Methods for attenuating influenza viruses and attenuated influenza virus strains, and uses thereof

Abstract

The present invention provides a method for attenuating influenza virus, an attenuated influenza virus strain, and its use. The attenuating method of the present invention includes a step of deleting random numbers and positions of bases in the transmembrane domain and cytoplasmic domain of the influenza virus conserved region of M2 protein to obtain an attenuated influenza virus strain with corresponding base deletions. The attenuated influenza virus strain obtained by the attenuating method of the present invention has good growth characteristics on MDCK cell lines expressing M2 protein. The high-dose virus strain can grow in MDCK cells or chicken embryos and has a higher chicken hemagglutination titer. Nasally immunized Balb / C mice show that the virus strain is non-pathogenic to mice compared with the parent virus IAV PR8. The random base deletion method of the present invention reduces the toxicity of influenza virus and lays the foundation for screening safer and more effective IAV attenuated live vaccines.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. 202210100598.6, filed on January 27, 2022, entitled "Method for attenuating influenza viruses and attenuated influenza virus strains, and uses thereof," the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present invention relates to the technical field of biopharmaceuticals, and in particular to a method for attenuating influenza viruses and attenuated influenza virus strains, and uses thereof. [Background technology]

[0003] Influenza A (IA) is a highly contagious acute respiratory disease caused by influenza A viruses (IAV), which belong to the Orthomyxoviridae family and contain segmented negative-strand RNA. Vaccination is the primary means of preventing IA infection, and currently available vaccines include inactivated and live-attenuated vaccines. Due to limited mucosal immunity and cytotoxic T cell responses, the protective effect of inactivated vaccines is short-lasting and requires annual administration. Conversely, intranasal immunization with live-attenuated IAV vaccines can induce strong mucosal and cellular immunity, resulting in longer-lasting protective effects. Currently, only two live-attenuated vaccines are commercially available, both of which are cold-adapted strains, limiting their use to individuals aged 2–49 years. Therefore, there is a need to develop safer and more effective live-attenuated vaccines to combat IAV infection. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention aims to provide a method for attenuating influenza viruses, an attenuated influenza virus strain, and uses thereof. The attenuated influenza virus strain (replication-restricted virus) obtained by the attenuation method of the present invention has good growth characteristics on MDCK cell lines expressing M2 protein and is non-pathogenic to mice compared with the parent virus, laying the foundation for screening safer and more effective attenuated live IAV vaccines; the attenuated influenza virus strain can grow well in MDCK cells or chicken embryos, and high-dose replication-restricted viruses can grow well in MDCK cells or chicken embryos, making it possible to mass-produce the attenuated virus using chicken embryos. [Means for solving the problem]

[0005] The present invention provides a method for attenuating influenza viruses, which comprises the steps of deleting bases at random positions and numbers in the transmembrane and cytoplasmic domains of the M2 protein in the influenza virus conserved regions, and obtaining attenuated influenza virus strains having the corresponding base deletions.

[0006] Preferably, the parent virus targeted by the attenuation method is A / Puerto Rico / 8 / 1934.

[0007] Preferably, the base deletions at random numbers and positions are selected from any of the following (a) to (f): (a) A total of 14 bases are deleted from positions 73 to 86 in the M2 protein coding region. (b) A deletion of 20 bases from positions 87 to 106 in the M2 protein coding region. (c) A deletion of 22 bases from positions 130 to 151 in the M2 protein coding region. (d) A deletion of 38 bases from positions 152 to 189 in the M2 protein coding region. (e) A deletion of 65 bases from positions 87 to 151 in the M2 protein coding region. (f) A total of 103 bases were deleted from positions 87 to 189 in the M2 protein coding region.

[0008] The present invention also provides an attenuated influenza virus strain prepared by the attenuation method described in the above technical means, which is prepared by deleting any one of the following bases (a) to (f) in the M2 protein of A / Puerto Rico / 8 / 1934, which is a parent virus: (a) A total of 14 bases are deleted from positions 73 to 86 in the M2 protein coding region. (b) A deletion of 20 bases from positions 87 to 106 in the M2 protein coding region. (c) A deletion of 22 bases from positions 130 to 151 in the M2 protein coding region. (d) A deletion of 38 bases from positions 152 to 189 in the M2 protein coding region. (e) A deletion of 65 bases from positions 87 to 151 in the M2 protein coding region. (f) A total of 103 bases were deleted from positions 87 to 189 in the M2 protein coding region.

[0009] Preferably, the nucleotide sequence of the coding region of the M2 protein of the attenuated influenza virus strain is set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0010] The present invention also provides a set of plasmids for constructing an attenuated influenza virus strain, wherein the plasmids comprise the nucleotides set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, respectively.

[0011] Preferably, the base plasmid used in constructing the plasmid contains the nucleotide sequence shown in SEQ ID NO:7.

[0012] The present invention also provides primers for constructing the plasmids described in the above technical means, and the base sequences of the primers are shown in SEQ ID NOs: 8 to 19.

[0013] The present invention also provides the use of the attenuation method described in the above technical means or the attenuated influenza virus strain described in the above technical means, or the plasmid described in the above technical means, or the primer described in the above technical means in the preparation of a live attenuated influenza vaccine. [Effects of the Invention]

[0014] The present invention provides a method for attenuating influenza viruses. The attenuated influenza virus strains (replication-restricted viruses) obtained by the attenuation method of the present invention have good growth characteristics in MDCK cell lines expressing M2 protein and are non-pathogenic to mice compared with the parent virus, laying the foundation for screening safer and more effective attenuated live IAV vaccines; the attenuated influenza virus strains can grow well in MDCK cells or chicken embryos, and high-dose replication-restricted viruses can grow well in MDCK cells or chicken embryos, making it possible to mass-produce the attenuated viruses using chicken embryos. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows agarose gel electrophoresis patterns of the extracted plasmids PR8-M2-del14, PR8-M2-del20, PR8-M2-del22, PR8-M2-del38, PR8-M2-del65, and PR8-M2-del103 according to the present invention. [Figure 2] 1 shows agarose gel electrophoresis patterns of RT-PCR identification of the replication-restricted viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 of the present invention. [Figure 3] 1 shows viral growth curves of the replication-restricted viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 of the present invention and the parent virus IAV PR8 strain. [Figure 4] Photographs showing cellular lesions 72 hours after infection of MDCK cells with the replication-restricted viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 of the present invention and the parental virus IAV PR8 strain at different MOIs. [Figure 5] This figure shows the hemagglutination titers 72 hours after infection of MDCK cells with the replication-restricted viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 of the present invention and the parental virus IAV PR8 strain at different MOIs. [Figure 6] 1 is a graph showing changes in mouse body weight within 14 days after immunization with the replication-restricted viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 of the present invention and the parent virus IAV PR8 strain. [Figure 7] 1 is a graph showing changes in mouse body weight 10 days after challenge with IAV PR8 strain. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides a method for attenuating influenza viruses, which comprises the steps of deleting bases at random positions and numbers in the transmembrane and cytoplasmic domains of the M2 protein in the influenza virus conserved regions, and obtaining attenuated influenza virus strains having the corresponding base deletions.

[0017] The present invention uses a reverse genetic engineering system to generate a series of random base deletions in the transmembrane domain (TM) and cytoplasmic domain (CT) regions, evaluates the toxicity of the resulting randomly deleted viruses, and helps to obtain a safe and effective live attenuated IAV vaccine.

[0018] In the present invention, the parent virus targeted by the attenuation method is preferably A / Puerto Rico / 8 / 1934 (abbreviated as "IAV PR8").

[0019] In the present invention, the base deletions at random numbers and positions are selected from any of the following (a) to (f): (a) A total of 14 bases are deleted from positions 73 to 86 in the M2 protein coding region, and the base sequence of the coding gene is shown in SEQ ID NO: 1; (b) a deletion of 20 bases in total from positions 87 to 106 of the M2 protein coding region, the base sequence of the coding gene of which is shown in SEQ ID NO: 2; (c) a deletion of 22 bases in total from positions 130 to 151 of the M2 protein coding region, the base sequence of the coding gene of which is shown in SEQ ID NO: 3; (d) a deletion of a total of 38 bases from positions 152 to 189 in the M2 protein coding region, the base sequence of which is shown in SEQ ID NO: 4; (e) a deletion of a total of 65 bases from positions 87 to 151 of the M2 protein coding region, the base sequence of which is shown in SEQ ID NO: 5; (f) A total of 103 bases from positions 87 to 189 in the M2 protein coding region are deleted, and the base sequence of the coding gene is shown in SEQ ID NO:6.

[0020] The attenuated influenza virus strains produced by the attenuation method of the present invention can grow well in MDCK cells or chicken embryos, and by observing weight changes, survival status, and detecting virus content in the nasal turbinates and lungs of nasally immunized Babl / C mice, the attenuated influenza virus strains were found to be non-pathogenic to mice compared with the parent IAV PR8 virus. The attenuation method of the present invention lays the foundation for screening for safer and more effective live attenuated IAV vaccines.

[0021] The present invention also provides an attenuated influenza virus strain prepared by the attenuation method described in the above technical means, which is prepared by deleting any one of the following bases (a) to (f) in the M2 protein of A / Puerto Rico / 8 / 1934, which is a parent virus: (a) A total of 14 bases are deleted from positions 73 to 86 in the M2 protein coding region. (b) A deletion of 20 bases from positions 87 to 106 in the M2 protein coding region. (c) A deletion of 22 bases from positions 130 to 151 in the M2 protein coding region. (d) A deletion of 38 bases from positions 152 to 189 in the M2 protein coding region. (e) A deletion of 65 bases from positions 87 to 151 in the M2 protein coding region. (f) A total of 103 bases were deleted from positions 87 to 189 in the M2 protein coding region.

[0022] In the present invention, the nucleotide sequence of the coding region of the M2 protein of the attenuated influenza virus strain is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0023] The present invention also provides a set of plasmids for constructing an attenuated influenza virus strain, each of which contains the nucleotides set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. The plasmids of the present invention are plasmids with random base deletions in the M2 gene. The present invention does not particularly limit the method for constructing deletion plasmids, and they can be constructed using conventional deletion plasmid construction methods well known to those skilled in the art. In the present invention, the base plasmid used to construct the plasmid preferably contains the base sequence set forth in SEQ ID NO:7. The present invention does not particularly limit the type of base plasmid, as long as it contains the base sequence set forth in SEQ ID NO:7.

[0024] The present invention also provides primers for constructing the plasmids described in the above technical means, and the base sequences of the primers are shown in SEQ ID NOs: 8 to 19.

[0025] The present invention also provides the use of the attenuation method described in the above technical means or the attenuated influenza virus strain described in the above technical means, or the plasmid described in the above technical means, or the primer described in the above technical means in the preparation of a live attenuated influenza vaccine.

[0026] The method for attenuating influenza viruses and the attenuated influenza virus strains of the present invention, as well as their uses, will be described in more detail below with reference to specific examples, and the technical means of the present invention include, but are not limited to, the following examples.

[0027] (Example) Example 1 Construction of a random deletion plasmid of the influenza virus M2 gene A plasmid expressing the M2 gene of the PR8 influenza virus strain (the present invention does not impose any particular limitations on the method for constructing this plasmid; a plasmid overexpressing M2 can be constructed according to conventional recombinant plasmid construction methods using commonly used plasmids and M genes well known to those skilled in the art) was extracted and subjected to PCR amplification using a series of base deletion primers (primer sequences are shown in Table 1). After confirming the correct molecular weight by agarose gel electrophoresis, the target band was recovered from the gel and recombined at 50°C for 15 minutes. The recombinant product was transformed into competent E. coli cells, and colonies were selected and sequenced to verify accuracy. Using an endotoxin-removing miniprep medium kit, deletion plasmids PR8-M2-del14, PR8-M2-del20, PR8-M2-del22, PR8-M2-del38, PR8-M2-del65, and PR8-M2-del103 were successfully prepared (Figure 1 shows the agarose gel electrophoresis patterns of the extracted plasmids PR8-M2-del14, PR8-M2-del20, PR8-M2-del22, PR8-M2-del38, PR8-M2-del65, and PR8-M2-del103).

[0028] [Table 1]

[0029] Example 2 Rescue and validation of replication-restricted influenza viruses HEK293T cells were spread on a special 6-well plate manufactured by Thermo Fisher Scientific, and after 12 hours, seven plasmids containing PR8 genes (pFlu-PR8-PB2, pFlu-PR8-PB1, pFlu-PR8-PA, pFlu-PR8-NP, pFlu-PR8-NS, pFlu-PR8-HA, pFlu-PR8-NA), the M2 gene random base deletion series plasmid constructed in Example 1, and a plasmid expressing the M2 protein were co-transfected into the HEK293T cells. The medium was changed 6 to 8 hours after transfection. 48 hours after transfection, the cell plates were frozen and thawed once, and the supernatant was collected and inoculated into T25 cell flasks of MDCK cells expressing M2 protein. 72 to 96 hours after inoculation, cell lesions were observed. The cell flasks were frozen and thawed once, and then centrifuged to collect the supernatants. These were named rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103, respectively.

[0030] The collected viral RNA was extracted using a viral RNA extraction kit and amplified by RT-PCR using the primers listed in Table 2. After confirming the correct molecular weight by agarose gel electrophoresis (Figure 2), sequencing of the PCR products demonstrated that the collected viruses were replication-restricted viruses with the corresponding base deletions. Figure 2 shows the agarose gel electrophoresis patterns of the RT-PCR identification of the replication-restricted viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103.

[0031] [Table 2]

[0032] Example 3 Replication-restricted influenza virus titer measurement MDCK cells expressing M2 protein were spread on a 96-well plate and grown until the cells covered a monolayer. 50 μL of replication-restricted influenza virus was then added to 450 μL of 1% TPCK opi-MEM and mixed well. This was used as system 1, with a final concentration of 10 -1 50 μL of system 1 was added to 450 μL of 1% TPCK opi-MEM, and after shaking well, this was used as system 2, with a final concentration of 10 -2 In order to -10 The medium in the 96-well plate was then discarded, the plate was washed with PBS, and 100 μL of the corresponding diluted virus solution was added to each well. Each gradient was repeated three times. The cells were then cultured in a 37°C, 5% CO2 incubator for 72 hours, after which cell lesions were observed and the TCID was calculated using the Reed-Muench method. 50 was calculated and the virus titers are shown in Table 3.

[0033] [Table 3]

[0034] Example 4 Measurement of growth curves of replication-restricted influenza viruses M2 protein-expressing MDCK cells were spread onto 48-well plates and grown until the cells covered a monolayer. The cells were then inoculated with the replication-restricted influenza viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 and the parental IAV PR8 virus at a multiplicity of infection (MOI) of 0.001 in triplicate and cultured at 37°C in a 5% CO2 incubator. Viruses were collected at 24, 48, 72, and 96 hours after infection. The virus solutions collected at different times were serially diluted 10-fold, and each dilution was repeated three times. These were then inoculated onto MDCK cells expressing M2 protein grown in a monolayer in a 96-well plate. After 72 hours of incubation in a cell incubator at 37°C and 5% CO2, cell lesions were observed and the TCID was calculated using the Reed-Muench method.50 After the calculation and data analysis were completed, growth curves of the replication-restricted influenza viruses (Figure 3) were plotted. As can be seen from Figure 3, rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del65, and WT-PR8 had similar growth characteristics and were able to reach similar viral titers 48 hours post-infection. rPR8-M2-del38 and rPR8-M2-del103 also had good growth characteristics on MDCK cells expressing the M2 protein.

[0035] Example 5 Measurement of replication status of replication-restricted influenza viruses in MDCK cells MDCK cells were spread onto 48-well plates and grown until the cells covered the monolayer. Then, replication-restricted influenza viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 and the parental virus IAV were cultured in 48-well plates. The PR8 strain was inoculated into cells at multiplicities of infection (MOI) of 0.001, 0.004, 0.016, 0.064, 0.256, 1.024, 4.096, and 16.384, and then cultured in an incubator at 37°C and 5% CO2. After 72 hours of culture in an incubator at 37°C and 5% CO2, cell lesions were observed and photographed (Figure 4). MDCK cells were infected with the PR8 strain at different MOIs (photographs showing cellular lesions 72 hours later). The cells were then frozen and thawed three times, and the supernatant was collected by centrifugation. A chicken erythrocyte hemagglutination test was performed to record the hemagglutination titer (Figure 5). The hemagglutination titer was measured 72 hours after infection of MDCK cells with the replication-restricted viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 and the parent IAV PR8 strain at different MOIs (Figure 5). The test results indicated that high doses of the replication-restricted influenza virus could grow well in MDCK cells. Figure 5 also shows that the attenuation effect of the rPR8-M2-del20 virus was more pronounced, indicating that the replication-restricted influenza virus obtained in this invention is not completely dependent on the MDCK cell line expressing the M2 protein.

[0036] Stock solutions of the replication-restricted influenza viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 were inoculated into four chicken embryos, respectively. After 72 hours, the allantoic fluid of the chicken embryos was collected and subjected to a chicken hemagglutination test. The hemagglutination titers were recorded (Table 4). The rPR8-M2-del20 virus, which had the most attenuated effect, was able to replicate in chicken embryos and achieve a higher chicken hemagglutination titer. This indicates that the replication-restricted influenza viruses of the present invention can be mass-produced using chicken embryos, reducing costs and increasing production volume.

[0037] [Table 4]

[0038] Example 6 Replication-restricted virus immunity experiments Balb / C female mice aged 4-5 weeks were divided into 7 groups of 8 mice each. 6 TCID 50 The mice were intranasally immunized with the viruses rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103 and the parent virus IAV PR8 strain at a dose of 100 mg / kg. Three days after immunization, three mice per group were sacrificed, and their nasal turbinates and lungs were crushed to measure the virus content in the crushed fluid. The measurement method was the same as in Example 3, using the Reed-Muench method. 50The viral titers were calculated, and the results are shown in Table 5. The weights of the remaining five mice were measured and recorded daily and observed until day 14. A graph of the changes in mouse weight (FIG. 6) was prepared and compared with that of the WT-PR8 strain. It was found that the weights of the mice immunized with the rPR8-M2-del14, rPR8-M2-del20, and rPR8-M2-del65 viruses initially decreased and then tended to increase. The weights of the mice immunized with the rPR8-M2-del22, rPR8-M2-del38, and rPR8-M2-del103 viruses fluctuated slightly, but all of the mice immunized with the WT-PR8 strain died on day 5 after immunization. The replication-restricted influenza virus of the present invention was nonpathogenic to mice, and the HI titers of the serum on day 14 after immunization were all 2. 6.5 ~2 10 (Table 6), indicating that the immunization effect of the weakened influenza virus was better. 6 TCID 50 The mice were challenged with a dose of virus at 100 mg / kg / day, and their weights were measured and recorded daily. They were then observed for 10 days, and a graph of their weight changes was created (Figure 7). As can be seen from the graph, the weights of mice immunized with rPR8-M2-del14, rPR8-M2-del22, and rPR8-M2-del38 viruses fluctuated slightly after challenge, indicating that these three attenuated influenza virus strains provided excellent protection. This lays the foundation for screening for safer and more effective live attenuated IAV vaccines.

[0039] [Table 5]

[0040] [Table 6]

[0041] It should be pointed out that the above are only preferred embodiments of the present invention, and those skilled in the art may make some improvements and modifications without departing from the principle of the present invention, and such improvements and modifications should be regarded as within the protection scope of the present invention.

[0042] (Addendum) (Appendix 1) A method for attenuating an influenza virus, comprising the steps of deleting bases at random numbers and positions in the transmembrane domain and cytoplasmic domain of the M2 protein in the influenza virus conserved region, and obtaining a attenuated influenza virus strain having the corresponding base deletions.

[0043] (Appendix 2) The method for attenuating influenza viruses described in Appendix 1, characterized in that the influenza viruses include A / Puerto Rico / 8 / 1934.

[0044] (Appendix 3) The weakening method described in Appendix 1, characterized in that the base deletions of random number and positions are selected from any of the following (a) to (f): (a) A total of 14 bases are deleted from positions 73 to 86 in the M2 protein coding region. (b) A total of 20 bases are deleted from positions 87 to 106 in the M2 protein coding region. (c) A total of 22 bases are deleted from positions 130 to 151 in the M2 protein coding region. (d) A total of 38 bases are deleted from positions 152 to 189 in the M2 protein coding region. (e) A total of 65 bases are deleted from positions 87 to 151 in the M2 protein coding region. (f) A total of 103 bases are deleted from positions 87 to 189 in the M2 protein coding region.

[0045] (Appendix 4) An attenuated influenza virus strain prepared by the attenuation method described in any one of appendices 1 to 3.

[0046] (Appendix 5) 5. The attenuated influenza virus strain described in Appendix 4, wherein the nucleotide sequence of the coding region of the M2 protein of the attenuated influenza virus strain is set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0047] (Appendix 6) A set of plasmids for constructing an attenuated influenza virus strain, each of which contains the nucleotides set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.

[0048] (Appendix 7) 7. The plasmid according to claim 6, wherein the base plasmid used in constructing the plasmid comprises the base sequence shown in SEQ ID NO: 7.

[0049] (Appendix 8) A primer for constructing the plasmid according to Appendix 6 or 7, characterized in that the base sequence of the primer is set forth in SEQ ID NOs: 8 to 19.

[0050] (Appendix 9) 1. Use of the attenuating method according to any one of appendices 1 to 3 or said attenuated influenza virus strain according to appendices 4 or 5 or the plasmid according to appendices 6 or 7 or the primer according to appendix 8 in the preparation of a live attenuated influenza vaccine.

[0051] (Appendix 10) 10. Use of an attenuated influenza virus strain as described in Supplementary Note 4 or 5 in the prophylaxis of influenza A.

Claims

1. A method for attenuating an influenza virus, comprising the steps of deleting bases in the following numbers and positions in the transmembrane domain and cytoplasmic domain of the M2 protein in an influenza virus conserved region, to obtain an attenuated influenza virus strain having the corresponding base deletions, wherein the base deletions in the following numbers and positions are selected from any of (a) to (f) below. (a) A total of 14 bases are deleted from positions 73 to 86 in the M2 protein coding region. (b) A total of 20 bases are deleted from positions 87 to 106 in the M2 protein coding region. (c) A total of 22 base deletions from positions 130 to 151 in the M2 protein coding region (d) A total of 38 base deletions from positions 152 to 189 in the M2 protein coding region (e) A deletion of 65 bases from positions 87 to 151 in the M2 protein coding region (f) A total of 103 bases are deleted from positions 87 to 189 in the M2 protein coding region.

2. 2. The method of claim 1, wherein the influenza virus is A / Puerto Rico / 8 / 1934.

3. 10. An attenuated influenza virus strain prepared by the attenuation method of claim 1.

4. The attenuated influenza virus strain according to claim 3, wherein the base sequence of the coding region of the M2 protein of the attenuated influenza virus strain is set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO:

6.

5. A set of plasmids for constructing an attenuated influenza virus strain, each of which contains the nucleotides set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:

6.

6. The plasmid according to claim 5, wherein the base plasmid used in constructing the plasmid contains the base sequence shown in SEQ ID NO:

7.

7. Primers for constructing the plasmid according to claim 5, wherein the base sequences of the primers are represented by SEQ ID NOs: 8 to 19, The primers having the nucleotide sequences shown in SEQ ID NO: 8 and 9 were used to construct a plasmid containing the nucleotide sequence shown in SEQ ID NO: 1, The primers having the nucleotide sequences shown in SEQ ID NO: 10 and 11 were used to construct a plasmid containing the nucleotide sequence shown in SEQ ID NO: 2, The primers having the nucleotide sequences shown in SEQ ID NOs: 12 and 13 were used to construct a plasmid containing the nucleotide sequence shown in SEQ ID NO: 3, The primers having the nucleotide sequences shown in SEQ ID NOs: 14 and 15 were used to construct a plasmid containing the nucleotide sequence shown in SEQ ID NO: 4, The primers having the nucleotide sequences shown in SEQ ID NOs: 16 and 17 were used to construct a plasmid containing the nucleotide sequence shown in SEQ ID NO: 5, A primer characterized in that the primers having the base sequences shown in SEQ ID NOs: 18 and 19 are used to construct a plasmid containing the nucleotide shown in SEQ ID NO:

6.

8. A method for producing a live attenuated influenza vaccine, comprising the step of preparing a live attenuated influenza vaccine by the attenuation method of claim 1.

9. Use of the attenuated influenza virus strain of claim 3 in the preparation of a live attenuated influenza vaccine.

10. 6. Use of the plasmid of claim 5 in the preparation of a live attenuated influenza vaccine.

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