Antibody-secreting cell responses to rotavirus proteins in gnotobiotic pigs inoculated with attenuated or virulent human rotavirus

Because of their similarities to infants in mucosal immune responses and th eir susceptibility to human rotavirus (HRV) diarrhea, gnotobiotic pigs prov ide a useful model for rotaviral disease. In this study, we performed quant itative enzyme-linked immunospot (ELISPOT) assays to measure local and syst emic isotype-specific antibody-secreting cell (ASC) responses to individual structural (VP4, VP6, and VP7) and nonstructural (NSP3 and NSP4) proteins of Wa HRV. The Spodoptera frugiperda cells expressing each recombinant bacu lovirus HRV protein were formalin fixed and used as antigen for ELISPOT ass ays. Neonatal gnotobiotic pigs were orally inoculated once with virulent Wa (WaV) or three times with attenuated Wa (WaA) HRV or mock inoculated (Mock ) and then were challenged with virulent Wa (WaV/PC) 28 days after the firs t inoculation. The ASCs from intestinal and systemic lymphoid tissues of pi gs from each group were quantitated by ELISPOT assay at the day of challeng e, at postinoculation day 28 (WaV, WaA, and Mock) or at postchallenge day ( PCD) 7 (WaV+WaV/PC, WaA+WaV/PC, and Mock+WaV/PC). In all virus-inoculated p igs, regardless of the inoculum, lymphoid tissue, or isotype, VP6 induced t he highest numbers of ASCs, followed by VP4; ASCs specific for VP7, NSP3, a nd NSP4 were less numerous. At challenge, total HRV- and HRV protein-specif ic immunoglobulin A (IgA) and IgG ASCs in intestinal lymphoid tissues were significantly greater in WaV- than in WaA-inoculated pigs, and WaV pigs wer e fully protected against diarrhea postchallenge, whereas the WaA pigs were partially protected. At PCD 7, there were no significant differences in AS C numbers for any HRV proteins between the WaV+WaV/PC and WaA+WaV/PC groups .