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Fluorescent biosensors are chemically or genetically encoded reporters of cellular processes, signaling pathways, or biomolecule concentration, whose output is quantified using fluorescence microscopy or fluorescence spectrometry. These biosensors can detect the target activity or metabolites via mechanisms including conversion between nonfluorescent and fluorescent forms, changes in reporter intensity, changes in the intensity ratio across fluorescence channels, alterations to the subcellular localization of the bioreporter, and by fluorescence resonance energy transfer. Here, we describe the use of a chemical photoconverting biosensor, and genetically encoded localization and ratiometric biosensors, for monitoring the cellular and signaling processes involved in pathogen-induced apoptosis and the resulting destruction of the pathogen. While this study uses biosensors to monitor responses to infection, these approaches can be readily translated to other cellular systems and other fluorescent biosensors.

Interstitial macrophages (IMs) are key regulators of allergic inflammation. We previously showed that the absence of semaphorin 3E (Sema3E) exacerbates asthma features in both acute and chronic asthma models. However, it has not been studied whether Sema3E, via its receptor plexinD1, regulates IM function in allergic asthma. Therefore, we investigated the role of plexinD1 deficiency on IMs in allergic asthma. We found that the absence of plexinD1 in IMs increased airway hyperresponsiveness, airway leukocyte numbers, allergen-specific IgE, goblet cell hyperplasia, and Th2/Th17 cytokine response in the house dust mite (HDM)–induced allergic asthma model. Muc5ac, Muc5b, and α-SMA genes were increased in mice with Plxnd1-deficient IMs compared with wild-type mice. Furthermore, plexinD1-deficient bone marrow–derived macrophages displayed reduced IL-10 mRNA expression, at both the baseline and following HDM challenge, compared with their wild-type counterpart mice. Our data suggest that Sema3E/plexinD1 signaling in IMs is a critical pathway that modulates airway inflammation, airway resistance, and tissue remodeling in the HDM murine model of allergic asthma. Reduced IL-10 expression by plexinD1-deficient macrophages may account for these enhanced allergic asthma features.

Background Oral cholera vaccine (OCV) containing killed Vibrio cholerae O1 and O139 organisms (Bivalent-OCV; Biv-OCV) are playing a central role in global cholera control strategies. OCV is currently administered in a 2-dose regimen (day 0 and 14). There is a growing body of evidence that immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae mediate protection against cholera. There are limited data on anti-OSP responses in recipients of Biv-OCV. We assessed serum antibody responses against O1 OSP, as well as antibody secreting cell (ASC) responses (a surrogate marker for mucosal immunity) and memory B cell responses in blood of adult recipients of Biv-OCV in Dhaka, Bangladesh. Methodology/Principal findings We enrolled 30 healthy adults in this study and administered two doses of OCV (Shanchol) at days 0 and 14. Blood samples were collected before vaccination (day 0) and 7 days after each vaccination (day 7 and day 21), as well as on day 44. Serum responses were largely IgA with minimal IgG and IgM responses in this population. There was no appreciable boosting following day 14 vaccination. There were significant anti-OSP IgA ASC responses on day 7 following the first vaccination, but none after the second immunization. Anti-OSP IgA memory B cell responses were detectable 30 days after completion of the vaccination series, with no evident induction of IgG memory responses. In this population, anti-Ogawa OSP responses were more prominent than anti-Inaba responses, perhaps reflecting impact of previous exposure. Serum anti-OSP responses returned to baseline within 30 days of completing the vaccine series. Conclusion Our results call into question the utility of the 2-dose regimen separated by 14 days in adults in cholera endemic areas, and also suggest that Biv-OCV-induced immune responses targeting OSP are largely IgA in this highly endemic cholera area. Studies in children in cholera-endemic areas need to be performed. Protective efficacy that extends for more than a month after vaccination presumably is mediated by direct mucosal immune response which is not assessed in this study. Our results suggest a single dose of OCV in adults in a cholera endemic zone may be sufficient to mediate at least short-term protection., Author summary Cholera, which can be a severe watery diarrheal illness caused by the non-invasive bacterium Vibrio cholerae, remains a global public health concern for many developing countries. Immune responses targeting the O-specific polysaccharide (OSP) of V. cholerae are involved in mediating protection against cholera. Evidence suggests that inactivated whole-cell oral cholera vaccine (OCV) that includes killed V. cholerae O1 and O139 organisms provides protection against cholera caused by V. cholerae O1, but the immune correlates of this protection are not fully defined. In this study, we specifically assessed induction of immune responses targeting V. cholerae O1 OSP following vaccination with the OCV in adults in a cholera endemic area, Bangladesh. Our results show that OCV induces serum, mucosal and memory anti-OSP responses, and that these responses are maximal following the first dose of vaccination, without evident boosting with the second dose. This result calls into question the day 0 and day 14 two dose regimen currently used among adults in a cholera-endemic zone.

Cholera caused by Vibrio cholerae O1 confers at least 3 to 10 years of protection against subsequent disease regardless of age, despite a relatively rapid fall in antibody levels in peripheral blood, suggesting that memory B cell responses may play an important role in protection. The V. cholerae O1-specific polysaccharide (OSP) component of lipopolysaccharide (LPS) is responsible for serogroup specificity, and it is unclear if young children are capable of developing memory B cell responses against OSP, a T cell-independent antigen, following cholera. To address this, we assessed OSP-specific memory B cell responses in young children (2 to 5 years, n = 11), older children (6 to 17 years, n = 21), and adults (18 to 55 years, n = 28) with cholera caused by V. cholerae O1 in Dhaka, Bangladesh. We also assessed memory B cell responses against LPS and vibriocidal responses, and plasma antibody responses against OSP, LPS, and cholera toxin B subunit (CtxB; a T cell-dependent antigen) on days 2 and 7, as well as days 30, 90, and 180 after convalescence. In all age cohorts, vibriocidal responses and plasma OSP, LPS, and CtxB-specific responses peaked on day 7 and fell toward baseline over the follow-up period. In comparison, we were able to detect OSP memory B cell responses in all age cohorts of patients with detectable responses over baseline for 90 to 180 days. Our results suggest that OSP-specific memory B cell responses can occur following cholera, even in the youngest children, and may explain in part the age-independent induction of long-term immunity following naturally acquired disease.

Background The mediators of protection against cholera, a severe dehydrating illness of humans caused by Vibrio cholerae, are unknown. We have previously shown that plasma IgA as well as memory B IgG cells targeting lipopolysaccharide (LPS) of Vibrio cholerae O1 correlate with protection against V. cholerae O1 infection among household contacts of cholera patients. Protection against cholera is serogroup specific, and serogroup specificity is defined by the O-specific polysaccharide (OSP) component of LPS. Therefore, we prospectively followed household contacts of cholera patients to determine whether OSP-specific immune responses present at the time of enrollment are associated with protection against V. cholerae infection. Methodology In this study, we enrolled two hundred forty two household contacts of one hundred fifty index patients who were infected with Vibrio cholerae. We determined OSP-specific memory B cells and plasma IgA, IgG and IgM antibody responses on study entry (day 2). Principle findings The presence of OSP-specific plasma IgA, IgM, and IgG antibody responses on study entry were associated with a decrease in the risk of infection in household contacts (IgA, p = 0.015; IgM, p = 0.01, and IgG, p = 0.024). In addition, the presence of OSP-specific IgG memory B cell responses in peripheral blood on study entry was also associated with a decreased risk of infection (44% reduction; 95% CI: 31.1 to 99.8) in contacts. No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cell responses. Conclusion These results suggest that immune responses that target OSP, both in plasma and memory responses, may be important in mediating protection against infection with V. cholerae O1., Author summary Vibrio cholerae is a non-invasive pathogen which causes watery diarrheal diseases both in adults and children. Natural infection with Vibrio cholerae provides protection against subsequent diseases and protection against cholera is serogroup specific. Serogroup specificity is defined by O-specific polysaccharide (OSP) of V. cholerae. In this study, we have found that uninfected household contacts had higher baseline OSP-specific plasma IgA, IgG and IgM antibody responses than infected contacts. These observations demonstrate those plasma antibodies responses against OSP are associated with a decrease of the risk of infection of household contacts of cholera patients. We also found that OSP-specific IgG memory B cells are associated with a decrease in the risk of infection in contacts of cholera patients. This result further supports the hypothesis that immune responses targeting V. cholerae OSP is a prime mediator of protection against cholera, and suggests that future work should focus on more detailed analysis of mucosal immune responses targeting OSP, as well as evaluation of potential mechanisms of how antibodies targeting V. cholerae OSP might mediate protection against cholera.

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.

Protective immunity to cholera is serogroup specific, and serogrouping is defined by the O-specific polysaccharide (OSP) of lipopolysaccharide (LPS). We characterized OSP-specific immune responses in adult recipients of an oral killed cholera vaccine (OCV WC-rBS) and compared these with responses in patients with cholera caused by Vibrio cholerae O1 Ogawa. Although vaccinees developed plasma immunoglobulin G (IgG), IgM, IgA antibody and antibody secreting cell (ASC, marker of mucosal response) to Ogawa OSP and LPS 7 days after vaccination, responses were significantly lower than that which occurred after cholera. Similarly, patients recovering from cholera had detectable IgA, IgM, and IgG memory B cell (MBC) responses against OSP and LPS on Day 30 and Day 90, whereas vaccinees only developed IgG responses to OSP 30 days after the second immunization. The markedly lower ASC and MBC responses to OSP and LPS observed among vaccinees might explain, in part, the lower protection of an OCV compared with natural infection.

Current oral cholera vaccines induce lower levels of protective efficacy and shorter durations of protection in young children than in adults. Immunity against cholera is serogroup specific, and immune responses to Vibrio cholerae lipopolysaccharide (LPS), the antigen that mediates serogroup-specific responses, are associated with protection against disease. Despite this, responses against V. cholerae O-specific polysaccharide (OSP), a key component of the LPS responsible for specificity, have not been characterized in children. Here, we report a comparison of polysaccharide antibody responses in children from a region in Bangladesh where cholera is endemic, including infants (6 to 23 months, n = 15), young children (24 to 59 months, n = 14), and older children (5 to 15 years, n = 23) who received two doses of a killed oral cholera vaccine 14 days apart. We found that infants and young children receiving the vaccine did not mount an IgG, IgA, or IgM antibody response to V. cholerae OSP or LPS, whereas older children showed significant responses. In comparison to the vaccinees, young children with wild-type V. cholerae O1 Ogawa infection did mount significant antibody responses against OSP and LPS. We also demonstrated that OSP responses correlated with age in vaccinees, but not in cholera patients, reflecting the ability of even young children with wild-type cholera to develop OSP responses. These differences might contribute to the lower efficacy of protection rendered by vaccination than by wild-type disease in young children and suggest that efforts to improve lipopolysaccharide-specific responses might be critical for achieving optimal cholera vaccine efficacy in this younger age group.

Vibrio cholerae O1 causes cholera, a dehydrating diarrheal disease. We have previously shown that V. cholerae -specific memory B cell responses develop after cholera infection, and we hypothesize that these mediate long-term protective immunity against cholera. We prospectively followed household contacts of cholera patients to determine whether the presence of circulating V. cholerae O1 antigen-specific memory B cells on enrollment was associated with protection against V. cholerae infection over a 30-day period. Two hundred thirty-six household contacts of 122 index patients with cholera were enrolled. The presence of lipopolysaccharide (LPS)-specific IgG memory B cells in peripheral blood on study entry was associated with a 68% decrease in the risk of infection in household contacts ( P = 0.032). No protection was associated with cholera toxin B subunit (CtxB)-specific memory B cells or IgA memory B cells specific to LPS. These results suggest that LPS-specific IgG memory B cells may be important in protection against infection with V. cholerae O1.

Children bear a large component of the global burden of cholera. Despite this, little is known about immune responses to cholera in children, especially those under 5 years of age. Cholera vaccine studies have demonstrated lower long-term protective efficacy in young children than in older children and adults. Memory B cell (MBC) responses may correlate with duration of protection following infection and vaccination. Here we report a comparison of immune responses in young children (3 to 5 years of age; n 17), older children (6 to 17 years of age; n 17), and adults (18 to 60 years of age; n 68) hospitalized with cholera in Dhaka, Bangladesh. We found that young children had lower baseline vibriocidal antibody titers and higher fold increases in titer between day 2 and day 7 than adults. Young children had higher baseline IgG plasma antibody levels to Vibrio cholerae antigens, although the magnitudes of responses at days 7 and 30 were similar across age groups. As a surrogate marker for mucosal immune responses, we assessed day 7 antibody-secreting cell (ASC) responses. These were comparable across age groups, although there was a trend for older age groups to have higher levels of lipopolysaccharide-specific IgA ASC responses. All age groups developed comparable MBC responses to V. cholerae lipopolysaccharide and cholera toxin B subunit at day 30. These findings suggest that young children are able to mount robust vibriocidal, plasma antibody, ASC, and MBC responses against V. cholerae O1, suggesting that under an optimal vaccination strategy, young children could achieve protective efficacy comparable to that induced in adults. Cholera is an acute dehydrating diarrheal disease caused predominantly by the Vibrio cholerae O1 or O139 serogroup. Globally, the vast majority of cholera is caused by V. cholerae O1. Cholera is endemic in over 50 countries and affects 3 to 5 million people each year, causing more than 100,000 deaths (2, 41). In areas of the world in which cholera is endemic, children under 5 years of age have the highest burden of disease (12, 31), and during cholera epidemics, children and adults are both at risk of dehydrating illness (21, 24, 34). In a rural area of Bangladesh in which cholera is endemic, 80% of the population had detectable vibriocidal antibodies by the age of 15 years (26). Similarly, in a recent observational study in urban Bangladesh, household contacts of patients with V. cholerae O1 infection who were 5 years of age had a significantly higher risk of developing infection than older family members in a 21-day observational period following identification of the index household case (15).

Background Multiple infections with diverse enterotoxigenic E. coli (ETEC) strains lead to broad spectrum protection against ETEC diarrhea. However, the precise mechanism of protection against ETEC infection is still unknown. Therefore, memory B cell responses and affinity maturation of antibodies to the specific ETEC antigens might be important to understand the mechanism of protection. Methodology In this study, we investigated the heat labile toxin B subunit (LTB) and colonization factor antigens (CFA/I and CS6) specific IgA and IgG memory B cell responses in Bangladeshi adults (n = 52) who were infected with ETEC. We also investigated the avidity of IgA and IgG antibodies that developed after infection to these antigens. Principal Findings Patients infected with ETEC expressing LT or LT+heat stable toxin (ST) and CFA/I group or CS6 colonization factors developed LTB, CFA/I or CS6 specific memory B cell responses at day 30 after infection. Similarly, these patients developed high avidity IgA and IgG antibodies to LTB, CFA/I or CS6 at day 7 that remained significantly elevated at day 30 when compared to the avidity of these specific antibodies at the acute stage of infection (day 2). The memory B cell responses, antibody avidity and other immune responses to CFA/I not only developed in patients infected with ETEC expressing CFA/I but also in those infected with ETEC expressing CFA/I cross-reacting epitopes. We also detected a significant positive correlation of LTB, CFA/I and CS6 specific memory B cell responses with the corresponding increase in antibody avidity. Conclusion This study demonstrates that natural infection with ETEC induces memory B cells and high avidity antibodies to LTB and colonization factor CFA/I and CS6 antigens that could mediate anamnestic responses on re-exposure to ETEC and may help in understanding the requirements to design an effective vaccination strategies., Author Summary Enterotoxigenic Escherichia coli (ETEC) is a non-invasive pathogen causing diarrhea in children as well as in adults and travelers in developing countries. After colonizing the intestine using colonization factors, the organisms secrete heat-stable (ST) and/or heat-labile (LT) enterotoxin to cause watery diarrhea. Natural infection with ETEC provides protection against subsequent infection; however, the precise mechanism is unknown. In this study, we have shown that adult patients with diarrhea infected with ETEC develop toxin (LTB) and colonization factor (CFA/I and CS6) specific memory B cell responses as well as highly avid antigen-specific antibodies. The antibody avidity indices were shown to be positively associated with memory B cell responses, suggesting that these processes may occur in concert. This study encourages further evaluation of such responses in children as well as in vaccinees.

Immunity againstVibrio choleraeO1 is serogroup specific, and serogrouping is defined by the O-specific polysaccharide (OSP) part of lipopolysaccharide (LPS). Despite this, human immune responses toV. choleraeOSP have not previously been characterized. We assessed immune responses againstV. choleraeOSP in adults with cholera caused byV. choleraeO1 El Tor serotype Inaba or Ogawa in Dhaka, Bangladesh, using O1 OSP-core–bovine serum albumin (OSPc:BSA) conjugates; responses targeted OSP in these conjugates. Responses of Inaba-infected patients to Inaba OSP and LPS increased significantly in IgG, IgM, and IgA isotypes from the acute to convalescent phases of illness, and the responses correlated well between OSP and LPS (R= 0.86, 0.73, and 0.91, respectively;P< 0.01). Plasma IgG, IgM, and IgA responses to Ogawa OSP and LPS in Ogawa-infected patients also correlated well with each other (R= 0.60, 0.60, and 0.92, respectively;P< 0.01). Plasma IgM responses to Inaba OSP and Ogawa OSP correlated with the respective serogroup-specific vibriocidal antibodies (R= 0.80 and 0.66, respectively;P< 0.001). Addition of either OSPc:BSA or LPS, but not BSA, to vibriocidal assays inhibited vibriocidal responses in a comparable and concentration-dependent manner. Mucosal IgA immune responses to OSP and LPS were also similar. Our study is the first to characterize anti-OSP immune responses in patients with cholera and suggests that responses targetingV. choleraeLPS, including vibriocidal responses that correlate with protection against cholera, predominantly target OSP. Induction of anti-OSP responses may be associated with protection against cholera, and our results may support the development of a vaccine targetingV. choleraeOSP.