α-Galactosylceramide-Reactive NKT Cells Increase IgG1 Class Switch against a Clostridioides difficile Polysaccharide Antigen 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, 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 IgG titers were evident in sera from immunized mice. The inclusion of α-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 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. Absence of NKT cells similarly had a modest effect on isotype switch, but ratios of IgG1/IgG2c decreased. These results indicate that α-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.

Keywords: Clostridioides difficile; Clostridium difficile; NKT cell; carbohydrate; humoral immunity.

FIG 1

Purification and characterization of the C. difficile PSII antigen. As described in Materials and Methods, a crude carbohydrate fraction was extracted from C. difficile using hot water and phenol. After dialysis, the crude carbohydrate preparation was applied to a GS50 size exclusion column. (A) Total carbohydrate profile of eluted fractions as determined by the sulfuric acid/phenol assay. PSII was concentrated in the peak indicated and as demonstrated in panel B, whereby ³¹P NMR analysis was used to confirm the predominance of PSII in the pooled column fractions. The image on the left depicts the entire PSII peak. (C) Four sequential subpools of those fractions, whereby the largest fraction numbers, or tail end of the peak (purple), have the PSIII removed. (D) Detection of PSII by ELISA using plates coated with PSII (left) or oxygen-killed C. difficile (center) as well as the correlation between the two assays (right). (E) Rabbit anti-PSII but not a control antiserum could detect PSII by dot blotting. Neither proteins nor nucleic acids could be detected in the column fractions where PSII was concentrated. Data are representative of at least 4 experiments.

FIG 2

NKT-influenced IgM and IgG production against PSII. B6 mice were immunized s.c. with 15 μg PSII or PSII plus 4 μg α-GC. After 1 week, mice were boosted with 7.5 μg PSII. Sera were collected after 28 days, and PSII-specific IgM, IgG1, IgG2b, IgG2c, and IgG3 were detected by ELISA. Each data point represents an individual mouse. A two-tailed t test was used to detect significant differences between groups.

FIG 3

PSII/α-GC vaccination leads to enhanced protection against C. difficile disease. (A to G) Age- and sex-matched naive B6 mice were cefoperazone treated and then mock infected or infected with 10⁴ live C. difficile spores. Mice previously immunized with PSII alone, PSII plus α-GC, or α-GC and PSII 1 week apart were also cefoperazone treated and infected. Graphs show the percentage of original weight, which was obtained daily and for the duration indicated. Data show the results of two pooled experiments with n = 5 for each group except for the naive-infected, α-GC- and then vehicle-treated, and PSII/α-GC groups (n = 10). Statistical significance was determined by repeated-measure ANOVA and Tukey’s multiple-comparison test (*, P < 0.05; **, P < 0.01). (H) Fecal pellets collected 2 days after infection were used to determine bacterial load. In group A, zero counts were detected (limit of detection in assay, 10²). (I) ELISA measurement of C. difficile-specific IgG1 in response to the various vaccination modalities used. In panels H and I, significance was determined by one-way ANOVA with Tukey’s multiple-comparison test (*, P < 0.05; **, P < 0.01). (J and K) Naïve (n = 4) and PSII/α-GC-immunized (n = 5) mice, antibiotic treated and infected with 5 × 10⁴ spores. Weight loss and survival are depicted. Numbers above bars on the left are the numbers of remaining live animals in the group. A Mantel-Cox log rank test was used to detect differences between groups subject to Kaplan-Meier survival analysis.

FIG 4

NKT cells are required for α-GC-enhanced protection but do not affect disease susceptibility of naive mice to C. difficile. Mice depicted were immunized with PSII plus α-GC and boosted with PSII before treatment with cefoperazone and infection with 5 × 10⁴ C. difficile spores per mouse. (A) Mean weight (± standard deviations) in NKT-sufficient controls (Traj18^+/+, Traj18^+/−, n = 7) and in 3 individual NKT-deficient Traj18^−/− littermate mice. (B) PSII-specific IgM, IgG1, IgG2b, IgG2c, and IgG3 with samples pooled from experiment shown in panel A and the Traj18^−/− samples shown in panel C. In panels A and B, significance was determined by two-tailed t test. (C) Weights in PSII/α-GC-immunized B6 (n = 5) and Traj18^−/− (n = 6) mice following challenge with 3 × 10⁵ spores per mouse. (D) Weights in naive B6 (n = 5) and Traj18^−/− (n = 6) mice following challenge with 5 × 10⁴ spores per mouse.