Rescued Influenza A Virus With Codon Deoptimized NS1 Gene Is Attenuated Both In Vitro And In Vivo

Influenza virus vaccines, such as killed and live attenuated vaccines, are still most effective mothods of prophylaxis. Trivalent inactivated vaccines delivered intramuscularly can elicit good serum antibody responses but induce poorly mucosal IgA antibody and cell-mediated immunity. Live attenuated vaccine(cold adapted vaccine) adiminstrated intranasally may elicit a long-lasting, broader immune (humoral and cellular) response, which more closely resembles natural immunity. It turns out that live attenuated vaccines are more convenient and effective than inactivated vaccines. By taking advantage of reverse genetics, researches developed many candidates to attenuate virulence and improve antigenicity for live attenuated vaccines, it would be a major breakthrough to develop safe and effective live attenuated vaccines.According to the codon usage bias of influenza A virus, we designed and synthesized a condon-deoptimized NS gene by substituting codons of 110 amino acids in the NS1 gene of A/Puerto Rico/8/34(H1N1) with unpreferred synonymous codons. The influenza A virus with the codon deoptimized NS1 gene (deoNS virus) was rescued by 12-plasmid based reverse genetics. Plaque forming assay and virus growth curve showed that the growth of deoNS virus was reduced about 1000 times in MDCK cells compared to that of the wild-type virus. Intranasal inoculation with deoNS virus did not cause death or evident disease in infected BALB/c mice. Furthermore, the virus titer in the lungs of mice infected with deoNS virus was significantly lower (i.e. 100~1000 times) than that of wild-type virus.We explored the feasibility to attenuate influenza virus by codon deoptimization of NS1, our results indicated that influenza virus could be effectively attenuated by synonymous codon deoptimization of NS1 gene. This strategy will be useful to develop new attenuated candidates for the production of live attenuated influenza vaccines.