Abstract: Deaths from cholera are again making news, this time in Angola. Drs. David Sack, Bradley Sack, and Claire-Lise Chaignat discuss whether there is hope for controlling cholera.

Abstract: Toxin-coregulated pilin A (TcpA) is the main structural subunit of a type IV bundle-forming pilus of Vibrio cholerae, the cause of cholera. Toxin-coregulated pilus is involved in formation of microcolonies of V. cholerae at the intestinal surface, and strains of V. cholerae deficient in TcpA are attenuated and unable to colonize intestinal surfaces. Anti-TcpA immunity is common in humans recovering from cholera in Bangladesh, and immunization against TcpA is protective in murine V. cholerae models. To evaluate whether transcutaneously applied TcpA is immunogenic, we transcutaneously immunized mice with 100 μg of TcpA or TcpA with an immunoadjuvant (cholera toxin [CT], 50 μg) on days 0, 19, and 40. Mice immunized with TcpA alone did not develop anti-TcpA responses. Mice that received transcutaneously applied TcpA and CT developed prominent anti-TcpA immunoglobulin G (IgG) serum responses but minimal anti-TcpA IgA. Transcutaneous immunization with CT induced prominent IgG and IgA anti-CT serum responses. In an infant mouse model, offspring born to dams transcutaneously immunized either with TcpA and CT or with CT alone were challenged with 106 CFU (one 50% lethal dose) wild-type V. cholerae O1 El Tor strain N16961. At 48 h, mice born to females transcutaneously immunized with CT alone had 36% ± 10% (mean ± standard error of the mean) survival, while mice born to females transcutaneously immunized with TcpA and CT had 69% ± 6% survival (P < 0.001). Our results suggest that transcutaneous immunization with TcpA and an immunoadjuvant induces protective anti-TcpA immune responses. Anti-TcpA responses may contribute to an optimal cholera vaccine.

Abstract: Objectives The effectiveness of vaccines in populations must consider both direct and indirect protection. This study reanalyses data from a large individually randomized oral cholera vaccine trial that was conducted in rural Bangladesh from 1985 to 1990. A recent analysis of the results of that trial showed that the proportion of people in household clusters who received the vaccine was inversely related to placebo incidence during the first year of surveillance, which was attributed to herd immunity. Methods In this study we measure the relationship between neighbourhood-level oral cholera vaccine coverage and protective efficacy (PE) during a 2 year follow-up period, controlling for known effect modifiers. We link trial data to a household geographic information system to facilitate the neighbourhood-level analysis. Findings Neighbourhood-level PE can be partially explained by vaccine coverage after adjusting for ecological variables.

Abstract: Recent analysis of results of the 1985 vaccine trial in Bangladesh showed that a killed oral cholera vaccine could provide herd protection (1), and this finding sheds new light on the potential utility of this vaccine and other oral cholera vaccines. Although there may be several mechanisms for herd protection, this finding of herd protection was somewhat unexpected. The nature of the herd protection with cholera vaccine is unlike that with live oral polio vaccine which can be excreted and can infect others, thereby immunizing persons who did not receive vaccine directly. By contrast, the cholera vaccine used in this study was inactivated, making it impossible for non-immunized persons to be immunized inadvertently. Another type of herd protection is seen with vaccine for Haemophilus influenzae type b in which the vaccination reduces respiratory carriage of the pathogen, thereby eradicating the reservoir and reducing transmission. Another example is that of measles vaccination which essentially stops transmission when the density of susceptible subjects is reduced below that needed to sustain transmission. Since cholera is transmitted directly from contaminated food or water, the finding of herd immunity seemed not entirely expected. This editorial reviews the evidence for herd protection and introduces new findings from the environmental studies on cholera to suggest a more complete understanding of the mechanisms for herd protection with cholera vaccine. Hopefully, by combining the observations of 'herd protection' with some newer concepts of 'herd amplification' coming from recent environmental studies, we may develop a better understanding of the most efficient ways to control cholera. A review of the evidence for herd protection The first suggestion of herd protection came from one analysis of the 1985 trial in which young children who had not received vaccine were about 50% less likely to be infected if their mothers had received vaccine (2). It was hypothesized that mothers receiving vaccine would have increased levels of antibody in their breastmilk and that these antibodies might protect their young children. However, the antibody titres in breastmilk of vaccinated mothers were not different from that in mothers who were given placebo. The protection observed without a corresponding elevation in breastmilk antibody titres suggested a different mechanism not associated specifically with protection from breastmilk antibodies. It seemed more likely that the immunized mothers were less likely to be colonized with Vibrio cholerae and pass this organism on to their children. Thus, the children had some protection from the vaccine, although they themselves were not vaccinated. More direct evidence of herd protection came from the recent re-analysis of the same 1985 field trial which combined GIS analysis with the data of cases occurring during the first year of the trial (1). The re-analysis included 89,596 people who were individually randomized and were immunized with B-subunit-whole cell (BS-WC) vaccine, the whole cell (WC) only vaccine, or a placebo. In this trial, both BS-WC and WC only vaccines were protective and thus, for this analysis, the vaccinated groups were combined. This new analysis was intended to determine if there was evidence of an indirect protective effect among the 6,423 geographic clusters depending on the proportion of persons in the clusters who had received one of the active vaccines (either the BS-WC or the WC only vaccine). It was hypothesized that an indirect effect of the vaccine would be manifested by lower incidence rates of cholera among recipients of placebo in clusters with higher levels of vaccine coverage. Since the randomization was by individual, the proportion of persons receiving an active vaccine varied widely between the different clusters, with proportions receiving active vaccine between 4% and 65% in the different clusters. For this analysis, the unit of the cluster is the bari which is a group of houses within the village. …

Abstract: In Expert Review of Vaccines 5(4), 483–494 (2006), Edward T Ryan and colleagues give an overview of live-attenuated cholera vaccines [1]. However, when referring to the killed whole-cell/cholera to...

Abstract: SUMMARY An oral cholera vaccine made up of heat-treated recombinant cholera toxin (rCT), V. cholerae lipopolysaccharide (LPS), and recombinant toxin-co-regulated pili subunit A (rTcpA), entrapped in liposomes in the presence of unmethylated bacterial CpG-DNA (ODN#1826) was used to orally immunize a group of eight week old rats. A booster dose was given 14 days later. Control rats received placebo (vaccine diluent). The kinetics of the immune response were investigated by enumerating the antigen specific-antibody secreting cells (ASC) in the blood circulation and intestinal lamina propria using the ELISPOT assay and a histo-immunofluorescence assay (IFA), respectively. ASC of all antigenic specificities were detected in the blood of the vaccinated rats as early as two days after the booster dose. The numbers of LPS-ASC and TcpA-ASC in the blood were at their peak at day 3 post booster while the number of CT-ASC was highest at day 4 after the booster immunization. At day 13 post im- munization, no ASC were detected in the blood. A several fold increase in the number of ASC of all antigenic specificities in the lamina propria above the background numbers of the control animals were found in all vacci- nated rats at days 6 and 13 post booster (earlier and later time points were not studied). Vibriocidal antibody and specific antibodies to CT, LPS and TcpA were detected in 57.1% and 52.4%, 14.3%, and 19.0% of the orally vacci- nated rats, respectively. The data indicated that rats orally primed with the vaccine could produce a rapid anam- nestic response after re-exposure to the V. cholerae antigens. Thus, a single dose of the vaccine is expected to elicit a similar anamnestic immune response in people from cholera endemic areas who have been naturally primed to V. cholerae antigens, while two doses at a 14 day interval should be adequate for a traveler to a disease endemic area.

Abstract: Problem Cholera is the paradigm for waterborne bacterial diseases. For over a 100 years, scientists have tried to develop a universally effective vaccine for cholera. We are hampered in our efforts because we do not know the details of the basic immune response to Vibrio cholerae antigens. What are the most proactive antigens? What special needs for immunization are engendered by previous exposure to cholera or the age of the individual? How long does immunity last, and is this immunity a classic immunologic memory or re-exposure and continual boosting? Method of study Immunization with synthetic derivatives of the carbohydrate moieties of V. cholerae lipopolysaccharide (LPS) coupled to different carrier proteins (neoglycoconjugates, NGC) has allowed dissection of the response to the disaccharide array of perosamine that represent either the Inaba or the Ogawa serotype. Studying serum anti-LPS endpoint titers and the serum vibriocidal response to NGC provides insight into the importance of LPS serotype-specific B-cell epitopes and how antibody response are influenced by the form of the LPS immunogen. Results We found that murine serum antibody responses to V. cholerae LPS are dynamic. The magnitude of serum anti-LPS antibody titers and the capacity to induced vibriocidal antibodies (immunoglobulin M) are influenced by the initial immunizing serotype of LPS, the structure of the LPS immunogen (native LPS versus NGC), and the order of serotype immunization in a prime boost immunization strategy. The dynamic of the immune response to LPS immunogens is typified by the fact that the host species can affect the immunization response. We found mice do not make vibriocidal antibody to Inaba NGC but rabbits do. This is in contrast to the Ogawa NGC that induced vibriocidal antibody in mice. Conclusion The results suggest that the host's B-cell repertoire can influence the immunization efficacy; therefore, the development of the new generation of NGC V. cholerae vaccines should focus on human volunteers and their ability to mount protective responses.

Abstract: Cholera remains an important public health threat. A cholera vaccine that provides durable protection at the mucosal surface, especially among children in endemic settings, is urgently needed. The availability of the complete genome sequence of a clinical isolate of Vibrio cholerae O1 El Tor has allowed for comparative and functional genomic approaches in the study of cholera. This work holds promise for the identification of bacterial targets of protective human immune responses and may contribute to the development of a new generation of cholera vaccines.

Abstract: Vibrio cholerae serogroups O1 and O139 Bengal produce cholera toxin (CT) a typical AB5 bacterial toxin comprising an ADP-ribosylation enzyme A-subunit (CTA) and a carbohydrate binding B-subunit (CTB). DUKORALR the inactivated oral cholera vaccine has recently been licensed for use in the European Union. This vaccine contains killed whole cells of V. cholerae and 1 mg of purified recombinant CTB (rCTB). DUKORALR has a good safety profile and there has been no indication that active CT is present. Nevertheless, an assay that confirms the absence of active CTA in the vaccine is advantageous to ensure vaccine safety. Conventional assays such as the Y-cell assay can not detect biologically active amounts of CT in DUKORALR because of the large amount of rCTB present. We have developed an assay based on a fluorescently labelled 11-mer peptide substrate that detects CTA activity despite the presence of excess rCTB.

Abstract: The live oral polio vaccine was the first mucosal vaccine accepted for general use. Since then, similar vaccines have been developed against typhoid fever, cholera and rotavirus infection, and a nasal vaccine against influenza has recently been registered in the USA. The only non-living mucosal vaccine on the market today is an oral cholera vaccine consisting of inactivated Vibrio cholerae and the B subunit of cholera toxin. Several groups of scientists are at present working on the development of other mucosal vaccines based on inactivated microbes or parts of them. Results from animal trials at the Norwegian Institute of Public Health, suggest that non-living nasal vaccines can provide protective immunity and may be combined with the same types of vaccines for injection. Clinical trials with nasal vaccines consisting of beta-propiolactone inactivated influenza particles, showed that it was possible to achieve serum concentrations of antibodies at levels providing protection against influenza. IgA antibodies, which were formed in nasal secretions, were specifically aimed at influenza and ought to hinder the spread of the disease. By optimizing the immunization regimes so that the immunological memory is better exploited, and by adding adjuvants to the formulations, it is probable that non-living mucosal vaccines can be realistic alternatives to several of the vaccines now given by injection.

Abstract: The WHO has issued a new set of recommendations for the control of cholera. And there, hidden in a forest of clauses of the kind more frequently seen in prenuptial agreements than in health recommendations, the careful reader will find the statement ‘... the use of OCV* in certain endemic situations should be recommended...’. Readers unfamiliar with the protracted battle over the use of vaccines for the control of cholera in endemic situations may read this sentence and miss a sea change. Cholera has a cachet in the lay world similar to tuberculosis or malaria. After all it is hard to imagine that Gabriel Garcia Marquez would have called his novel ‘love in a time of shigellosis’ or any other enteric disease; (Marquez 1988). There remains a justified horror of cholera outbreaks among people in less fortunate conditions. A cholera outbreak in Europe or the US is hard to imagine but so is a malaria outbreak. The reason why we do not have a world cholera day is probably because of the low mortality associated with cholera – if it is treated appropriately. A disease which can be cured within several days with rehydration and oral antibiotics does not register when we assess the impact in disability-adjusted life years (DALYs). And herein lies a major problem. DALYs were a major breakthrough. Bill Gates reportedly called the World Development Report 1993: Investing in Health (World Bank 1993), which brought the concept of DALYs to a wider public, a terrific read. Yet DALYs fail to capture the significance of a disease like cholera. Having lived through a single cholera outbreak is probably sufficient to fully understand the chain of catastrophic events: initially, curiosity about the first cases of watery diarrhoea admitted; the nosocomical infection of most or all patients on the ward; the closure of the ward; rigorous but belated disease-control measures; hospitals besieged by patients with watery diarrhoea; excess mortality not so much among the cholera patients but among other patients who cannot access the care they require. These are just the direct consequences. Family members have to look after their sick, shops and banks stay closed and the normal functions of a community come to a halt. At which stage the media catch on to the news and draw attention to the catastrophe affecting the community. In due time the government points out the damage caused by these unsubstantiated reports, and where an opposition exists the blame game can begin. This constitutes another unusual aspect of cholera. Not only can the damage not be assessed in DALYs, cholera remains a highly political issue. No matter where we met with affected communities, invariably the government was considered at least partially responsible and consequently highly sensitive. For example: Thailand does not report cholera cases. Public health experts are aware that ‘acute watery diarrhoea’ cases in Thailand are likely to be thinly disguised cholera cases. But before we single out Thailand, the fact that no cholera case has been reported from the People’s Republic of China has less to do with the absence of cholera than with the complete clampdown on reporting of cholera cases. Younger public health professionals early in their training become aware of the individual stigma associated with AIDS. Communities reporting cholera outbreaks are well aware of the stigma associated with cholera outbreaks. Besides the intangible damage cause by a tarnished reputation (who wants to live in a city thought to be cholera-ridden?) other negative effects should be measurable. The two economic sectors principally affected are seafood exports and tourism. While it is possible that Vibrio are inadvertently allowed to contaminate the water surrounding seafood during packaging, there is no evidence that Vibrio infect shrimp or fish. Yet the export market of small nations can collapse after reports of a cholera outbreak. Similarly, most tourists have no chance of experiencing a cholera episode as long as they follow the most basic rules of hygiene (only drink bottled or boiled water, only eat well-cooked food and fruit you have peeled yourself). It may therefore not come as a surprise that the countries currently reporting cholera outbreaks derive a larger income from humanitarian aid than from seafood exports and tourism: 94% of the 101 383 cholera cases and 99% of the 2345 related deaths registered with the WHO in 2004 occurred in sub-Saharan Africa (Sack et al. 2006). So what is a government official in charge of preventing the next cholera outbreak in her country supposed to do? If *Oral cholera vaccine. Tropical Medicine and International Health doi:10.1111/j.1365-3156.2006.01771.x

Abstract: This paper investigated the impact of interviewers on respondent self-reported willingness to pay for cholera vaccines in three study sites: Kolkata, India; Matlab, Bangladesh; and Beira, Mozambique. Specifically examined were the relationships between interviewer gender and respondent gender, age, and education level. The analysis also measured the overall variance in responses caused by individual interviewers. It was found that only individual interviewer effects are present in the Kolkata and Beira studies, while interviewer gender effects and gender-gender, -age, and –education interactions are present in the Matlab study. When controlling for individual interviewer effects in the Matlab analysis, only the gender-education interaction is still significant. Despite the high levels of significance of individual interviewer effects in each study, the magnitude of these effects on response variance as indicated by their coefficients is still fairly small. These results suggest that these CVM studies may be more resistant to interviewer characteristic effects than other studies that a) focus on gender-sensitive issues, b) use smaller numbers of interviewers or c) use smaller sample sizes. The results also suggest that while whole interviewer effects are present in all three studies, the size of the interviewer teams prevents the few aberrant interviewers from causing large amounts of variance across the whole sample, keeping the CVM results reliable and likely to be generalizable to their respective populations.

Abstract: To the Editor: Two recent articles indicate the continued availability to clinicians of a choice of three available cholera vaccines. 1,2 Production and sale of whole‐cell …

Abstract: To the Editor: In their editorial “Caring for the Caregivers” Katz and colleagues give arguments for why a team of two physicians and two nurses from Israel assisting in a cholera epidemic in Equatorial Guinea was not vaccinated against cholera, although they recognized the protective efficacy …

Abstract: Cholera is an acute diarrheal disease caused by the serogrups O1 and O139 of the enteropathogenic bacterium Vibrio cholerae that continuous causing epidemic outbreaks in many underdeveloped countries where this disease is endemic. The most promising strategy for developing an effective cholera vaccine has focused in the use of V. cholerae live attenuated strains as oral vaccines. The behavior of these genetically modified strains in the environment during vaccination campaigns is unpredictable. Therefore, there is the risk of acquisition of virulent genes through horizon- tal gene transfer mechanisms mediated by other microorganisms. In order to decrease the chance of occurrence of these undesired genetic events this work describes the construction of new vaccine strain candidates with diminished capacity to proliferate in the environment. The new strains were generated by introducing a defined mutation in the lysA gene of the previously obtained V. cholerae vaccine strain candidate 1333. This gene encodes the enzyme diaminopimelate descarboxylase, which catalizes the last step in the lysine biosynthetic pathway. The resultant mutant strains are lysine auxotrophs and for that reason they have diminished capacity to proliferate in lysine-deficient conditions. One of the mutants, 1333L2-2, was compared with its parental strain 1333 regarding morphology, in vitro growing rate, motility and colonization in the infant mouse cholera model and these features were similar in both strains.

Abstract: Live, orally administered, attenuated vaccine strains of Vibrio cholerae have many theoretical advantages over killed vaccines. A single oral inoculation could result in intestinal colonization and rapid immune responses, obviating the need for repetitive dosing. Live V. cholerae organisms can also respond to the intestinal environment and immunological exposure to in vivo expressed bacterial products, which could result in improved immunological protection against wild-type V. cholerae infection. The concern remains that live oral cholera vaccines may be less effective among partially immune individuals in cholera endemic areas as pre-existing antibodies can inhibit live organisms and decrease colonization of the gut. A number of live oral cholera vaccines have been developed to protect against cholera caused by the classical and El Tor serotypes of V. cholerae O1, including CVD 103-HgR, Peru-15 and V. cholerae 638. A number of live oral cholera vaccines have also been similarly developed to protect against cholera caused by V. cholerae O139, including CVD 112 and Bengal-15. Live, orally administered, attenuated cholera vaccines are in various stages of development and evaluation.

Abstract: Cholera, a diarrheal disease, is known for explosive epidemics that can quickly kill thousands. Endemic cholera is a seasonal torment that also has a significant mortality. Not all nations with extensive rural communities can achieve the required infrastructure or behavioral changes to prevent epidemic or endemic cholera. For some communities, a single-dose cholera vaccine that protects those at risk is the most efficacious means to reduce morbidity and mortality. It is clear that our understanding of what a protective cholera immune response is has not progressed at the rate our understanding of the pathogenesis and molecular biology of cholera infection has. This review addresses V. cholerae lipopolysaccharide (LPS)-based immunogens because LPS is the only immunogen proven to induce protective antibody in humans. We discuss the role of anti-LPS antibodies in protection from cholera, the importance and the potential role of B cell subsets in protection that is based on their anatomical location and the intrinsic antigen-receptor specificity of various subsets is introduced.