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Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes with a semi-conserved TCRα, activated by the presentation of vitamin B metabolites by the MHC-I related protein, MR1, and with diverse innate and adaptive effector functions. The role of MAIT cells in acute intestinal infections, especially at the mucosal level, is not well known. Here, we analyzed the presence and phenotype of MAIT cells in duodenal biopsies and paired peripheral blood samples, in patients during and after culture-confirmed Vibrio cholerae O1 infection. Immunohistochemical staining of duodenal biopsies from cholera patients (n = 5, median age 32 years, range 26-44, 1 female) identified MAIT cells in the lamina propria of the crypts, but not the villi. By flow cytometry (n = 10, median age 31 years, range 23-36, 1 female), we showed that duodenal MAIT cells are more activated than peripheral MAIT cells (p < 0.01 across time points), although there were no significant differences between duodenal MAIT cells at day 2 and day 30. We found fecal markers of intestinal permeability and inflammation to be correlated with the loss of duodenal (but not peripheral) MAIT cells, and single-cell sequencing revealed differing T cell receptor usage between the duodenal and peripheral blood MAIT cells. In this preliminary report limited by a small sample size, we show that MAIT cells are present in the lamina propria of the duodenum during V. cholerae infection, and more activated than those in the blood. Future work into the trafficking and tissue-resident function of MAIT cells is warranted., Competing Interests: The authors have declared that no competing interests exist.

Background: Blood collection, transportation and storage remain a problem in countries where infrastructure, laboratory facilities and skilled manpower are scarce. This limits evaluation of immune responses in natural infections and vaccination in field studies. We developed methods to measure antigen specific antibody responses using dried blood spot (DBS) in cholera, ETEC and typhoid fever patients as well as recipients of oral cholera vaccine (OCV)., Methodology/principle Findings: We processed heparinized blood for preparing DBS and plasma specimens from patients with, cholera, ETEC and typhoid as well as OCV recipients. We optimized the conventional vibriocidal method to measure vibriocidal antibody response in DBS eluates. We measured responses in DBS samples and plasma (range of titer of 5 to 10240). Vibriocidal titer showed strong agreement between DBS eluates and plasma in cholera patients (ICC = 0.9) and in OCV recipients (ICC = 0.8) using the Bland-Altman analysis and a positive correlation was seen (r = 0.7, p = 0.02 and r = 0.6, p = 0.006, respectively). We observed a strong agreement of lipopolysaccharide (LPS) and cholera toxin B (CTB)-specific antibody responses between DBS eluates and plasma in cholera patients and OCV recipients. We also found agreement of heat labile toxin B (LTB) and membrane protein (MP)-specific antibody responses in DBS eluates and plasma specimen of ETEC and typhoid patients respectively., Conclusion: Our results demonstrate that dried blood specimens can be used as an alternate method for preservation of samples to measure antibody responses in enteric diseases and vaccine trials and can be applied to assessment of responses in humanitarian crisis and other adverse field settings., Competing Interests: The authors have declared that no competing interests exist.

Although much focus is placed on cholera epidemics, the greatest burden occurs in settings in which cholera is endemic, including areas of South Asia, Africa and now Haiti 1,2 . Dhaka, Bangladesh is a megacity that is hyper-endemic for cholera, and experiences two regular seasonal outbreaks of cholera each year 3 . Despite this, a detailed understanding of the diversity of Vibrio cholerae strains circulating in this setting, and their relationships to annual outbreaks, has not yet been obtained. Here we performed whole-genome sequencing of V. cholerae across several levels of focus and scale, at the maximum possible resolution. We analyzed bacterial isolates to define cholera dynamics at multiple levels, ranging from infection within individuals, to disease dynamics at the household level, to regional and intercontinental cholera transmission. Our analyses provide a genomic framework for understanding cholera diversity and transmission in an endemic setting.

Environmental enteropathy (EE) is a poorly understood condition that refers to chronic alterations in intestinal permeability, absorption, and inflammation, which mainly affects young children in resource-limited settings. Recently, EE has been linked to suboptimal oral vaccine responses in children, although immunological mechanisms are poorly defined. The objective of this study was to determine host factors associated with immune responses to an oral cholera vaccine (OCV). We measured antibody and memory T cell immune responses to cholera antigens, micronutrient markers in blood, and EE markers in blood and stool from 40 Bangladeshi children aged 3-14 years who received two doses of OCV given 14 days apart. EE markers included stool myeloperoxidase (MPO) and alpha anti-trypsin (AAT), and plasma endotoxin core antibody (EndoCab), intestinal fatty acid binding protein (i-FABP), and soluble CD14 (sCD14). We used multiple linear regression analysis with LASSO regularization to identify host factors, including EE markers, micronutrient (nutritional) status, age, and HAZ score, predictive for each response of interest. We found stool MPO to be positively associated with IgG antibody responses to the B subunit of cholera toxin (P = 0.03) and IgA responses to LPS (P = 0.02); plasma sCD14 to be positively associated with LPS IgG responses (P = 0.07); plasma i-FABP to be positively associated with LPS IgG responses (P = 0.01) and with memory T cell responses specific to cholera toxin (P = 0.01); stool AAT to be negatively associated with IL-10 (regulatory) T cell responses specific to cholera toxin (P = 0.02), and plasma EndoCab to be negatively associated with cholera toxin-specific memory T cell responses (P = 0.02). In summary, in a cohort of children 3-14 years old, we demonstrated that the majority of biomarkers of environmental enteropathy were positively associated with immune responses after vaccination with an OCV., Competing Interests: The authors have declared that no competing interests exist.

Background: Cholera is endemic in Bangladesh, with outbreaks reported annually. Currently, the majority of epidemic cholera reported globally is El Tor biotype Vibrio cholerae isolates of the serogroup O1. However, in Bangladesh, outbreaks attributed to V. cholerae serogroup O139 isolates, which fall within the same phylogenetic lineage as the O1 serogroup isolates, were seen between 1992 and 1993 and in 2002 to 2005. Since then, V. cholerae serogroup O139 has only been sporadically isolated in Bangladesh and is now rarely isolated elsewhere., Methods: Here, we present case histories of four cholera patients infected with V. cholerae serogroup O139 in 2013 and 2014 in Bangladesh. We comprehensively typed these isolates using conventional approaches, as well as by whole genome sequencing. Phenotypic typing and PCR confirmed all four isolates belonging to the O139 serogroup., Findings: Whole genome sequencing revealed that three of the isolates were phylogenetically closely related to previously sequenced El Tor biotype, pandemic 7, toxigenic V. cholerae O139 isolates originating from Bangladesh and elsewhere. The fourth isolate was a non-toxigenic V. cholerae that, by conventional approaches, typed as O139 serogroup but was genetically divergent from previously sequenced pandemic 7 V. cholerae lineages belonging to the O139 or O1 serogroups., Conclusion: These results suggest that previously observed lineages of V. cholerae O139 persist in Bangladesh and can cause clinical disease and that a novel disease-causing non-toxigenic O139 isolate also occurs.

Vibrio cholerae, the cause of cholera, induces both innate and adaptive immune responses in infected humans. Leptin is a hormone that plays a role in both metabolism and mediating immune responses. We characterized leptin levels in 11 children with cholera in Bangladesh, assessing leptin levels on days 2, 7, 30, and 180 following cholera. We found that patients at the acute stage of cholera had significantly lower plasma leptin levels than matched controls, and compared with levels in late convalescence. We then assessed immune responses to V. cholerae antigens in 74 children with cholera, correlating these responses to plasma leptin levels on day 2 of illness. In multivariate analysis, we found an association between day 2 leptin levels and development of later anti-cholera toxin B subunit (CtxB) responses. This finding appeared to be limited to children with better nutritional status. Interestingly, we found no association between leptin levels and antibody responses to V. cholerae lipopolysaccharide, a T cell-independent antigen. Our results suggest that leptin levels may be associated with cholera, including the development of immune responses to T cell-dependent antigens., (© The American Society of Tropical Medicine and Hygiene.)

Vibrio cholerae O1 causes dehydrating diarrhea with a high mortality rate if untreated. The infection also elicits long-term protective immunity. Since V. cholerae is noninvasive, mucosal immunity is likely important for protection. In this study, we compared humoral immune responses in the duodenal mucosa and blood of cholera patients at different time points after the onset of disease and compared them with those of healthy controls (HCs). Immune responses to lipopolysaccharide (LPS) and the recombinant cholera toxin B subunit (rCTB) were assessed by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Significant increases in V. cholerae LPS-specific IgA and IgG antibody levels were seen in duodenal extracts on day 30, but the levels decreased to baseline by day 180; plasma V. cholerae LPS-specific IgA levels remained elevated longer. Levels of mucosal CTB antibodies also peaked on day 30, but the increase reached statistical significance only for IgG. A significant correlation was found between the CTB antibody-secreting cell (ASC) response in the circulatory system on day 7 and subsequent CTB-specific IgA levels in duodenal extracts on day 30 and the numbers of CTB-specific IgA ASCs in duodenal tissues on day 180. The proportion (0.07%) of mucosal V. cholerae LPS IgA ASCs peaked on day 30 and remained elevated through day 180 compared to that of HCs (P = 0.03). These results suggest that protective immunity against V. cholerae is not likely mediated by the constitutive secretion of antibodies at the mucosal surface; our results are consistent with those of other studies that suggest instead that anamnestic immune responses of mucosal lymphocytes may play a major role in protection against cholera.

Rice-based vaccines do not require high-cost purification. They are stable at room temperature, can eliminate the risk of attenuated vaccine strains, and are resistant to gastrointestinal degradation. We tested the applicability of an oral delivery system for tuberculosis (TB) and cholera antigens in transgenic rice for induction of immune responses in the mucosal compartment as well as in the systemic circulation. For vaccine development, we selected mycobacterial Ag85B antigen and immunoprotective P4 epitope of TcpA fused to the nontoxic cholera toxin B (CTB) subunit for immunization against TB and cholera, respectively, in independent constructs. The expression levels of CTB, CTB-TcpA, and CTB-Ag85B in transgenic lines containing stably integrated, chimeric genes showed up to 0.64%, 0.34%, and 0.02% of total rice seed protein, respectively. Oral immunization of mice with each of the three seed lines resulted in significantly increased levels of both anti-CTB IgG and IgA responses in the serum and IgA responses in the bronchoalveolar lavage (BAL) fluid. This indicated the capacity for oral immunization to elicit immune responses in the respiratory mucosal compartment. Plant-expressed TcpA could be detected in immunoblot analysis by using TcpA-specific commercial antibody, while there was no recognition of rice-expressed Ag85B by the commercial antibody raised against the latter antigen, where both antibodies were produced against the antigens expressed in the bacterial system. This study focused on identifying antigens resistant to both posttranslational modifications in plants and immunogenic under the proposed delivery system in animals for boosting the mucosal and systemic humoral immune response against enteric as well as respiratory pathogens. [ABSTRACT FROM AUTHOR]