The role of the cell-mediated immune response to rotavirus infection
The objective. of this project was to determine the protein specificity of the cytotoxic T- lymphocyte (CTL) response to rotavirus infection in mice and to assess the rotavirus serotype/strain independent nature of this response. Previous work, involving the rotavirus group at Warwick, had shown that the outer shell glycoprotein VP7 is a major target antigen for a CTL response that is virus serotype-independent. However, that work did not cover all twelve rotavirus proteins, was confined to one strain of adult mice (C57BL/6, H- 2b) and covered only two of the fourteen VP7 serotypes (serotypes 3 and 6) (Offit et al., 1994). Recombinant vaccinia viruses expressing individual rotavirus UKtc proteins VP2, VP3, NS26 and NS12 were constructed to complete a set of recombinant vaccinia viruses covering the full complement of rotavirus proteins from the bovine UKtc strain. These were used to define rotavirus proteins eliciting a CTL response in three different mouse haplotypes. UKtc NS53 and UKtc VP7 stimulated a strong CTL response only in the H-2b MHC class I haplotype (UKtc NS53 and UKtc VP7 were restricted at H-2Db and H-2K b, respectively). Conversely, UKtc VP3 stimulated a strong CTL response in the H-2d and H-2k (but not in H-2b) MHC class I haplotypes. Work using congenic mouse strains was used to verify that the VP7 protein specific CTL response is restricted solely by the MHC class I antigens. Rotavirus RRV NS53 was not only found to elicit a CTL response in the H-2b MHC class I haplotype, similar to UKtc NS53, but also in the H-2d MHC class I haplotype. Thus, the individual rotavirus protein that elicits a CTL response not only depends on the MHC class I haplotype, but also on the actual rotavirus strain being tested. Many of the previous studies looking at the CTL response-to individual rotavirus proteins have, unlike this study, used several different rotavirus strains and, therefore, may have given an inaccurate representation of the rotavirus proteins that elicit a CTL response. Recombinant vaccinia viruses were also used to examine the serotype/strain independent nature of the CTL response against the major target antigens. The analysis was extended to cover VP3 from two different strains, NS53 from three different strains and VP7 from seven of the fourteen serotypes. VP3 and NS53 were found to elicit a strain- dependent response whereas the serotype-independent nature of the CTL response to VP7 was confirmed. Since the serotype-independent nature of the rotavirus VP7-specific CTL response was found to cross-protect between half of the VP7 serotypes, irrespective of the immunising. serotype, it would be reasonable to speculate that the CTL response is serotype-independent between all the VP7 serotypes. Finally, recombinant vaccinia viruses were used to locate CTL epitopes on NS53 and VP7. Recombinant vaccinia virus expressing a UKtc NS53 deletant mutant (P9DM5) showed there to be at least one strain specific epitope in the first 150 amino acids of the UKtc NS53 protein. Recombinant vaccinia viruses expressing four different UKtc VP7 fragments spanning 46% of this protein were examined. It was found that the fragment spanning the restriction enzyme sites at nucleotide 90 (CIaI) and nucleotide 196 (Hhal), i. e. between amino acids 13 and 48 of the mature UKtc VP7 protein, contained a serotype- dependent CTL epitope. The finding suggests that the immunodominant epitope identified in the same region of VP7 by Franco et al. (1993) was not the serotype-independent epitope.