NKT cells induce IgG1 class switch against C. difficile carbohydrate antigens and enhance immunity against a live pathogen challenge

Abstract All clinical Clostridioides difficile strains identified to date express a surface capsule-like polysaccharide structure known as polysaccharide II (PSII). The PSII antigen is immunogenic and when conjugated to a protein carrier and induces a protective antibody response in animal models. Given that CD1d-restricted Natural Killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1d-binding glycolipid adjuvant could lead to enhanced protection against a live C. difficile challenge. We purified PSII from a clinical isolate of C. difficile and immunized B6 mice with PSII alone or PSII plus the CD1d-binding glycolipid α-galactosylceramide (α-GC). PSII-specific IgM and IgG3 titers were evident in sera from immunized mice, but the inclusion of α-GC led to additional isotype switch and production of IgG1. Enhanced protection against C. difficile disease was achieved by inclusion of the α-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Traj18−/− mice were not protected by the PSII/α-GC immunization modality. These results indicate that α-GC-driven NKT cells stimulate a protective IgG1 response directed at the C. difficile PSII antigen. This study suggests that NKT activation represents a pathway for additional B cell help that could be used to supplement existing efforts to develop a C. difficile vaccine.