Functional mapping of protective domains and epitopes in the rotavirus VP6 protein

The purpose of this study was to determine which regions of the VP6 protein of the murine rotavirus strain EDIM are able to elicit protection against rotavirus shedding in the adult mouse model following intranasal (i.n.) immunization with fragments of VP6 and a subsequent oral EDIM challenge. In the initial experiment, the first (fragment AB), middle (BC), or last (CD) part of VP6 that was genetically fused to maltose-binding protein (MBP) and expressed in Escherichia coli was examined. Mice (BALB/c) immunized with two 9-mug doses of each of the chimeras and 10 mug of the mucosal adjuvant LT(R192G) were found to be protected against EDIM shedding (80, 92, and nearly 100% reduction, respectively; P less than or equal to 0.01) following challenge. Because CD produced almost complete protection, we prepared four E. coli-expressed, MBP-fused chimeras containing overlapping fragments of the CD region (i.e., CD1, CD2, CD3, and CD4) whose lengths ranged from 61 to 67 amino acid residues. Following i.n. immunization, CD1, CD2, and CD4 induced significant (P less than or equal to 0.004) protection (88, 84, and 92% reduction, respectively). In addition, 11 peptides (18 to 30 residues) of the CD region with between 0 and 13 overlapping amino acids were synthesized. Two 50-mug doses of each peptide with LT(R192G) were administered i.n. to BALB/c mice. Five peptides were found to elicit significant (P less than or equal to 0.02) protection. Moreover, a 14-amino-acid region within peptide 6 containing a putative CD4(+) T-cell epitope was found to confer nearly complete protection, suggesting a protective role for CD4(+) T cells. Mice that were protected by fragments BC and CD1 and four of the five protective synthetic peptides did not develop measurable rotavirus antibodies in serum or stool, implying that protection induced by these domains was not dependent on antibody. Together, these observations suggest that multiple regions of VP6 can stimulate protection, a region of VP6 as small as 14 amino acids containing a CD4(+) T-cell epitope can stimulate nearly complete protection, and protection mediated by a subset of epitopes in the VP6 protein does not require antibodies in BALB/c mice.