Infection with V. cholerae
provides long-term protection against subsequent disease (5
). Although the mechanism(s) of protective immunity is not well understood, protection may be mediated by anamnestic immune responses to V. cholerae
). In support of this, memory-B-cell responses to V. cholerae
protein antigens remain detectable for at least 1 year after infection, longer than circulating antibodies or circulating memory-B-cell responses to V. cholerae
O1 lipopolysaccharide (16
). Because helper T cells play a critical role in the development of long-lived memory B cells directed against protein antigens (8
), we have hypothesized that the initial CD4+
T-cell response to V. cholerae
infection may contribute to long-term protection against cholera.
Previously, we observed that patients recovering from cholera develop gut-homing central and effector memory CD4+
T-cell immune responses to V. cholerae
protein antigens that peak in the circulation 7 days after cholera (3
). In this study, we characterized the functions of the cellular immune responses to V. cholerae
O1 in cholera patients and vaccinees by measuring the lineage-specific CD4+
T-cell responses to ex vivo
The paradigmatic cellular immune response to mucosal infection involves Th2-regulated IgA production; however, there are examples where Th1 or Th17 responses may be critical determinants of protective immunity against potentially invasive mucosal pathogens (12
). Because cholera is prototypical of a noninflammatory, toxin-mediated diarrhea (17
), it was plausible that the disease would induce a CD4+
T-cell response skewed predominantly toward a noninflammatory Th2 response to V. cholerae
However, the findings of this study do not support this hypothesis. Instead, infection with V. cholerae
primed Th1 and Th17 responses, with a shift toward Th1 to Th2 CD4+
T-cell responses. Despite the absence of overt pyogenic infection and gross histological changes in the gut during cholera, these responses are preceded by a mucosal innate immune response to infection that includes increases in IL-1β, IL-6, and IL-8 secretion by lamina propria lymphocytes. These results are consistent with increasing evidence that early responses to V. cholerae
O1 infection include the upregulation of expression of proteins with presumed bacterial inhibitory properties, including lactoferrin and long palate, lung, and nasal epithelium clone 1 (LPLUNC1) (14
), and the migration of neutrophils to the lamina propria and epithelium during acute cholera (14
). It was previously shown that IL-1β expression is increased during acute cholera, a finding reproduced in this study. However, the finding that IL-6, a critical bridge between innate and adaptive immunity through the promotion of B-cell IgA class switch differentiation and T-cell differentiation (Th17 lineage), was increased in acute cholera is an extension of these findings with potential implications for the development of long-term immunologic memory.
Despite the similar antigenic composition of the WC-CTB vaccine and live V. cholerae O1 organisms encountered in natural infection, we observed significant differences in the CD4+ T-cell response in cholera patients and vaccinees. Compared to natural infection with V. cholerae O1, vaccination resulted in limited priming of cellular immune responses to V. cholerae antigens. In addition, while subjects recovering from cholera developed a shift toward increased Th1/Th2 CD4+ T-cell responses, a comparable shift in the Th1/Th2 ratio of T-cell cytokine production was not observed in subjects who received two doses of the vaccine.
The finding that natural cholera induces proinflammatory CD4+
T-cell responses greater than those seen in WC-CTB vaccine recipients is consistent with in vitro
and murine models of the mechanisms of the adjuvant effects of CT holotoxin. In mice, the effectiveness of CT as an adjuvant is dependent on the presence of CD4+
T cells (18
). The CT holotoxin induces a broad spectrum of CD4+
T-cell responses, including Th1, Th2, Th17, and regulatory T-cell responses, to coadministered antigens (20
). Recent evidence, also from a murine model, suggests that the induction of Th17 responses is essential for the mucosal adjuvant activity of CT holotoxin; specifically, the induction of an IL-17 response by CT is required to generate protective mucosal IgA antibody responses to coadministered antigens (11
). Our clinical observations here similarly demonstrate that humans with natural cholera (and, hence, exposure to CT holotoxin) also develop V. cholerae
antigen-specific mucosal IL-17 responses, despite the absence of overt pyogenic infection.
In contrast to the adjuvant effect of the CT holotoxin, the mucosal application of only CTB, the GM1 ganglioside-binding portion of the toxin, results in the differentiation of both adaptive and natural regulatory T cells that produce IL-10 and suppress Th1, Th2, and Th17 responses and promote tolerance to coadministered antigens (10
). Thus, the presence of CTB in the WC-CTB vaccine may provide a possible mechanism by which vaccine-induced CD4+
T-cell responses to V. cholerae
MP may be diminished or skewed toward the development of a Th2 T-cell phenotype. This may also explain why in initial clinical trials, the protective efficacy of the whole-cell-only vaccine (without CTB) against cholera was more persistent and ultimately afforded protection that exceeded the protection afforded by the WC-CTB vaccine by the third year of follow-up (62% for the whole-cell-only vaccine compared to 40% for the WC-CTB vaccine) (6
). Because the more recently licensed Shanchol vaccine does not include CTB, it may elicit a different profile of cellular immune responses to V. cholerae
protein antigens than the WC-CTB vaccine and requires additional study.
In this study, we observed robust CD4+
T-cell immune responses in cholera patients that fell outside the predicted model for a noninvasive mucosal infection. However, in future studies, it will be critical to gauge the significance of these early CD4+
T-cell responses to V. cholerae
. Because memory-B-cell responses to protein antigens persist longer than serum antibody responses, it may be useful to evaluate the association between early T-cell responses and the subsequent development and maintenance of long-term memory B cells. In addition, future studies are needed to examine the role of the CXCR5-expressing follicular helper T cells, which likely play a particularly important role in regulating B-cell proliferation and class switching (24
). These studies may help further define the role of early T-cell responses in determining longlasting immunity to cholera.