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

Abstract

The only licensed live attenuated influenza A virus 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 types 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, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were 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. IMPORTANCEThe live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. 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 into 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 that novel strategies to continue to improve LAIV vaccine safety should be investigated.A nnual influenza epidemics are caused by influenza A (H1N1 and H3N2) and influenza B viruses that infect 5% to 10% of adults and 20% to 30% of children (1). Disease outcomes range from subclinical infections to severe infections that result in very high mortality. The estimated 3 to 5 million cases of severe illness and 250,000 to 500,000 deaths each year, as well as significant socioeconomic losses, warrant continuous improvements in influenza preventi...

Increasing the Safety Profile of the Master Donor Live Attenuated Influenza Vaccine

“…The current MDV A/AA/6/60 LAIV has mutations mainly located in areas of the polymerase complex. There is always a concern that, with enough pressure, the MDV A/AA/6/60 LAIV could introduce mutations to compensate and revert back to a pathogenic strain [47]. By introducing mutations into additional viral genes (PA and NS1), the probability of reversion to a more virulent strain is decreased even further, adding to the increased safety profile desired for an MDV LAIV.…”

MiRNA Targeted NP Genome of Live Attenuated Influenza Vaccines Provide Cross-Protection against a Lethal Influenza Virus Infection

“…A large number of studies have shown that LAIV can induce a cross-protective response against both homogenous and heterologous viruses challenge [7,35]. TS IAV is safe and effective, and can induce humoral, mucosal and cellular immunity which can protect humans from influenza virus infection [23,36,37]. The results in our study also found that 192t-6, 192t-9 and TS IAV could protect mice from PR8 homologous virus infection and reduce the replication of influenza virus in mice, and 192t-6 and 192t-9 had similar protective effects on mice as TS IAV.…”

“…We next determined the virulence of miR-192 targeted viruses in vivo. Meanwhile, a temperature-sensitive (TS IAV) constructed from PR8 virus was used as a positive control [23]. Mice were i.n.…”

MicroRNA-Based Attenuation of Influenza Virus across Susceptible Hosts

“…LAIV platforms offer improved immunogenicity through both attenuated replication and mucosal delivery, which more closely resembles natural infection. However, the potential for LAIVs to resort to virulence has raised safety concerns (38). This is particularly concerning within livestock where, due to the potential for reassortment and creation of novel strains, only killed vaccines are recommended (9).…”