Antigen structure affects cellular routing through DC-SIGN.

Dendritic cell (DC) lectins mediate the recognition, uptake, and processing of antigens, but they can also be coopted by pathogens for infection. These distinct activities depend upon the routing of antigens within the cell. Antigens directed to endosomal compartments are degraded, and the peptides are presented on major histocompatibility complex class II molecules, thereby promoting immunity. Alternatively, HIV-1 can avoid degradation, as virus engagement with C-type lectin receptors (CLRs), such as DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin) results in trafficking to surface-accessible invaginated pockets. This process appears to enable infection of T cells in trans We sought to explore whether antigen fate upon CLR-mediated internalization was affected by antigen physical properties. To this end, we employed the ring-opening metathesis polymerization to generate glycopolymers that each display multiple copies of mannoside ligand for DC-SIGN, yet differ in length and size. The rate and extent of glycopolymer internalization depended upon polymer structure-longer polymers were internalized more rapidly and more efficiently than were shorter polymers. The trafficking, however, did not differ, and both short and longer polymers colocalized with transferrin-labeled early endosomes. To explore how DC-SIGN directs larger particles, such as pathogens, we induced aggregation of the polymers to access particulate antigens. Strikingly, these particulate antigens were diverted to the invaginated pockets that harbor HIV-1. Thus, antigen structure has a dramatic effect on DC-SIGN-mediated uptake and trafficking. These findings have consequences for the design of synthetic vaccines. Additionally, the results suggest strategies for targeting DC reservoirs that harbor viral pathogens.

Modular Polymer Antigens To Optimize Immunity.

Subunit vaccines can have excellent safety profiles, but their ability to give rise to robust immune responses is often compromised. For glycan-based vaccines, insufficient understanding of B and T cell epitope combinations that yield optimal immune activation hinders optimization. To determine which antigen features promote desired IgG responses, we synthesized epitope-functionalized polymers using ring-opening metathesis polymerization (ROMP) and assessed the effect of B and T cell epitope loading. The most robust responses were induced by polymers with a high valency of B and T cell epitopes. Additionally, IgG responses were greater for polymers with T cell epitopes that are readily liberated upon endosomal processing. Combining these criteria, we used ROMP to generate a nontoxic, polymeric antigen that elicited stronger antibody responses than a comparable protein conjugate. These findings highlight principles for designing synthetic antigens that elicit strong IgG responses against inherently weak immune targets such as glycans.

CH-π Interactions Are Required for Human Galectin-3 Function.

Glycan-binding proteins, or lectins, recognize distinct structural elements of polysaccharides, to mediate myriad biological functions. Targeting glycan-binding proteins involved in human disease has been challenging due to an incomplete understanding of the molecular mechanisms that govern protein-glycan interactions. Bioinformatics and structural studies of glycan-binding proteins indicate that aromatic residues with the potential for CH-π interactions are prevalent in glycan-binding sites. However, the contributions of these CH-π interactions to glycan binding and their relevance in downstream function remain unclear. An emblematic lectin, human galectin-3, recognizes lactose and N-acetyllactosamine-containing glycans by positioning the electropositive face of a galactose residue over the tryptophan 181 (W181) indole forming a CH-π interaction. We generated a suite of galectin-3 W181 variants to assess the importance of these CH-π interactions to glycan binding and function. As determined experimentally and further validated with computational modeling, variants with smaller or less electron-rich aromatic side chains (W181Y, W181F, W181H) or sterically similar but nonaromatic residues (W181M, W181R) showed poor or undetectable binding to lactose and attenuated ability to bind mucins or agglutinate red blood cells. The latter functions depend on multivalent binding, highlighting that weakened CH-π interactions cannot be overcome by avidity. Two galectin-3 variants with disrupted hydrogen bonding interactions (H158A and E184A) showed similarly impaired lactose binding. Molecular simulations demonstrate that all variants have decreased binding orientation stability relative to native galectin-3. Thus, W181 collaborates with the endogenous hydrogen bonding network to enhance binding affinity for lactose, and abrogation of these CH-π interactions is as deleterious as eliminating key hydrogen bonding interactions. These findings underscore the critical roles of CH-π interactions in carbohydrate binding and lectin function and will aid the development of novel lectin inhibitors.

Glycan-costumed virus-like particles promote type 1 anti-tumor immunity.

Cancer vaccine development is inhibited by a lack of strategies for directing dendritic cell (DC) induction of effective tumor-specific cellular immunity. Pathogen engagement of DC lectins and toll-like receptors (TLRs) shapes immunity by directing T cell function. Strategies to activate specific DC signaling pathways via targeted receptor engagement are crucial to unlocking type 1 cellular immunity. Here, we engineered a glycan-costumed virus-like particle (VLP) vaccine that delivers programmable peptide antigens to induce tumor-specific cellular immunity in vivo. VLPs encapsulating TLR7 agonists and decorated with a selective mannose-derived ligand for the lectin DC-SIGN induced robust DC activation and type 1 cellular immunity, whereas VLPs lacking this key DC-SIGN ligand failed to promote DC-mediated immunity. Vaccination with glycan-costumed VLPs generated tumor antigen-specific Th1 CD4+ and CD8+ T cells that infiltrated solid tumors, inhibiting tumor growth in a murine melanoma model. Thus, VLPs employing lectin-driven immune reprogramming provide a framework for advancing cancer immunotherapies.

Inhibition of LILRB2 by a Novel Blocking Antibody Designed to Reprogram Immunosuppressive Macrophages to Drive T-Cell Activation in Tumors.

Tumor-associated macrophages (TAM) play an important role in maintaining the immunosuppressive state of the tumor microenvironment (TME). High levels of CD163+ TAMs specifically are associated with poor prognosis in many solid tumor types. Targeting TAMs may represent a key approach in development of the next generation of cancer immune therapeutics. Members of the leukocyte immunoglobulin-like receptor B (LILRB) family, including LILRB2 (ILT4), are known to transmit inhibitory signals in macrophages and other myeloid cells. Leveraging bulk and single cell RNA-sequencing datasets, as well as extensive immunophenotyping of human tumors, we found that LILRB2 is highly expressed on CD163+ CD11b+ cells in the TME and that LILRB2 expression correlates with CD163 expression across many tumor types. To target LILRB2, we have developed JTX-8064, a highly potent and selective antagonistic mAb. JTX-8064 blocks LILRB2 binding to its cognate ligands, including classical and nonclassical MHC molecules. In vitro, JTX-8064 drives the polarization of human macrophages and dendritic cells toward an immunostimulatory phenotype. As a result, human macrophages treated with a LILRB2 blocker are reprogrammed to increase the activation of autologous T cells in co-culture systems. Furthermore, JTX-8064 significantly potentiates the activity of anti-PD-1 in allogeneic mixed lymphocyte reaction. In a human tumor explant culture, pharmacodynamic activity of JTX-8064 was observed in monotherapy and in combination with anti-PD-1. Collectively, our work provides strong translational and preclinical rationale to target LILRB2 in cancer.

Simple, direct conjugation of bacterial O-SP-core antigens to proteins: development of cholera conjugate vaccines.

Bacterial O-SP-core antigens can be conjugated to proteins in the same, simple way as synthetic, linker-equipped carbohydrates by applying squaric acid chemistry. Introduction of spacers (linkers) to either O-SP-core antigens or protein carriers, which is involved in commonly applied protocols, is not required. The newly developed method described here consists of preparation of a squaric acid monoester derivative of O-SP-core antigen, utilizing the amino group inherent in the core, and reaction of the monoester with the carrier protein. The intermediate monoester can be easily purified; its conjugation can be monitored by SELDI-TOF mass spectrometry and, thus, readily controlled, since the conjugation can be terminated when the desired carbohydrate-protein ratio is reached. Here, we describe production of conjugates containing the O-SP-core antigen of Vibrio cholerae O1, the major cause of cholera, a severe dehydrating diarrheal disease of humans. The resultant products are recognized by convalescent phase sera from patients recovering from cholera in Bangladesh, and anti-O-SP-core-protein responses correlate with plasma antilipopolysaccharide and vibriocidal responses, which are the primary markers of protection from cholera. The results suggest that such conjugates have potential as vaccines for cholera and other bacterial diseases.

Glycan-Modified Virus-like Particles Evoke T Helper Type 1-like Immune Responses.

Dendritic cells (DCs) are highly effective antigen-presenting cells that shape immune responses. Vaccines that deliver antigen to the DCs can harness their power. DC surface lectins recognize glycans not typically present on host tissue to facilitate antigen uptake and presentation. Vaccines that target these surface lectins should offer improved antigen delivery, but their efficacy will depend on how lectin targeting influences the T cell subtypes that result. We examined how antigen structure influences uptake and signaling from the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin or CD209). Virus-like particles (VLPs) were engineered from bacteriophage Qß to present an array of mannoside ligands. The VLPs were taken up by DCs and efficiently trafficked to endosomes. The signaling that ensued depended on the ligand displayed on the VLP: only those particles densely functionalized with an aryl mannoside, Qß-Man540, elicited DC maturation and induced the expression of the proinflammatory cytokines characteristic of a T helper type 1 (TH1)-like immune response. This effect was traced to differential binding to DC-SIGN at the acidic pH of the endosome. Mice immunized with a VLP bearing the aryl mannoside, and a peptide antigen (Qß-Ova-Man540) had antigen-specific responses, including the production of CD4+ T cells producing the activating cytokines interferon-γ and tumor necrosis factor-α. A TH1 response is critical for intracellular pathogens (e.g., viruses) and cancer; thus, our data highlight the value of targeting DC lectins for antigen delivery and validate the utility of DC-targeted VLPs as vaccine vehicles that induce cellular immunity.

Development of immunoglobulin M memory to both a T-cell-independent and a T-cell-dependent antigen following infection with Vibrio cholerae O1 in Bangladesh.

Vibrio cholerae O1 can cause severe watery diarrhea that can be life-threatening without treatment. Infection results in long-lasting protection against subsequent disease. Development of memory B cells of the immunoglobulin G (IgG) and IgA isotypes to V. cholerae O1 antigens, including serotype-specific lipopolysaccharide (LPS) and the B subunit of cholera toxin (CTB), after cholera infection has been demonstrated. Memory B cells of the IgM isotype may play a role in long-term protection, particularly against T-cell-independent antigens, but IgM memory has not been studied in V. cholerae O1 infection. Therefore, we assayed acute- and convalescent-phase blood samples from cholera patients for the presence of memory B cells that produce cholera antigen-specific IgM antibody upon polyclonal stimulation in in vitro culture. We also examined the development of serological and antibody-secreting cell responses following infection. Subjects developed significant IgM memory responses by day 30 after infection, both to the T-cell-independent antigen LPS and to the T-cell-dependent antigen CTB. No significant corresponding elevations in plasma IgM antibodies or circulating IgM antibody-secreting cells to CTB were detected. In 17 subjects followed to day 90 after infection, significant persistence of elevated IgM memory responses was not observed. The IgM memory response to CTB was negatively correlated with the IgG plasma antibody response to CTB, and there was a trend toward negative correlation between the IgM memory and IgA plasma antibody responses to LPS. We did not observe an association between the IgM memory response to LPS and the vibriocidal titer.

Concomitant enterotoxigenic Escherichia coli infection induces increased immune responses to Vibrio cholerae O1 antigens in patients with cholera in Bangladesh.

Vibrio cholerae O1 and enterotoxigenic Escherichia coli (ETEC) are major bacterial pathogens that cause dehydrating disease requiring hospitalization of children and adults. The cholera toxin (CT) produced by V. cholerae O1 and the heat-labile toxin (LT) and/or heat-stable toxin (ST) of ETEC are responsible for secretory diarrhea. We have observed that about 13% of hospitalized diarrheal patients are concomitantly infected with V. cholerae O1 and ETEC. In order to understand the outcome of such dual infections on the clinical and immunological responses in cholera patients, we studied patients infected with V. cholerae O1 (group VC; n = 25), those infected with both V. cholerae O1 and ETEC (group VCET; n = 25), and those infected with ETEC only (group ET; n = 25). The VCET group showed more severe dehydration and had a higher intake of intravenous fluid and more vomiting than the ETEC group (P = 0.01 to 0.003). The VCET patients showed higher vibriocidal responses and increased antibody titers to cholera toxin and lipopolysaccharide (LPS) in plasma than did the V. cholerae O1 patients (P = 0.02 to <0.001). All responses in the V. cholerae O1 and in the VCET groups were more robust than those seen in the group infected with ETEC only (P = 0.01 to <0.001). We thus show that concomitant colonization with ETEC induces immune responses to V. cholerae antigens that are more robust than those seen with V. cholerae O1 infection alone. It is possible that LT or other factors expressed by ETEC may play a role as a mucosal adjuvant in enhancing the immune responses to V. cholerae O1.

Zinc influences innate immune responses in children with enterotoxigenic Escherichia coli-induced diarrhea.

Information is limited on the effect of zinc on immune responses in children with diarrhea due to enterotoxigenic Escherichia coli (ETEC), the most common bacterial pathogen in children. We studied the immunological effect of zinc treatment (20 mg/d) and supplementation (10 mg/d) in children with diarrhea due to ETEC. A total of 148 children aged 6-24 mo were followed up for 9 mo after a 10-d zinc treatment (ZT; n = 74) or a 10-d zinc treatment plus 3-mo supplementation (ZT+S; n = 74), as well as 50 children with ETEC-induced diarrhea that were not treated with zinc (UT). Fifty control children (HC) of the same age group from the same location were also studied. Serum zinc concentrations were higher in both the ZT (P < 0.001) and ZT+S groups (P < 0.001) than in the UT group but did not differ from the HC group. We found higher serum complement C3 immediately after zinc administration in both ZT (P < 0.001) and ZT+S (P < 0.001) groups than in the UT group. Phagocytic activity in children in both ZT (P < 0.01) and ZT+S (P < 0.01) groups was greater than in the UT group. However, oxidative burst capacity was lower in zinc-receiving groups (ZT, P < 0.001 and ZT+S, P < 0.001) than in the UT group. The naïve:memory T cell ratio in both ZT (P < 0.05) and ZT+S (P < 0.01) groups was higher than in the UT group from d 2 to 15. Increased responses, including complement C3, phagocytic activity, and changes in T cell phenotypes, suggest that zinc administration enhances innate immunity against ETEC infection in children.

A Cholera Conjugate Vaccine Containing O-specific Polysaccharide (OSP) of V. cholerae O1 Inaba and Recombinant Fragment of Tetanus Toxin Heavy Chain (OSP:rTTHc) Induces Serum, Memory and Lamina Proprial Responses against OSP and Is Protective in Mice.

BACKGROUND: Vibrio cholerae is the cause of cholera, a severe watery diarrhea. Protection against cholera is serogroup specific. Serogroup specificity is defined by the O-specific polysaccharide (OSP) component of lipopolysaccharide (LPS). METHODOLOGY: Here we describe a conjugate vaccine for cholera prepared via squaric acid chemistry from the OSP of V. cholerae O1 Inaba strain PIC018 and a recombinant heavy chain fragment of tetanus toxin (OSP:rTTHc). We assessed a range of vaccine doses based on the OSP content of the vaccine (10-50 µg), vaccine compositions varying by molar loading ratio of OSP to rTTHc (3:1, 5:1, 10:1), effect of an adjuvant, and route of immunization. PRINCIPLE FINDINGS: Immunized mice developed prominent anti-OSP and anti-TT serum IgG responses, as well as vibriocidal antibody and memory B cell responses following intramuscular or intradermal vaccination. Mice did not develop anti-squarate responses. Intestinal lamina proprial IgA responses targeting OSP occurred following intradermal vaccination. In general, we found comparable immune responses in mice immunized with these variations, although memory B cell and vibriocidal responses were blunted in mice receiving the highest dose of vaccine (50 µg). We found no appreciable change in immune responses when the conjugate vaccine was administered in the presence or absence of immunoadjuvant alum. Administration of OSP:rTTHc resulted in 55% protective efficacy in a mouse survival cholera challenge model. CONCLUSION: We report development of an Inaba OSP:rTTHc conjugate vaccine that induces memory responses and protection against cholera in mice. Development of an effective cholera conjugate vaccine that induces high level and long-term immune responses against OSP would be beneficial, especially in young children who respond poorly to polysaccharide antigens.

In silico predicted mycobacterial epitope elicits in vitro T-cell responses.

Epitope-based vaccines permit the selection of only a specific subset of epitopes to induce the necessary immune response, thus providing a rational alternative to conventional design approaches. Using a range of immunoinformatics tools, we identified a novel, contiguous 28 amino acid multi-epitope cluster within the highly conserved secretory protein Ag85B of Mycobacterium tuberculosis, the causative agent of TB. This cluster, named Ep85B, is composed of epitopes which bind to three HLA Class I and 15 Class II molecules, and harbors the potential to generate 99% population coverage in TB-endemic regions. We experimentally evaluated the capacity of Ep85B to elicit T-cell immune responses using whole blood cells and, as predicted, observed significant increases in populations of both CD4+ and memory CD4+ CD45RO+ T-cells. Our results demonstrate the practical utility of an epitope-based design methodology - a strategy that, following further evaluation, may serve as an additional tool for the development of novel vaccine candidates against TB and other diseases.

Antigen-specific memory B-cell responses to enterotoxigenic Escherichia coli infection in Bangladeshi adults.

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.

Evaluation in mice of a conjugate vaccine for cholera made from Vibrio cholerae O1 (Ogawa) O-specific polysaccharide.

BACKGROUND: Protective immunity against cholera is serogroup specific. Serogroup specificity in Vibrio cholerae is determined by the O-specific polysaccharide (OSP) of lipopolysaccharide (LPS). Generally, polysaccharides are poorly immunogenic, especially in young children. METHODOLOGY: Here we report the evaluation in mice of a conjugate vaccine for cholera (OSP:TThc) made from V. cholerae O1 Ogawa O-Specific Polysaccharide-core (OSP) and recombinant tetanus toxoid heavy chain fragment (TThc). We immunized mice intramuscularly on days 0, 21, and 42 with OSP:TThc or OSP only, with or without dmLT, a non-toxigenic immunoadjuvant derived from heat labile toxin of Escherichia coli. PRINCIPAL FINDINGS: We detected significant serum IgG antibody responses targeting OSP following a single immunization in mice receiving OSP:TThc with or without adjuvant. Anti-LPS IgG responses were detected following a second immunization in these cohorts. No anti-OSP or anti-LPS IgG responses were detected at any time in animals receiving un-conjugated OSP with or without immunoadjuvant, and in animals receiving immunoadjuvant alone. Responses were highest following immunization with adjuvant. Serum anti-OSP IgM responses were detected in mice receiving OSP:TThc with or without immunoadjuvant, and in mice receiving unconjugated OSP. Serum anti-LPS IgM and vibriocidal responses were detected in all vaccine cohorts except in mice receiving immunoadjuvant alone. No significant IgA anti-OSP or anti-LPS responses developed in any group. Administration of OSP:TThc and adjuvant also induced memory B cell responses targeting OSP and resulted in 95% protective efficacy in a mouse lethality cholera challenge model. CONCLUSION: We describe a protectively immunogenic cholera conjugate in mice. Development of a cholera conjugate vaccine could assist in inducing long-term protective immunity, especially in young children who respond poorly to polysaccharide antigens.

Immune responses to O-specific polysaccharide and lipopolysaccharide of Vibrio cholerae O1 Ogawa in adult Bangladeshi recipients of an oral killed cholera vaccine and comparison to responses in patients with cholera.

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.

Study of avidity of antigen-specific antibody as a means of understanding development of long-term immunological memory after Vibrio cholerae O1 infection.

The avidity of antibodies to specific antigens and the relationship of avidity to memory B cell responses to these antigens have not been studied in patients with cholera or those receiving oral cholera vaccines. We measured the avidity of antibodies to cholera toxin B subunit (CTB) and Vibrio cholerae O1 lipopolysaccharide (LPS) in Bangladeshi adult cholera patients (n = 30), as well as vaccinees (n = 30) after administration of two doses of a killed oral cholera vaccine. We assessed antibody and memory B cell responses at the acute stage in patients or prior to vaccination in vaccinees and then in follow-up over a year. Both patients and vaccinees mounted CTB-specific IgG and IgA antibodies of high avidity. Patients showed longer persistence of these antibodies than vaccinees, with persistence lasting in patients up to day 270 to 360. The avidity of LPS-specific IgG and IgA antibodies in patients remained elevated up to 180 days of follow-up. Vaccinees mounted highly avid LPS-specific antibodies at day 17 (3 days after the second dose of vaccine), but the avidity waned rapidly to baseline by 30 days. We examined the correlation between antigen-specific memory B cell responses and avidity indices for both antigens. We found that numbers of CTB- and LPS-specific memory B cells significantly correlated with the avidity indices of the corresponding antibodies (P < 0.05; Spearman's ρ = 0.28 to 0.45). These findings suggest that antibody avidity after infection and immunization is a good correlate of the development and maintenance of memory B cell responses to Vibrio cholerae O1 antigens.

Vaccine specific immune response to an inactivated oral cholera vaccine and EPI vaccines in a high and low arsenic area in Bangladeshi children.

BACKGROUND: Immune responses to the inactivated oral whole cell cholera toxin B (CTB) subunit cholera vaccine, Dukoral(®), as well as three childhood vaccines in the national immunization system were compared in children living in high and low arsenic contaminated areas in Bangladesh. In addition, serum complement factors C3 and C4 levels were evaluated among children in the two areas. VACCINATIONS: Toddlers (2-5 years) were orally immunized with two doses of Dukoral 14 days apart. Study participants had also received diphtheria, tetanus and measles vaccines according to the Expanded Program on Immunization (EPI) in Bangladesh. RESULTS: The mean level of arsenic in the urine specimens in the children of the high arsenic area (HAA, Shahrasti, Chandpur) was 291.8µg/L while the level was 6.60µg/L in the low arsenic area (LAA, Mirpur, Dhaka). Cholera specific vibriocidal antibody responses were significantly increased in the HAA (87%, P<0.001) and the LAA (75%, P<0.001) children after vaccination with Dukoral, but no differences were found between the two groups. Levels of CTB specific IgA and IgG antibodies were comparable between the two groups, whereas LPS specific IgA and IgG were higher in the LAA group, although response rates were comparable. Diphtheria and tetanus vaccine specific IgG responses were significantly higher in the HAA compared to the LAA group (P<0.001, P=0.048 respectively), whereas there were no differences in the measles specific IgG responses between the groups. Complement C3 and C4 levels in sera were higher in participants from the HAA than the LAA groups (P<0.001, P=0.049 respectively). CONCLUSIONS: The study demonstrates that the oral cholera vaccine as well as the EPI vaccines studied are immunogenic in children in high and low arsenic areas in Bangladesh. The results are encouraging for the potential use of cholera vaccines as well as the EPI vaccines in arsenic endemic areas.

Antibody avidity in humoral immune responses in Bangladeshi children and adults following administration of an oral killed cholera vaccine.

Antibody avidity for antigens following disease or vaccination increases with affinity maturation and somatic hypermutation. In this study, we followed children and adults in Bangladesh for 1 year following oral cholera vaccination and measured the avidity of antibodies to the T cell-dependent antigen cholera toxin B subunit (CTB) and the T cell-independent antigen lipopolysaccharide (LPS) in comparison with responses in other immunological measurements. Children produced CTB-specific IgG and IgA antibodies of high avidity following vaccination, which persisted for several months; the magnitudes of responses were comparable to those seen in adult vaccinees. The avidity of LPS-specific IgG and IgA antibodies in vaccinees increased significantly shortly after the second dose of vaccine but waned rapidly to baseline levels thereafter. CTB-specific memory B cells were present for only a short time following vaccination, and we did not find significant memory B cell responses to LPS in any age group. For older children, there was a significant correlation between CTB-specific memory T cell responses after the second dose of vaccine and CTB-specific IgG antibody avidity indices over the subsequent year. These findings suggest that vaccination induces a longer-lasting increase in the avidity of antibodies to a T cell-dependent antigen than is measured by a memory B cell response to that antigen and that early memory T cell responses correlate well with the subsequent development of higher-avidity antibodies.

Identification of immunogenic Salmonella enterica serotype Typhi antigens expressed in chronic biliary carriers of S. Typhi in Kathmandu, Nepal.

BACKGROUND: Salmonella enterica serotype Typhi can colonize and persist in the biliary tract of infected individuals, resulting in a state of asymptomatic chronic carriage. Chronic carriers may act as persistent reservoirs of infection within a community and may introduce infection to susceptible individuals and new communities. Little is known about the interaction between the host and pathogen in the biliary tract of chronic carriers, and there is currently no reliable diagnostic assay to identify asymptomatic S. Typhi carriage. METHODOLOGY/PRINCIPAL FINDINGS: To study host-pathogen interactions in the biliary tract during S. Typhi carriage, we applied an immunoscreening technique called in vivo-induced antigen technology (IVIAT), to identify potential biomarkers unique to carriers. IVIAT identifies humorally immunogenic bacterial antigens expressed uniquely in the in vivo environment, and we hypothesized that S. Typhi surviving in the biliary tract of humans may express a distinct antigenic profile. Thirteen S. Typhi antigens that were immunoreactive in carriers, but not in healthy individuals from a typhoid endemic area, were identified. The identified antigens included a number of putative membrane proteins, lipoproteins, and hemolysin-related proteins. YncE (STY1479), an uncharacterized protein with an ATP-binding motif, gave prominent responses in our screen. The response to YncE in patients whose biliary tract contained S. Typhi was compared to responses in patients whose biliary tract did not contain S. Typhi, patients with acute typhoid fever, and healthy controls residing in a typhoid endemic area. Seven of 10 (70%) chronic carriers, 0 of 8 bile culture-negative controls (0%), 0 of 8 healthy Bangladeshis (0%), and 1 of 8 (12.5%) Bangladeshis with acute typhoid fever had detectable anti-YncE IgG in blood. IgA responses were also present. CONCLUSIONS/SIGNIFICANCE: Further evaluation of YncE and other antigens identified by IVIAT could lead to the development of improved diagnostic assays to identify asymptomatic S. Typhi carriers.

Immune responses to the O-specific polysaccharide antigen in children who received a killed oral cholera vaccine compared to responses following natural cholera infection in Bangladesh.

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.