Vibriocidal Antibody Responses to a Bivalent Killed Whole-Cell Oral Cholera Vaccine in a Phase III Trial in Kolkata,India

Background

During the development of a vaccine, identification of the correlates of protection is of paramount importance for establishing an objective criterion for the protective performance of the vaccine. However, the ascertainment of correlates of immunity conferred by any vaccine is a difficult task.

Methods

While conducting a phase three double-blind, cluster-randomized, placebo-controlled trial of a bivalent killed whole-cell oral cholera vaccine in Kolkata, we evaluated the immunogenicity of the vaccine in a subset of participants. Randomly chosen participants (recipients of vaccine or placebo) were invited to provide blood samples at baseline, 14 days after the second dose and one year after the first dose. At these time points, serum geometric mean titers (GMT) of vibriocidal antibodies and seroconversion rates for vaccine and placebo arms were calculated and compared across the age strata (1 to 5 years, 5 to 15 years and more than 15 years) as well as for all age groups.

Results

Out of 137 subjects included in analysis, 69 were vaccinees and 68 received placebo. There were 5•7 and 5•8 geometric mean fold (GMF) rises in titers to Vibrio cholerae Inaba and Ogawa, respectively at 14 days after the second dose, with 57% and 61% of vaccinees showing a four-fold or greater titer rise, respectively. After one year, the titers to Inaba and Ogawa remained 1•7 and 2•8 fold higher, respectively, compared to baseline. Serum vibriocidal antibody response to V. cholerae O139 was much lower than that to Inaba or Ogawa. No significant differences in the GMF-rises were observed among the age groups.

Conclusions

The reformulated oral cholera vaccine induced a statistically significant anti-O1 Inaba and O1 Ogawa vibriocidal antibody response 14 days after vaccination, which although declined after one year remained significantly higher than baseline. Despite this decline, the vaccine remained protective five years after vaccination.     

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.     

Major Shift of Toxigenic V. cholerae O1 from Ogawa to Inaba Serotype Isolated from Clinical and Environmental Samples in Haiti

In October of 2010, an outbreak of cholera was confirmed in Haiti for the first time in more than a century. A single clone of toxigenic Vibrio cholerae O1 biotype El Tor serotype Ogawa strain was implicated as the cause. Five years after the onset of cholera, in October, 2015, we have discovered a major switch (ranging from 7 to 100%) from Ogawa serotype to Inaba serotype. Furthermore, using wbeT gene sequencing and comparative sequence analysis, we now demonstrate that, among 2013 and 2015 Inaba isolates, the wbeT gene, responsible for switching Ogawa to Inaba serotype, sustained a unique nucleotide mutation not found in isolates obtained from Haiti in 2012. Moreover, we show that, environmental Inaba isolates collected in 2015 have the identical mutations found in the 2015 clinical isolates. Our data indicate that toxigenic V. cholerae O1 serotype Ogawa can rapidly change its serotype to Inaba, and has the potential to cause disease in individuals who have acquired immunity against Ogawa serotype. Our findings highlight the importance of monitoring of toxigenic V. cholerae O1 and cholera in countries with established endemic disease.     

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.     

A duplex vibriocidal assay to simultaneously measure bactericidal antibody titers against Vibrio cholerae O1 Inaba and Ogawa serotypes

Currently available cholera vaccines are formulated with killed-whole cells of Vibrio cholerae O1 Inaba and Ogawa serotypes. A serum vibriocidal assay has been widely used to evaluate the immunogenicity of cholera vaccines in clinical trials. In this study, we developed a duplex vibriocidal assay to obtain vibriocidal antibody titers against both serotypes simultaneously. Initially, serial dilutions of serum from vaccinees were incubated with guinea pig complements along with both streptomycin-resistant Inaba and ampicillin-resistant Ogawa strains for 1 h. The mixture was then inoculated on separate agar plates containing each antibiotic to selectively culture each corresponding serotype and incubated at 37 °C for 16 to 20 h. Bacterial colonies were enumerated using an automated colony counting system to obtain the vibriocidal antibody titers defined by the reciprocal of serum dilution inhibiting bacterial growth by 50%. Performance of the duplex vibriocidal assay was examined by comparison with a single serotype vibriocidal assay using 20 clinical sera consisting of ten-paired sera prepared at pre- and post-vaccination. Both assays showed a good correlation for vibriocidal titers against the two serotypes, respectively, as determined by Pearson correlation coefficient (r) and regression coefficient (β) analyses; r = 0.998, β = 1.003 for Inaba and r = 0.997, β = 0.999 for Ogawa, respectively. The duplex vibriocidal assay can diminish the amount of sera required for the assay and enhance assay efficiency in terms of time, labor intensity, and expenditure.     

Clinical Isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata,India: Preponderance of SXT Element and Presence of Haitian ctxB Variant

Background

Increase in the number of multidrug resistant pathogens and the accompanied rise in case fatality rates has hampered the treatment of many infectious diseases including cholera. Unraveling the mechanisms responsible for multidrug resistance in the clinical isolates of Vibrio cholerae would help in understanding evolution of these pathogenic bacteria and their epidemic potential. This study was carried out to identify genetic factors responsible for multiple drug resistance in clinical isolates of Vibrio cholerae O1, serotype Ogawa, biotype El Tor isolated from the patients admitted to the Infectious Diseases Hospital, Kolkata, India, in 2009.

Methodology/Principal Findings

One hundred and nineteen clinical isolates of V. cholerae were analysed for their antibiotic resistance phenotypes. Antibiogram analysis revealed that majority of the isolates showed resistance to co-trimoxazole, nalidixic acid, polymixin B and streptomycin. In PCR, SXT integrase was detected in 117 isolates and its sequence showed 99% identity notably to ICEVchInd5 from Sevagram, India, ICEVchBan5 from Bangladesh and VC1786ICE sequence from Haiti outbreak among others. Antibiotic resistance traits corresponding to SXT element were transferred from the parent Vibrio isolate to the recipient E. coli XL-1 Blue cells during conjugation. Double-mismatch-amplification mutation assay (DMAMA) revealed the presence of Haitian type ctxB allele of genotype 7 in 55 isolates and the classical ctxB allele of genotype 1 in 59 isolates. Analysis of topoisomerase sequences revealed the presence of mutation Ser83 → Ile in gyrA and Ser85→ Leu in parC. This clearly showed the circulation of SXT-containing V. cholerae as causative agent for cholera in Kolkata.

Conclusions

There was predominance of SXT element in these clinical isolates from Kolkata region which also accounted for their antibiotic resistance phenotype typical of this element. DMAMA PCR showed them to be a mixture of isolates with different ctxB alleles like classical, El Tor and Haitian variants.     

Variable gene family usage of protective and non‐protective anti‐Vibrio cholerae O1 LPS antibody heavy chains

Vibrio cholerae causes cholera, an enteric disease of humans that is a worldwide problem. The O1 serogroup of Vibrio cholerae contains two predominant serotypes (Inaba and Ogawa) of LPS, a proven protective antigen for humans and experimental animals. We generated B‐cell hybridomas from mice immunized with either: (i) two doses of purified Inaba LPS; (ii) two doses of an Inaba hexasaccharide conjugate (terminal six perosamine bound to a protein carrier), (iii) four doses of purified Inaba LPS; or (iv) a low dose of purified Inaba LPS followed by a booster with the Inaba conjugate. We showed previously that the first and third immunization protocols induce vibriocidal antibodies, as does the fourth; the second protocol induces antibodies that bind Inaba and Ogawa LPS but are not vibriocidal. Anti‐LPS mAbs derived from hybridomas resulting from each immunization protocol were characterized for binding to Inaba and Ogawa LPS, their vibriocidal or protective capacity, and the variable heavy chain family they expressed. LPS immunogens selected different LPS‐specific B cells expressing six different Vh chain families. Protective and non‐protective mAbs could express variable regions from the same family. One mAb was specific for Inaba LPS, the other mAbs were cross‐reactive with both LPS serotypes. Sequence comparison suggests that the pairing of a specific light chain, somatic mutation, or the specific VDJ recombination can modulate the protective capacity of mAbs that express a common variable heavy chain family member.     

Use of a Cholera Rapid Diagnostic Test during a Mass Vaccination Campaign in Response to an Epidemic in Guinea, 2012

Background

During the 2012 cholera outbreak in the Republic of Guinea, the Ministry of Health, supported by Médecins Sans Frontières - Operational Center Geneva, used the oral cholera vaccine Shanchol as a part of the emergency response. The rapid diagnostic test (RDT) Crystal VC, widely used during outbreaks, detects lipopolysaccharide antigens of Vibrio cholerae O1 and O139, both included in Shanchol. In the context of reactive use of a whole-cell cholera vaccine in a region where cholera cases have been reported, it is essential to know what proportion of vaccinated individuals would be reactive to the RDT and for how long after vaccination.

Methodology/Principal Findings

A total of 108 vaccinated individuals, selected systematically among all persons older than one year, were included at vaccination sites and 106 were included in the analysis. Stools samples of this cohort of vaccinated participants were collected and tested with the RDT every day until the test was negative for two consecutive visits or for a maximum of 7 days. A total of 94.3% of cholera vaccine recipients had a positive test after vaccination; all except one of these positive results were reactive only with the O139 antigen. The mean time to become negative in those with an initial positive result after vaccination was 3.8 days, standard deviation 1.1 days.

Conclusions/Significance

The RDT Crystal VC becomes positive in persons recently vaccinated against cholera, although almost exclusively to the O139 antigen. This reactivity largely disappeared within five days after vaccination. These results suggest that the test can be used normally as soon as 24 hours after vaccination in a context of O1 epidemics, which represent the vast majority of cases, and after a period of five days in areas where V. cholerae O139 is present. The reason why only O139 test line became positive remains to be investigated.     

The characteristics of the reactogenicity and immunological activity of a new cholera bivalent chemical vaccine based on the results of controlled trials]

The reactogenic properties and immunological potency of modified cholera chemical vaccine (choleragen-toxoid + O-antigens Inaba and Ogawa) were tested in 278 volunteers aged 18 years and over in comparison with those of a commercial batch of monovalent cholera vaccine (choleragen-toxoid + O-antigen Inaba). The cholera vaccine, enriched with O-antigen Ogawa, was found to be safe; vaccination with this vaccine was not accompanied by the development of systemic and local reactions whose frequency and intensity met the requirements for the reactogenic properties of commercial cholera vaccine. The immunological potency of the bivalent vaccine with respect to strain Inaba was not inferior to that of the commercial vaccine; at the same time in persons immunized with the new preparation the titers of vibriocidal antibodies to strain of serovar Inaba were five-fold higher. The conclusion on the expediency of using cholera chemical vaccine enriched with O-antigen Ogawa was made.     

Development of Stable Vibrio cholerae O1 Hikojima Type Vaccine Strains Co–Expressing the Inaba and Ogawa Lipopolysaccharide Antigens

We describe here the development of stable classical and El Tor V. cholerae O1 strains of the Hikojima serotype that co–express the Inaba and Ogawa antigens of O1 lipopolysaccharide (LPS). Mutation of the wbeT gene reduced LPS perosamine methylation and thereby gave only partial transformation into Ogawa LPS on the cell surface. The strains express approximately equal amounts of Inaba– and Ogawa–LPS antigens which are preserved after formalin–inactivation of the bacteria. Oral immunizations of both inbred and outbred mice with formalin–inactivated whole–cell vaccine preparations of these strains elicited strong intestinal IgA anti–LPS as well as serum vibriocidal antibody responses against both Inaba and Ogawa that were fully comparable to the responses induced by the licensed Dukoral vaccine. Passive protection studies in infant mice showed that immune sera raised against either of the novel Hikojima vaccine strains protected baby mice against infection with virulent strains of both serotypes. This study illustrates the power of using genetic manipulation to improve the properties of bacteria strains for use in killed whole–cell vaccines.     

Mass Vaccination with a New,Less Expensive Oral Cholera Vaccine Using Public Health Infrastructure in India: The Odisha Model

Introduction

The substantial morbidity and mortality associated with recent cholera outbreaks in Haiti and Zimbabwe, as well as with cholera endemicity in countries throughout Asia and Africa, make a compelling case for supplementary cholera control measures in addition to existing interventions. Clinical trials conducted in Kolkata, India, have led to World Health Organization (WHO)-prequalification of Shanchol, an oral cholera vaccine (OCV) with a demonstrated 65% efficacy at 5 years post-vaccination. However, before this vaccine is widely used in endemic areas or in areas at risk of outbreaks, as recommended by the WHO, policymakers will require empirical evidence on its implementation and delivery costs in public health programs. The objective of the present report is to describe the organization, vaccine coverage, and delivery costs of mass vaccination with a new, less expensive OCV (Shanchol) using existing public health infrastructure in Odisha, India, as a model.

Methods

All healthy, non-pregnant residents aged 1 year and above residing in selected villages of the Satyabadi block (Puri district, Odisha, India) were invited to participate in a mass vaccination campaign using two doses of OCV. Prior to the campaign, a de jure census, micro-planning for vaccination and social mobilization activities were implemented. Vaccine coverage for each dose was ascertained as a percentage of the censused population. The direct vaccine delivery costs were estimated by reviewing project expenditure records and by interviewing key personnel.

Results

The mass vaccination was conducted during May and June, 2011, in two phases. In each phase, two vaccine doses were given 14 days apart. Sixty-two vaccination booths, staffed by 395 health workers/volunteers, were established in the community. For the censused population, 31,552 persons (61% of the target population) received the first dose and 23,751 (46%) of these completed their second dose, with a drop-out rate of 25% between the two doses. Higher coverage was observed among females and among 6–17 year-olds. Vaccine cost at market price (about US$1.85/dose) was the costliest item. The vaccine delivery cost was $0.49 per dose or $1.13 per fully vaccinated person.

Discussion

This is the first undertaken project to collect empirical evidence on the use of Shanchol within a mass vaccination campaign using existing public health program resources. Our findings suggest that mass vaccination is feasible but requires detailed micro-planning. The vaccine and delivery cost is affordable for resource poor countries. Given that the vaccine is now WHO pre-qualified, evidence from this study should encourage oral cholera vaccine use in countries where cholera remains a public health problem.     

Investigation towards bivalent chemically defined glycoconjugate immunogens prepared from acid-detoxified lipopolysaccharide of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O1, serotype Inaba

A free amino group present on the acid-detoxified lipopolysaccharide (pmLPS) of V. cholerae O1 serotype Inaba was investigated for site-specific conjugation. Chemoselective pmLPS biotinylation afforded the corresponding mono-functionalized derivative, which retained antigenicity. Thus, pmLPS was bound to carrier proteins using thioether conjugation chemistry. Induction of an anti-LPS antibody (Ab) response in BALB/c mice was observed for all conjugates. Interestingly, the sera had vibriocidal activity against both Ogawa and Inaba strains opening the way to a possible bivalent vaccine. However, the level of this Ab response was strongly affected by both the nature of the linker and of the carrier. Furthermore, no switch from IgM to IgG, i.e. from a T cell-independent to a T cell-dependent immune response was detected, a result tentatively explained by the possible presence of free polysaccharide in the formulation. Taken together, these results encourage further investigation towards the development of potent pmLPS-based neoglycoconjugate immunogens, fully aware of the challenge faced in the development of a cholera vaccine that will provide efficient serogroup coverage.     

吸附霍乱菌苗免疫后人群血清杀弧菌抗体观察

本文介绍了用霍乱杀弧菌抗体微量法,检测经Ｏ１型吸附霍乱菌苗免疫前后人群的Ｏ１型霍乱和Ｏ１３９型霍乱的血清杀弧菌抗体。结果表明,免前人群的Ｉｎａｂａ、Ｏｇａｗａ、Ｏ１３９型三种自然杀弧菌抗体很低（ＧＭＴ在５．４９以下）,免后Ｉｎａｂａ和Ｏｇａｗａ型杀弧菌抗体大幅度增加,ＧＭＴ分别达到７８２和５９２、Ｏ１３９型霍乱杀弧菌抗体免疫前后无变化     

Age-Dependent Association among Helicobacter pylori Infection,Serum Pepsinogen Levels and Immune Response of Children to Live Oral Cholera Vaccine CVD 103-HgR

Background

Through its effects on gastric secretion, we hypothesized that Helicobacter pylori infection may influence oral immunization. Accordingly, we examined the association between H. pylori infection, serum pepsinogen (PG) (measures for H. pylori gastritis) and vibriocidal antibody (a correlate of protection) seroconversion following oral immunization with CVD 103-HgR live cholera vaccine among children of different ages.

Methods

Sera from 422 Chilean children who were vaccinated with a single dose of CVD 103-HgR were tested by ELISA for serum IgG antibodies to H. pylori, PG I and PG II levels and antibodies to Shigella flexneri 2a lipopolysaccharide and hepatitis A virus (as markers of low socioeconomic status and exposure to enteric pathogens).

Results

The likelihood of vibriocidal antibody seroconversion following vaccination with CVD 103-HgR was significantly decreased in H. pylori-seropositive children age 6 months to 4 years with PG II>8 µg/L (adjusted OR 0.14 (95% CI 0.03–0.61; P = 0.009), and also in H. pylori seropositives with lower PG II level (adjusted OR 0.34, 95% CI 0.14–0.83; P = 0.017), compared to H. pylori-seronegatives. H. pylori-seropositive children aged 5–9 years with serum PG I>30 µg/L (indicating more severe gastritis) had higher odds of vibriocidal seroconversion than those with lower PG I levels (adjusted OR 4.41, 95%CI 1.26–15.38; P = 0.02). There was no significant association between exposures to S. flexneri 2a or hepatitis A virus and vibriocidal seroconversion.

Conclusions

As H. pylori gastritis progresses with increasing pediatric age in developing country venues, changes in gastric secretion ensue that we believe explain the observed differences in age-related immune responses to immunization with live oral cholera vaccine. The effect of H. pylori and changes of gastric acid secretion on the immunogenicity of various oral vaccines should be studied in different developing, transitional and industrialized country settings.     

O1群霍乱Ogawa血清型结合疫苗免疫学效果研究

霍乱是经粪口传播的烈性传染病,相应的疫苗研究已逾百年,但目前还没有理想的疫苗。实验中以霍乱O1群小川血清型的脂多糖为目标抗原,用不同方法制备了其四种霍乱结合疫苗,通过小鼠模型验证了各结合物的免疫学效果。结果显示,不同结合物免疫学效果不一,其中增大多糖分子量后制备的结合物免疫效果较好,氨还原法制备的结合物多针免疫后也可诱导特异性抗体产生,而且具有针对小川和稻叶两种血清型的杀弧菌活性。     

Susceptibility to Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh

Background

Despite recent progress in understanding the molecular basis of Vibrio cholerae pathogenesis, there is relatively little knowledge of the factors that determine the variability in human susceptibility to V. cholerae infection.

Methods and Findings

We performed an observational study of a cohort of household contacts of cholera patients in Bangladesh, and compared the baseline characteristics of household members who went on to develop culture-positive V. cholerae infection with individuals who did not develop infection. Although the vibriocidal antibody is the only previously described immunologic marker associated with protection from V. cholerae infection, we found that levels of serum IgA specific to three V. cholerae antigens—the B subunit of cholera toxin, lipopolysaccharide, and TcpA, the major component of the toxin–co-regulated pilus—also predicted protection in household contacts of patients infected with V. cholerae O1, the current predominant cause of cholera. Circulating IgA antibodies to TcpA were also associated with protection from V. cholerae O139 infection. In contrast, there was no association between serum IgG antibodies specific to these three antigens and protection from infection with either serogroup. We also found evidence that host genetic characteristics and serum retinol levels modify susceptibility to V. cholerae infection.

Conclusions

Our observation that levels of serum IgA (but not serum IgG) directed at certain V. cholerae antigens are associated with protection from infection underscores the need to better understand anti–V. cholerae immunity at the mucosal surface. Furthermore, our data suggest that susceptibility to V. cholerae infection is determined by a combination of immunologic, nutritional, and genetic characteristics; additional factors that influence susceptibility to cholera remain unidentified.     

Individuals with Le(a+b-) blood group have increased susceptibility to symptomatic vibrio cholerae O1 infection

Background

Human genetic factors such as blood group antigens may affect the severity of infectious diseases. Presence of specific ABO and Lewis blood group antigens has been shown previously to be associated with the risk of different enteric infections. The aim of this study was to determine the relationship of the Lewis blood group antigens with susceptibility to cholera, as well as severity of disease and immune responses to infection.

Methodology

We determined Lewis and ABO blood groups of a cohort of patients infected by Vibrio cholerae O1, their household contacts, and healthy controls, and analyzed the risk of symptomatic infection, severity of disease if infected and immune response following infection.

Principal Findings

We found that more individuals with cholera expressed the Le(a+b−) phenotype than the asymptomatic household contacts (OR 1.91, 95% CI 1.03–3.56) or healthy controls (OR 1.90, 95% CI 1.13–3.21), as has been seen previously for the risk of symptomatic ETEC infection. Le(a–b+) individuals were less susceptible to cholera and if infected, required less intravenous fluid replacement in hospital, suggesting that this blood group may be associated with protection against V. cholerae O1. Individuals with Le(a–b−) blood group phenotype who had symptomatic cholera had a longer duration of diarrhea and required higher volumes of intravenous fluid replacement. In addition, individuals with Le(a–b−) phenotype also had lessened plasma IgA responses to V. cholerae O1 lipopolysaccharide on day 7 after infection compared to individuals in the other two Lewis blood group phenotypes.

Conclusion

Individuals with Lewis blood type Le(a+b−) are more susceptible and Le(a–b+) are less susceptible to V. cholerae O1 associated symptomatic disease. Presence of this histo-blood group antigen may be included in evaluating the risk for cholera in a population, as well as in vaccine efficacy studies, as is currently being done for the ABO blood group antigens.     

Detection and molecular characterization of Vibrio cholerae O1 Inaba biotype El Tor strain in Kerala, S. India

The resurgence of enteric pathogen Vibrio cholerae, the causative organism of epidemic cholera, remains a major health problem in many developing countries. The outbreaks of cholera follow a seasonal pattern in regions of endemicity. The southern Indian state of Kerala is endemic to cholera. A V. cholerae strain isolated from the stool sample of a patient in Piravam, Kerala, South India, was analysed. However, this case occurred at a time not associated with cholera outbreaks, leading to concern among the State health officials. We compared the virulence potential of the isolate with that of the standard or reference strains, that have been widely used as positive control. The isolate was identified as V. cholerae O1 biotype El Tor serotype Inaba. The resistance pattern of the isolate to common antibiotics was examined and it was found to be multi-drug resistant in nature. The strain was analysed for the presence of the CTX genetic element, which encodes genes for cholera toxin and other important regulatory genes. It was found to be positive for all the genes tested. In Kerala, most of the cholera outbreaks have been reported to be caused by V. cholerae O1 El Tor belonging to Ogawa serotype. Interestingly, the V. cholerae strain isolated from this case has been found to be of Inaba serotype, which is rarely reported.     

Evolutionary perspective on the origin of Haitian cholera outbreak strain

Cholera epidemic has not been reported in Haiti for at least 100?years, although cholera has been present in Latin America since 1991. Surprisingly, the recent cholera epidemic in Haiti (October 2010) recorded more than 250,000 cases and 4000 deaths in the first 6?months and became one of the most explosive and deadly cholera outbreak in recent history. In the present study, we conducted genomic analyses of pathogenicity islands of three Haitian Vibrio cholerae strains and compared them with nine different V. cholerae O1 El Tor genomes. Although CIRS101 is evolutionarily most similar to the Haitian strains, our study also provides some important differences in the genetic organization of pathogenicity islands of Haitian strains with CIRS101. Evolutionary analysis suggests that unusual functional constraints have been imposed on the Haitian strains and we hypothesize that amino acid substitution is more deleterious in Haitian strains than in nonHaitian strains.     

The Population Structure of Vibrio cholerae from the Chandigarh Region of Northern India

Background

Cholera infection continues to be a threat to global public health. The current cholera pandemic associated with Vibrio cholerae El Tor has now been ongoing for over half a century.

Methodology/Principal Findings

Thirty-eight V. cholerae El Tor isolates associated with a cholera outbreak in 2009 from the Chandigarh region of India were characterised by a combination of microbiology, molecular typing and whole-genome sequencing. The genomic analysis indicated that two clones of V. cholera circulated in the region and caused disease during this time. These clones fell into two distinct sub-clades that map independently onto wave 3 of the phylogenetic tree of seventh pandemic V. cholerae El Tor. Sequence analyses of the cholera toxin gene, the Vibrio seventh Pandemic Island II (VSPII) and SXT element correlated with this phylogenetic position of the two clades on the El Tor tree. The clade 2 isolates, characterized by a drug-resistant profile and the expression of a distinct cholera toxin, are closely related to the recent V. cholerae isolated elsewhere, including Haiti, but fell on a distinct branch of the tree, showing they were independent outbreaks. Multi-Locus Sequence Typing (MLST) distinguishes two sequence types among the 38 isolates, that did not correspond to the clades defined by whole-genome sequencing. Multi-Locus Variable-length tandem-nucleotide repeat Analysis (MLVA) identified 16 distinct clusters.

Conclusions/Significance

The use of whole-genome sequencing enabled the identification of two clones of V. cholerae that circulated during the 2009 Chandigarh outbreak. These clones harboured a similar structure of ICEVchHai1 but differed mainly in the structure of CTX phage and VSPII. The limited capacity of MLST and MLVA to discriminate between the clones that circulated in the 2009 Chandigarh outbreak highlights the value of whole-genome sequencing as a route to the identification of further genetic markers to subtype V. cholerae isolates.