Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate

Abstract

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.

Mechanism of enterovirus VP0 maturation cleavage based on the structure of a stabilised assembly intermediate

“…CryoEM grids were prepared, data collected and processed as previously described [32], with some small modifications. Briefly, 3 µl of concentrated sample was applied to ultrathin continuous carbon-coated lacey carbon 400-mesh copper grids (Agar Scientific, UK) following glow discharge in air (10 mA, 30 s).…”

An enterovirus A71 virus-like particle with replaced loops confers partial cross-protection in mice

“…Several studies have been conducted to develop VLPs-based multivalent or multiplex vaccines ( Lei et al., 2020 ; Wu et al., 2019 ; Xu et al., 2020 ; Zhao et al., 2015 ). Co-expression of EV P1 and 3CD genes in yeast or insect cells can form EV VLPs ( Kingston et al., 2023 ; Li et al., 2013 ). Using EV-A71 viral capsid as the backbone and replacing its VP1 G-H loop with CV-A16 VP1 G-H loop to construct a bivalent chimeric EV-A71 inactivated vaccine or VLPs vaccine could protect neonatal mice against lethal doses of EV-A71 and CV-A16 infection ( Yang et al., 2016 ; Zhao et al., 2015 ).…”