Defining Polysaccharide-Specific Antibody Targets against Vibrio cholerae O139 in Humans following O139 Cholera and following Vaccination with a Commercial Bivalent Oral Cholera Vaccine, and Evaluation of Conjugate Vaccines Targeting O139.

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.

Protection afforded by previous Vibrio cholerae infection against subsequent disease and infection: A review.

BACKGROUND: Cholera is an acute, diarrheal disease caused by Vibrio cholerae O1 or 139 that is associated with a high global burden. METHODS: We analyzed the estimated duration of immunity following cholera infection from available published studies. We searched PubMed and Web of Science for studies of the long-term immunity following cholera infection. We identified 22 eligible studies and categorized them as either observational, challenge, or serological. RESULTS: We found strong evidence of protection at 3 years after infection in observational and challenge studies. However, serological studies show that elevated humoral markers of potential correlates of protection returned to baseline within 1 year. Additionally, a subclinical cholera infection may confer lower protection than a clinical one, as suggested by 3 studies that found that, albeit with small sample sizes, most participants with a subclinical infection from an initial challenge with cholera had a symptomatic infection when rechallenged with a homologous biotype. CONCLUSIONS: This review underscores the need to elucidate potential differences in the protection provided by clinical and subclinical cholera infections. Further, more studies are warranted to bridge the gap between the correlates of protection and cholera immunity. Understanding the duration of natural immunity to cholera can help guide control strategies and policy.

Immunohistochemical, histological and ultrastructural evaluation of protection provided by cholera vaccine against V. cholerae O139.

In our previous study, complete protection was observed in rabbit immunized with 1 × 1010 CFU of live attenuated VCUSM21P vaccine against challenge with 1 × 109 CFU Vibrio cholerae O139. In the present study, we investigated whether the vaccines can effectively protect immunized animals from any pathologic changes using histological, immunohistochemical and ultrastructural techniques. Severe pathology is evident in wild type injected ileum in non-immunized, showing extensive villous destruction, edema, necrosis and inflammation with infiltration of large numbers of inflammatory cells, extensive damage to the villi and microvilli with pore formation. Histology of ileum injected with wild type in immunized rabbit shows no significant pathological changes except for a few inflammatory cells in lamina propria with mild edema in mucosa and submucosa. immunohistochemical staining revealed O139 antigens of wild type are seen in the lamina propria of edematous villi, muscularis mucosa and submucosa with weak presence in the muscle coat in non-immunized rabbit after challenged with wild type in non-immunized rabbits, but in immunized rabbit localisation of the O139 LPS antigen is seen at the tips of the intact villi, within lamina propria and muscularis mucosa only. These observations suggest that the vaccine can effectively protect animals from any pathologic changes and eliminate V. cholerae O139 from the immunized animals.

Induction of systemic, mucosal and memory antibody responses targeting Vibrio cholerae O1 O-specific polysaccharide (OSP) in adults following oral vaccination with an oral killed whole cell cholera vaccine in Bangladesh.

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.

Safety and immunogenicity of a killed bivalent (O1 and O139) whole-cell oral cholera vaccine in adults and children in Vellore, South India.

This open-label study assessed the safety and immunogenicity of two doses (14 days apart) of an indigenously manufactured, killed, bivalent (Vibrio cholerae O1 and O139), whole-cell oral cholera vaccine (SHANCHOL; Shantha Biotechnics) in healthy adults (n = 100) and children (n = 100) in a cholera endemic area (Vellore, South India) to fulfill post-licensure regulatory requirements and post-World Health Organization (WHO) prequalification commitments. Safety and reactogenicity were assessed, and seroconversion rates (i.e. proportion of participants with a ≥ 4-fold rise from baseline in serum vibriocidal antibody titers against V. cholerae O1 Inaba, O1 Ogawa and O139, respectively) were determined 14 days after each vaccine dose. No serious adverse events were reported during the study. Commonly reported solicited adverse events were headache and general ill feeling. Seroconversion rates after the first and second dose in adults were 67.7% and 55.2%, respectively, against O1 Inaba; 47.9% and 45.8% against O1 Ogawa; and 19.8% and 20.8% against O139. In children, seroconversion rates after the first and second dose were 80.2% and 68.8%, respectively, against O1 Inaba; 72.9% and 67.7% against O1 Ogawa; and 26.0% and 18.8% against O139. The geometric mean titers against O1 Inaba, O1 Ogawa, and O139 in both adults and children were significantly higher after each vaccine dose compared to baseline titers (P < 0.001; for both age groups after each dose versus baseline). The seroconversion rates for O1 Inaba, O1 Ogawa, and O139 in both age groups were similar to those in previous studies with the vaccine. In conclusion, the killed, bivalent, whole-cell oral cholera vaccine has a good safety and reactogenicity profile, and is immunogenic in healthy adults and children. Trial Registration: ClinicalTrials.gov NCT00760825; CTRI/2012/01/002354.

Immunogenicity of a Glycoconjugate from Hydrazine-Detoxified Lipopolysaccharide of Vibrio cholerae O139.

A glycoconjugate (dLPS-CBSA) from hydrazine-detoxified lipopolysaccharide (LPS) of Vibrio cholerae O139 and BSA was used for immunization of BALB/c mice. The immunogenicity (concentration of induced IgG antibodies) of the conjugate was determined by enzyme-linked immunosorbent assay. The highest level of LPS-specific IgGs was found in sera collected 2 weeks after the third immunization. Both IgM and IgG antibodies present in the anti-V. cholerae O139-specific sera showed long-lasting vibriocidal activity. The glycoconjugate also stimulated production of IL-4, IL-6, IFNγ, TNFα and IL-10. Immunization with the conjugate induced significant production of both Th1 and Th2 cytokine sets, indicating balanced Th1/Th2 immune response polarization. The quantitative analysis of total and LPS-specific IgM and IgG antibodies production by B lymphocytes (from both the spleen and the bone marrow) was performed by ELISPOT assay. From the analysis of antibodies produced directly by B lymphocytes in the bone marrow (of dLPS-CBSA immunized mice), we were able to identify the IgM-IgG switch in the antibodies produced.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139.

Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL-1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL-1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

A Randomized, Placebo-Controlled Trial Evaluating Safety and Immunogenicity of the Killed, Bivalent, Whole-Cell Oral Cholera Vaccine in Ethiopia.

Killed whole-cell oral cholera vaccine (OCV) has been a key component of a comprehensive package including water and sanitation measures for recent cholera epidemics. The vaccine, given in a two-dose regimen, has been evaluated in a large number of human volunteers in India, Vietnam, and Bangladesh, where it has demonstrated safety, immunogenicity, and clinical efficacy. We conducted a double-blind randomized placebo-controlled trial in Ethiopia, where we evaluated the safety and immunogenicity of the vaccine in 216 healthy adults and children. OCV was found to be safe and elicited a robust immunological response against Vibrio cholerae O1, with 81% adults and 77% children demonstrating seroconversion 14 days after the second dose of vaccine. This is the first study to evaluate safety and immunogenicity of the vaccine in a population outside Asia using a placebo-controlled, double-blind, randomized study design.

Detection of Vibrio cholerae O1 and O139 in environmental waters of rural Bangladesh: a flow-cytometry-based field trial.

Presence of Vibrio cholerae serogroups O1 and O139 in the waters of the rural area of Matlab, Bangladesh, was investigated with quantitative measurements performed with a portable flow cytometer. The relevance of this work relates to the testing of a field-adapted measurement protocol that might prove useful for cholera epidemic surveillance and for validation of mathematical models. Water samples were collected from different water bodies that constitute the hydrological system of the region, a well-known endemic area for cholera. Water was retrieved from ponds, river waters, and irrigation canals during an inter-epidemic time period. Each sample was filtered and analysed with a flow cytometer for a fast determination of V. cholerae cells contained in those environments. More specifically, samples were treated with O1- and O139-specific antibodies, which allowed precise flow-cytometry-based concentration measurements. Both serogroups were present in the environmental waters with a consistent dominance of V. cholerae O1. These results extend earlier studies where V. cholerae O1 and O139 were mostly detected during times of cholera epidemics using standard culturing techniques. Furthermore, our results confirm that an important fraction of the ponds' host populations of V. cholerae are able to self-sustain even when cholera cases are scarce. Those contaminated ponds may constitute a natural reservoir for cholera endemicity in the Matlab region. Correlations of V. cholerae concentrations with environmental factors and the spatial distribution of V. cholerae populations are also discussed.

Preparation and immunogenicity of conjugate based on hydrazine-treated lipopolysaccharide antigen of Vibrio cholerae O139.

A glycoconjugate construct was based on attachment of V. cholerae O139 hydrazine-treated lipopolysaccharide (LPS) to carboxylated bovine serum albumin (CBSA) via its amino group. The immunological properties of the glycoconjugate were tested using BALB/c mice, injected subcutaneously without any adjuvant three times at 2 weeks interval. The immunogenicity of the conjugate was estimated by enzyme-linked immunosorbent assay, testing of anti-LPS IgG, IgM, and IgA antibodies. The conjugate elicited a statistically significant increase of LPS-specific IgG levels in mice (p < 0.001). The specific anti-LPS IgG and IgA response after the second booster dose was significantly higher compared with reference and unconjugated detoxified LPS response. Antibodies elicited by the dLPS-CBSA conjugate were vibriocidal.

Immunogenicity of a killed bivalent (O1 and O139) whole cell oral cholera vaccine, Shanchol, in Haiti.

BACKGROUND: Studies of the immunogenicity of the killed bivalent whole cell oral cholera vaccine, Shanchol, have been performed in historically cholera-endemic areas of Asia. There is a need to assess the immunogenicity of the vaccine in Haiti and other populations without historical exposure to Vibrio cholerae. METHODOLOGY/PRINCIPAL FINDINGS: We measured immune responses after administration of Shanchol, in 25 adults, 51 older children (6-17 years), and 47 younger children (1-5 years) in Haiti, where cholera was introduced in 2010. A≥4-fold increase in vibriocidal antibody titer against V. cholerae O1 Ogawa was observed in 91% of adults, 74% of older children, and 73% of younger children after two doses of Shanchol; similar responses were observed against the Inaba serotype. A≥2-fold increase in serum O-antigen specific polysaccharide IgA antibody levels against V. cholerae O1 Ogawa was observed in 59% of adults, 45% of older children, and 61% of younger children; similar responses were observed against the Inaba serotype. We compared immune responses in Haitian individuals with age- and blood group-matched individuals from Bangladesh, a historically cholera-endemic area. The geometric mean vibriocidal titers after the first dose of vaccine were lower in Haitian than in Bangladeshi vaccinees. However, the mean vibriocidal titers did not differ between the two groups after the second dose of the vaccine. CONCLUSIONS/SIGNIFICANCE: A killed bivalent whole cell oral cholera vaccine, Shanchol, is highly immunogenic in Haitian adults and children. A two-dose regimen may be important in Haiti, and other populations lacking previous repeated exposures to V. cholerae.

Construction and characterization of a Vibrio cholerae serogroup O139 vaccine candidate by genetic engineering.

The present study aimed to construct and evaluate the live attenuated Vibrio cholerae serogroup O139 vaccine candidate, in which genes encoding protective antigens were integrated into the chromosomal DNA. Using the initial strain, O139-ZJ9693, the toxin-linked cryptic (TLC) and cholera toxin (CTX) genetic elements and repeats in the toxin (RTX) gene cluster were deleted from its chromosomal DNA, and the cholera toxin genes, ctxB and rstR, were transferred into the chromosome to construct the candidate vaccine strain. The expression of ctxB and the vaccine virulence were then examined. Polymerase chain reaction (PCR), enzymatic digestion and electrophoresis were performed to confirm that TLC, CTX and RTX were deleted, and that ctxB and rstR were transferred into the vaccine candidate DNA. According to the preliminary evaluation, the ctxB gene exhibited cholera toxin subunit B expression, and no enterotoxigenic or cytotoxic effects were observed in this strain. In conclusion, a recombinant strain containing genes encoding protective antigens that replaced virulence-associated genes was successfully constructed in the present study; this candidate strain may have the potential to be utilized to further evaluate the immune response.

Development of a 5-minute rapid test for detecting Vibrio cholerae O139.

Vibrio cholerae O139 is an etiology of cholera in Thailand. We determined to prepare a rapid test to detect V cholerae O139 using an immunochromatographic method to be used for surveillance and use in community laboratories. We conjugated murine monoclonal antibodies specific to the lipopolysaccharide of V. cholerae O139 with colloidal gold particles. The sensitivity of the test was determined using 10-fold dilutions of V. cholerae O139. The lowest number of bacterial cells detected by the test was 10(6) cfu/ml. The specificity was determined using 51 isolates of pure cultures, including V. cholerae serogroups O1, O139, and non-O1/non-O139 and other enteric bacteria; 27 rectal swab specimens and 100 specimens of enriched alkaline peptone water, 50 of which contained V. cholerae O1 and the other did not contain V. cholerae O1. The rapid test showed specificity to V cholerae O139 and no cross reaction with V cholerae O1, V. cholerae non-O1/ non-O139, and other enteric bacteria. The test is rapid, simple and easy to use.

Development and evaluation of a highly sensitive immunochromatographic strip test using gold nanoparticle for direct detection of Vibrio cholerae O139 in seafood samples.

A strip test for the detection of Vibrio cholerae O139 was developed using two monoclonal antibodies (MAbs), namely VC-273 and VC-812, which specifically bind to the lipopolysaccharide and capsular polysaccharide of V. cholerae O139. The MAb VC-273 gold nanoparticle conjugate was sprayed onto a glass fiber pad that was placed adjacent to a sample chamber. MAb VC-812 and the goat anti-mouse immunoglobulin G (GAM) antibody were sprayed onto a nitrocellulose membrane in strips at positions designated as T and C, respectively. The test strips were assessed for their ability to directly detect V. cholerae O139 using samples dispersed in application buffer, and a 100 µL aliquot of sample was applied to the sample chamber. The results were observable within 20 min after application of the sample. In samples containing V. cholerae O139, the antigen was bound to the colloidal gold-conjugated MAb to form an antibody-antigen complex. This complex was captured by the MAbs at the T test line, resulting in the appearance of a reddish-purple band at the T position. The sensitivity of the test was determined to be 104 cfu mL⁻¹. Direct detection of V. cholerae O139 in various fresh seafood samples could be accomplished with similar sensitivities. The detection limit was substantially improved to 1 cfu mL⁻¹ of the original bacterial content after pre-incubation of the sample in alkaline peptone water for 12 h. The V. cholerae strip test provides several advantages over other methods, including the speed and simplicity of use because there is no requirement for sophisticated equipment.

Cholera vaccine: new preventive tool for endemic countries.

Cholera is a major global public health problem and remains an important threat in almost every developing country, especially in areas where population overcrowding and poor sanitation are common, such as slums and refugee camps. Cholera is one of the most dreaded diseases in the world, in some cases leading to death within 24 h if left untreated. Without treatment, severe infection has a mortality rate of 30-50%. In 2007, WHO recorded 177,963 cholera cases and 4,031 deaths worldwide. However, the estimated actual burden of cholera is in the vicinity of 3 to 5 million cases and 100,000 to 130,000 deaths per year. The disease is endemic to parts of Africa, Asia, the Middle East and South America. Large outbreaks are common after natural disasters or in populations displaced by war, where there is inadequate sewage disposal and contaminated water. In India, during the 10-y period (1997-2006) studied, the states having the highest number of reported outbreaks were West Bengal, Orissa, Maharashtra and Kerala, which together accounted for 60% of all reported outbreaks. A review of cholera cases in India reported to WHO from 2003-2007 showed that the numbers were in the few thousands with a case fatality rate of < 1%. However, it is believed that the number of cholera cases and deaths occurring annually in India is much greater than the number reported. A literature review covering a four-year period from 2003 to 2006 found reported cholera outbreaks in 18 of the 35 States and Union Territories of India. Of these, 11 had cholera outbreaks reported for multiple years. Vietnam has produced a cheaper variant of killed whole-cell vaccine devoid of the B subunit. This vaccine contains both Vibrio cholerae O1 and O139, and provides 50 per cent protection for at least three years after vaccination. For endemic cholera, population-level immunity is relatively high, making control possible with relatively low vaccine coverage levels. This vaccine should be used in areas where cholera is endemic, particularly in those at risk of outbreaks, in conjunction with other prevention and control strategies.

TLP01, an mshA mutant of Vibrio cholerae O139 as vaccine candidate against cholera.

No commercially live vaccine against cholera caused by Vibrio cholerae O139 serogroup is available and it is currently needed. Virulent O139 strain CRC266 was genetically modified by firstly deleting multiple copies of the filamentous phage CTXφ, further tagging by insertion of the endoglucanase A coding gene from Clostridium thermocellum into the hemagglutinin/protease gene and finally deleting the mshA gene, just to improve the vaccine biosafety. One of the derived strains designated as TLP01 showed full attenuation and good colonizing capacity in the infant mouse cholera model, as well as highly immunogenic properties in the adult rabbit and rat models. Since TLP01 lacks MSHA fimbriae, it is refractory to infection with another filamentous phage VGJφ and therefore protected of acquiring CTXφ from a recombinant hybrid VGJφ/CTXφ. This strategy could reduce the possibilities of stable reversion to virulence out of the human gut. Furthermore, this vaccine strain was impaired to produce biofilms under certain culture conditions, which might have implications for the strain survival in natural settings contributing to vaccine biosafety as well. The above results has encouraged us to consider TLP01 as a live attenuated vaccine strain having an adequate performance in animal models, in terms of attenuation and immunogenicity, so that it fulfills the requirements to be evaluated in human volunteers.

Immune responses to cholera in children.

Cholera is a severe acute dehydrating diarrheal disease caused by Vibrio cholerae O1 or O139 infection, and is associated with significant mortality and morbidity globally. Although young children bear a high burden of the disease, currently available oral vaccines give a lower efficacy and shorter duration of protection in this group than in adults. According to the studies of natural infection, young children achieve comparable systemic anti-V. cholerae antigen-specific antibody, gut-homing antibody-secreting cell and memory B-cell responses as adults. Studies on innate and cell-mediated immune responses are lacking in children, and may offer important insights into differences in vaccine efficacy. The impact of host factors such as malnutrition, genetics and coinfection with other pathogens also remains to be fully defined.

[The optimization of conditions of diagnostic fluorescent monoclonal immunoglobulins production to identify comma bacillus of serogroups O1 and O139].

The article considers, the issue of producing the species-specific fluorescent monoclonal immunoglobulins to detect comma bacillus of serogrups O1 and O139 in the reaction of direct immunfluorescence. It is established that not only ascitic but culture fluid too can be the source of monoclonal antibodies for producing fluorescent conjugates. The optimal conditions are selected to produce the fluorescent monoclonal immunoglobulins-monoclonal antibodies. The corresponding producing techology can be reproduced at any time in view of availability of hybrid-producers of monoclonal antibodies O1 and monoclonal antibodies O139 in the institute cryodepositoty. The results of testing the fluorescent preparations on homologous and heterologous strains demonstrated their strict specificiy and high sensibility regarding comma bacillus of serogroups O1 and O139. The new preparations favor significant increase of effectiveness of diagnostics of V. cholerae O1 and O139.

Safety and immunogenicity study of a killed bivalent (O1 and O139) whole-cell oral cholera vaccine Shanchol, in Bangladeshi adults and children as young as 1 year of age.

BACKGROUND: Safety and immunogenicity study of an oral, killed, bivalent whole-cell, cholera vaccine, Shanchol was carried out in Bangladeshi participants. This study was conducted prior to initiating a feasibility study in Bangladesh. STUDY PARTICIPANTS: The double-blind, randomized placebo controlled study was carried out in adults (18-45 years), toddlers (2-5 years) and younger children (12-23 months). Two doses of the vaccine/placebo were given 14 days apart. RESULTS: Shanchol did not elicit major adverse events in any age group. Vibriocidal antibody responses in adults were 60% against Vibrio cholerae O1 Inaba, 72% against V. cholerae O1 Ogawa and 21% against V. cholerae O139. In toddlers, responses were 84%, 75% and 64% and in younger children it was 74%, 78% and 54% against Inaba, Ogawa and O139 serotypes. The responses in all ages were higher in vaccinees compared to pre-immune titers or to responses in placebo recipients (P<0.001). Plasma IgA antibody response to O1 Inaba LPS was seen in 61%, 73% and 45% of adults, toddlers and younger children, respectively. CONCLUSIONS: The safety and immunogenicity data for Shanchol is promising and warrants future use in large scale trial in cholera endemic areas, high risk Bangladeshi population and in other countries in the region.

Synthesis of the conjugation ready, downstream disaccharide fragment of the O-PS of Vibrio cholerae O:139.

The linker-equipped disaccharide, 8-amino-3,6-dioxaoctyl 2,6-dideoxy-2-acetamido-3-O-ß-D-galactopyranosyluronate-ß-D-glucopyranoside (10), was synthesized in eight steps from acetobromogalactose and ethyl 4,6-O-benzylidene-2-deoxy-2-trichloroacetamido-1-thio-ß-D-glucopyranoside. The hydroxyl group present at C-4(II) in the last intermediate, 8-azido-3,6-dioxaoctyl 4-O-benzyl-6-bromo-2,6-dideoxy-2-trichloroacetamido-3-O-(benzyl 2,3-di-O-benzyl-ß-D-galactopyranosyluronate)-ß-D-glucopyranoside (9), is positioned to allow further build-up of the molecule and, eventually, construction of the complete hexasaccharide. Global deprotection (9→10) was done in one step by catalytic hydrogenolysis over palladium-on-charcoal.