Cholera intoxication of human enteroids reveals interplay between decoy and functional glycoconjugate ligands.

Prior research on cholera toxin (CT) binding and intoxication has relied on human colonic cancer derived epithelial cells. While these transformed cell lines have been beneficial, they neither derive from small intestine where intoxication occurs, nor represent the diversity of small intestinal epithelial cells (SI-ECs) and variation in glycoconjugate expression among individuals. Here, we used human enteroids, derived from jejunal biopsies of multipledonors to study CT binding and intoxication of human non-transformed SI-ECs. We modulated surface expression of glycosphingolipids, glycoproteins and specific glycans to distinguish the role of each glycan/glycoconjugate. Cholera-toxin-subunit-B (CTB) mutants were generated to decipher the preference of each glycoconjugate to different binding sites and the correlation between CT binding and intoxication. Human enteroids contain trace amounts of GM1, but other glycosphingolipids may be contributing to CT intoxication. We discovered that inhibition of either fucosylation or O-glycosylation sensitize enteroids to CT-intoxication. This can either be a consequence of the removal of fucosylated "decoy-like-ligands" binding to CTB's non-canonical site and/or increase in the availability of Gal/GalNAc-terminating glycoconjugates binding to the canonical site. Furthermore, simultaneous inhibition of fucosylation and O-glycosylation increased the availability of additional Gal/GalNAc-terminating glycoconjugates but counteracted the sensitization in CT intoxication caused by inhibiting O-glycosylation because of reduction in fucose. This implies a dual role of fucose as a functional glycan and a decoy, the interplay of which influences CT binding and intoxication. Finally, while the results were similar for enteroids from different donors, they were not identical, pointing to a role for human genetic variation in determining sensitivity to CT.

A thermostable, dry formulation inactivated Hikojima whole cell/cholera toxin B subunit oral cholera vaccine.

The feared diarrheal disease cholera remains an important global health problem. Use of oral cholera vaccine (OCV) from a global stockpile against both epidemic and endemic cholera is a cornerstone in the World Health Organisations (WHOs) global program for "Ending cholera by 2030". Three liquid inactivated whole-cell OCVs (Dukoral®, ShancholTM, and Euvichol-Plus®) are WHO prequalified and have proved to be safe and effective. However, their multicomponent composition and cold-chain requirement increase manufacturing, storage and transport costs. ShancholTM and Euvichol-Plus® OCVs used in WHOs global vaccine stockpile also lack the protective cholera toxin B-subunit (CTB) antigen present in Dukoral®, which results in suboptimal efficacy. WHOs Global Task Force on Cholera Control (GTFCC) has identified a thermostable, dry formulation vaccine as a priority for further OCV development. We describe here the development of such a vaccine, based on a lyophilized mixture of a single strain of formalin-killed Hikojima bacteria together with a low-cost, recombinantly produced CTB. The new vaccine, which is easy and inexpensive to manufacture, could be stored for at least 26 months at 25 °C and for at least 8 months at 40 °C with preservation of cell morphology and with no loss of protective Ogawa and Inaba lipopolysaccharides or CTB. It also proved to be well tolerated and to have equivalent oral immunogenicity in mice as ShancholTM and Dukoral® OCVs with regard to both serum and intestinal-mucosal antibody responses.

Bacterial travellers' diarrhoea: A narrative review of literature published over the past 10 years.

Travellers' diarrhoea (TD) is the most frequent illness experienced by international travellers to lower-income countries with bacterial agents considered to account for 80-90% of cases. In this review, we summarise evidence published on bacterial TD over the past 10 years, focusing on the epidemiology and aetiology of TD. Diarrhoeagenic Escherichia coli (DEC) continue to be the most commonly implicated bacteria in TD, although Enteropathogenic E. coli (EPEC) and Enteroaggregative E. coli (EAEC) now appear to be predominant where Enterotoxigenic E. coli (ETEC) was previously considered most prevalent globally. Where fluroquinolone resistance had primarily been documented for Campylobacter in Southeast Asia, widespread resistance has been observed in most regions of the world for multiple enteropathogens, including Shigella, Salmonella, ETEC and EAEC. Implementation of novel molecular methods for pathogen detection has led to identification of bacterial pathogens, including Clostridium difficile (with and without the use of prior antibiotics), Arcobacter species and Bacteroides fragilis, as aetiological agents in TD. The widespread resistance to first-line antibiotics in multiple bacterial enteropathogens warrants continued surveillance and re-evaluation of current treatment practices. Further investigations are required to determine the prevalence and geographical distribution of bacterial enteropathogens that have been more recently implicated in TD.

Characterization of the ganglioside recognition profile of Escherichia coli heat-labile enterotoxin LT-IIc.

The heat-labile enterotoxins of Escherichia coli and cholera toxin of Vibrio cholerae are related in structure and function. Each of these oligomeric toxins is comprised of one A polypeptide and five B polypeptides. The B-subunits bind to gangliosides, which are followed by uptake into the intoxicated cell and activation of the host's adenylate cyclase by the A-subunits. There are two antigenically distinct groups of these toxins. Group I includes cholera toxin and type I heat-labile enterotoxin of E. coli; group II contains the type II heat-labile enterotoxins of E. coli. Three variants of type II toxins, designated LT-IIa, LT-IIb and LT-IIc have been described. Earlier studies revealed the crystalline structure of LT-IIb. Herein the carbohydrate binding specificity of LT-IIc B-subunits was investigated by glycosphingolipid binding studies on thin-layer chromatograms and in microtiter wells. Binding studies using a large variety of glycosphingolipids showed that LT-IIc binds with high affinity to gangliosides with a terminal Neu5Acα3Gal or Neu5Gcα3Gal, e.g. the gangliosides GM3, GD1a and Neu5Acα3-/Neu5Gcα3--neolactotetraosylceramide and Neu5Acα3-/Neu5Gcα3-neolactohexaosylceramide. The crystal structure of LT-IIc B-subunits alone and with bound LSTd/sialyl-lacto-N-neotetraose d pentasaccharide uncovered the molecular basis of the ganglioside recognition. These studies revealed common and unique functional structures of the type II family of heat-labile enterotoxins.

A phase I/II study to evaluate safety, tolerability and immunogenicity of Hillchol®, an inactivated single Hikojima strain based oral cholera vaccine, in a sequentially age descending population in Bangladesh.

BACKGROUND: The World Health Organization (WHO) recommends the use of oral cholera vaccines (OCVs) as part of an integrated control program, both in highly endemic settings and during cholera epidemics. The available and internationally recommended WHO-prequalified OCVs (Dukoral, Shanchol, Euvichol) contain multiple heat and formalin-killed V. cholerae strains of Inaba and Ogawa serotypes. MSD Wellcome Trust Hilleman Laboratories Pvt. Ltd. in technical collaboration with University of Gothenburg, Sweden has developed a new single strain OCV, Hillchol. This vaccine consists of formaldehyde-inactivated whole cell El Tor V. cholerae O1 bacteria engineered into the Hikojima serotype for stable expression of both the Ogawa (AB) and Inaba (AC) LPS antigens on the bacterial surface. We evaluated the safety and immunogenicity of this novel and potentially much less expensive OCV in comparison with Shanchol. METHODS: We conducted a randomized, non-inferiority, age-descending clinical trial of OCV (Hillchol vs. Shanchol) in the Mirpur area of Dhaka city from July 2016 to May 2017. This study was carried out in three different age cohorts (1-<5, 5-17 and ≥18 years old). Two doses of vaccine were given at 14 days intervals to 560 healthy participants. FINDINGS: No serious adverse events were reported. There were no significant differences in the rates of adverse events between the test vaccine (Hillchol) and the comparator (Shanchol) group. Serum vibriocidal antibody responses in all age groups combined were comparable for all the O1 Ogawa (59% vs. 67%; 90% CI of difference: -14.55, -0.84) and Inaba (70% vs. 71%; 90% CI of difference: -7.24, 5.77) serotypes, showing that the Hillchol vaccine was non-inferior to Shanchol. This new vaccine was also non-inferior to Shanchol in the different age strata. CONCLUSION: The safety and immunogenicity profile of the new OCV Hillchol is comparable to Shanchol in persons residing in a cholera-endemic setting. ClinicalTrials.gov number: NCT02823899.

Immune responses against oxidized LDL as possible targets for prevention of atherosclerosis in systemic lupus erythematosus.

Patients suffering from systemic lupus erythematosus (SLE) are at increased risk of developing cardiovascular disease (CVD) and traditional therapies including statins provide insufficient protection. Impaired removal of apoptotic material is a common pathogenic mechanism in both SLE and atherosclerosis and is considered to be a key factor in the development of autoimmunity. Since oxidized LDL and apoptotic material bind to the same receptors, we aimed to investigate if targeting the oxidized LDL autoimmunity can affect atherosclerosis in SLE. To investigate the possible role of oxidized LDL autoimmunity in the accelerated atherosclerosis associated with SLE we used a hypercholesterolemic SLE mouse model (B6.lpr.ApoE-/- mice). Promoting LDL tolerance through mucosal immunization with an apolipoprotein B-100 peptide p45 (amino acids 661-680) and cholera toxin B-subunit fusion protein increased regulatory T cells and B cells in mesenteric lymph nodes and reduced plaque development in the aorta by 33%. Treatment with the oxidized LDL-specific antibody Orticumab reduced aortic atherosclerosis by 43%, subvalvular plaque area by 50% and the macrophage content by 31%. The present study provides support for oxLDL as a possible target for prevention of cardiovascular complications in SLE.

Development of Hillchol®, a low-cost inactivated single strain Hikojima oral cholera vaccine.

Cholera remains an important global health problem with up to 4 million cases and 140,000 deaths annually. Oral cholera vaccines (OCVs) are now a cornerstone of the WHOs "Ending Cholera - A Global Roadmap to 2030" global program for the eventual elimination of cholera. There are currently three WHO prequalified OCVs available, Dukoral®, Shanchol® and Euvichol-Plus®. These vaccines are effective but due to a multiple strain composition and two different methods of inactivation, are complex and costly to manufacture. We describe here the characterization and industrial scale development of Hillchol®; a novel, likely affordable single-component OCV for low and middle-income countries. Hillchol® consists of formalin-inactivated bacteria of a stable recombinant Vibrio cholerae O1 El Tor Hikojima serotype strain expressing approximately 50% each of Ogawa and Inaba O1 LPS antigens. The novel OCV can be manufactured on an industrial scale at a low cost. Hillchol® was well tolerated in animal toxicology studies and shown to have non-inferior oral immunogenicity in mice for both intestinal-mucosal and serological immune responses when compared with a WHO-prequalified OCV. The optimized production of this single component OCV will reduce cost of OCV production and thus substantially increase vaccine availability. Based on these results, Hillchol® has been produced at a GMP facility and used successfully for clinical phase I/II studies.

A New Strategy Toward B Cell-Based Cancer Vaccines by Active Immunization With Mimotopes of Immune Checkpoint Inhibitors.

Therapeutic monoclonal antibodies (mAbs), targeting tumor antigens, or immune checkpoints, have demonstrated a remarkable anti-tumor effect against various malignancies. However, high costs for mono- or combination therapies, associated with adverse effects or possible development of resistance in some patients, warrant further development and modification to gain more flexibility for this immunotherapy approach. An attractive alternative to passive immunization with therapeutic antibodies might be active immunization with mimotopes (B-cell peptides) representing the mAbs' binding epitopes, to activate the patient's own anti-tumor immune response following immunization. Here, we identified and examined the feasibility of inducing anti-tumor effects in vivo following active immunization with a mimotope of the immune checkpoint programmed cell death 1 (PD1), alone or in combination with a Her-2/neu B-cell peptide vaccine. Overlapping peptides spanning the extracellular domains of human PD1 (hPD1) were used to identify hPD1-derived mimotopes, using the therapeutic mAb Nivolumab as a proof of concept. Additionally, for in vivo evaluation in a tumor mouse model, a mouse PD1 (mPD1)-derived mimotope was identified using an anti-mPD1 mAb with mPD1/mPDL-1 blocking capacity. The identified mimotopes were characterized by in vitro assays, including a reporter cell-based assay, and their anti-tumor effects were evaluated in a syngeneic tumor mouse model stably expressing human Her-2/neu. The identified PD1-derived mimotopes were shown to significantly block the mAbs' capacity in inhibiting the respective PD1/PD-L1 interactions. A significant reduction in tumor growth in vivo was observed following active immunization with the mPD1-derived mimotope, associated with a significant reduction in proliferation and increased apoptotic rates in the tumors. Particularly, combined vaccination with the mPD1-derived mimotope and a multiple B-cell epitope Her-2/neu vaccine potentiated the vaccine's anti-tumor effect. Our results suggest active immunization with mimotopes of immune checkpoint inhibitors either as monotherapy or as combination therapy with tumor-specific vaccines, as a new strategy for cancer treatment.

Alpha-galactosylceramide enhances mucosal immunity to oral whole-cell cholera vaccines.

Cholera is a severe diarrheal disease caused by the bacterium Vibrio cholerae (V. cholerae) that results in 3-4 million cases globally with 100,000-150,000 deaths reported annually. Mostly confined to developing nations, current strategies to control the spread of cholera include the provision of safe drinking water and improved sanitation and hygiene, ideally in conjunction with oral vaccination. However, difficulties associated with the costs and logistics of these strategies have hampered their widespread implementation. Specific challenges pertaining to oral cholera vaccines (OCVs) include a lack of safe and effective adjuvants to further enhance gut immune responses, the complex and costly multicomponent vaccine manufacturing, limitations of conventional liquid formulation and the lack of an integrated delivery platform. Herein we describe the use of the orally active adjuvant α-Galactosylceramide (α-GalCer) to strongly enhance intestinal bacterium- and toxin-specific IgA responses to the OCV, Dukoral® in C57BL/6 and BALB/c mice. We further demonstrate the mucosal immunogenicity of a novel multi-antigen, single-component whole-cell killed V. cholerae strain and the enhancement of its immunogenicity by adding α-GalCer. Finally, we report that combining these components and recombinant cholera toxin B subunit in the SmPill® minisphere delivery system induced strong intestinal and systemic antigen-specific antibody responses.

Requirement for Cyclic AMP/Protein Kinase A-Dependent Canonical NFκB Signaling in the Adjuvant Action of Cholera Toxin and Its Non-toxic Derivative mmCT.

Cholera toxin (CT) is widely used as an effective adjuvant in experimental immunology for inducing mucosal immune responses; yet its mechanisms of adjuvant action remain incompletely defined. Here, we demonstrate that mice lacking NFκB, compared to wild-type (WT) mice, had a 90% reduction in their systemic and mucosal immune responses to oral immunization with a model protein antigen [Ovalbumin (OVA)] given together with CT. Further, NFκB-/- mouse dendritic cells (DCs) stimulated in vitro with CT showed reduced expression of MHCII and co-stimulatory molecules, such as CD80 and CD86, as well as of IL-1ß, and other pro-inflammatory cytokines compared to WT DCs. Using a human monocyte cell line THP1 with an NFκB activation reporter system, we show that CT induced NFκB signaling in human monocytes, and that inhibition of the cyclic AMP-protein kinase A (cAMP-PKA) pathway abrogated the activation and nuclear translocation of NFκB. In a human monocyte-CD4+ T cell co-culture system we further show that the strong Th17 response induced by CT treatment of monocytes was abolished by blocking the classical but not the alternative NFκB signaling pathway of monocytes. Our results indicate that activation of classical (canonical) NFκB pathway signaling in antigen-presenting cells (APCs) by CT is important for CT's adjuvant enhancement of Th17 responses. Similar findings were obtained using the almost completely detoxified mmCT mutant protein as adjuvant. Altogether, our results demonstrate that activation of the classical NFκB signal transduction pathway in APCs is important for the adjuvant action of both CT and mmCT.

Proteomic analysis of cholera toxin adjuvant-stimulated human monocytes identifies Thrombospondin-1 and Integrin-ß1 as strongly upregulated molecules involved in adjuvant activity.

Cholera Toxin (CT) as well as its related non-toxic mmCT and dmLT mutant proteins have been shown to be potent adjuvants for mucosally administered vaccines. Their adjuvant activity involves activation of cAMP/protein kinase A (PKA) signaling and inflammasome/IL-1ß pathways in antigen presenting cells (APC). To get a further understanding of the signal transduction and downstream pathways activated in APCs by this group of adjuvants we have, employing quantitative proteomic analytic tools, investigated human monocytes at various time points after treatment with CT. We report the activation of three main biological pathways among upregulated proteins, peaking at 16 hours of CT treatment: cellular organization, metabolism, and immune response. Specifically, in the further analyzed immune response pathway we note a strong upregulation of thrombospondin 1 (THBS1) and integrin ß1 (ITGB1) in response to CT as well as to mmCT and dmLT, mediated via cAMP/PKA and NFKB signaling. Importantly, inhibition in vitro of THSB1 and ITGB1 in monocytes or primary dendritic cells using siRNA abrogated the ability of the treated APCs to promote an adjuvant-stimulated Th17 cell response when co-cultured with peripheral blood lymphocytes indicating the involvement of these molecules in the adjuvant action on APCs by CT, mmCT and dmLT.

B cells treated with CTB-p210 acquire a regulatory phenotype in vitro and reduce atherosclerosis in apolipoprotein E deficient mice.

OBJECTIVE: Intranasal immunization with a fusion protein of the ApoB100-derived peptide p210 and the cholera toxin B subunit (CTB-p210) has previously been shown to induce mucosal tolerance and reduce atherosclerosis development, but the exact mode of action remains to be elucidated. Recent studies have indicated an important role for B cells in mucosal tolerance, in particular by induction of regulatory B (Bregs) and T cells (Tregs). In this study, we aimed to investigate if transfer of B cells pulsed with CTB-p210 can protect against atherosclerosis. METHOD AND RESULTS: First, we studied if CTB-p210 can induce Bregs and Tregs in vitro. After pulsing B cells from Apobtm2Sgyldlr-/- or Apoe-/- mice with CTB-p210 for 1 h and co-culturing them with naïve T cells for 48 h, we observed increased expression of membrane bound TGFß/latency-associated peptide (mTGFß/LAP) on B cells and an increased proportion of CD25hiFoxP3+ Tregs. Adoptive transfer of B cells pulsed with CTB-p210 into high-fat diet-fed Apoe-/- mice at 8, 10 and 12 weeks of age, reduced the plaque area in the aorta at 20 weeks of age as compared with control-treated (CTB-pOVA treated B cells or PBS) mice. Moreover, mice receiving p210-CTB treated B cells had increased levels of anti-p210 IgG antibodies. CONCLUSION: Our observations suggest that CTB-p210 pulsed B cells acquire a regulatory phenotype and induce Tregs in vitro. Adoptive transfer of CTB-p210, but not control-treated, B cells into Apoe-/- mice decreased atherosclerosis development.

Preclinical immunogenicity and protective efficacy of an oral Helicobacter pylori inactivated whole cell vaccine and multiple mutant cholera toxin: A novel and non-toxic mucosal adjuvant.

Mucosal vaccines against Helicobacter pylori consisting of either whole cell bacteria or recombinant antigens can induce immune protection against challenge in mice only when co-administrated with a strong mucosal adjuvant such as cholera toxin (CT) or Escherichia coli heat labile enterotoxin (LT). The strong enterotoxicity of these adjuvants however preclude their use in human vaccines. The recently developed multiple mutant CT (mmCT) is a strong, yet practically non-toxic novel mucosal adjuvant which here was admixed with a formalin-inactivated H. pylori whole cell vaccine (WCV) as a potential vaccine candidate against H. pylori infection. We report that intragastric immunizations with H. pylori WCV together with mmCT, similar to immunization with WCV together with CT, resulted in 50-125-fold reduction in colonization of H. pylori in the stomach of mice associated with rises in both serum IgG and intestinal-mucosal IgA anti-H. pylori antibody responses and strong T cell and IFNγ and IL-17A cytokine responses. Data presented in this study also supports that the proposed vaccine can be grown in a bioreactor and would be effective against infection caused by a multitude of pathogenic H. pylori strains isolated from patients from various continents. The results warrant immunization studies in humans to evaluate the safety, immunogenicity and efficacy of the proposed H. pylori WCV and mmCT.

GM1 ganglioside-independent intoxication by Cholera toxin.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.

A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae.

Antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of the lgt gene encoding a (pro)lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in Escherichia coli and other Gram-negative organisms. The lgt gene was deleted from E. coli and complemented by the Vibrio cholerae-derived gene provided in trans on a temperature-sensitive plasmid, allowing cells to grow at 30°C but not at 37°C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed, whereby transformants were selected by growth at 39°C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting the lgt gene from V. cholerae and complementing the lesion with the corresponding gene from E. coli The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, the lgt system described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using the E. coli-derived lgt gene for complementation.IMPORTANCE Many recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues.

Surface expression of Helicobacter pylori HpaA adhesion antigen on Vibrio cholerae, enhanced by co-expressed enterotoxigenic Escherichia coli fimbrial antigens.

Helicobacter pylori infection can cause peptic ulceration and is associated with gastric adenocarcinoma. This study aimed to construct and characterize a non-virulent Vibrio cholerae O1 strain, which grows more rapidly than H. pylori, as vector for H. pylori antigens for possible use as a vaccine strain against H. pylori. This was done by recombinant expression of the H. pylori adhesion antigen HpaA alone or, as a proof of principle, together with different colonization factor (CF) antigens of enterotoxigenic Escherichia coli (ETEC) which may enhance immune responses against HpaA. A recombinant V. cholerae strain co-expressing HpaA and a fimbrial CF antigens CFA/I or CS5, but not the non-fimbrial CF protein CS6, was shown to express larger amounts of HpaA on the surface when compared with the same V. cholerae strain expressing HpaA alone. Mutations in the CFA/I operon showed that the chaperon, possibly together with the usher, was involved in enhancing the surface expression of HpaA. Oral immunization of mice with formaldehyde-inactivated recombinant V. cholerae expressing HpaA alone or together with CFA/I induced significantly higher serum antibody responses against HpaA than mice similarly immunized with inactivated HpaA-expressing H. pylori bacteria. Our results demonstrate that a non-virulent V. cholerae strain can be engineered to allow strong surface expression of HpaA, and that the expression can be further increased by co-expressing it with ETEC fimbrial antigens. Such recombinant V. cholerae strains expressing HpaA, and possibly also other H. pylori antigens, may have the potential as oral inactivated vaccine candidates against H. pylori.

Retrospective Analysis of Serotype Switching of Vibrio cholerae O1 in a Cholera Endemic Region Shows It Is a Non-random Process.

Genomic data generated from clinical Vibrio cholerae O1 isolates collected over a five year period in an area of Kolkata, India with seasonal cholera outbreaks allowed a detailed genetic analysis of serotype switching that occurred from Ogawa to Inaba and back to Ogawa. The change from Ogawa to Inaba resulted from mutational disruption of the methyltransferase encoded by the wbeT gene. Re-emergence of the Ogawa serotype was found to result either from expansion of an already existing Ogawa clade or reversion of the mutation in an Inaba clade. Our data suggests that such transitions are not random events but rather driven by as yet unidentified selection mechanisms based on differences in the structure of the O1 antigen or in the serotype-determining wbeT gene.

Biochemical and structural characterization of the novel sialic acid-binding site of Escherichia coli heat-labile enterotoxin LT-IIb.

The structurally related AB5-type heat-labile enterotoxins of Escherichia coli and Vibrio cholerae are classified into two major types. The type I group includes cholera toxin (CT) and E. coli LT-I, whereas the type II subfamily comprises LT-IIa, LT-IIb and LT-IIc. The carbohydrate-binding specificities of LT-IIa, LT-IIb and LT-IIc are distinctive from those of cholera toxin and E. coli LT-I. Whereas CT and LT-I bind primarily to the GM1 ganglioside, LT-IIa binds to gangliosides GD1a, GD1b and GM1, LT-IIb binds to the GD1a and GT1b gangliosides, and LT-IIc binds to GM1, GM2, GM3 and GD1a. These previous studies of the binding properties of type II B-subunits have been focused on ganglio core chain gangliosides. To further define the carbohydrate binding specificity of LT-IIb B-subunits, we have investigated its binding to a collection of gangliosides and non-acid glycosphingolipids with different core chains. A high-affinity binding of LT-IIb B-subunits to gangliosides with a neolacto core chain, such as Neu5Gcα3- and Neu5Acα3-neolactohexaosylceramide, and Neu5Gcα3- and Neu5Acα3-neolactooctaosylceramide was detected. An LT-IIb-binding ganglioside was isolated from human small intestine and characterized as Neu5Acα3-neolactohexaosylceramide. The crystal structure of the B-subunit of LT-IIb with the pentasaccharide moiety of Neu5Acα3-neolactotetraosylceramide (Neu5Ac-nLT: Neu5Acα3Galß4GlcNAcß3Galß4Glc) was determined providing the first information for a sialic-binding site in this subfamily, with clear differences from that of CT and LT-I.

Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae.

There is an urgent need for new adjuvants that are effective with mucosally administered vaccines. Cholera toxin (CT) is the most powerful known mucosal adjuvant but is much too toxic for human use. In an effort to develop a useful mucosal adjuvant we have generated a novel non-toxic mutant CT molecule that retains much of the adjuvant activity of native CT. This was achieved by making the enzymatically active A subunit (CTA) recalcitrant to the site-specific proteolytic cleavage ("nicking") required for toxicity, which was found to require mutations not only in the two residues rendering the molecule resistant to trypsin but also in neighboring sites protecting against cleavage by Vibrio cholerae proteases. This multiple-mutated CT (mmCT) adjuvant protein could be efficiently produced in and purified from the extracellular medium of CT-deleted V. cholerae. The mmCT completely lacked detectable enterotoxicity in an infant mouse model and had >1000-fold reduced cAMP inducing activity compared to native CT in a sensitive mammalian target cell system. It nonetheless proved to have potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to mucosally co-administered antigens including oral cholera and intranasal influenza vaccines. We conclude that mmCT is an attractive novel non-toxic mucosal adjuvant for enhancing immune responses to co-administered mucosal vaccines.

Cholera toxin, and the related nontoxic adjuvants mmCT and dmLT, promote human Th17 responses via cyclic AMP-protein kinase A and inflammasome-dependent IL-1 signaling.

We have examined the molecular pathways involved in the adjuvant action of cholera toxin (CT) and two novel nontoxic molecules, multiple-mutated CT (mmCT) and double-mutant heat-labile toxin (dmLT) on human T cell responses. Human PBMCs or isolated monocytes were stimulated in vitro with CT, mmCT, or dmLT plus a polyclonal stimulus (staphylococcal enterotoxin B) or specific bacterial Ags, and effects on expression of cytokines and signaling molecules were determined. CT, mmCT, and dmLT strongly enhanced IL-17A and to a lesser extent IL-13 responses, but had little effect on IFN-γ production or cell proliferation. Intracellular cytokine staining revealed that the enhanced IL-17A production was largely confined to CD4(+) T cells and coculture experiments showed that the IL-17A promotion was effectively induced by adjuvant-treated monocytes. Relative to CT, mmCT and dmLT induced at least 100-fold lower levels of cAMP, yet this cAMP was enough and essential for the promotion of Th17 responses. Thus, inhibition of cAMP-dependent protein kinase A was abolished, and stimulation with a cAMP analog mimicked the adjuvant effect. Furthermore, CT, mmCT, and dmLT induced IL-1ß production and caspase-1 activation in monocytes, which was associated with increased expression of key proinflammatory and inflammasome-related genes, including NLRP1, NLRP3, and NLRC4. Inflammasome inhibition with a specific caspase-1 inhibitor, or blocking of IL-1 signaling by IL-1 receptor antagonist, abrogated the Th17-promoting effect. We conclude that CT, mmCT, and dmLT promote human Th17 responses via cAMP-dependent protein kinase A and caspase-1/inflammasome-dependent IL-1 signaling.