Molecular and immunobiological characterization of VP8*, N-terminal trypsin cleavage product of bovine rotavirus VP4

by Lee, Joong Bok

It is the objective of this thesis to contribute to the understanding of the molecular pathogenesis of rotavirus infection and the development of a subunit vaccine for the control of rotavirus infection. Rotavirus, a member of the family Reoviridae, is a major etiological agent of diarrhea in animals. It has been shown that VP8*, the N-terminal trypsin cleavage product of structural protein VP4 is responsible for virus binding to MA-104 cells and human type 0 erythrocytes. The VP8* gene was cloned and expressed in bacteria. This protein (rVP8* 1 was immunoprecipitated with antiserum to bovine rotavirus C486 (BRV). To study the interaction between rVP8* and cells, binding and inhibition assays were performed. The rVP8.* bound to MA-104 cells and the viral protein binding was competitively inhibited by BRV. Permissive cell binding was not disrupted by neuraminidase treatment of the MA-104 cell monolayers, which suggested that the binding of BRV to MA-104 cell is sialic acid independent. To study the interaction between VP8* and erythrocytes, rVP8* was solubilized and hemagglutination (HA) and hemagglutination inhibition (HI) assays were performed. The rVPB* protein agglutinated erythrocytes and its HA was inhibited by antiserum to BRV. Molecular and biochemical characterization of the HA demonstrated that rVP8* formed dimers, and its binding to erythrocytes required an a2-8 linkage between sialic acids and was independent of acetylation of sialic acid residues. To map the neutralizing epitopes on VP8*, we developed a panel of monoclonal antibodies inhibiting hemagglutination of VP8* protein. One of these antibodies (2E8) was used to generate a mutant virus resistant to neutralization by the monoclonal antibody. The antibody escape mutant was found to contain one point mutation at amino acid position 116 (Glu -> Asp). To investigate the effect of this escape mutation on the cellular binding and hemagglutination activities, the VP8* gene of the escape mutant was expressed in bacteria, and its functional activities were compared with those of the parental strains. The escape mutant protein showed HA and binding to nucleated cells. The HA of the parental protein was inhibited by 2E8, whereas the HA of the. escape mutant was not. This data indicated that the epitope recognized by neutralizing monoclonal antibody 2E8 is located in the HA domain, and the 2E8 epitope is not involved in rotavirus attachment to nucleated cells. To understand the mechanism of virus neutralization by 2E8, radiolabeled BRV was incubated with 2EB and the complex purified by centrifugation through a CsCl gradient. The distribution of radioactivity was analysed, and the peak fractions were observed by electron microscopy. The radioactivity profile of the virus was altered, and its structural integrity destroyed by 2E8. iii The virus was not affected by non-neutralizing monoclonal antibodies when compared to the radioactivity profile by BRV alone. This result suggests that 2E8 binds to BRV VPS*, affecting the stability of the particle, and may thereby neutralize virus infectivity. To examine if this antigen could induce neutralizing antibody responses in vivo, different species of animals were immunized with rVP8*. The rVP8* antigen produced a neutralizing immune response in rabbits. The rVPB* antigen also was shown to prime the murine immune system, and to induce an immune response. A field trial in cattle showed a statistically significant difference in humoral immunity between rVP8+-vaccinated and unvaccinated groups of cows. The milk from the vaccinated group was found to contain sufficient antibodies to protect calves for the first ten days of their lives.