Expression of polyprotein and 3D polymerase protein in Sf9 cells and immunogenicity against enterovirus A71B5 (Thailand strain)

Abstract

Enterovirus 71 B5 subgenotype (EV-A71B5) is a primary human pathogen of hand, foot, and mouth disease. In this study, we aimed to prepare a novel recombinant protein polyprotein (P1) and 3D polymerase (3Dpol) protein of EV-A71B5 and determine mice immunogenicity and neutralization activity against EV-A71 of the B5 subgenotype commonly found in Thailand. Using a dual promoter system and investigating its expression in Sf9 cells, we constructed a novel recombinant protein containing P1 and 3Dpol protein. The purified P1-3Dpol was observed by western blotting and transmission electron microscopy (TEM) to determine the particle size. Furthermore, we determined the immunogenicity and neutralization activity against EV-A71 of the B5 subgenotype using concatenation of Bagg and Albino (BALB/c) mice. The results revealed that P1-3Dpol was expressed in Sf9 cells. We used TEM to visualize the particle size of P1-3Dpol to be approximately 33 nm. P1-3Dpol had the potential to elicit the production of immunoglobulin G and immunoglobulin M antibodies and the T helper type 1-dominant cytokine interferon-γ after immunizing BALB/c mice and inducing neutralization antibodies against EV-A71B5. Our results demonstrated that Sf9 cells successfully produced P1-3Dpol, which can leverage immune efficacy in BALB/c mice and be used to develop vaccines against the EV-A71B5 strain prevalent in Thailand.