Cholera toxin (CT) and heat-labile enterotoxin (LT) are powerful mucosal adjuvants whose cellular targets and mechanism of action are unknown. There is emerging evidence that dendritic cells (DC) are one of the principal cell types that mediate the adjuvant effects of these toxins in vivo. Here we investigate the effects of CT and LT on the maturation of human monocyte-derived DC (MDDC) in vitro. We found that an enzymatically active A domain is necessary for both CT and LT to induce the maturation of MDDC and that this activation is strictly cyclic AMP (cAMP) dependent. ADP-ribosylation-defective derivatives of these toxins failed to induce maturation of MDDC, whereas dibutyryl-cyclic-3,5-AMP and Forskolin mimic the maturation of MDDC induced by CT and LT. In addition, an inhibitor of cAMP-dependent kinases, Rp-8-Br-cAMPs, blocked the ability of CT, LT, and Forskolin to activate MDDC. CT, LT, dibutyryl-cyclic-3,5-AMP, and Forskolin also dominantly inhibit interleukin 12 and tumor necrosis factor alpha production by MDDC in the presence of saturating concentrations of lipopolysaccharide. Taken together, these results show that the effects of CT and LT on MDDC are mediated by cAMP.Cholera toxin (CT) and heat-labile enterotoxin (LT) are AB5 enterotoxins produced by Vibrio cholerae and enteropathic Escherichia coli, the primary causative agents of cholera and traveler's diarrhea, respectively. CT and LT consist of a 27-kDa catalytic A domain anchored in a ring of five identical, 11.7-kDa B subunits (18). The B pentamers of these toxins bind to gangliosides on cell membranes (12). The B pentamer of CT (CTB) binds exclusively to GM1 gangliosides, while the B pentamer of LT (LTB) binds to other gangliosides in addition to GM1 (9). These toxins exploit the host protein retention and degradation pathways to gain access to the cytoplasm, reviewed in reference 11. In the cytosol, their A1 subunits catalyze the transfer of an ADP-ribose from NAD to stimulatory ␣-subunits of G proteins (Gs␣). After ADP-ribosylation, Gs␣ binds to adenylate cyclase and constitutively activates it, leading to a sustained increase in intracellular cyclic AMP (cAMP) concentration (4).CT and LT are also powerful mucosal immunogens and adjuvants, reviewed in reference 14. In mice, antibody responses to CT and bystander antigens can last up to 2 years (15, 26). CT has been shown to induce primarily Th2 responses, characterized by CD4 ϩ T cells producing interleukin 4 (IL-4), IL-5, IL-6, and IL-10 and by the production of immunoglobulin G1 (IgG1), IgA, and IgE antibodies (17,29). By contrast, LT has been reported to induce mixed Th1 and Th2 responses (24). It has been proposed that differences in the ganglioside binding specificities of their B pentamers contribute to their discordant Th1/Th2 patterns (30, 31). Although these toxins are extensively used as adjuvants in animal models, their toxicity makes them unsuitable for human use. For this reason, a number of investigators have attempted to identify nontoxic derivatives of CT and LT that retain ...

A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.

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