This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Rotaviruses are the most common etiological agents of diarrhea in infants and young children worldwide. There are no commercial vaccines available at present. Since we recently established non-human primate model of rotavirus infection, we are currently working with objective to identify cytotoxic (CTL) and T helper (TH) epitopes in rotavirus intermediate layer protein (VP6) major surface immunogen. Our study is carried out with a broader objective to utilize identified epitopes in future studies with novel vaccine candidates. Based on studies published by several groups with murine models of rotavirus infection and human studies, we hypothesize that rotavirus VP6 contains CTL and TH epitopes that can be utilized for development of novel diagnostic and prophylactic tools. Recently published DNA sequence of our rhesus rotavirus isolate (TUCH) VP6 protein was used to synthesize a set of overlapping peptides. To identify the CTL and TH epitopes, peptide pools and/or individual peptides are being used to stimulate in vitro the T cells obtained from serologically pre-selected rotavirus convalescent macaques to measure a) production of IFN-g by CD3+CD8+ T cells and b) expression of Ki-67 lymphocyte proliferation marker by CD3+CD4+ T cells. Epitope mapping is carried out in the context of rhesus MHC class I molecule (Mamu-A*01+ allele) with the objective to utilize the identified CTL epitopes for preparation of tetrameric complexes for quantitation of rotavirus-specific CTLs by flow cytometry. So far, we identified 1-2 oligomers in TUCH VP6 C-terminus as T cell epitope candidates.
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