Cytokine and Antibody Responses in Gnotobiotic Pigs after Infection with Human Norovirus Genogroup II.4 (HS66 Strain)

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Abstract
A human norovirus genogroup II.4 strain HS66 (HuNoV-HS66) infects and causes mild diarrhea in gnotobiotic (Gn) pigs (S. Cheetham, M. Souza, T. Meulia, S. Grimes, M. G. Han, and L. J. Saif, J. Virol. 80:10372-10381, 2006). In this study we evaluated systemic and intestinal humoral and cellular immune responses to HuNoV-HS66 in orally inoculated pigs. Antibodies and type I interferon (IFN-I or IFN-α), proinflammatory interleukin-6 (IL-6), Th1 (IL-12 and IFN-γ), Th2 (IL-4), and Th2/regulatory T ([Treg] IL-10) cytokine profiles in serum and intestinal contents (IC) of the HuNoV-HS66-inoculated pigs and controls were assessed by enzyme-linked immunosorbent assay at selected postinoculation days (0 to 28). Using an enzyme-linked immunospot assay, we evaluated immunoglobulin M (IgM), IgA, and IgG antibody-secreting cells (ASC) and cytokine-secreting cells (CSC) in intestine, spleen, and blood. In the HuNoV-inoculated pigs, antibody titers in serum and IC were generally low, and 65% seroconverted. Pigs with higher diarrhea scores were more likely to seroconvert and developed higher intestinal IgA and IgG antibody titers. The numbers of IgA and IgG ASC were higher systemically than in the gut. In serum, HuNoV induced persistently higher Th1 (low transient IFN-γ and high IL-12) than the other cytokines, but also low Th2 (IL-4) and Th2/Treg(IL-10) levels; low, transient proinflammatory (IL-6) cytokines; and, notably, a delayed IFN-α response. In contrast, intestinal innate (IFN-α early and late) and Th1 (IL-12 late) cytokines were significantly elevated postinfection. HuNoV-HS66 also elicited higher numbers of Th1 (IL-12 and IFN-γ) CSC than Th2 (IL-4) and proinflammatory (IL-6) CSC, with the latter responses low in blood and intestine, reflecting low intestinal inflammation in the absence of gut lesions. These data provide insights into the kinetics of cytokine secretion in serum and IC of HuNoV-inoculated Gn pigs and new information on intestinal humoral and cellular immune responses to HuNoV that are difficult to assess in human volunteers.