Ricin Toxin B Subunit Enhancement of Rotavirus NSP4 Immunogenicity in Mice

Abstract

A 90–amino acid peptide from the simian rotavirus SA-11 nonstructural protein, NSP4 was linked to the N-terminus of the Ricinus communis A-B toxin B subunit protein (RTB) and synthesized inEscherichia coli. Recombinant RTB and the NSP4₉₀::RTB fusion protein bound artificial receptor glycoprotein asialofetuin in an in vitro enzyme-linked immunosorbent assay (ELISA), demonstrating biological activity of the recombinant protein. Mice co-inoculated with purified recombinant RTB plus NSP4₉₀ peptide proteins or heat denatured NSP4₉₀::RTB fusion protein generated higher titers of serum anti-NSP4₉₀ IgG antibodies than mice immunized with NSP4₉₀ peptide alone, indicating the presence of adjuvant functions for N-terminal linked RTB. Serum anti-NSP4₉₀ IgG titers were highest in mice immunized with native recombinant NSP4₉₀::RTB fusion protein, confirming the immunostimulatory function of RTB. Results of experiments described here demonstrate the feasibility of using RTB mediated adjuvant functions for stimulation of the antigenicity of a rotavirus nonstructural protein. The ability of recombinant NSP4₉₀::RTB fusion protein synthesized in E. coli to bind glycoprotein receptor molecules effectively indicates that protein linkage to the RTB N-terminus and synthesis of the recombinant NSP4₉₀::RTB fusion protein in bacteria do not interfere with the immunostimulatory properties of the RTB subunit.