Th1 and Th17 Responses to LTB and Colonization Factors Following Oral ETEC Vaccination

T helper cells (Th) are central to mucosal IgA induction and key targets for modulation by vaccine adjuvants. To improve understanding of cellular mechanisms underlying mucosal vaccine-induced immunity, we analyzed antigen-specific peripheral blood Th responses elicited by the oral enterotoxigenic Escherichia coli (ETEC) vaccine ETVAX, administered with or without the double mutant heat-labile toxin (dmLT) adjuvant. ETVAX, consisting of inactivated E. coli overexpressing colonization factors CFA/I, CS3, CS5, and CS6 with a heat-labile toxin B-subunit toxoid, was given orally in two doses to adult volunteers, either alone or with 10 or 25 µg dmLT. Antigen-specific Th-associated cytokine responses were assessed in stimulated peripheral blood mononuclear cells isolated from 15 to 18 individuals/group using ELISA and electrochemiluminescence assays. ETVAX predominantly induced Th1 (IFN-γ) and Th17 (IL-17A) responses, with minimal Th2-associated cytokines. Responses were markedly reduced after CD4+ T-cell depletion, supporting a Th cell origin. The strongest responses targeted LTB and CS3, with IFN-γ responses detected in 60-80% and IL-17A in 40-60% across all vaccinees. Responses to CFA/I, CS5 and CS6 were generally weaker. Exploratory comparisons suggested broader IFN-γ responses and more consistent IFN-γ and IL-17A responses to lower-dose antigens, particularly CS6, in recipients receiving vaccine plus 10 µg dmLT. These trends paralleled IgA antibody-secreting cell response patterns, with significantly enhanced IgA responses to CS6 in the vaccine plus 10 µg dmLT group. In conclusion, ETVAX induces antigen-specific Th1- and Th17-type responses in peripheral blood, supporting a role for cellular immunity in mucosal responses to oral ETEC vaccines.