Association of Gamma Interferon and Interleukin-17 Production in Intestinal CD4⁺T Cells with Protection against Rotavirus Shedding in Mice Intranasally Immunized with VP6 and the Adjuvant LT(R192G)

Abstract

Mucosal immunization of mice with chimeric, Escherichia coli-expressed VP6, the protein that comprises the intermediate capsid layer of the rotavirus particle, together with attenuated E. coli heat-labile toxin LT(R192G) as an adjuvant, reduces fecal shedding of rotavirus antigen by >95% after murine rotavirus challenge, and the only lymphocytes required for protection are CD4 ؉ T cells. Because these cells produce cytokines with antiviral properties, the cytokines whose expression is upregulated in intestinal memory CD4؉ T cells immediately after rotavirus challenge of VP6/LT(R192G)-immunized mice may be directly or indirectly responsible for the rapid suppression of rotavirus shedding. This study was designed to identify which cytokines are significantly upregulated in intestinal effector sites and secondary lymphoid tissues of intranasally immunized BALB/c mice after challenge with murine rotavirus strain EDIM. Initially, this was done by using microarray analysis to quantify mRNAs for 96 murine common cytokines. With this procedure, the synthesis of mRNAs for gamma interferon (IFN-␥) and interleukin-17 (IL-17) was found to be temporarily upregulated in intestinal lymphoid cells of VP6/LT(R192G)-immunized mice at 12 h after rotavirus challenge. These cytokines were also produced in CD4؉ T cells obtained from intestinal sites specific to VP6/LT(R192G)-immunized mice after in vitro exposure to VP6 as determined by intracellular cytokine staining and secretion of cytokines. Although genetically modified mice that lack receptors for either IFN-␥ or IL-17 remained protected after immunization, these results provide suggestive evidence that these cytokines may play direct or indirect roles in protection against rotavirus after mucosal immunization of mice with VP6/LT(R192G).

Virus-specific intestinal IFN-γ producing T cell responses induced by human rotavirus infection and vaccines are correlated with protection against rotavirus diarrhea in gnotobiotic pigs

“…Our study demonstrated a direct correlation between the existence of extralymphoid IFN-γ producing T cells and protection at the site of virus entry (intestine). In studies of mice, intestinal IFN-γ producing CD4+ T cells were suggested to be the only lymphocytes required for protection against rotavirus infection after the mice were immunized with a chimeric VP6 vaccine using attenuated E. coli labile toxin as an adjuvant [15,28,46,47]. Their data and ours collectively emphasize the role of intestinal IFN-γ producing CD4+ T cells as correlates of rotavirus protective immunity, possibly more important than CD8+ T cells.…”

The A Subunit of Escherichia coli Heat-Labile Enterotoxin Functions as a Mucosal Adjuvant and Promotes IgG2a, IgA, and Th17 Responses to Vaccine Antigens

“…Experimental evidence has shown that LT, some mutant LTs, and CT promote Th17 cell development (6,17,35,37,43,53). To assess the contribution of the A subunit to Th17 responses in these animals, we evaluated splenocytes from the OVA-immunized mice for secretion of IL-17 in an antigen restimulation assay.…”

“…Subsequently, antigen-specific adaptive immune responses develop, including IgG1/IgG2a antibodies, mucosal IgA, and mixed Th1/ Th2/Th17/Treg cellular responses, depending upon the intrinsic nature of the coadministered antigen, the type of attenuating mutation to the LT (if any), and the route of immunization (3,6,13,24,37,41,42,53,55). The mechanisms through which LT functions as a mucosal adjuvant are not well understood, and conflicting results have been obtained by different investigators using different preparations of LT, different antigens, and different routes of immunization.…”

Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax

“…Indeed, a recent report observed that CT is able to generate Th17 cells in an IL-6-dependent manner (11), and vaccines containing CT or Escherichia coli heat-labile toxin also induced IL-17 (36)(37)(38)(39), but the mechanisms involved and the role of IL-17 in mediating adjuvant effects (i.e., antibody responses and protection against infection) were not explored. In addition, our data show that a natural biological molecule such as CT induces DCs to secrete a complex mixture of products that are required to act in combination to determine T-cell fate (Fig.…”