Live-attenuated virus vaccines are highly effective in preventing viral disease but carry intrinsic risks of residual virulence and reversion to pathogenicity. The classically derived Candid#1 virus protects seasonal field workers in Argentina against zoonotic infection by Junín virus (JUNV) but is not approved in the United States, in part due to the potential for reversion at the attenuating locus, a phenylalanine-to-isoleucine substitution at position 427 in the GP2 subunit of the GPC envelope glycoprotein. Previously, we demonstrated facile reversion of recombinant Candid#1 (rCan) in cell culture and identified an epistatic interaction between the attenuating I427 and a secondary K33S mutation in the stable signal peptide (SSP) subunit of GPC that imposes an evolutionary barrier to reversion. The magnitude of this genetic barrier is manifest in our repeated failures to rescue the hypothetical revertant virus. In this study, we show that K33S rCan is safe and attenuated in guinea pigs and capable of eliciting potent virus-neutralizing antibodies. Immunized animals are fully protected against lethal challenge with virulent JUNV. In addition, we employed a more permissive model of infection in neonatal mice to investigate genetic reversion. RNA sequence analysis of the recovered virus identified revertant viruses in pups inoculated with the parental rCan virus and none in mice receiving K33S rCan (P < 0.0001). Taken together, our findings support the further development of K33S rCan as a safe second-generation JUNV vaccine. IMPORTANCE Our most successful vaccines comprise weakened strains of virus that initiate a limited and benign infection in immunized persons. The live-attenuated Candid#1 strain of Junín virus (JUNV) was developed to protect field workers in Argentina from rodent-borne hemorrhagic fever but is not licensed in the United States, in part due to the likelihood of genetic reversion to virulence. A single-amino-acid change in the GPC envelope glycoprotein of the virus is responsible for attenuation, and a single nucleotide change may regenerate the pathogenic virus. Here, we take advantage of a unique genetic interaction between GPC subunits to design a mutant Candid#1 virus that establishes an evolutionary barrier to reversion. The mutant virus (K33S rCan) is fully attenuated and protects immunized guinea pigs against lethal JUNV infection. We find no instances of reversion in mice inoculated with K33S rCan. This work supports the further development of K33S rCan as a second-generation JUNV vaccine.
Keywords: Junín virus; arenavirus; attenuation; evolution; live-attenuated vaccine; reversion.