Molecular mechanisms underlying attenuation of live attenuated Japanese encephalitis virus vaccine SA14-14-2

ABSTRACT The live attenuated Japanese encephalitis virus vaccine SA14-14-2 demonstrated ≥ 95 % efficacy and is today the vaccine of choice against JEV globally. Relative to its parent strain SA14, SA14-14-2 carries 46 nucleotide and 24 amino acid alterations, with 8 of the latter located within the envelope glycoprotein. The vaccine strain also fails to synthesize the nonstructural protein NS1’ owing to a silent mutation that abrogates a-1-frameshifting event close to the 5’ end of the NS2A coding sequence. Previous studies employing reverse genetics and mouse models implicated both absence of NS1’ and mutated E, in attenuation of SA14-14-2. We demonstrate progressive reduction in ER stress sensor PERK levels and increased expression of CEBP-homologous protein (CHOP), accompanied by dephosphorylation of eIF2α, inhibition of autophagy maturation and necroptosis following infection of cultured cells with wild-type JEV strain P20778. Autonomous expression of NS1’ caused constitutive up-regulation of CHOP and loss of PERK. Conversely, infection with SA14-14-2 led to significantly increased IRE-1α activation, ER chaperone levels and autophagy. We report labile conformational epitopes accompanied by drastically reduced folding kinetics of intracellular SA14-14-2 envelope protein engendered by sluggish oxidation of cysteine sulfhydryl groups to form disulfide bonds within the endoplasmic reticulum along with altered envelope epitopes in extracellular SA14-14-2 viral particles. We also demonstrate near total conversion of prM to pr and M in SA14-14-2 virus particles. These alterations were accompanied by enhanced activation of mouse and human antigen presenting cells by SA14-14-2 along with superior CD8 + recall T cell responses to viral structural proteins in volunteers vaccinated with SA14-14-2. Author Summary The random process of cell culture passage adopted in generation of most live attenuated virus vaccines leads to fixation of multiple nucleotide changes in their genomes and renders it difficult if not impossible to pinpoint those mutations primarily responsible for their attenuated phenotype. Identifying the precise attenuating mutations and their modi operandi should aid in developing rationally attenuated vaccines for other viruses. We discovered that wild type (WT) JEV uses the nonstructural protein NS1’ to take over the host protein synthesis machinery to produce viral proteins. Loss of NS1’ in SA14-14-2 deprives the vaccine strain of this ability. Viruses uniformly target host death pathways to avoid generating potent antiviral immune responses. WT JEV prevents autophagy maturation. Conversely the SA14-14-2 vaccine activates autophagy due to unresolved ER stress caused by inability of its envelope glycoprotein to fold promptly post synthesis. Combined with enhanced proteolytic cleavage of the viral prM protein in SA14-14-2, this resulted in altered envelope epitopes on extracellular SA14-14-2 virus particles. These changes culminated in enhanced activation of innate and adaptive immune responses by SA14-14-2.