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Cholera caused by Vibrio cholerae O139 could reemerge, and proactive development of an effective O139 vaccine would be prudent. To define immunoreactive and potentially immunogenic carbohydrate targets of Vibrio cholerae O139, we assessed immunoreactivities of various O-specific polysaccharide (OSP)-related saccharides with plasma from humans hospitalized with cholera caused by O139, comparing responses to those induced in recipients of a commercial oral whole-cell killed bivalent (O1 and O139) cholera vaccine (WC-O1/O139). We also assessed conjugate vaccines containing selected subsets of these saccharides for their ability to induce protective immunity using a mouse model of cholera. We found that patients with wild-type O139 cholera develop IgM, IgA, and IgG immune responses against O139 OSP and many of its fragments, but we were able to detect only a moderate IgM response to purified O139 OSP-core, and none to its fragments, in immunologically naive recipients of WC-O1/O139. We found that immunoreactivity of O139-specific polysaccharides with antibodies elicited by wild-type infection markedly increase when saccharides contain colitose and phosphate residues, that a synthetic terminal tetrasaccharide fragment of OSP is more immunoreactive and protectively immunogenic than complete OSP, that native OSP-core is a better protective immunogen than the synthetic OSP lacking core, and that functional vibriocidal activity of antibodies predicts in vivo protection in our model but depends on capsule thickness. Our results suggest that O139 OSP-specific responses are not prominent following vaccination with a currently available oral cholera vaccine in immunologically naive humans and that vaccines targeting V. cholerae O139 should be based on native OSP-core or terminal tetrasaccharide. IMPORTANCE Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP). Little is known about immunity to O139 OSP. In this study, we used synthetic fragments of the O139 OSP to define immune responses to OSP in humans recovering from cholera caused by V. cholerae O139, compared these responses to those induced by the available O139 vaccine, and evaluated O139 fragments in next-generation conjugate vaccines. We found that the terminal tetrasaccharide of O139 is a primary immune target but that the currently available bivalent cholera vaccine poorly induces an anti-O139 OSP response in immunologically naive individuals.

Cholera infection can result in severe dehydration that may lead to death within a short period of time if not treated immediately. Vaccination is an important strategy to prevent the disease., Cholera remains a major public health problem in resource-limited countries. Vaccination is an important strategy to prevent cholera, but currently available vaccines provide only 3 to 5 years of protection. Understanding immune responses to cholera antigens in naturally infected individuals may elucidate which of these are key to longer-term protection seen following infection. We recently identified Vibrio cholerae O1 sialidase, a neuraminidase that facilitates binding of cholera toxin to intestinal epithelial cells, as immunogenic following infection in two recent high-throughput screens. Here, we present systemic, mucosal, and memory immune responses to sialidase in cholera index cases and evaluated whether systemic responses to sialidase correlated with protection using a cohort of household contacts. Overall, we found age-related differences in antisialidase immune response following cholera. Adults developed significant plasma anti-sialidase IgA, IgG, and IgM responses following infection, whereas older children (≥5 years) developed both IgG and IgM responses, and younger children only developed IgM responses. Neither older children nor younger children had a rise in IgA responses over the convalescent phase of infection (day 7/day 30). On evaluation of mucosal responses and memory B-cell responses to sialidase, we found adults developed IgA antibody-secreting cell (ASC) and memory B-cell responses. Finally, in household contacts, the presence of serum anti-sialidase IgA, IgG, and IgM antibodies at enrollment was associated with a decrease in the risk of subsequent infection. These data show cholera patients develop age-related immune responses against sialidase and suggest that immune responses that target sialidase may contribute to protective immunity against cholera. IMPORTANCE Cholera infection can result in severe dehydration that may lead to death within a short period of time if not treated immediately. Vaccination is an important strategy to prevent the disease. Oral cholera vaccines provide 3 to 5 years of protection, with 60% protective efficacy, while natural infection provides longer-term protection than vaccination. Understanding the immune responses after natural infection is important to better understand immune responses to antigens that mediate longer-term protection. Sialidase is a neuraminidase that facilitates binding of cholera toxin to intestinal epithelial cells. We show here that patients with cholera develop systemic, mucosal, and memory B-cell immune responses to the sialidase antigen of Vibrio cholerae O1 and that plasma responses targeting this antigen correlate with protection.

Enteric fever remains a significant global problem, and control programs are significantly limited by the lack of an optimal assay for identifying individuals with acute infection. This is especially critical considering the recently released World Health Organization (WHO) position paper endorsing the role of the typhoid conjugate vaccine in communities where enteric fever is endemic. A reliable diagnostic test is needed to assess and evaluate typhoid intervention strategies and determine which high-burden areas may benefit most from a vaccine intervention. Our collaborative team has developed and evaluated a point-of-care serodiagnostic assay based on detection of anti-HlyE and LPS IgA. Our finding of the high diagnostic accuracy of the DPP Typhoid System for the rapid detection of enteric fever has the potential to have significant public health impact by allowing for improved surveillance and for control and prevention programs in areas with limited laboratory capacity., There is a critical need for an improved rapid diagnostic for enteric fever. We have previously demonstrated that serum IgA responses targeting Salmonella enterica serovar Typhi hemolysin E (HlyE) and lipopolysaccharide (LPS) are able to discriminate patients with acute typhoid from healthy controls in areas where enteric fever is endemic (healthy endemic controls) and from patients with other bacterial infections. We now have data demonstrating that IgA antibody responses against these antigens also work well for identifying patients with acute S. Paratyphi A infection. To develop a test for acute enteric fever detection, we have adapted a point-of-care immunochromatographic dual-path platform technology (DPP), which improves on the traditional lateral flow technology by using separate sample and conjugate paths and a compact, portable reader, resulting in diagnostics with higher sensitivity and multiplexing abilities. In this analysis, we have compared our standard enzyme-linked immunosorbent assay (ELISA) method to the DPP method in detecting acute phase plasma/serum anti-HlyE and anti-LPS IgA antibodies in a cohort of patients with culture-confirmed S. Typhi (n = 30) and Paratyphi A infection (n = 20), healthy endemic controls (n = 25), and febrile endemic controls (n = 25). We found that the DPP measurements highly correlated with ELISA results, and both antigens had an area under the curve (AUC) of 0.98 (sensitivity of 92%, specificity of 94%) with all controls and an AUC of 0.98 (sensitivity of 90%, specificity of 96%) with febrile endemic controls. Our results suggest that the point-of-care DPP Typhoid System has high diagnostic accuracy for the rapid detection of enteric fever and warrants further evaluation. IMPORTANCE Enteric fever remains a significant global problem, and control programs are significantly limited by the lack of an optimal assay for identifying individuals with acute infection. This is especially critical considering the recently released World Health Organization (WHO) position paper endorsing the role of the typhoid conjugate vaccine in communities where enteric fever is endemic. A reliable diagnostic test is needed to assess and evaluate typhoid intervention strategies and determine which high-burden areas may benefit most from a vaccine intervention. Our collaborative team has developed and evaluated a point-of-care serodiagnostic assay based on detection of anti-HlyE and LPS IgA. Our finding of the high diagnostic accuracy of the DPP Typhoid System for the rapid detection of enteric fever has the potential to have significant public health impact by allowing for improved surveillance and for control and prevention programs in areas with limited laboratory capacity.

An episode of cholera provides better protection against reinfection than oral cholera vaccines, and the reasons for this are still under study. To better understand this, we compared the immune responses of human cells exposed to live Vibrio cholerae with those of cells exposed to heat-killed V. cholerae (similar to the contents of oral cholera vaccines). We also compared the effects of active cholera toxin and the inactive cholera toxin B subunit (which is included in some cholera vaccines). One key immune signaling molecule, IL-23, was uniquely produced in response to the combination of live bacteria and active cholera holotoxin. Stimulation with V. cholerae that did not produce the active toxin or was killed did not produce an IL-23 response. The stimulation of IL-23 production by cholera toxin-producing V. cholerae may be important in conferring long-term immunity after cholera., Vibrio cholerae infection provides long-lasting protective immunity, while oral, inactivated cholera vaccines (OCV) result in more-limited protection. To identify characteristics of the innate immune response that may distinguish natural V. cholerae infection from OCV, we stimulated differentiated, macrophage-like THP-1 cells with live versus heat-inactivated V. cholerae with and without endogenous or exogenous cholera holotoxin (CT). Interleukin 23A gene (IL23A) expression was higher in cells exposed to live V. cholerae than in cells exposed to inactivated organisms (mean change, 38-fold; 95% confidence interval [95% CI], 4.0 to 42; P