Cellular and Humoral Immune Responses to Noncanonical Enterotoxigenic Escherichia coli Virulence Factors

A broadly protective licensed vaccine against enterotoxigenic Escherichia coli (ETEC) remains unavailable. To inform rational ETEC vaccine design, we characterized immune responses to the noncanonical antigens EatA mucinase, EtpA adhesin, and YghJ metalloprotease following experimental ETEC infection. We analyzed longitudinal cellular and humoral responses to EatA, EtpA, and YghJ in 30 volunteers infected with ETEC. Antigen-specific CD4⁺ T cell responses were assessed using an activation-induced marker (AIM) assay detecting CD69, CD134, CD137, and CD154 expression after antigen stimulation. Antigen-specific IgA and IgG responses in serum and IgA responses in intestinal lavage were quantified using bead-based multiplex flow cytometry. Antigen-reactive CD69⁺CD134⁺ CD4⁺ T cells increased on Day 10 for all three antigens, with EtpA also inducing CD134⁺CD137⁺ responses. Serum anti-EatA IgA and IgG increased on Days 10 and 28, accompanied by increased intestinal lavage anti-EatA IgA on Day 10. Mucosal IgA responses to EatA and EtpA correlated strongly with systemic IgG. Immune responses did not seem to be associated with ETEC colonization or host blood group. This first integrated characterization of human immune responses against the ETEC antigens EatA, EtpA, and YghJ demonstrates their immunogenicity in both cellular and humoral compartments, and supports their relevance as potential vaccine targets.