Peripheral markers of humoral immunity to cholera decrease to baseline levels before protective immunity against cholera ends. Possible explanations are that constitutive antibody secretion at the mucosal surface maintains protection upon reexposure and that increased levels of mucosal antibody secretion persist longer than detectable increases in circulating antibodies. However, in direct opposition, we found that the increases in circulating CTB- and LPS-specific antibodies actually persist for a longer time than the increases in the levels of these antibodies in duodenal tissue following cholera infection. This finding suggests that it is unlikely that protective immunity to cholera is mediated by constitutive antibody secretion at the mucosal surface.
In contrast, we did find that cholera patients had persistent increases in V. cholerae
LPS-specific ASCs in duodenal tissue after cholera infection. V. cholerae
LPS-specific IgA ASCs remained at significantly increased levels compared to those for healthy controls at least until day 180, long after circulating LPS-specific IgA ASCs returned to baseline levels. Although V. cholerae
LPS-specific ASCs on day 180 represented only 0.07% of all lamina propria IgA ASCs, the lamina propria harbors 80% of all ASCs in humans (6
), and it is possible that even a relatively small proportion of V. cholerae
LPS-specific ASCs in the lamina propria may allow protective immunity against cholera.
Interestingly, the persistent increase in V. cholerae
LPS-specific ASCs remained detectable despite the absence of detectable increases in IgA antibodies in mucosal tissues. This finding is of potential interest, since it has been recently demonstrated that IgA plasma cells may not secrete antibodies until induced to do so by the cessation of ongoing stimulation with the cytokine B cell-activating factor (5
) or upon an encounter with an antigen (22
). The lack of correlation between the proportion of LPS-specific ASCs in the lamina propria and tissue antibody levels seen in our study raises the possibility that some of the persistent V. cholerae
LPS-specific ASCs detected by ELISPOT assay in the lamina propria may produce substantial quantities of antibodies only in response to certain stimuli on reexposure.
While such long-lasting V. cholerae LPS-specific mucosal ASCs may contribute to protective immunity, an alternative possibility is that memory B cells that are capable of both proliferation and differentiation into plasma cells may mediate anamnestic responses that result in the amplification of humoral immunity on reexposure to an antigen. We have already found evidence that V. cholerae antigen-specific memory B cells persist in the blood much longer than antibodies and thus may contribute to long-term protection upon reexposure. However, we were not able to measure memory B cells in the duodenal biopsy specimens in this study because of the limited numbers of cells obtained.
In our previous study of memory B cell responses in blood after cholera infection (8
), there were significant differences in the development of memory B cells into the T-cell-independent antigen LPS and the T-cell-dependent antigen CTB. We previously observed that peripheral IgG memory B cell responses to the protein antigen CTB persisted for a full year after cholera infection, but IgG memory B cell responses to LPS in blood waned more rapidly. Here, in mucosal tissues, we found the converse: V. cholerae
LPS-specific IgA ASC responses in lamina propria persisted longer than IgA ASC responses to CTB in lamina propria. The lamina propria is a site of T-cell-independent antigen-induced IgA class switch recombination in the intestine, while T-cell-dependent IgA class switching is restricted to follicular lymphoid tissues (4
). Our results suggest that LPS-specific IgA ASCs, derived from T-cell-independent IgA class switch recombination, may contribute to repopulating the lamina propria for a longer time than ASCs derived from follicular T-cell-dependent class switch recombination.
We found that the levels of circulating CT IgG and LPS IgG ASCs increased significantly at day 7 compared to those at the acute stage of infection and in healthy controls and then declined by day 30. However, we were not able to see any major differences in IgG ASC responses against CT in the gut. It has been shown previously that CTB IgG responses increased in the circulatory system after oral immunization with killed whole-cell enterotoxigenic Escherichia coli
(ETEC) vaccine containing toxoid antigen (15
), as well as after natural cholera infection (16
) or ETEC diarrhea (16
). Moreover, Quiding et al. (18
) observed striking differences between duodenal and peripheral CTB ASC responses after oral immunization with B-subunit whole-cell (B-WC) cholera vaccine (18
), with responses higher in the duodenum than in the circulatory system after vaccination. However, here we saw a different response following severe cholera infection. Further studies using intestinal immunocytes of the IgG antibody isotype (20
) are needed.
The lack of correlation between concomitant measures of plasma V. cholerae antigen-specific IgA and ASCs and mucosal-tissue IgA and ASCs underscores the difficulty in identifying circulating markers of a protective immune response that is primarily dependent on anamnestic mucosal immune responses. Based on this study, the initial peak in CTB-specific ASCs is the best marker for a subsequent mucosal response to CTB. However, the T-cell-independent response to LPS appears to have different kinetics, and the initial ASC response in blood is not predictive of the ultimate magnitude of the mucosal anti-LPS response. The development of novel noninvasive assays for measuring anamnestic mucosal immune responses to antigenic challenge (somewhat analogous to the use of a tuberculin skin test) may ultimately provide better measurements of protective immunity against cholera and other mucosal infections than the measurement of circulating antibodies or ASC responses.
By flow cytometry, we have previously shown that the level of gut-homing B cells in the blood increases on day 7 compared to those in healthy controls and on day 2 of the illness (2
). In this study, we found that the numbers of gut-homing CD19+
cells also increased significantly in the lamina propria late after infection, on days 30 and 180, compared to those on day 2 and in healthy controls. This finding suggests that gut-homing CD19+
cells may take some time to home back to the gut from the circulatory system. The baseline levels of CD19+
and gut-homing CD19+
cells in biopsy specimens were also relatively high in our healthy Bangladeshi controls, perhaps triggered by infection with other pathogens and the resulting recruitment of lymphocytes to this site (3
). Further studies are needed to better understand the kinetics of antigen-specific B cell responses in the duodenal mucosa of V. cholerae
Taken together, our data are consistent with those of previous studies and provide additional evidence suggesting that protective immunity to cholera is not likely to be mediated by constitutive antibody secretion at the mucosal surface at the time of reinfection with V. cholerae. However, our data do not differentiate between the contributions to protective immunity of persistent V. cholerae LPS-specific IgA ASCs in the lamina propria or memory B cells capable of anamnestic immune responses upon reexposure. Additional studies are needed to address whether canonical memory B cells are present in mucosal tissue and whether these and/or circulating memory B cells contribute to protective immunity after cholera infection.