Expression of rotavirus proteins and virus-like particles in bacterial and insect cells

Abstract

Rotavirus infection remains a leading cause of severe gastroenteritis affecting infants and young children. Currently, only live-attenuated (LA) vaccines are licensed and globally administered. These vaccines are not as effective in low- and middle-income countries such as those in Sub-Saharan Africa and Asia compared to high-income countries. This immunity gap could be bridged by using non-replicatingsubunits vaccines such as virus-like particles (VLPs). In this study, proteins VP2, VP6, and VP7 from two rotavirus strains, the South African RVA/Human-wt/ZAF/GR10924/1999/G9P[6] strain and SA11 RVA/Simian-tc/ZAF/SA11-H96/1958/G3P5B[2] strain, were expressed in bacteria and insect cells. We attempted to reduce the toxicity associated with VP7 expression in bacteria by removing the endoplasmic reticulum leader peptide (amino acids 1-50). Although the bacteria expressed a soluble protein, a deletion of 159 bp (amino acids 121-174) occurred in the middle of the VP7 open reading frame (ORF). VP2 and VP6 was expressed in bacteria but the proteins were not soluble. The ORFs of SA11 VP2/6/4 and VP7 were insect-cell codon-optimised. The ORFs of VP2 and VP6 were cloned into the pFBdual donor plasmid for double-layered particle (DLP) production. The ORFs of SA11 VP4 and VP7 were cloned in separate pFBdual donor plasmids for flexible transcapsidation. The Bac-to-Bac expression system was used for the generation of recombinant baculoviruses. Overexpression of VP2 could be observed with Coomassie-stained SDS-PAGE and VP6 expression through western blot. VP2/6 expression was confirmed through a novel immunofluorescent monolayer assay (IFMA) protocol and SDS-PAGE. DLP assembly was confirmed with transmission electron microscopy (TEM). VP4 expression was confirmed through PAGE. However, no VP7 expression could be detected on PAGE. On TEM, no triple-layers particles (TLPs) were detected even with the addition of 20 mM calcium supplemented in the media and buffers.