Functional mapping of protective epitopes within the rotavirus VP6 protein in mice belonging to different haplotypes.

We recently used "functional mapping" to locate protective epitopes in the carboxyl terminus (aa 197-397) of the VP6 protein (designated CD) of the EDIM strain of murine rotavirus [J. Virol. 74 (2000) 11574]. For this, H-2(d) BALB/c mice were given two intranasal (i.n.) immunizations (separated by 2 weeks) with VP6 or CD genetically-fused to maltose-binding protein, or with overlapping synthetic CD peptides, along with LT(R192G), a genetically-attenuated E. coli heat-labile toxin. The protective efficacies, i.e., percentage reductions in rotavirus shedding relative to control mice during 7 days following oral challenge with EDIM, were determined 4 weeks after the second immunization. Five of the 11 overlapping CD peptides stimulated significant protection (57-85%, P<0.05). Furthermore, chimeric VP6, the CD fragment, and a 14-amino-acid VP6 peptide within CD (RLSFQLMRPPNMTP), identified as a H-2(d)-restricted CD4 T cell epitope, were highly protective (93-98%, P<0.05). In this study, we continued to utilize functional mapping to show that the 14-mer peptide elicited significant protection (97.0%, P<0.05) in another H-2(d) mouse strain (DBA/2) but partial protection in H-2(b) 129 (39.2%) and C57Bl/6 (53.6%) as well as H-2(k) C3H (44.6%) mice. The first 13 amino acids of this 14-mer were necessary to induce maximal protection in H-2(d) mice. In addition, the H-2(b) 129 mice were immunized intranasally (i.n.) with 10 of the synthetic CD peptides and 5 were found to induce significant protection (90-97%, P<0.05). We also performed functional mapping to identify MHC class I epitopes in rotavirus proteins. A class I-binding epitope for H-2(b) C57Bl/6 mice had previously been mapped by ex vivo CTL assays within the VP6 protein and two additional class I epitopes were identified by computer-based prediction. When examined for their protective efficacies by functional mapping, two of the three were found to be partially but not significantly protective (44 and 46%, P>0.05). To better determine the usefulness of our in vivo methods to identify MHC class I-binding epitopes, four epitopes from the outer capsid VP7 rotavirus protein determined in ex vivo assays were evaluated for their protective efficacies and two were found to be partially protective. Together, these studies show that functional mapping is useful in locating epitopes that are relevant to the development of subunit rotavirus vaccines.