Characterization of mutations associated with cold adapted properties in DelNS1 influenza viruses; potential for new live attenuated influenza vaccine development

Abstract

BACKGROUND: Our previous study identified an A14U substitution in the M segment non-coding region that supports replication of NS1-deleted (DelNS1) virus derived from the A/WSN/33 strain. Construction of DelNS1 versions of other influenza strains revealed that different strains require different adaptive mutations to restore virus replication when NS1 is deleted. METHOD: Using a similar strategy, a panel of DelNS1 viruses derived from WSN H1N1, 2009 H1N1, H5N1 and H7N9 were constructed and characterized. These DelNS1 viruses are able to replicate in both MDCK cells and eggs, are avirulent in mice, and can provide cross protection against lethal challenge with heterosubtypic viruses in animals. In the DelNS1 virus derived from 2009 H1N1 virus, we also identified substitutions which facilitate virus replication at lower temperatures (30-33oC) but limit virus replication at higher temperatures (37-39oC). We found this cold adapted DelNS1 2009 H1N1 virus to be able to replicate to comparable titers to the wild type virus in MDCK cells and embryonated chicken eggs, but to cause no disease symptoms in mice, even at the highest dose tested. Furthermore, mice receiving nasal immunization with cold adapted DelNS1 2009 H1N1 virus are protected from lethal challenges with H1N1, H5N1 and H7N9 viruses. The DelNS1 2009 H1N1 virus appears to provide better protection than the currently available cold adapted live attenuated H1N1 virus vaccine strain in animal experiments, suggesting DelNS1 may confer an advantage augment the effectiveness of live attenuated flu vaccines , promoting the induction of stronger and broader immunity to influenza virus infection. Finally, we extended the scope of our DelNS1 system to construct a recombinant virus containing the RBD domain of MERS-CoV in the place of NS1, and found that it is able to protect against infection with MERS-CoV in the DPP4-transgenic mouse model. CONCLUSION: This study characterizes the substitutions required to support virus replication at lower temperature in the absence of NS1 protein in various influenza virus strains, and demonstrates that the combination of DelNS1 and cold adapted properties may have potential as a strategy to develop better and safer live attenuated influenza vaccines.