EVA71 is a medically important virus that is commonly associated with HFMD, resulting in periodic outbreaks, significant economic impact, and loss of life. Vaccination offers the potential to curtail future outbreaks, and vaccine development has been increasingly the focus of global research efforts.

Enterovirus A71 (EVA71) infection can result in paralysis and may be fatal. In common with other picornaviruses, empty capsids are produced alongside infectious virions during the viral lifecycle. These empty capsids are antigenically indistinguishable from infectious virus, but at moderate temperatures they are converted to an expanded conformation. In the closely related poliovirus, native and expanded antigenic forms of particle have different long-term protective efficacies when used as vaccines. The native form provides long-lived protective immunity, while expanded capsids fail to generate immunological protection. Whether this is true for EVA71 remains to be determined. Here, we selected an antigenically stable EVA71 virus population using successive rounds of heating and passage and characterized the antigenic conversion of both virions and empty capsids. The mutations identified within the heated passaged virus were dispersed across the capsid, including at key sites associated with particle expansion. The data presented here indicate that the mutant sequence may be a useful resource to address the importance of antigenic conformation in EVA71 vaccines.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation. We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilized virus-like particles (VLPs) in Pichia pastoris. The stabilized VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilization, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralize virus in vitro. Therefore, anti-EVA71 neutralizing antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.