Enterovirus A71 (EV-A71) is one of the major etiological agents of hand, foot, and mouth disease, and its infection occasionally leads to fatal neurological complications in children. However, only inactivated whole virus vaccines against EV-A71 are commercially available in Mainland China. Furthermore, the mechanisms underlying the infectivity and pathogenesis of EV-A71 remain to be better understood. By adaptation of an EV-A71 B5 strain in monkey Vero cells in the presence of brilliant black BN (E151), an anti-EV-A71 agent, a double mutant with VP1-V238A,K244R emerged to be E151 enhanced infection. The growth of reverse genetics (RG) mutant RG/B5-VP1-V238A,K244R (RG/B5-AR) was promoted by E151 in Vero cells, but inhibited in other human and murine cells; while its parental wild type RG/B5-wt was strongly prevented by E151 from infection in all tested cells. In the absence of E151, RG/B5-AR exhibited defective cell entry/exit, resulting in reduced viral transmission and growth
in vitro
. It had augmented binding affinity to sulfated glycans, cells and tissue/organs, which probably functioned as decoys to restrict viral dissemination and infection. RG/B5-AR was also attenuated with 355 times higher 50% lethal dose and shorter timing of virus clearance than RG/B5-wt in suckling AG129 mice. It, however, remained highly immunogenic in adult AG129 mice to protect their suckling mice from lethal EV-A71 challenges through maternal neutralizing antibodies. Overall, discovery of the attenuated mutant RG/B5-AR contributes to better understanding of virulence determinants of EV-A71 and further development of novel vaccines against EV-A71.
Importance
Enterovirus A71 (EV-A71) is highly contagious in children and has been responsible for thousands of deaths in Asia-Pacific region since 1990s. Unfortunately, the virulence determinants and pathogenesis of EV-A71 are not fully clear. We discovered that a novel EV-A71 mutant VP1-V238A,K244R showed growth attenuation with reduced efficiency of cell entry/exit. In Vero cell line which has been approved for manufacturing EV-A71 vaccines, the growth defects of the mutant were compensated by a food dye brilliant black BN. The mutant was also with augmented binding affinity to sulfated glycans and other cellular components which probably restricted the viral infection and dissemination. Therefore, it was virulence attenuated in a mouse model but still retained its immunogenicity. Our findings suggest the mutant as a promising vaccine candidate against EV-A71 infection.
Neutralizing Antibodies of Inactivated Thai Enterovirus A71 Strain in Mice for Development of Enterovirus A71 Vaccine
