Gromowski Gregory D, Firestone Cai-Yen, Whitehead Stephen S
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
J Virol. 2015 Jun;89(12):6328-37. doi: 10.1128/JVI.00219-15. Epub 2015 Apr 8.
The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vaccine immunogenicity. Here, we generated recombinant JEV (rJEV) strains containing JEV SA14-14-2 vaccine-specific mutations that are located in the untranslated regions (UTRs) and seven protein genes or are introduced from PCR-amplified regions of the JEV SA14-14-2 genome. The resulting mutant viruses were evaluated in tissue culture and in mice. The authentic JEV SA14-14-2 (E) protein, with amino acid substitutions L107F, E138K, I176V, T177A, E244G, Q264H, K279M, A315V, S366A, and K439R relative to the wild-type rJEV clone, was essential and sufficient for complete attenuation of neurovirulence. Individually, the nucleotide substitution T39A in the 5' UTR (5'-UTR-T39A), the capsid (C) protein amino acid substitution L66S (C-L66S), and the complete NS1/2A genome region containing 10 mutations each significantly reduced virus neuroinvasion but not neurovirulence. The levels of peripheral virulence attenuation imposed by the 5'-UTR-T39A and C-L66S mutations, individually, were somewhat mitigated in combination with other vaccine strain-specific mutations, which might be compensatory, and together did not affect immunogenicity. However, a marked reduction in immunogenicity was observed with the addition of the NS1/2A and NS5 vaccine virus genome regions. These results suggest that a second-generation recombinant vaccine can be rationally engineered to maximize levels of immunogenicity without compromising safety.
The live-attenuated JEV SA14-14-2 vaccine has been vital for controlling the incidence of disease caused by JEV, particularly in rural areas of Asia where it is endemic. The vaccine was developed >25 years ago by passaging wild-type JEV strain SA14 in tissue cultures and rodents, with intermittent tissue culture plaque purifications, to produce a virus clone that had adequate levels of attenuation and immunogenicity. The vaccine and parent virus sequences were later compared, and mutations were identified throughout the vaccine virus genome, but their contributions to attenuation were never fully elucidated. Here, using reverse genetics, we comprehensively defined the impact of JEV SA14-14-2 mutations on attenuation of virulence and immunogenicity in mice. These results are relevant for quality control of new lots of the current live-attenuated vaccine and provide insight for the rational design of second-generation, live-attenuated, recombinant JEV vaccine candidates.
减毒活日本脑炎病毒（JEV）SA14 - 14 - 2疫苗的安全性和有效性归因于其衍生过程中病毒基因组积累的突变。然而，对于这些突变中大多数对毒力减弱和疫苗免疫原性的贡献知之甚少。在此，我们构建了重组JEV（rJEV）毒株，其包含位于非翻译区（UTR）和七个蛋白基因中的JEV SA14 - 14 - 2疫苗特异性突变，或从JEV SA14 - 14 - 2基因组的PCR扩增区域引入的突变。对产生的突变病毒进行了组织培养和小鼠实验评估。相对于野生型rJEV克隆，具有氨基酸替换L107F、E138K、I176V、T177A、E244G、Q264H、K279M、A315V、S366A和K439R的真实JEV SA14 - 14 - 2（E）蛋白对于完全减弱神经毒力是必需且充分的。单独来看，5'UTR中的核苷酸替换T39A（5'-UTR-T39A）、衣壳（C）蛋白氨基酸替换L66S（C-L66S）以及包含10个突变的完整NS1/2A基因组区域均显著降低了病毒的神经侵袭能力，但未降低神经毒力。5'-UTR-T39A和C-L66S突变单独施加的外周毒力减弱水平，与其他疫苗株特异性突变组合时会有所减轻，这可能具有补偿作用，且共同作用时不影响免疫原性。然而，添加NS1/2A和NS5疫苗病毒基因组区域后，免疫原性显著降低。这些结果表明，可以合理设计第二代重组疫苗，在不影响安全性的前提下最大化免疫原性水平。
减毒活JEV SA14 - 14 - 2疫苗对于控制由JEV引起的疾病发病率至关重要，特别是在亚洲流行该病的农村地区。该疫苗于25多年前通过在组织培养物和啮齿动物中传代野生型JEV毒株SA14，并进行间歇性组织培养蚀斑纯化而研发出来，以产生具有足够减毒水平和免疫原性的病毒克隆。后来对疫苗和亲本病毒序列进行了比较，并在整个疫苗病毒基因组中鉴定出了突变，但其对减毒的贡献从未得到充分阐明。在此，我们利用反向遗传学全面定义了JEV SA14 - 14 - 2突变对小鼠毒力减弱和免疫原性的影响。这些结果与当前减毒活疫苗新批次的质量控制相关，并为第二代减毒活重组JEV候选疫苗的合理设计提供了见解。
