Abstract
Enterovirus 71 (EV71) is one of the 69 enterovirus serotypes within the picornavirus family and has identified as the major etiological agent responsible for the outbreaks of severe neurological diseases in the past decades. The study of virus-like particles (VLPs) could provide further insight into the virus structure and virus assembly pathway that are crucial for antiviral drug design and could serve as a potential vaccine. We have previously cloned the genes encoding P1 polyprotein and 3CD protease into two separate baculoviruses (Bac-P1 and Bac-3CD) and demonstrated that the co-expression of P1 and 3CD in insect cells resulted in the cleavage of P1 by 3CD into individual structural proteins (VP1, VP3 and VP0) and subsequently formation of EV71 VLP. In this study, we further cloned the two genes into one baculovirus (vBac-P1-3CD) and expressed P1 and 3CD proteins by single-infection. The formation and aggregation of VLP within the infected insect cells were visualized by ultrathin-sectioning and transmission electron microscopy (TEM). These particles measured 25-27 nm in diameter and appeared icosahedral, thus resembling the authentic enterovirus in size and appearance. The VLPs were further purified by sucrose and CsCl gradient ultracentrifugation, and the purity of the sample was greater than 90%. The purified VLPs comprised VP0, VP3 and VP1 of roughly equal molar ratios, and implied the similarity between the VLP and the authentic virus in composition. Nonetheless, after purification the size and morphology of VLPs appeared indistinguishable from those of poliovirus VLP. More importantly, the immunogold labeling confirmed the presence of VP1 epitopes on the VLP surface. After characterizing the properties of VLP, we further evaluated the potential of VLP as a vaccine by comparing the humoral and cellular immune responses elicited by the purified VLP, denatured VLP and heat-inactivated EV71 virus. After immunization of BALB/c mice, EV71 VLP induced potent and long-lasting humoral immune responses as evidenced by the high total IgG titer and neutralization titer. The splenocytes collected from the VLP-immunized mice exhibited significant cell proliferation and produced high levels of IFN-γ, IL-2 and IL-4 after stimulation, indicating the induction of Th1 and Th2 immune responses by VLP immunization. More importantly, the VLP immunization of mother mice conferred protection (survival rate up to 89%) to neonatal mice against the lethal (1000 LD50) viral challenge. Compared with the VLP immunization, immunization with denatured VLP and heat-inactivated EV71 elicited lower neutralization titers and conferred less effective protection to newborn mice, although they induced comparable levels of total IgG and cellular immune responses. These data collectively indicate the importance of the preservation of VLP structure and implicate the potential of VLP as a vaccine to prevent EV71 infection.