alpha-Galactosylceramide-Reactive NKT Cells Increase IgG1 Class Switch against a Clostridioides difficile Polysaccharide Antigen and Enhance Immunity against a Live Pathogen Challenge

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, induces a protective antibody response in animal models. Given that CD1 d-restricted natural killer T (NKT) cells promote antibody responses, including those against carbohydrates, we tested the hypothesis that immunization with PSII and a CD1 d-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 CD1 d-binding glycolipid alpha-galactosylceramide (alpha-GC). PSII -specific 1gM and IgG titers were evident in sera from immunized mice. The inclusion of alpha-GC had a modest influence on isotype switch but increased the IgG1/IgG2c ratio. Enhanced protection against C. difficile disease was achieved by inclusion of the alpha-GC ligand and was associated with reduced bacterial numbers in fecal pellets. In contrast, NKT-deficient Trap18(-/-) mice were not protected by the PSII/alpha-GC immunization modality. Absence of NKT cells similarly had a modest effect on isotype switch, but ratios of IgG1/IgG2c decreased. These results indicate that alpha-GC-driven NKT cells move the humoral immune response against C. difficile PSII antigen toward Th2-driven IgG1 and may contribute to augmented protection. This study suggests that NKT activation represents a pathway for additional B-cell help that could be used to supplement existing efforts to develop vaccines against polysaccharides derived from C. difficile and other pathogens.