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Secreted toxin B (TcdB) substantially contributes to the pathology observed during Clostridium difficile infection. To be successfully incorporated into a vaccine, TcdB-based immunogens must stimulate the production of neutralizing antibody (Ab)-encoding memory B cells (Bmem cells). Despite numerous investigations, a clear analysis of Bmem cellular responses following vaccination against TcdB is lacking. B6 mice were therefore used to test the ability of a nontoxigenic C-terminal domain (CTD) fragment of TcdB to induce Bmem cells that encode TcdB-neutralizing antibody. CTD was produced from the historical VPI 10463 strain (CTD1) and from the hypervirulent strain NAP1/BI/027 (CTD2). It was then demonstrated that CTD1 induced strong recall IgG antibody titers, and this led to the development of functional Bmem cells that could be adoptively transferred to naive recipients. Bmem cell-driven neutralizing Ab responses conferred protection against lethal challenge with TcdB1. Further experiments revealed that an experimental adjuvant (Imject) and a clinical adjuvant (Alhydrogel) were compatible with Bmem cell induction. Reactivity of human Bmem cells to CTD1 was also evident in human peripheral blood mononuclear cells (PBMCs), suggesting that CTD1 could be a good vaccine immunogen. However, CTD2 induced strong Bmem cell-driven antibody titers, and the CTD2 antibody was neutralizing in vitro, but its protection against lethal challenge with TcdB2 was limited to delaying time to death. Therefore, CTD from different C. difficile strains may be a good immunogen for stimulating B cell memory that encodes in vitro neutralizing Ab but may be limited by variable protection against intoxication in vivo., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The CD1d-binding glycolipid α-galactosylceramide exerts potent adjuvant effects on T-dependent humoral immunity. The mechanism is driven by cognate interaction between CD1d-expressing B cells and TCR-expressing type I CD1d-restricted NKT cells. Thus, far positive effects of alpha-galactosylceramide have been observed on initial and sustained antibody titers as well as B-cell memory. Following vaccination, each of these features is desirable, but good B-cell memory is of paramount importance for long-lived immunity. We therefore tested the hypothesis that CD1d expression in vivo differentially affects initial antibody titers versus B-cell memory responses. CD1d(+/+) and CD1d(+/-) mice were generated and immunized with antigen plus CD1d ligand before analysis of cytokine expression, CD40L expression, initial and longer term antibody responses and B-cell memory. As compared with CD1d(+/+) controls, CD1d(+/-) mice had equivalent numbers of total NKT cells, lower cytokine production, fewer CD40L-expressing NKT cells, lower initial antibody responses, similar long-term antibody responses and higher B-cell memory. Our data indicate that weak CD1d antigen presentation may facilitate good B-cell memory without compromising antibody responses. This work may impact vaccine design since over-stimulation of NKT cells at the time of vaccination may not lead to optimal B-cell memory.

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (alpha-galactosylceramide [alpha-GC]) at the time of immunization improves PA-specific Ab responses. We observed that alpha-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d(-/-) mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jalpha281(-/-) mice expressing CD1d but lacking type I alpha-GC-reactive NKT cells, alpha-GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

Activation of natural killer-like T (NKT) cells with the CD1d ligand alpha-galactosylceramide enhances T-dependent humoral immune responses against coadministered T-dependent Ag. At present, there is little information on the mechanisms involved other than a dependence on CD1d expression by antigen-presenting cells and/or development of the NKT subset. We therefore tested the hypothesis that direct presentation of alpha-GC by B cells was required for NKT-enhanced Ab responses against T-dependent Ag. We reconstituted B cell-deficient microMT mice with B cells from C57Bl/6 donors or CD1d(-/-) donors before immunization with NP-KLH alone or NP-KLH mixed with alpha-GC. We made the surprising observation that B-cell expression of CD1d is absolutely required for the NKT-enhanced Ab response. Our data show that the mechanism by which NKT cells enhance humoral immune responses involves interaction with CD1d-expressing B cells.

CD1 molecules are non-polymorphic major histocompatibility complex class I-related proteins that bind and present glycolipid antigens to T-cell antigen receptors (TCR) expressed by alphabeta T cells or natural killer-like T cells (NKT). Anti-metastatic properties of NKT cells reactive to the CD1d-binding antigen alpha-galactosylceramide (alpha-GalCer) are now being explored as a contributor to tumour cell killing. In this study, we tested the hypothesis that presentation of alpha-GalCer by murine CD1d (mCD1d) to mCD1d-restricted NKT cells was facilitated by plasma membrane glycolipid rafts. Confocal microscopy of mCD1d-transfected A20 B cells (A20mCD1d) demonstrated that mCD1d was raft-localized. This observation was confirmed by immunoblotting of raft fractions isolated on sucrose density gradients. Raft disruption by the cholesterol-binding agent nystatin, or short-chain ceramides, inhibited presentation of low concentrations of alpha-GalCer to NKT cells. Inhibition of antigen presentation was reversed by treatment of A20mCD1d cells with higher alpha-GalCer concentrations, or removal of raft-disrupting agents. These data indicate that partitioning of mCD1d into membrane rafts increases the capacity of antigen-presenting cells to present limiting quantities of glycolipid antigens, perhaps by stabilizing mCD1d/antigen structures on the plasma membrane and optimizing TCR engagement on NKT cells.