EXPRESSION OF ENTEROVIRUS 71 VP1 ANTIGENS IN CHINESE HAMSTER OVARY AND BLASTOCYSTIS CELLS

Abstract

Enterovirus 71 (EV71), belonging to Picornaviridae family, is a major cause of Hand-Foot-and-Mouth Disease (HFMD) in young children with worldwide distribution. Large outbreaks of the disease with cases of neurological complications and fatalities have occurred throughout the Asia-Pacific region including Singapore. HFMD is considered as an important emerging public health threat particularly due to its total economic burden. Presently, there is no licensed vaccine to protect children against EV71 infection; only supportive treatment is provided. Although vaccine development studies against EV71 are reported, there is still need for further development of safe vaccines that can confer broad immunogenicity and protection against EV71 infection. The main immunogenic capsid protein of EV71, VP1, has been the focus of EV71 subunit vaccine development by many researchers. The aim of this project is to investigate the feasibility of VP1 vaccine antigen production in two novel expression systems, i.e. Chinese hamster ovary (CHO) cells and Blastocystis sp. This may lead to development of new vaccine candidates against EV71, namely CHO-derived VP1 as subunit vaccine for parenteral immunization, and Blastocystis-derived VP1 as live recombinant vaccine for oral immunization. A set of pcDNA3.1 mammalian expression vectors containing GFP tag and myc/His tag were constructed by cloning the VP1 antigen fused to CHO adiponectin and Blastocystis legumain signal peptides. VP1 expression in CHO cells was analyzed by Western blotting. GFP reporter expression in Blastocystis was assessed by confocal microscopy and flow cytometric analyses. In this project, His-tagged VP1 production in CHO cells was successfully achieved. However, further protein over-expression strategies including the creation of stable recombinant CHO cell lines and secretion engineering are needed to enhance and scale up the VP1 production yield. These strategies will facilitate the subsequent purification of VP1 by Immobilized Metal Affinity Chromatography (IMAC) for end-use CHO-derived VP1 vaccine. Although electroporation conditions were optimized empirically for Blastocystis cells to attain higher cell viability, multiple attempts to establish Blastocystis cells expressing the constructed mammalian-based GFP expression vectors resulted in relatively low GFP expression. Considering the numerous unknowns and drawbacks for establishment of heterologous gene expression in Blastocystis, further stepwise investigations are required to characterize an efficient transfection method with maximum DNA uptake efficiency and cell viability. Furthermore, bioinformatics analyses of Blastocystis promoters can facilitate design and construction of improved specialized expression vectors.