Mucosal immunization with a ricin toxin B subunit-rotavirus NSP4 fusion protein stimulates a Th1 lymphocyte response

The castor-oil plant Ricinus communis A-B dimeric toxin B subunit (RTB) was genetically linked at its N-terminus with a 90 amino acid peptide from simian rotavirus SA-11 non-structural protein NSP4(90) and produced in Escherichia coli BL21 cells. Biologically active recombinant NSP4(90)-RTB fusion protein was shown to bind glycoprotein asialofetuin receptor molecules in an in vitro enzyme-linked immunosorbent assay (ELISA). Oral inoculation of the purified NSP4(90)-RTB ligand-antigen fusion protein delivered the chimeric protein to intestinal epidermal cells for mucosal immunization against rotavirus infection. Mice fed the NSP4(90)-RTB fusion protein generated higher humoral and intestinal antibody titers than mice inoculated with NSP4(90) alone. Titers of serum IgG2a antibodies were significantly higher than IgG1 titers suggesting a dominant Th1 lymphocyte immune response. ELISA measurement of cytokines secreted from splenocyte isolated from immunized mice confirmed NSP4(90)-RTB fusion protein stimulates a strong Th1 cell-mediated immune response. The experimental results demonstrate that the ricin toxin B subunit N-terminus can be used as a site for delivery of virus antigens to the gut associated lymphoid tissues for RTB-mediated immune stimulation of antiviral mucosal immune responses.