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, entitled "Method for attenuating influenza virus and attenuated influenza virus strain, and use thereof," filed on January 27, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present invention relates to the technical field of biopharmaceuticals, 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 are segmented negative-strand RNA viruses belonging to the Orthomyxoviridae family. Vaccination is the main means of preventing IA infection, and currently there are inactivated and live attenuated vaccines. Due to limited mucosal immunity and cytotoxic T cell responses, the preventive effect of inactivated vaccines lasts only for a short period of time and must be administered once a year. Conversely, intranasal immunization with live attenuated IAV vaccines can induce strong mucosal and cellular immunity, so the preventive effect can last longer. Currently, there are only two types of live attenuated vaccines on the market, both of which are cold-adapted strains, and the use of these vaccines is limited to people aged 2 to 49 years. Therefore, it is necessary to develop a safer and more effective live attenuated vaccine to resist IAV infection. Summary of the Invention [Problem to be solved by the invention]

[0004] The object of the present invention is to provide a method for weakening influenza virus, and an attenuated influenza virus strain, as well as its use. The attenuated influenza virus strain (replication-restricted virus) obtained by the attenuation method of the present invention has good growth characteristics on MDCK cell line expressing M2 protein, and is non-pathogenic to mice compared with the parent virus, which can lay the foundation for screening safer and more effective IAV attenuated live vaccines; the attenuated influenza virus strain can grow well in MDCK cells or chicken embryos, and high-dose replication-restricted virus can grow well in MDCK cells or chicken embryos, and the attenuated virus can be mass-produced using chicken embryos. [Means for solving the problem]

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

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

[0007] Preferably, the random number and position of base deletions are selected from any of the following (a) to (f): (a) A deletion of 14 bases 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 position 87 to position 151 in the M2 protein coding region, (f) A deletion of 103 bases 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. The attenuated influenza virus strain 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 deletion of 14 bases 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 position 87 to position 151 in the M2 protein coding region, (f) A deletion of 103 bases 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 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.

[0010] The present invention also provides a set of plasmids for constructing an attenuated influenza virus strain, said plasmids comprising 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 base 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 attenuated 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. Effect of the Invention

[0014] The present invention provides a method for attenuating influenza virus. The attenuated influenza virus strain (replication-restricted virus) obtained by the attenuation method of the present invention has good growth characteristics on MDCK cell line expressing M2 protein, and is non-pathogenic to mice compared with the parent virus, which can lay the foundation for screening safer and more effective IAV attenuated live vaccines; the attenuated influenza virus strain can grow well in MDCK cells or chicken embryos, and high-dose replication-restricted virus can grow well in MDCK cells or chicken embryos, and the attenuated virus can be mass-produced using chicken embryos. [Brief description 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. [Diagram 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. [Diagram 3] Virus 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] These are 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 parent virus IAV PR8 strain at different MOIs. [Diagram 5] FIG. 1 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 parent 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 PREFERRED EMBODIMENTS

[0016] The present invention provides a method for attenuating influenza viruses, comprising the steps of deleting bases at random numbers and positions in the transmembrane and cytoplasmic domains of influenza virus conserved regions of the M2 protein, 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, and evaluates the toxicity of the generated randomly deleted viruses, which is helpful in obtaining a safe and effective live attenuated IAV vaccine.

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

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

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

[0021] The present invention also provides an attenuated influenza virus strain prepared by the attenuation method described in the above technical means. The attenuated influenza virus strain 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 deletion of 14 bases 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 position 87 to position 151 in the M2 protein coding region, (f) A deletion of 103 bases 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, the plasmids comprising the nucleotides 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, respectively. The plasmids of the present invention are random base deletion plasmids of the M2 gene. The present invention is not particularly limited to the method of constructing the deletion plasmid, and can be constructed using conventional deletion plasmid construction methods well known to those skilled in the art. In the present invention, the basic plasmid used in constructing the plasmid preferably comprises the base sequence shown in SEQ ID NO:7. The present invention is not particularly limited to the type of the basic plasmid, as long as it comprises the base sequence shown 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 attenuated 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 virus and the attenuated influenza virus strain of the present invention, as well as their uses, are 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 random deletion plasmids of influenza virus M2 gene A plasmid expressing the M2 gene of the PR8 influenza virus strain (the present invention is not particularly limited in the method of constructing this plasmid, and 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 PCR amplification was performed using a series of base deletion primers (primer sequences are shown in Table 1). After confirming that the molecular weight was correct by agarose gel electrophoresis, the target band was recovered from the gel and recombined for 15 minutes under a temperature condition of 50 ° C. The recombinant product was transformed into E. coli competent cells, colonies were selected and sequenced to verify the accuracy, and the deletion plasmids PR8-M2-del14, PR8-M2-del20, PR8-M2-del22, PR8-M2-del38, PR8-M2-del65, and PR8-M2-del103 were successfully prepared using an endotoxin-removal miniprep medium kit (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 are shown in Figure 1).

[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, and after 12 hours, the plasmids of seven genes including PR8 (pFlu-PR8-PB2, pFlu-PR8-PB1, pFlu-PR8-PA, pFlu-PR8-NP, pFlu-PR8-NS, pFlu-PR8-HA, pFlu-PR8-NA), the plasmid of the M2 gene random base deletion series constructed in Example 1, and the plasmid expressing the M2 protein were co-transfected into the HEK293T cells, and the transfection was performed. The medium was replaced 6 to 8 hours after transfection, and 48 hours after transfection, the cell plate was frozen and thawed once, the supernatant was collected and inoculated into a T25 cell flask of MDCK cells expressing M2 protein, and 72 to 96 hours after inoculation, the cell lesions were observed, and the cell flask was frozen and thawed once, and then centrifuged to collect the supernatant, which were named rPR8-M2-del14, rPR8-M2-del20, rPR8-M2-del22, rPR8-M2-del38, rPR8-M2-del65, and rPR8-M2-del103, respectively.

[0030] The viral RNA collected above was extracted using a viral RNA extraction kit, and RT-PCR amplification was performed with the primers in Table 2. After confirming that the molecular weight was correct by agarose gel electrophoresis (Figure 2), sequencing of the PCR product showed that the collected virus was a replication-restricted virus with the corresponding base deletion. Figure 2 shows the 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.

[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 the cells were grown until they covered the monolayer. Then, 50 μL of the replication-restricted influenza virus was added to 450 μL of 1% TPCK opi-MEM, and mixed well. The resulting mixture 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 made into system 2, with a final concentration of 10 -2 In order to make -10 The medium in the 96-well plate was then discarded, the plate was washed with PBS, and 100 μL of the corresponding diluted solution of the virus solution was added to each well, and each gradient was repeated three times. After 72 hours of culture in a cell culture incubator at 37°C and 5% CO2, the cell lesions were observed and the TCID 50 The viral titers were calculated and are shown in Table 3.

[0033] [Table 3]

[0034] Example 4 Measurement of growth curves of replication-restricted influenza viruses MDCK cells expressing M2 protein were spread in 48-well plates and grown until the cells covered the 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 in a 37°C, 5% CO2 incubator. Viruses were collected at 24, 48, 72, and 96 hours after infection. The virus fluids collected at different times were serially diluted 10-fold, and each dilution was repeated three times. The diluted virus was then inoculated into MDCK cells expressing M2 protein grown in a monolayer in a 96-well plate. After incubation in a 37°C, 5% CO2 cell incubator for 72 hours, cell lesions were observed and the TCID50 After the data analysis was completed, the growth curves of the replication-restricted influenza viruses (Figure 3) were drawn. 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 could reach similar virus titers 48 hours after infection. rPR8-M2-del38, rPR8-M2-del103 also had good growth characteristics on MDCK cells expressing M2 protein.

[0035] Example 5 Measurement of replication status of replication-restricted influenza viruses in MDCK cells MDCK cells were spread in 48-well plates, grown until the cells covered the monolayer, and then cultured 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 parent virus IAV. The PR8 strain was inoculated into cells at a multiplicity 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, the cellular lesions were observed and photographed (Figure 4). 5 shows the cellular lesions 72 hours after infection of MDCK cells with PR8 strain at different MOIs (photographs showing the cellular lesions 72 hours after infection of MDCK cells with PR8 strain at different MOIs), then the cells were frozen and thawed three times, and the supernatant was collected by centrifugation to perform chicken erythrocyte agglutination test, and the hemagglutination titer (hemagglutination titer 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, rPR8-M2-del103 and the parent virus IAV PR8 strain at different MOIs in FIG. 5) was recorded. The test results show that high doses of the replication-restricted influenza virus can grow well on MDCK cells, and FIG. 5 shows that the weakening effect of the rPR8-M2-del20 virus is more obvious, indicating that the replication-restricted influenza virus obtained in the present invention is not completely dependent on the MDCK cell line expressing M2 protein.

[0036] The 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 for chicken hemagglutination test, and the hemagglutination titers were recorded (Table 4). The rPR8-M2-del20 virus with the highest attenuation effect can replicate in chicken embryos and reach a higher chicken hemagglutination titer, indicating that the replication-restricted influenza virus of the present invention can be mass-produced using chicken embryos, which can reduce costs and increase production.

[0037] [Table 4]

[0038] Example 6 Replication-restricted virus immunity experiments Balb / C female mice aged 4 to 5 weeks were divided into 7 groups of 8 mice each. 6 TCID 50 The mice were intranasally immunized with 100 μg / kg of 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. Three days after immunization, three mice from each group were sacrificed, and their nasal turbinates and lungs were crushed to measure the virus content in the crushed liquid. The measurement method was the same as in Example 3, and the Reed-Muench method was used to measure the virus content. 50The viral titers are shown in Table 5. The weights of the remaining 5 mice were measured and recorded every day, and observed until the 14th day. A graph of the changes in mouse weight (FIG. 6) was prepared and compared with the WT-PR8 strain. It was found that the weights of the mice immunized with rPR8-M2-del14, rPR8-M2-del20, and rPR8-M2-del65 viruses initially decreased and then tended to increase, while the weights of the mice immunized with 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 the 5th day after immunization. The replication-restricted influenza virus of the present invention was non-pathogenic to mice, and the HI titers of the sera 14 days after immunization were all 2. 6.5 ~2 10 (Table 6), indicating that the immunization effect of the weakened influenza virus is better. 6 TCID 50 The mice were challenged with the virus at a dose of 100 mg / kg / day, and their weights were measured and recorded daily. After 10 days of observation, a graph of the changes in the mice's weight (Figure 7) was drawn. As can be seen from the graph, the weights of the mice immunized with rPR8-M2-del14, rPR8-M2-del22, and rPR8-M2-del38 viruses fluctuated slightly after virus challenge, indicating that these three strains of weakened influenza viruses have excellent protective power. 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 noted that the above are only preferred embodiments of the present invention, but 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] (Additional Note) (Appendix 1) A method for attenuating an influenza virus, comprising the steps of deleting bases at random numbers and positions in the transmembrane and cytoplasmic domains of the M2 protein in a conserved region of the influenza virus, to obtain a attenuated influenza virus strain having the corresponding base deletions.

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

[0044] (Appendix 3) The weakening method described in Appendix 1, characterized in that the random number and position of base deletions 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 position 87 to position 151 in the M2 protein coding region (f) A deletion of 103 bases from positions 87 to 189 in the M2 protein coding region

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

[0046] (Appendix 5) The attenuated influenza virus strain described in Appendix 4, characterized in that 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.

[0047] (Appendix 6) A set of plasmids for constructing an attenuated influenza virus strain, characterized in that the plasmids contain 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.

[0048] (Appendix 7) A plasmid according to Appendix 6, characterized in that the basic plasmid used in constructing the plasmid contains the base sequence shown in SEQ ID NO:7.

[0049] (Appendix 8) A primer for constructing the plasmid according to appended claim 6 or 7, characterized in that the base sequence of the primer is set forth in SEQ ID NO:8 to 19.

[0050] (Appendix 9) 10. The use of a method of attenuation according to any one of appendices 1 to 3 or an attenuated influenza virus strain as defined in appendices 4 or 5, or a plasmid as defined in appendices 6 or 7, or a primer as defined in appendix 8, in the preparation of a live attenuated influenza vaccine.

[0051] (Appendix 10) 13. Use of an attenuated influenza virus strain as described in appendix 4 or 5 in the prophylaxis of influenza A.

Claims

1. A method for attenuating an influenza virus, comprising the steps of deleting bases at random numbers and positions in the transmembrane and cytoplasmic domains of the M2 protein in a conserved region of the influenza virus, to obtain a attenuated influenza virus strain having the corresponding base deletions.

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

3. The method for weakening according to claim 1, wherein the random number and position of base deletions are selected from any of the following (a) to (f). (a) A total of 14 base deletions 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 in total from positions 87 to 151 in the M2 protein coding region (f) A deletion of 103 bases from positions 87 to 189 in the M2 protein coding region

4. An attenuated influenza virus strain prepared by the attenuation method according to any one of claims 1 to 3.

5. The attenuated influenza virus strain according to claim 4, characterized in that 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.

6. A set of plasmids for constructing an attenuated influenza virus strain, characterized in that the plasmids each contain the nucleotides 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.

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.

8. A primer for constructing the plasmid according to claim 6 or 7, characterized in that the base sequence of the primer is represented by SEQ ID NO: 8 to 19.

9. Use of the attenuated method according to any one of claims 1 to 3 or said attenuated influenza virus strain according to claim 4 or 5 or the plasmid according to claim 6 or 7 or the primer according to claim 8 in the preparation of a live attenuated influenza vaccine.

10. Use of the attenuated influenza virus strain according to claim 4 or 5 in the prevention of influenza A.

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