Initiative for Vaccine Research (IVR)

Diarrhoeal Diseases (Updated February 2009)

Cholera

Introduction

Cholera is a substantial health burden in many countries in Africa, Asia, and South and Central America, where it is endemic. The exact scale of the problem is however uncertain because of weaknesses in the existing surveillance system, difficulties to clinically distinguish mild-to-moderate case of cholera from other causes of acute diarrhoea and failures to report cases or even outbreaks to WHO, which acknowledges that only around 5%-10% of cholera cases are actually reported [57].

Disease Burden

Cholera is an acutely dehydrating, watery diarrhoeal disease with vomiting, caused by intestinal infection with Vibrio cholerae. The acute form of the disease (cholera gravis) leads within hours to hypovolemia, acidosis, and potassium deficiency from the loss of fluid and electrolytes [58] [59]. Complications include renal failure, pulmonary oedema, abortion in pregnant women, and profound hypoglycemia and seizures in young children. Before the advent of effective rehydration therapy, cholera epidemics were associated with CFRs exceeding 40% and led to tens of thousands of deaths [60]. Rapid administration of fluid replacement therapy and supportive treatment have reduced mortality to around 4%.

Cholera probably has existed on the Indian subcontinent for thousands of years as judged from ancient manuscripts. The disease was repeatedly one of the most dreaded pandemic diseases in history, being able to spread rapidly to large numbers of people, with a high CFR. Seven cholera pandemics have been recorded since the early nineteenth century. The current “seventh” cholera pandemic started in 1961 and is still continuing. This pandemic, due to the emergence of V cholerae biotype El Tor, first appeared in Indonesia and has since spread worldwide, leading to the re-emergence of cholera in Africa in the early 1970s and in Central and South America in the early 1990s. While cholera no longer poses a threat to countries with minimum standards of hygiene, it remains a challenge to countries where access to safe water and proper sanitation are not guaranteed.

Cholera is transmitted via the faecal-oral route, with epidemic outbreaks often occurring after wars or civil unrests or after natural disasters including flooding when water and/or food supplies become contaminated in crowded population settings with limited sanitation. The disease is now endemic in many parts of Africa and Asia. Explosive outbreaks usually occur in areas with inadequate sanitation, poor hygiene, and lack of safe water supplies, whereas in some countries, a seasonal rhythm for cholera epidemics has been observed [61] [62] . Recent outbreaks of cholera in several countries including Iraq, India, and Sudan illustrate the fact that cholera today remains an important threat in almost every developing country in the world.

Vibrios can also persist for long periods of time in environmental waters where they associate with plankton, shellfish and algae, constituting a long-term reservoir. During epidemics, coastal waters become heavily contaminated with V cholerae from infected humans and can be at the origin of cases through drinking of contaminated water or consumption of contaminated seafood, particularly undercooked shellfish [63].

The number of cholera cases reported to WHO annually has remained relatively constant since 1995, varying from 100,000 to 300,000 cases per year, with Africa accounting for >94% of the total. In 2006, a total of 236,896 cases were notified to WHO from 52 countries; 31 out of 46 African countries experienced an outbreak of cholera and reported a total number of cases of 202,407 with 5,259 deaths [64] . These numbers however appear to be grossly underestimated, as many countries in the Indian subcontinent and southeast Asia do not report their cholera cases [65] . As an example, no cholera cases have been reported from Thailand since 1994, although V cholerae was readily isolated from stool samples in 5.4% of cases of bacterial diarrhoeal disease between January 1995 and December 2000 in hospitalized children in Bangkok [66] . Similarly, there is no report of cholera cases from Bangladesh, whereas experts estimate that there might be as many as 1 million cases in the country every year. A recent estimate puts the number of people who die from cholera each year at about 120,000, and the total number of yearly cholera cases worldwide at 3-5 million [67] [68].

The number of imported cholera cases notified to WHO likely also represents a substantial underestimate of the true burden of disease. That number was only 68 during 2005, but many additional cases could readily be identified from sources other than WHO [65]. The overall risk for travellers to contract cholera is often reported to be in the order of 2 to 3 cases per 1 million travellers. Recent estimates have put it at approximately 5 cases per 100,000 travellers [69] and it could be as high as 5 per 1000 for travellers visiting countries in which a cholera outbreak is occurring.

Bacteriology

V cholerae was discovered by Robert Koch in the early 1880s in the faeces of a patient with the disease. Only enterotoxigenic V. cholerae serogroup O1 and new serogroup O139, which emerged in the 1990s in Bangladesh and India, are known to cause epidemics of cholera. Other serogroups of V. cholerae (O5, O37, O141) can cause isolated cases of watery diarrhoea, but do not cause epidemics. Isolates of V. cholerae serogroup O1 are classified, on the basis of phenotypic characteristics, into two biotypes, El Tor and classical. Currently, the El Tor biotype is responsible for virtually all the cholera cases throughout the world, and classical isolates have not been encountered since the mid-1990s in Bangladesh. V. cholerae O1 can be further classified into two serotypes, Inaba and Ogawa, based on serum agglutination. A possible third serotype, Hikojima, has been described, but is very rare. Immunity to V. cholerae infection is serogroup-specific, so that vaccines that target serogroup O1 do not protect from infection with serogroup O139.

Steps in the pathogenicity of cholera include colonization of the small intestinal mucosa, production of the pilus structure and elaboration of the enterotoxin cholera toxin (CT), an 84 kD multimeric protein consisting of a central active A subunit bound to five surrounding B subunits [70] . The B subunit is responsible for the binding of the toxin to the GM1 ganglioside receptors on epithelial cell surface, whereas the A subunit, an ADP-rybosilating enzyme, is responsible for the toxicity of the toxin through stimulation of the target cell adenylate cyclase, leading to hypersecretion of fluids and loss of electrolytes.

Vaccine

The historical whole-cell injectable vaccine that was developed by Haffkine in 1894 in India, induced a mediocre 48% protection that lasted for only about three months. The vaccine, which was made of killed V. cholerae strains of both Inaba and Ogawa serotypes, never was recommended by WHO, but still may be currently available in some countries. Two types of cholera vaccines have been developed since then, a killed oral vaccine and a live attenuated oral vaccine; both have been shown to be safe, immunogenic and efficacious [71] [72].

Killed oral cholera vaccines

The killed oral vaccine (DukoralTM, licensed by SBL Vaccine, Sweden, to Crucell, Holland), which is recommended since 1999 by WHO as a tool to prevent cholera in populations at risk of an epidemic in emergency situations, consists of a mixture of four preparations of heat- or formalin-killed whole-cell V. cholerae O1, representing both serotypes (Inaba and Ogawa) and both biotypes (classical and El Tor), that are then added with purified recombinant cholera toxin B subunit (CTB). Because CT cross-reacts with E coli LT, the vaccine also provides short-term protection against ETEC (see below), which is of added benefit for travellers [73] [74].

The DukoralTM whole cell/recombinant B subunit (WC/rBS) vaccine - given orally with buffer to neutralize stomach acidity - was found, in field trials in Bangladesh and Peru, to confer 80-90% protection during 6 months in all age groups after administration of 2 doses 1-2 weeks apart. In Bangladesh, protection declined rapidly in young children after 6 months, but was still about 60% in older children and adults after three years [75] [76] . The vaccine was also successfully used for mass vaccination in refugee camps in Uganda [77] , Darfour and Indonesia (Aceh) to protect at-risk populations from potential cholera outbreaks. In a field trial in Mozambique, the vaccine demonstrated 89% protection against severe diarrhoea with dehydration and 77% protection against milder forms of the disease [78] . An individually randomized, placebo-controlled trial of killed oral cholera vaccines in 89,596 children more than 2 years of age and women in Bangladesh showed that mass vaccination could provide herd immunity, as protection was also found in children less than 2 years of age [79].

The vaccine is currently administered in a 3-doses schedule to 2-6 years old children, with a boost every 6 months; and as a 2-dose regimen to older children and adults, with boosting every 2 years. A variant of the Dukoral vaccine containing no recombinant CTB-subunit has been produced and tested in Viet Nam [80] [81] . It is administered in two doses, 1-2 weeks apart. A field trial conducted in Nha-Trang, Viet Nam, showed an efficacy of 66% against V. cholerae El Tor after 8 months in all age groups tested. The vaccine (ORC-VaxTM) is being used for public health interventions in Viet Nam [82] . A bivalent O1 and O139 whole-cell oral vaccine without CTB has also been developed in Viet Nam and shown to be safe and immunogenic in both adults and children, generating 90% anti-O1 and 68% anti-O139 vibriocidal antibody responses after a two-dose regimen.

Production of the bivalent O1 and O139 ORC-VaxTM vaccine was recently licensed by VaBiotech (Vietnam) to Shantha Biotechnics (India) and BioFarma (Indonesia), which have undertaken a new, complete preclinical and clinical development. A large-scale, randomized, placebo-controlled Phase III trial on 70,000 persons is currently ongoing in urban Kolkata, India, with the help of the International Vaccine Initiative (IVI) in Seoul [83].

Attenuated live oral cholera vaccine

This type of vaccine consists of a live attenuated, genetically modified V. cholerae O1 Inaba strain (CVD103-HgR), which has been engineered to produce the B subunit (CTB) but not the A subunit of CT. The vaccine, OrocholTM (Berna Biotech, now Crucell, Switzerland) was given orally along with buffer to neutralize stomach acidity. It was available in two formulations, a low dose formulation for developed countries and a 10-fold higher dose formulation for developing countries. Placebo-controlled trials in a number of South American and Asian countries demonstrated the safety and immunogenicity of a single dose of OrocholTM [84] . Protection efficacy against experimental challenge given 3 months after vaccination with V. cholerae O1 (of either El Tor or classical biotype) in adult volunteers in the USA was found to be about 80% against all cases of diarrhoeas and 90% against severe diarrhoea. However, in a subsequent large-field trial performed in cholera-endemic Indonesia on 67 000 volunteers, the vaccine failed to demonstrate protection [85] , in part due to the limited number of cholera cases recorded during the trial. Although the vaccine retrospectively showed protective efficacy when used for the control of an ongoing outbreak in the Federated States of Micronesia [86] , the manufacturer discontinued its production in 2004.

Other cholera vaccines in development

Candidate vaccines in development include:

  • a live attenuated, single-dose, oral vaccine (V cholerae 638) developed in Cuba, already tested in Phase II trials in Mozambique [87] ; building on the success of strain 638, Cuban investigators have constructed an analogous attenuated vaccine candidate derived from an O139 strain that should go into clinical trials shortly;
  • a live attenuated O1 El Tor strain (Peru-15) developed as an oral vaccine by AVANT Immunotherapeutics (USA) under the name CholeraGardeTM, which elicited a 62% protection against V cholerae challenge in North American volunteers [88] and was found to be safe and immunogenic in a Phase II trial in Bangladesh [89] . Efficacy studies (Phase IIb) were reportedly imminent. Meanwhile, Peru-15 was engineered to express and secrete high levels of CTB by transfection with a recombinant plasmid carrying the CTB gene under the transcriptional control of a strong constitutive promoter. The resulting strain, Peru-15pCTB, was shown to secrete approximately 30-fold more CTB than Peru-15, was genetically stable, and elicited high anti-CTB, LT-neutralizing antibody titers and high vibriocidal antibody titers when administered by the oral route to rabbits or by the intranasal route to mice. Peru-15pCTB will therefore replace Peru-15 as an oral, single-dose, bivalent cholera/ETEC vaccine candidate [90] . The vaccine currently is undergoing PhaseI/II clinical trials;
  • Bengal 15, similar to Peru-15, and CVD112, are live attenuated strains of V cholerae O139, which have been shown to be safe and immunogenic in Phase I trials in human volunteers [91];
  • a parenteral O-antigen-conjugated vaccine, in preclinical development at the Pasteur Institute in Paris;
  • a parenteral plasmid DNA vaccine, in development at the Putra University in Malaysia and the Malaysia National Biotechnology Directorate;
  • a rice-based oral vaccine made from transgenic rice seeds that express 30 mµg CTB per seed in protein storage organelles where CTB appears to be stable for more than 1.5 year at room temperature. When fed to mice, the transgenic seeds elicited anti-CTB serum IgG and mucosal IgA antibodies that completely blocked LTB-binding to GM1 ganglioside and protected the animals from oral challenge with CT [92].
  • A proteoliposome based formulation administered by the nasal route that elicits vibriocidal antibodies in mice [93].
  • The concern remains that live oral cholera vaccines may be less effective among partially immune individuals in cholera endemic areas as pre-existing antibodies could decrease colonization of the gut, as was observed in the case of many other live bacterial oral vaccines [94].

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Diarrhoeal Diseases

  1. Introduction
  2. Caliciviruses
  3. Campylobacter
  4. Cholera
  5. Enterotoxigenic Escherichia coli (ETEC)
  6. Rotavirus
  7. Shigellosis
  8. Typhoid Fever
  9. References