC AGATAGAGCT CGCCGTCCTG CTATCCAACGAAGGCATTAT TAACAGCGAG GACGAGCACC TCCTTGCTCT AGAAAGGAAA CTAAAGAAGA TGATTCATTCAGCGATCGATCGATCGATCGATTGATTGATT CGAGACGAAA CATAAGTGTA ATCAAACGTG TCTTGACCGAATTGCTGCCG GGACCTTCGA TGCTGGTGAA TTTTCACTGC CCACATTCGA CTCTTTGAAC ATAACTGCAGCTTCACTGAA TGACGATGGA CTGGACAACC ATACTATACT CCTGTATTAT AGCAGCCACCTG CAGCCACCCTG CTGATGATTG CTATTTTCGT CGTTTATATG GTGTCCCGTG ACAACGTTTC CTGTTCAATCTGTTTA

[0156] SEQ ID NO.9:

[0157] saRNA 4 Read:

[0158] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0159] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0160] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0161] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0162] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0163] QEAINKITKN LNSLSELEVK NLQRLSGAMP ELHNEILELD EKVDDLRADT ISSQIELAVLLSNEGIINSE DEHLLALERK

[0164] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0165] LMIAIFVVYM VSRDNVSCSI CL

[0166] SEQ ID NO.10:

[0167] DNA sequence of saRNA 4 mutant:

[0168] ATGAAGGCGA TAATCGTTCT TCTGATGGTG GTGACTTCTA ATGCCGATCG GATTTGCACCGGCATCACTT CTTCAAACAG TCCTCACGTC GTGAAAACCG CTACTCAAGG AGAGGTTAAT GTAACGGGTGTTATACCCTT GACTACAACT CCCACTAAAT CACACTTTGC CAACTTGAAA GGTACTGAGA CCCGAGGAAAACTATGTCCA AAATGCTTGA ATTGCACTGA CTTGGACGTT GCCTTAGGGC GGCCAAAATG CACAGGGAAGATTCCCTCCG CTCGAGTAAG TATATTGCAT GAGGTTCGAC CTGTAACGAG CGGGTGTTTT CCGATAATGCATGATCGGAC AAAGATTCGT CAGCTTCCAA ATCTACTACG CGGTTATGAG CATGTAAGAC TTTCTACCCACAATGTCATC AACACAGAGG ATGCGCCGGG AGGCCCTTAC GAGATAGGTA CATCTGGCTC ATGTCTAAATATCACGAATG GGAAGGGATT TTTCGCGACA ATGGCATGGG CAGTCCCGAA GAATAAAACA GCAACAAACCCCTTAACAAT TGAGGTCCCT TATATTTGCA CGGAGGAAGA AGACCAAATA ACCGTTTGGG GGTTTCACTCGGATGATGAG ACTCAAATGG CTAGGCTCTA CGGAGACTCA AAGCCGCAGA AGTTTACCAG CTCCGCCAACGGGGTGACGA CGCATTACGT TTCGCAGATT GGGGGCTTCC CCAATCAAAC TGAAGACGGT GGTTTACCGCAGTCAGGCAG GATCGTAGTG GACTACATGG TGCAAAAATC GGGTAAGACT GGTACGATCA CTTATCAACGCGGTATCTTA TTACCCCAGA AGGTCTGGTG CGCGTCGGGC AAGTCTAAGG TCATAAAGGG CTCACTTCCACTTATTGGAG AGGCCGATTGCTTGCATGAG AAGTACGGGG GGTTGAACAA GAGCAAGCCG TACTACACGGGGGAACACGC GAAGGCAATC GGTAACTGCC CCATCTGGGT TAAAACTCCT CTCAAGTTAG CCAATGGAACAAAGTATCGG CCACCAGCAA AGCTACTTAA AGAGAGATTGACTGGGCGGTTGATCGG ATTTTTGGAAGGAGGGATGGG AGGGTATGAT CGCCGGGTGG CATGGTTATA CAAGCCACGG CGCGCACGGA GTGGCCGTCGCCGCTGACCT CAAGTCGACA CAGGAAGCGA TAAACAAAAT AACGAAAAAC CTTAACTCAC TATCTGAATTTGACGAAGTTGAGTTGAGTTGAGCCGAGGCCGA GAGCTTCATA ATGAAATTCT TGAGTTGGACGAGAAGGTCG ATGACCTGCG CGCGGACACT ATAAGCTCTC AGATAGAACT GGCTGTTCTA TTATCCAATGAAGGGATCAT CAATTCCGAA GATGAACATC TTCTCGCACT GGAGCGTAAG CTCAAAAAGA TGCCGGAGGACTGAGGAGTTGATT ACGGGTGCTT CGAGACTAAG CATAAGTGCA ATCAAACTTG CCTTGATCGCATTGCCGCCG GTACTTTCGA CGCGGGAGAG TTCTCTCTCC CTACATTTGA TAGCCTAAAT ATAACCGCGGCATCGTTAAA TGACCGACGGA CTTGACAATC ACACTATACT ATCCACTTACTGCTGCT CCTCGTCGCTGGCGGTAACT TTAATGATCG CCATCTTCGT GGTCTACATG GTTTCACGTG ACAATGTTTC TTGTTCTATTTGTCTA

[0169] SEQ ID NO.11:

[0170] saRNA 5 in the following:

[0171] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0172] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0173] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0174] GGFPNQTEDG GLPQSGRIVV DYMVQKSCKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0175] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0176] QEAINKITKN LNSLSELEVK NLQRLSGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLSNEGIINSE DEHLLALERK

[0177] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0178] LMIAIFVVYM VSRDNVSCSI CL

[0179] SEQ ID NO.12:

[0180] DNA sequence of the saRNA 5 mutant:

[0181] ATGAAAGCGA TAATCGTGCT CTTAATGGTC GTGACGAGTA ACGCAGATCG AATCTGTACAGGCATTACGA GTTCCAATAG TCCTCACGTG GTCAAAACTG CAACACAAGG AGAAGTTAAT GTAACAGGCGTGATCCCGTT AACGACAACA CCGACAAAAT CTCAATGATTGACTGACTGACTGTA CGCGCGGCAAGCTGTGCCCT AAGTGTTTAA ACTGCACCGA TCTTGATGTC GCTCTAGGGC GACCAAAGTG CACGGGCAAATCCTTCTG CTCGTGTGTC CATACTCCAT GAGGTCAGGC CAGTTACGTC TGGGTGCTTC CCCATTTATGCATGATCGGAC CATTAGATGCCTAGA CGGGTATGAG CACGTTCGAC TTAGCACGCATAACGTAATT AACACAGAAG ACGCGCCGGG TGGGCCGTAT GAAATCGGTA CTTCGGGCAG TTGCTTAAACATCACCAATG GGAAGGGATT TTTTGCCACT ATGGCATGGG CTGTGCCTAA AAACAAACCAACCAACCAACCAACCAACCAACCAG TACATTTGTA CGGAAGAAGA GGATCAGATC ACCGTCTGGG GATTCCACTCTGACGACGAG ACTCAAATGG CGCGGCTGTA TGGAGATAGC AAAACCGCAGA AGTTTACCAG CTCTGCGAATGGGGTGACGA CGCACTATGT GTCCCAGATC GGAGGTAACCAGGACCAGCAGCCAG GGTCTACCGCAAAGTGGCCG TATAGTGGTA GATTACATGG TGCAAAAGTC ATGTAAAACG GGTACCATCA CCTACCAGCGTGGGATTCTG TTACCGCAGA AGGTTTGGTG CGCCTCGGGT AAATCAAAGG TCATTAAAGG ATCGCTTCCCTTGATCGGCGATTTCTCCATGAA AAATACGGCG GACTGAACAA AAGCAAGCCC TATTATACGGGAGAGCATGC TAAGGCTATA GGCAACTGCC CGATTTGGGT AAAGACTCCT CTCAAGCTGG CAAACGGTACAAAATACAGA CCCCCTGCTA AATTGCTCAA GGAAAGGGGGGGGTTGGGGTTGCG TTTCCTCGAGGGAGGATGGG AAGGCATGAT CGCCGGCTGG CATGGTTACA CGTCGCACGG CGCTCACGGA GTTGCAGTGGCGGCAGATCT TAAGAGTACT CAGGAGGCTA TCAATAAGAT AACAAAGAAC CTTAATAGCT TAAGTGAACTTGAGGTTAAA AACTTCATCGACCGTCGACCGCC GAGCTTCATA ACGAAATTTT AGAGCTGGATGAGAAGGTTG ACGATCTGCG GGCTGACACT ATATCTTCCC AGATTGAACT GGCTGTTCTA TTGAGCAACGAGGGTATTAT CAACTCGGAG GATGAACACT TGCTGGCACT CGAACGTAAG TTAAAAAAGAGAGAGGCTGAGAGTTGAGTTGAGTT ACGGCTGTTT CGAGACGAAG CACAAATGCA ATCAAACGTG TCTGGATCGAATAGCAGCTG GTACCTTCGA TGCCGGGGAA TTTTCGTTAC CAACATTCGA TTCTCTAAAC ATTACCGCGGCATCTCTTAA CGATGACGGT CTAGATAACC ATACTATTCT TCTGATCGATCGCCTAGCCTAGCCTAGCTGACTGAG TTGATGATAG CGATCTTTGT GGTATACATG GTATCCCGTG ACAATGTTAG CTGTAGTATATGTTTG

[0182] SEQ ID NO.13:

[0183] saRNA 6 Read:

[0184] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTACQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0185] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0186] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0187] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0188] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0189] QEAINKITKN LNSLSELEVK NLQRLSGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLSNEGIINSE DEHLLALERK

[0190] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0191] LMIAIFVVYM VSRDNVSCSI CL

[0192] SEQ ID NO.14:

[0193] DNA sequence of the saRNA 6 mutant:

[0194] ATGAAGGCCA TTATTGTTCT GTTGATGGTG GTGACATCCA ATGCGGACCG GATCTGTACCGGCATTACAA GTTCAAACTC ACCCCACGTC GTGAAGACTG CTTGTCAGGG GGAGGTAAAT GTTACGGGAGTAATCCCGCT CACCACCACG CCGACTAAGT CGCACTTCGC GAATTTGAAA GGGACTGAAA CACGTGGTAAGCTGTGTCCA AAGTGCCTTA ACTGCACGGA CCTTGACGTA GCGCTCGGCC GCCCAAAGTG CACCGGTAAGATACCTTCGG CACGAGTGAG TATACTCCAT GAGGTCCGTC CGGTGACTAG TGGGTGCTTC CCTATTATGCATGACCGTAC AAAAATTAGG CAGCTACCCA ATTTGCTCCG CGGCTACGAA CACGTTCGTC TGTCGACCCACAATGTCATT AATACCGAGG ACGCACCTGG GGGCCCATAT GAGATCGGCA CTAGTGGGAG CTGCCTAAATATCACAAACG GGAAAGGGTT TTTTGCAACA ATGGCTTGGG CCGTACCAAA AAACAAGACA GCTACTAATCCCCTTACCAT CGAAGTTCCA TACATTTGCA CAGAGGAAGA GGACCAGATT ACAGTTTGGG GCTTTCATTCAGACGATGAG ACACAGATGG CGCGATTGTA CGGAGATAGC AAGCCTCAAA AATTCACGTC TTCTGCGAACGGGGTTACCA CACATTACGT CTCTCAGATC GGCGGGTTTC CTAATCAGAC CGAGGACGGA GGCCTGCCACAATCCGGGCG GATAGTGGTC GATTATATGG TGCAAAAATC AGGGAAGACT GGGACCATAA CCTACCAGAGAGGTATTCTC TTGCCACAGA AAGTGTGGTG TGCAAGTGGG AAGAGTAAGG TGATTAAGGG AAGCCTGCCCCTAATCGGCG AAGCGGATTGTCTCCATGAA AAATACGGAG GGTTGAATAA GTCGAAACCG TATTATACGGGAGAGCACGC GAAGGCCATC GGCAATTGCC CCATCTGGGT AAAAACACCC CTGAAGCTGG CAAATGGTACAAAATATCGG CCTCCTGCAA AGTTGTTAAA AGAGCGAGGTGTTTTCGGA GTTCCGG CTTCTTGGAAGGGGGTGGG AAGGTATGAT TGCGGGGTGG CATGGTTATA CCAGCCACGG GGCACACGGG GTCGCTGTTGCTGCGGACCT GAAGAGCACC CAAGAAGCCA TAAACAAAAT CACAAAGAAC TTAAATTCTC TGTCAGAGCTGAGAGTAGGAGTTGAGCTGCTGGGTT AGCAATGGAC GAATTACACA ACGAGATCTT AGAACTGGATGAAAAGGTAG ATGATTTACG AGCAGACACG ATCTCCAGTC AGATTGAGCT CGCAGTATTA CTTAGCAACGAGGGCATTAT AAACTCAGAA GATGAGCATT TATTGGCTTT GGAGGAGAAGGAGGAGAGCTGAGCCTT GGAGGAAGGAGCTGAGGAGCC GAGATAGGAA ATGGCTGCTT TGAGACGAAG CACAAGTGCA ACCAAACGTG TCTTGACCGGATCGCTGCTG GCACATTCGA CGCAGGAGAG TTTAGCTTGC CCACTTTTGA CAGTCTAAAT ATTACGGCTGCATCGTTGAA CGATGATGGG CTACCGATACCTACCTAGTTGATT ACCTACGTTGTTG CTAGTTCCCTGGCAGTCACC TTGATGATCG CAATCTTTGT CGTGTACATG GTCTCACGAG ATAATGTCTC ATGCTCCATATGCCTG

[0195] SEQ ID NO.15:

[0196] saRNA 7 in the following:

[0197] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0198] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0199] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0200] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0201] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0202] QEAINKITKN LNSLSELEVK NLQRLSGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLSNCGIINSE DEHLLALERK

[0203] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0204] LMIAIFVVYM VSRDNVSCSI CL

[0205] SEQ ID NO.16:

[0206] DNA sequence of the saRNA 7 mutant:

[0207] ATGAAGGCCA TCATAGTGTT GCTGATGGTA GTTACCTCAA ACGCTGATAG AATTTGTACAGGAATTACGT CGTCAAATAG TCCACATGTT GTTAAAACTG CCACGCAGGG AGAAGTAAAT GTGACTGGCGTAATCCCATT AACTACGACC CCCACAAAAA GCCACTTCGC AAACTTGAAG GGCACAGAGA CCCGTGGTAAACTCTGCCCG AAATGTCTCA ATTGCACGGA TTTGGACGTG GCTTTGGGAC GACCCAAGTG CACGGGCAAAATTCCAAGCG CGAGAGTGTC GATACTCCAC GAAGTACGCC CGGTAACAAG CGGGTGTTTT CCCATCATGCATGACCGGAC CAAAATTCGA CAATTGCCTA ACCTTTTGCG TGGTTATGAA CATGTGCGAC TTTCGACGCATAATGTTATA AACACCGAAG ACGCACCGGG TGGTCCATAT GAGATCGGCA CTAGCGGTTC CTGTCTGAACATTACAAATG GAAAAGGGTT CTTCGCTACT ATGGCATGGG CTGTGCCGAA GAACAAAACC GCAACTAATCCACTAACAAT TGAAGTACCT TACATATGCA CGGAGGAAGA AGATCAAATA ACCGTATGGG GATTCCACAGTGATGATGAA ACTCAGATGG CTAGGCTATA CGGTGATTCC AAGCCGCAGA AGTTTACCTC CTCCGCCAATGGAGTGACCA CCCATTACGT ATCGCAGATC GGCGGGTTTC CGAACCAGAC GGAGGATGGT GGCTTGCCCCAGTCCGGGCG CATAGTCGTC GACTATATGG TACAGAAGTC TGGAAAAACC GGAACAATCA CTTATCAGCGAGGGATACTG CTTCCACAAA AGGTTTGGTG CGCTTCCGGG AAGTCTAAGG TGATAAAGGG ATCATTACCACTAATCGGCG AGGCAGATTGCTTGCATGAA AAATATGGCG GCCTGAATAA AAGTAAGCCC TATTATACCGGCGAGCACGC TAAGGCTATC GGTAACTGCC CCATATGGGT CAAAACGCCG CTGAAACTGG CCAATGGGACGAAATACAGG CCACCTGCTA AACTTCTGAA GGAACGGGGAGGA TTCTTGAGGGGGGG CTTTCTCGAAGGGGGTTGGG AGGGGATGAT AGCCGGTTGG CATGGTTACA CCTCCCATGG TGCGCACGGT GTAGCGGTTGCCGCGGATCT AAAAAGTACA CAAGAGGCTA TAAACAAAAT AACCAAGAAT CTTAACTCAC TCAGCGAGCTGACCAGGTCCACCTACCTACCGATT AGCCATGGAC GAGTTACACA ATGAGATACT AGAGCTTGATGAAAAAGTAG ATGATTTGCG TGCGGATACA ATTTCGTCTC AAATTGAGTT GGCCGTACTT CTGAGTAATTGTGGCATTAT TAATAGTGAG GATGAGCACC TCTTAGCCCT TGAGCAGTAAG GCTGATCGAGGCTCGTCGA GAGATTGGTA ACGGCTGCTT CGAGACAAAG CACAAATGTA ACCAGACATG TTTGGACAGGATAGCCGCAG GTACTTTCGA CGCGGGGGAA TTCAGTTTAC CCACCTTTGA TAGCCTGAAT ATCACTGCAGCCAGCCTCAA TGACGATGGC CTACCGATAATTC AGTTACTGATT GTACCTTATTT CGTCGAGTTTAGCAGTTACT CTGATGATCG CCATCTTCGT GGTATATATG GTATCGCGTG ATAACGTTAG CTGTTCTATTTGCCTG

[0208] SEQ ID NO.17:

[0209] BV-HA 01 Registration:

[0210] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTATQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0211] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0212] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0213] GGFPNQTEDG GLPQSGRIVV DYMVQKSGKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0214] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0215] QEAINKITKN LNSLSELEVK NLQRLPGAMP ELHNEILELD EKVDDLRADT ISSQIELAVLLFNEGIINDE DEHLLALERK

[0216] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0217] LMIAIFVVYM VSRDNVSCSI CL

[0218] SEQ ID NO.18:

[0219] DNA sequence of BV-HA 01 mutant:

[0220] ATGAAGGCGA TTATCGTTTT GCTGATGGTA GTAACTTCAA ATGCAGACAG GATCTGTACCGGGATAACGA GTTCCAATAG TCCCCACGTG GTTAAGCAAG CACAAGAG TGAAGTAAT GTCACCGGAGTAATCCCAGACA TACAACAACA CCGATTCAGCAAGT CGCGCGGCAAACTCTGCCCT AAATGCTTAA ACTGTACGGA TCTCGACGTT GCGCTGGGGC GCCCGAAATG CACTGGGAAAATCCCCGAGTG CACGTGTATC CATCCTTCAT GAGGTTAGGC CCGTCACATC GGGGTGTTTC CCTAGACTATGCCCTAAGGATCCAAA AGGATATGAA CATGTGCGTC TATCCACCCATAACGTCATT AACACCGAAG ACGCCCCTGG CGGCCCCTAC GAAATCGGGA CGTCTGGGTC ATGTTTGAATTACAAACG GCAAGGGATT TTTTGCAACG ATGGCCTGGG CGGTAACTGACCCACTAAGTAACACAAA TACATATGCA CGGAAAGA GGACCAAATC ACCGTATGGG GTTTCCACAGTGACACGAA ACACAAATGG CCCGACTATA CGGGGACAGC AAGCCACAAA AATTTACCTC CAGCGCCAATGGTGTAACAA CGCACTACGT AAGTCAAATA CGACGATGCATTCC GGATTGCCGCAGTCGGGGAG AATCGTAGTA GATTACATGG TACAGAAGTC GGGAAAAACG GGTACTATAA CTTATCAAAGGGGTATCCTG CTCCCTCAGA AAGTTTGGTG CGCGTCGGGT AAAAGCAAGG TAATCAAGGCT CTAGCCGACCCTGAATTTGCACGAA AAATATGGGG GGTTGAATAA AAGTAAACCT TACTATACCGGCGAGCATGC AAAGGCGATT GGGAATTGTC CGATATGGGT GAAGACCCCA CTAAAGCTGG CAAACGGGACCAAATATAGA CCGCCTGCAA AGCTCCTCAA AGAGCGGCCGATTGCCAGGGCCGATT ATTTTTAGAAGGCGGCTGGG AGGGGATGAT AGCCGGCTGG CATGGTTATA CGTCCCACGG AGCTCATGGG GTGGCCGTAGCGGCCGATCT TAAATCTACA CAGGAAGCTA TCAACAAAAT CACGAAGAAT TTGAATTCGC TCTCTGAATTGGAGGGACCATCACCGACCGAGGAGGA GAGTTACATA ACGAAATACT CGAGTTAGACGAAAAGGTTG ATGACCTAAG AGCAGACACA ATTTCAAGCC AAATTGAGCT AGCAGTACTT CTCTTCAATGAGGGCATCAT TAATGACGAG GACGAGCATT TGCTGGCGCT GGAAAGAAAG CTTAAGAAGA TGCTTGAGGGGGTTGGTT ATGGGTGCTT TGAGACTAAG CATAAATGTA ACCAGACTTG TTTAGACCGGATTGCAGCCG GGACCTTTGA CGCGGGTGAA TTTAGCCTGC CGACGTTCGA TTCTTTGAAC ATAACGGCGGCTTCCTTGAA CGATGACGGG CTCGATAACC ATACCATTC ACTTACGTTGCT CAAGCTCGCTTGCGGTAACC CTCATGATTG CCATCTTTGT CGTGTATATG GTTAGCCGAG ATAACGTGTC GTGCTCTATTTGTCTT

[0221] SEQ ID NO.19:

[0222] BV-HA 02 Registration:

[0223] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTACQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0224] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0225] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0226] GGFPNQTEDG GLPQSGRIVV DYMVQKSCKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0227] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0228] QEAINKITKN LNSLSELEVK NLQRLSGAMD ELHNEILELD EKVDDLRADT ISSQIELAVLLSNCGIINSE DEHLLALERK

[0229] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0230] LMIAIFVVYM VSRDNVSCSI CL

[0231] SEQ ID NO.20:

[0232] DNA sequence of BV-HA 02 mutant:

[0233] ATGAAGGCGA TTATCGTGCT ATTGATGGTA GTCACGTCGA ACGCTGACCG GATATGTACGGGTATAACGA GTTCCAACTC ACCGCATGTT GTTAAAACCG CCTGCCAAGG TGAAGTTAAT GTGACAGGCGTGATACCACT CACTACAACG CCCACAAAGT CCCACTTCGC AAACCTCAAA GGAACCGAGA CACGAGGGAAACTCTGTCCC AAATGTCTGA ATTGCACAGA TCTCGACGTA GCTCTGGGGC GGCCGAAGTG CACGGGAAAAATCCCTTCAG CCAGAGTGTC AATTCTCCAC GAGGTTCGAC CAGTTACGTC GGGATGCTTC CCAATAATGCATGACCGGAC CAAGATAAGA CAATTACCTA ATCTTTTGCG AGGATACGAG CATGTACGTC TTTCCACTCATAACGTAATC AACACGGAGG ACGCGCCCGG CGGCCCTTAC GAGATAGGCA CTTCTGGTAG CTGCTTAAACATCACTAATG GAAAAGGTTT TTTCGCGACA ATGGCCTGGG CGGTCCCAAA AAACAAGACA GCCACAAACCCGTTGACTAT CGAAGTGCCC TACATCTGCA CTGAAGAGGA GGACCAAATT ACAGTTTGGG GATTTCACTCGGATGATGAG ACACAGATGG CTAGGCTCTA TGGCGACAGC AAACCTCAGA AGTTCACTTC TTCCGCCAACGGAGTTACCA CACATTACGT CTCGCAGATC GGCGGATTCC CAAATCAAAC GGAAGACGGT GGGTTGCCGCAGAGCGGTCG TATAGTAGTG GATTATATGG TACAGAAATC ATGCAAAACG GGCACGATTA CCTACCAAAGGGGTATATTG CTACCCCAGA AAGTATGGTG CGCATCGGGT AAATCTAAAG TGATAAAAGG TTCCCTACCCCTGATTGGTG AAGCTGATTGTTTGCATGAA AAATACGGGG GTCTTAACAA GTCAAAACCT TATTACACAGGTGAGCATGC TAAGGCCATT GGTAATTGTC CAATTTGGGT CAAGACGCCG TTGAAACTGG CCAATGGTACAAAATACAGA CCACCGGCAA AGCTTCTTAA GGAGCGTGGA TTTTTTGGCG CAATAGCTGG CTTCCTGGAGGGTGGATGGG AAGGGATGAT CGCAGGTTGG CACGGGTATA CCTCCCACGG CGCGCACGGG GTAGCAGTTGCAGCCGATCT TAAGTCAACG CAGGAGGCGA TAAACAAAAT TACAAAGAAC CTGAATTCGT TGTCTGAGCTGGAGGTCAAA AATCTCCAGC GTCTCTCCGG AGCGATGGAT GAACTACATA ATGAAATACT AGAATTGGACGAAAAAGTAG ACGATCTAAG AGCTGATACA ATATCTTCCC AAATCGAGTT GGCCGTTTTG CTCTCCAATTGTGGAATCAT AAATTCCGAG GACGAGCATC TACTTGCGTT AGAGAGGAAA CTAAAAAAGA TGCTAGGCCCATCGGCAGTG GAAATAGGAA ACGGGTGCTT CGAAACCAAG CACAAGTGTA ATCAAACGTG CCTCGACCGCATAGCTGCTG GTACTTTTGA TGCGGGAGAA TTTAGTTTAC CAACCTTTGA TAGCCTCAAT ATCACTGCCGCATCTCTGAA TGATGATGGC CTGGATAACC ATACAATTCT TCTTTACTAC AGCACTGCAG CATCTTCGCTCGCTGTGACA

[0234] TTGATGATTG CGATTTTTGT GGTCTACATG GTGAGCAGGG ATAACGTCTC CTGTTCAATTTGTCTG

[0235] SEQ ID NO.21:

[0236] Amino acid sequence of BV-HA 03 mutant:

[0237] MKAIIVLLMV VTSNADRICT GITSSNSPHV VKTACQGEVN VTGVIPLTTT PTKSHFANLKGTETRGKLCP KCLNCTDLDV

[0238] ALGRPKCTGK IPSARVSILH EVRPVTSGCF PIMHDRTKIR QLPNLLRGYE HVRLSTHNVINTEDAPGGPY EIGTSGSCLN

[0239] ITNGKGFFAT MAWAVPKNKT ATNPLTIEVP YICTEEEDQI TVWGFHSDDE TQMARLYGDSKPQKFTSSAN GVTTHYVSQI

[0240] GGFPNQTEDG GLPQSGRIVV DYMVQKSCKT GTITYQRGIL LPQKVWCASG KSKVIKGSLPLIGEADCLHE KYGGLNKSKP

[0241] YYTGEHAKAI GNCPIWVKTP LKLANGTKYR PPAKLLKERG FFGAIAGFLE GGWEGMIAGWHGYTSHGAHG VAVAADLKST

[0242] QEAINKITKN LNSLSELEVK NLQRLPGAMP ELHNEILELD EKVDDLRADT ISSQIELAVLLFNCGIINDE DEHLLALERK

[0243] LKKMLGPSAV EIGNGCFETK HKCNQTCLDR IAAGTFDAGE FSLPTFDSLN ITAASLNDDGLDNHTILLYY STAASSLAVT

[0244] LMIAIFVVYM VSRDNVSCSI CL

[0245] SEQ ID NO.22:

[0246] DNA sequence of BV-HA 03 mutant:

[0247] ATGAAGGCTA TTATTGTCCT GTTGATGGTT GTGACGTCTA ATGCGGACAG AATATGTACGGGGATTACCT CCTCCAACTC GCCCCATGTA GTGAAAACGG CATGCCAAGG GGAAGTAAAC GTTACGGGTGTAATACCCCCT CACGACGACG CCCACAAAGTGATCAATCAATCGATTAAGCGATT CTCGTGGCAAGCTGTGCCCC AAGTGCCTGA ATTGTACAGA CCTAGATGTG GCGCTCGGGA GGCCTAAATG CACAGGGAAGATCCCTTCGG CTAGGGTCTC CATCCTTCAC GAAGTACGTC CGGTTACGAG TGGTTGCTTT CCTATCATGCACGATAGGAC AGAATCACCG CGGTTACGAG CACGTCCGCT TGTCAACGCATAACGTGATT AACACTGAAG ACGCTCCAGG AGGTCCCTAC GAAATTGGAA CCTCCGGAAG TTGCTTGAACATCACTAACG GCAAAGGTTT CTTTGCAACT ATGGCTTGGG CTGTACCAAA AAATAAACCAT GCCTTACGACTGACTGACTGACTGACT CTGAGGAAGA AGATCAGATA ACAGTCTGGG GATTCCACTCGGATGATGAG ACTCAGATGG CACGACTCTA TGGTGACAGT AAACCTCAGA AATTCACATC GTCCGCGAACGGGGTGACAA CACACTACGT ATCCCAGATA GGTGGGTTTC CGAACCAAAC GAGGACCGAGAGCGGCGC CATCGTAGTC GATTACATGG TCCAAAAGAG TTGTAAGACA GGGACAATTA CATACCAGCGAGGTATATTTA CTCCCCCAGA AAGTGTGGTG TGCAAGCGGA AAATCGAAGG TAATTAAAGG GAGCTTGCCCCTAATCGGGG AAGCCGACTGCTTACACGAG AAGTACGGCG GCCTAAACAA GTCGAAGCCG TATTATACCGGCGAGCACGC AAAAGCCATA GGCAATTGTC CTATCTGGGT GAAGACACCT CTGAAATTGG CTAACGGAACCAAATACAGG CCACCCGGCAA AGTTGCTCAA GGAGAGAGGG TTCTTTGGAG CAATAGCGGG GTTCCTAGAAGGGATGAG AAGGAATGAT TGCGGGCTGG CACGGTTACA CATCACATGG GGCCCATGGC GTAGCCGTCGCCGCGATTT AAAAGTCCA CAAGAGGCCA TCAATAAAAT CACGAAAAAT TTGAATTCCC TCTCTGAGCTAGAGGTGAAGGTGAAG AATCTGCAAC GTTTACCAGG CGCTATGCCC GAGTTGCACA ACGAAATTCT CGAGCTTGATGAAAAGGTTG ACGACTTACG GGCAGACACC ATTTCAAGCC AGATCGAATT GGCGGTACTG CTGTTTAACTGTGGGATTAT CAACGATG GATGAGCACT TACTTGCGCT TGAACGAAAG CTTAAGAAGA TGCTTGGGCCGTCTGCCGTG GAGATTGGAA ACGGGTGTTT CGAGACAAA CATAAGTGCA ACCAGACATG TCTAGATCGTATCGCGGCGG GAACGTTCGA CGCTGGAGAA TTCTCTCTAC CGACGTTCGA CTCACTCAAC ATAACCGCTGCAAGCTTAAA CGATGACGGT CTTGATAATC ATACTATTCT CCTATACTAC TCTACGGCCG CTTCGTCGCTCGCGGTGACT CTCATGATAG CAATCTTCGT CGTCTATATG GTAAGCCGCG ATAACGTTTC GTGTTCAATTTGTTTG

Claims

1. A mutant of influenza B virus HA protein, characterized in that: Based on the sequence of wild-type influenza B virus HA protein and having mutation sites, the mutation sites include any one or more of S469D, S462F, S426P, D430P, G268C, T35C, and E464C.

2. The mutant according to claim 1, characterized in that A combination of any 3, 4 or 7 amino acid sequence mutation sites including S469D, S462F, S426P, D430P, G268C, T35C and E464C.

3. The mutant according to claim 2, characterized in that The wild-type influenza B virus is the Victoria strain, and the amino acid sequence of its HA protein is shown in SEQ ID NO.

1.

4. The mutant according to claim 2, characterized in that It includes four mutation sites, namely S469D, S462F, S426P and D430P, and has an amino acid sequence as shown in SEQ ID NO.

17.

5. The mutant according to claim 2, characterized in that It includes three mutation sites, G268C, T35C, and E464C, and has an amino acid sequence as shown in SEQ ID NO.

19.

6. The mutant according to claim 2, characterized in that It includes seven mutation sites, namely S469D, S462F, S426P, D430P, G268C, T35C and E464C, and has an amino acid sequence as shown in SEQ ID NO.

21.

7. An influenza vaccine, characterized in that: The method comprises the mutant according to any one of claims 1 to 6.

8. The influenza vaccine according to claim 7, wherein Including one or more of traditional single-stranded mRNA vaccines, self-replicating RNA vaccines, circular RNA vaccines, DNA vaccines or protein subunit vaccines.

9. The influenza vaccine according to claim 8, characterized in that Including self-replicating RNA vaccines.

10. Use of a mutant for preparing an agent for improving the efficacy against influenza B virus, characterized in that: The mutant is based on the sequence of wild-type influenza B virus HA protein and has mutation sites, and the mutation sites include any one or more of S469D, S462F, S426P, D430P, G268C, T35C, and E464C.

11. Use of a mutant for preparing a reagent for improving the stability of influenza B virus HA protein, characterized in that: The mutant is based on the sequence of wild-type influenza B virus HA protein and has mutation sites, and the mutation sites include any one or more of S469D, S462F, S426P, D430P, G268C, T35C, and E464C.

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