Human Enterovirus 71 (EV71), an emerging neurotropic virus is recognized as the main causative agent of hand, foot, and mouth disease (HFMD). Due to large number of outbreaks and increasing incidence of fatalities, EV71 infections have become a growing public health concern. However, to date, no clinically proven effective antiviral drugs or vaccines are available for prevention or treatment of the disease.
In this study, the aim was to investigate the potential of developing a yeast-based vaccine by producing virus like particles (VLP) of EV71 in Kluyveromyces lactis due to its numerous advantages such as non-toxicity to human and ability to grow to very high cell densities. The project was designed in view of producing VLP by the co-expression of (a) P1 and 3CD genes, and (b) VP1, VP0 and VP3 capsid proteins. Each gene was cloned into multiple cloning site of the pKLAC1 vector. The construction of a yeast strain for stable VLP expression was approached by integrating the expression cassette of pKLAC1 vector into the host genome via homologous recombination. Gene cassettes were then co-transformed and K. lactis transformants comprising of strains bearing multicopy tandem insertions were selected on media supplemented with acetamide. As acetamide is known to enhance the yield of K. lactis transformants bearing multicopy tandem insertions, this approach was exploited to construct expression strains with multiple heterologous genes.
The expression strains were successfully constructed and the presence of the genes encoding the VLP proteins confirmed by PCR. The selected constructs were then subjected to protein expression in YCB media with galactose. Total soluble proteins of the host and purified proteins of interest were analysed by SDS-PAGE and immunoassays, and preliminary results evidenced expression of the subunit proteins. Further analysis and optimization will provide valuable information on the potential of this strategy to produce vaccine candidates.