Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus

Bin Zhou, Victoria A. Meliopoulos, Wei Wang, Xudong Lin, Karla M. Stucker, Rebecca A. Halpin, Timothy B. Stockwell, Stacey Schultz-Cherry, David E. Wentworth

The only licensed live attenuated influenza A vaccines (LAIVs) in the United States (FluMist®) are created using internal protein coding gene segments from the cold-adapted temperature sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature sensitive phenotype can revert, and if so, the type of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations including PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the role of individual mutations was characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild type 2009 H1N1 pandemic virus. The live attenuated influenza vaccine FluMist® has been proven safe and effective and are widely used in the USA. The phenotype and genotype of the vaccine virus are believed to be very stable and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights on the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and novel strategies to continue to improve LAIV vaccine safety should be investigated.