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The cholera toxin B subunit (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on Ag-specific CD4(+) T cells has remained largely unexplored. Here, using tetramer analysis, we investigated how oral delivery of CTB fused to two CD4(+) T-cell epitopes, the BDC-2.5 T-cell 2.5 mi mimotope and glutamic acid decarboxylase (GAD) 286-300, affected diabetogenic CD4(+) T cells in nonobese diabetic (NOD) mice. When administered i.p., CTB-2.5 mi activated 2.5 mi(+) T cells and following intragastric delivery generated Ag-specific Foxp3(+) Treg and Th2 cells. While 2.5 mi(+) and GAD-specific T cells were tolerized in diabetes-resistant NODxB6.Foxp3(EGFP) F1 and nonobese resistant (NOR) mice, this did not occur in NOD mice. This indicated that NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB-fused epitopes. In contrast to NODxB6.Foxp3(EGFP) F1 mice, oral treatment in NOD mice lead to strong 2.5 mi(+) T-cell activation and the sequestration of these cells to the effector-memory pool. Oral treatment of NOD mice with CTB-2.5 mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CD103⁺ dermal dendritic cells (dDCs) are a recently described DC subset of the skin shown to be the principal migratory DCs capable of efficiently cross-presenting antigens and activating CD8⁺ T cells. Harnessing their activity would promote vaccine efficacy, but it has been unclear how this can be achieved. We tested a panel of adjuvants for their ability to affect dDCs. In comparison to the other adjuvants tested, the capacity of cholera toxin (CT) to induce the migration of dDCs was unique. Within 24 h of CT injection, large numbers of highly activated dDCs (including CD103⁺ dDCs) migrated to the draining lymph nodes and cross-presented coinjected antigens, potently activating naïve CD8⁺ T cells. Peptide vaccines adjuvanted with CT induced T-cell responses uniquely characterized by dynamic cytokine responses including the production of IL-2, and such vaccines were protective in situations reliant on CD8⁺ T-cell responses, including liver-stage Plasmodium challenge, or tumor challenge. This study is the first to examine the effects of adjuvants on CD103⁺ dDCs and identifies CT as a prototypical adjuvant for the activation of CD103⁺ dDCs, opening the way to development of vaccines and adjuvants that specifically target dDCs and generate effective CD8⁺ T-cell responses., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cholera toxin (CT) binds to GM1-ganglioside receptors present on all nucleated cells. Despite this, it is a very potent mucosal adjuvant that has a dramatic impact on immune cells, as well as nerve and epithelial cells, causing diarrhea. This fact has hampered our understanding of whether the adjuvanticity of CT is direct or indirect, as cells that bind CT may or may not be involved in its adjuvant function. The mucosal barrier is maintained by tight junctions between epithelial cells but dendritic cells (DCs) can protrude luminal dendrites. Here we investigated which cells are involved in the immune augmenting effect of CT. We explored oral immunizations with ovalbumin (OVA) and CT in bone marrow chimeric mice deficient in GM1-ganglioside in defined cellular subsets. We found that chimeric mice lacking GM1 in nonhematopoietic cells, including epithelial cells, mounted an unaltered intestinal IgA response. In contrast, chimeric mice lacking GM1-expressing hematopoietic cells in general, or specifically GM1-expressing conventional DCs (cDCs), largely failed to elicit anti-OVA adaptive immune responses. Therefore, the adjuvanticity of CT does not require epithelial activation, but is directly dependent on the binding of CT to gut cDCs via GM1-ganglioside. These results could have important implications for the generation of novel oral adjuvants., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The nature of CD4(+) T-cell responses after skin immunization and the role of migrating DCs in the presence of adjuvants in the elicited response are interesting issues to be investigated. Here, we evaluated the priming of CD4(+) T cells following ear immunization with low doses of model antigens in combination with either cholera toxin (CT) or the non-toxic β CT subunit (CTB) as an adjuvant. Following immunization with CT, we found efficient antigen presentation that is reflected in the production of IFN-γ and IL-17 by CD4(+) T cells over IL-4 or IL-5 production. The CTB-induced activation of DCs in the ear occurred without visible inflammation, which reflects a similar type of CD4(+) T-cell differentiation. In both cases, the elicited response was dependent on the presence of migrating skin cells. Remarkably, immunization with CT or with CTB led to the induction of a delayed-type hypersensitivity (DTH) response in the ear. The DTH response that was induced by CT immunization was dependent on IL-17 and partially dependent on IFN-γ activity. These results indicate that both CT and CTB induce an efficient CD4(+) T-cell response to a co-administered antigen following ear immunization that is dependent on migrating DCs., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Immunizations via the i.n. and intravaginal (ivag) routes effectively generate strong genital tract antibody-mediated immunity. To what extent the same is true for T-cell responses is incompletely known. Therefore, we set out to investigate optimal conditions for stimulation of genital tract CD4(+) T-cell responses, using adoptive transfer of mouse DO11.10 TCR transgenic T cells specific for OVA and OVA conjugated to cholera toxin (CT) as an immunogen. We observed that progesterone was required for a T-cell response following ivag immunization, whereas estradiol prevented a response. Although i.n. immunization stimulated OVA-specific CD4(+) T-cell responses in the draining LNs, it was substantially less effective compared to ivag. More importantly, an ivag booster immunization was absolutely required to attract T cells to the genital tract mucosa itself. While clinical use of CT is precluded because of its toxicity, we developed a combined adjuvant vector based on a non-toxic derivative of CT and immune-stimulating complexes. The CTA1-DD/immune-stimulating complexes (ISCOMs) adjuvant together with major outer membrane protein was effective at stimulating genital tract CD4(+) T-cell immunity and protection against a live chlamydial infection, which holds promise for the development of mucosal vaccines against sexually transmitted infections., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oral administration of the uveitogenic peptide (aa 336-351) derived from human HSP60 induced clinical and histological manifestations of uveitis in 65.8% (48/73) of Lewis rats. Uveitis was significantly decreased to 16.7% (11/66) in parallel experiments with the peptide linked to recombinant cholera toxin B subunit (rCTB), also given by mouth (chi(2)=34.2, p<0.0001). The protective efficacy between tolerized and immunized animals was 74.7%. Adoptive transfer of mesenteric lymph node cells from tolerized rats prevented the development of uveitis. A significantly higher proportion of regulatory CD4(+)CD45RC(low)RT6(+) subset of Th2 memory cells were found in the mesenteric lymph nodes (p<0.005) and spleens (p

Adjuvants that can improve mucosal vaccine efficacy are much warranted. In this comparative study between cholera toxin (CT) and immune-stimulating complexes (ISCOM) we found that, contrary to CT, ovalbumin (OVA)-ISCOM were poor inducers of mucosal anti-OVA IgA responses, but induced similar or better systemic immunity following oral immunizations. The addition of CT to the oral OVA-ISCOM protocol did not stimulate local anti-OVA IgA immunity, nor did it change the quality or magnitude of the systemic responses. Both vectors recruited strong innate immunity, but only OVA-ISCOM could directly induce IL-12, demonstrable at the protein and mRNA levels. CT had no inhibitory effects on lipopolysaccharide/IFN-gamma-induced IL-12 mRNA expression or IL-12 production. Furthermore, adjuvanticity of CT was unaffected in IL-12-deficient mice, while OVA-ISCOM showed partly impaired adjuvant effects by the lack of IL-12. CT abrogated the induction of oral tolerance stimulated by antigen feeding in these mice. In addition, CT did not alter TGF-beta levels, suggesting that the immunomodulating effect of CT was independent of IL-12 as well as TGF-beta production. Taken together, these findings indicate that mucosal adjuvanticity of CT and ISCOM are differently dependent on IL-12, suggesting that separate and distinct antigen-processing pathways are involved.

Cholera toxin (CT) is an effective mucosal adjuvant but causes significant intestinal secretion which limits its usefulness. In the present study we developed a new multiple emulsion (ME) delivery system into which antigen and CT could be incorporated and asked whether CT would retain its mucosal adjuvanticity when sequestered within emulsion particles. ME were selectively taken up into Peyer's patches, and those containing antigen plus CT generated intestinal secretory IgA and serum IgG antibody responses in mice comparable quantitatively and qualitatively to those occurring after oral immunization with soluble antigen plus CT. The ME particles containing CT did not cause intestinal secretion. The adjuvanticity of CT within ME was due to the CT present in the inner aqueous phase of the ME and was lost if CT binding was blocked by pre-incubation with GM1 ganglioside. Proteins incorporated in ME were protected from external acid, protease, and bile. We conclude that CT sequestered in ME, although unable to bind to the epithelium and thus stimulate intestinal secretion, still retains its mucosal adjuvanticity. Thus, the ability of CT to bind to enterocytes is not obligatory for its mucosal adjuvanticity.

BALB/c mice were immunized intranasally with either soluble ovalbumin (OVA) or OVA entrapped in liposomes. The effect of adding Sigma cholera toxin B subunit (sCT-B), which contained low amounts of cholera holotoxin (CT), or recombinant CT-B (rCT-B) which was free from CT, as mucosal adjuvants was also investigated. The mucosal [lung enzyme-linked immunospot assay (ELISPOT), lung washing] and systemic (serum antibody and spleen ELISPOT) responses of immunized mice to OVA and CT-B were determined. Results showed that soluble OVA and liposome-entrapped OVA were poor inducers of mucosal or systemic responses unless CT-B was added as adjuvant. The types of responses augmented by sCT-B and rCT-B were different. CT-B containing low levels of CT (i.e. sCT-B) boosted both mucosal and systemic IgA and IgG responses, whereas rCT-B only increased IgG responses, unless antigen was entrapped in liposomes. Although rCT-B was unable to adjuvant IgA responses against soluble OVA, it was able to induce IgA responses against itself. These data show that mucosal responses can be increased by addition of CT-B containing low levels of CT to antigen preparations given intranasally, suggesting a direct role for CT-A in isotype switching. Furthermore, the ability of CT-B to adjuvant IgA responses against added antigens and its ability to induce responses against itself appear to be separate phenomena. The results from this study should assist the rational formulation of mucosal vaccines which induce potent mucosal and systemic immune responses.