Application Of Reverse Genetics Technology In The Preparation Of Cold-adapted Influenza Virus Vaccine Strains

Vaccination is one of the most effective means to prevent the occurrence and spread of influenza disease.Currently,the widely used influenza vaccines include split vaccines,subunit vaccines and live attenuated vaccines.Compared with traditional inactivated vaccines,live attenuated vaccines are made of cold-adapted influenza attenuated strains and are immunized through the nasal mucosal route.They have the advantages of easy mass vaccination and mimic natural infection,and can fully activate humoral and cellular immune responses.They produce lasting and cross-immune protection,and directly induce respiratory mucosal immunity,secrete IgA antibodies,and establishe the first line of defense against infection.In addition,the vaccine technology using influenza attenuated vaccine strains as a carrier is one of the five technical routes for the research and development of new crown vaccines in my country.Clinical studies have shown that it has a good dual immune effect against influenza and new crowns.Therefore,the live attenuated influenza vaccine shows great potentialBased on the high variability of influenza virus,methods for preparing live attenuated influenza vaccine production strains that match the current epidemic include classical reassortment technology and reverse genetic technology.Compared with the time-consuming serological reassortment method adopted by WHO,Reverse genetics technology can respond quickly to new viruses in a timely manner,construct antigen-matched vaccine strains more efficiently,and genetically modify individual genes.In order to establish a reverse genetics technology platform for the rapid construction of cold-adapted influenza attenuated vaccine strains,this study adopted an 8-plasmid reverse genetics operating system,and cold-adapted A/Ann Arbor/6/60（H2N2）influenza virus strains as backbone donors,constructed a cold-adapted influenza virus vaccine strain AA-PR8 containing A/Puerto Rico/8/34（H1NI）virus HA and NA surface antigens,and its temperature sensitivity,cold-adapted phenotype,genetic stability and attenuation properties and other biological characteristics were identified,and the immunogenicity and protective effect of the vaccine strain were preliminarily evaluated in mice.The first part is the preparation of cold-adapted influenza virus vaccine strains using reverse genetics.Based on the principle of reverse genetics technology,the cold-adapted donor strain A/Leningrad/134/17/57（H2N2）from Russia and the cold-adapted donor strain A/Ann Arbor/6/60（H2N2）from the United States were constructed respectively.The 6 backbone plasmids were co-transfected with the HA and NA antigen plasmids of the PR8 strain,respectively,into 293T and MTY6 cells for virus rescue.Results Cold-adapted influenza virus vaccine strain could not be rescued by A/Leningrad/134/17/57（H2N2）backbone,but cold-adapted influenza virus vaccine strain was successfully prepared by A/Ann Arbor/6/60（H2N2）backbone.AA-PR8,and based on this,4 cold-adapted strains of H1N1 and H3N2 prevalent in the northern hemisphere in 2020-2022 were successfully constructed,and a cold-adapted strain with A/Ann Arbor/6/60（H2N2）as the skeleton was initially established.Preparation platform for influenza virus vaccine strains.The second part is the preliminary evaluation of AA-PR8 cold-adapted vaccine strain.The biological characteristics of the AA-PR8 vaccine strain were identified,and the results showed that AA-PR8 grew well after inoculation with chicken embryos,and the virus titer could reach 8.10 lgEID₅₀/m L;Can not grow at 39℃,has temperature sensitivity and cold-adapted phenotype;5 consecutive passages,all gene amino acid sites are not mutated,with good genetic stability;mice inoculated with the same dose of PR8 wild virus Compared with the mice inoculated with the AA-PR8 strain,the weight loss of the mice was significantly reduced,no animal death occurred,and the viral load in the lungs decreased by 3.83 lgEID₅₀/g on the 3rd day,no abnormal inflammatory response was observed,and it had attenuation characteristics;and further The immunogenicity and protective effect of the AA-PR8 vaccine strain in mice were evaluated.One or two vaccinations of the AA-PR8 vaccine strain in mice could effectively stimulate the mice’s humoral immunity and produce IgA and IgG antibodies.The mice were challenged with PR8 wild virus 14 days after the primary immunization,and the protection rate of mice was 83.33%（5/6）,and the body weight decreased.The mice were challenged 14 days after the second immunization,and the protection rate of the mice was 100%（6/6）,the body weight was basically not reduced,and no virus was detected in the lungs.Significantly reduces the viral load in the lungs of mice after challenge,improves the survival rate,and has a better immune protection effect.In conclusion,in this study,the cold-adapted attenuated A/Ann Arbor/6/60（H2N2）strain was used as the backbone,and the AA-PR8 cold-adapted influenza virus containing PR8 virus HA and NA surface antigens was successfully constructed by reverse genetics technology.vaccine strain.The vaccine strain has high yield,temperature sensitivity and cold-adapted phenotype,good genetic stability,and exhibits obvious attenuation properties in mouse models.It can effectively stimulate mice to produce higher titers of IgA and IgG antibodies,showing better immunogenicity and protection.