The cholera genome: an advance for science or for medicine?

genome sequence will help in developing protection against the disease. Robert Walgate discovers that it might - but perhaps not in the most obvious ways.

By | August 8, 2000

LONDON, 8 August (Science Analysed) So, we have the genome sequence of Vibrio cholerae, a fascinating organism for research, with its two circular chromosomes, its complex estuarine and marine ecology involving plankton and bacteriophage, a history of gene hopping and its links not only to many gut bacteria — including Escherichia coli — but also to a wide variety of organisms important in the marine biota. This will clearly be a treasure trove for science.

But is it of use to medicine, as some media reports and some scientists have hinted? Let's get some perspective. A quarter of a million cases of cholera, 3.6% of which (9135) were fatal, were reported to WHO in 1999. But those figures exclude Bangladesh, historically the prime focus of the disease, which does not report its cholera cases to WHO, and where experts estimate there are probably 400,000–600,000 cases a year and maybe 5000 deaths.

According to Dr Claire-Lise Chaignat, Head of WHO's Cholera Task Force, the official WHO figures may be 5–10-fold underestimates of the whole. So, we are talking of 1–2 million cases of the disease a year and perhaps 50,000–100,000 deaths.

This is not comparable with malaria, which kills a million infants in Africa each year, but cholera is much easier to prevent and treat, and there's less excuse for inaction. And there is a growing risk of epidemics as Third World cities explode in size and sanitation lags, says Chaignat. Cholera comes with bad hygiene, linked to poverty, disruption, migration and war. Prevention is by the simplest methods of public health: authorities must filter and chlorinate water, and provide good sanitation and education. This is largely a matter of wealth. Economic development almost entirely removes the threat; last year Europeans, for example, suffered 16 cases, all of which were imported, and no deaths.

Treatment could hardly be simpler: the patient must drink sufficient water fortified with widely available oral rehydration salts (ORT) to stop dehydration. This can reduce the death rate to 1% of cases. But response must be fast: "you lose water so fast that within 24–48 hours you can be on the verge of dying" says Chaignat. "It's very dramatic, I can tell you — but the treatment is also dramatic. Patients recover so quickly with ORT alone it's fantastic." Very severe cases can be treated with intravenous rehydration; antibiotics are usually unnecessary.

"Of course the research is fantastic. If we don't advance with science, we'll never change things. But the genome's not going to change anything right now for the patients, for treatment or prevention. ORT is widely available in primary health care settings in Africa and Asia. And it's much more available than any new treatment that may come along that's bound to be more expensive" Chaignat says.

There has been some talk of the implications of the new sequence information for vaccine development, although cholera vaccines seem more appropriate for travellers than for people living in endemic areas. But the estimated risk of cholera in European or North American travellers to endemic areas is only one or two cases per million trips. And as vaccination against cholera cannot prevent the introduction of the infection into a country — it arrives in contaminated food or water — cholera vaccination is not required of any traveller. Nevertheless, there are travellers at risk: health professionals working for long periods in endemic areas or aid workers in refugee camps.

As for the market, which affects investment in new vaccines, there's no clear published estimate but according to Frost & Sullivan — an international marketing consulting and training company — existing travel vaccines for hepatitis A, typhoid, yellow fever, Japanese encephalitis and cholera accrued a combined sale of US$339.6 million in 1998.

There does seem to be some room for a company to create or improve a vaccine or treatment for this disease. And, in fact, three fairly effective new oral cholera vaccines are already available, or nearly available, replacing a relatively ineffective 50-year-old injectable vaccine, which is not recommended by the WHO. "It has a lot of side effects and a very limited protection," Chaignat says. "But presently the new oral ones are very expensive for Third World use." One, Dukoral, is composed of whole killed V. cholerae plus recombinant cholera toxin. It was developed by Jan Holmgren and colleagues at the University of Gothenburg and is produced by the Swedish company SDL. The other (called Orachol, or Mutachol in the USA and Canada) is a live attenuated vaccine given in a single dose, created by Myron Levine and colleagues at the Center for Vaccine Development of the University of Maryland, USA.

Both are effective, but the Phase III trial of Orachol in Indonesia failed as the incidence of the disease was too low during the trial to give statistically significant results. Moreover, Manfred Schroeder, Deputy Head of the Medical Department of the Swiss Serum and Vaccine Institute (SSVI) in Berne, which produces Orachol, told BioMed Central that as his institute had recently undergone wholesale reorganization, there was some doubt about whether a new trial will be done at all; it would be a tremendous investment and, although he personally would wish to continue, the SSVI might make a strategic decision to get out of travel medicine, he says.

There is a third vaccine, a Vietnamese version of the Swedish vaccine — produced by a technology transfer agreement — to which a new virulent strain of V. cholerae, 0139, which is now causing about 17% of the cases in Bangladesh, has been added. But so far it is restricted to use in Vietnam.

From the public health perspective, what would be a perfect vaccine? "The oral vaccines are 80–90% effective. So you still have 10–20% of people unprotected. And they must become cheaper," says Chaignat. As for duration, the Swedish vaccine is 50% protective after three years "which is pretty good... Of course, it's not like tetanus or polio where you know you have 10 years."

One day such vaccines might be useful for public health, says Chaignat,"... if they get cheaper and once we know in which public health settings they would be useful. The first public health interventions with these vaccines will have to be monitored very carefully."

As for new vaccines, the development of the existing vaccines generated a great deal of knowledge about the genetics and expression of virulence factors of V. cholerae, including the discovery of virulence transported by infection of the bacteria by filamentous bacteriophage. This raises the possibility that attenuated strains could become virulent, and the information now provided by the genome sequence seems bound to make the development of additional attenuated vaccine strains easier.

However, new vaccines are by no means the most important likely benefit to come out of knowledge of the V. cholerae genome, according to John Mekalanos, Professor of Microbiology and Molecular Genetics at Harvard Medical School, one of the authors of last week's Nature paper.

"We think the genome will contribute at the level of therapy, prevention and vaccines — but the way it can help us with the environmental microbiology of cholera might be profound," says Mekalanos. "We might find ways of controlling cholera at the endemic or epidemic level."

"The first six global pandemics since the early 19th century were due to the classical biotype, which tended to cause epidemic rather than endemic cholera, even where sanitation and water quality were extremely poor. Cholera blew through Latin America in the late 19th century and then disappeared for 100 years. Why was that?"

"So one way the genome is going to be a tremendous influence — and this is a prediction I'm going to go way out on — is this: I believe there is an inherent difference in the environmental fitness of the organism that allows it to maintain itself in aquatic environments, that are related to its ability to establish endemicity. And that level of fitness is probably right down at the level of a genetic locus, that will be present in the El Tor [recent] strain, but absent in the classical one."

To study this and related questions, Mekalanos and colleagues are building a V. cholerae chip that will display every sequence, "so we'll be able to look at classical biotypes and the environmental strains, the ones that aren't causing disease because they lack the virulence genes but are maintaining themselves in aquatic environments. And ask 'what gives?' " If his hunch is right, they will find genes for endemicity.

Then you could imagine, for example, that you might find the environmental organism needs a particular carbohydrate that's affected by human activities in some way. If you could change the human activity, the environmental cholera strain, with its constant risk of converting to virulence, would be gone.

Also, V. cholerae's colonization of the human intestine has been traced to a filamentous structure called the Toxin co-regulated (TCP) pilus, which is expressed simultaneously with cholera toxin (which causes the diarrhoea and transmits the organism onward to its next host). The toxin genes are on a phage, and work by Mekalanos and Mathew Waldor showed that this phage only infects V. cholerae that are expressing the pili. It uses the pili to enter the bacterium. "So the phage is extraordinarily smart and strategic." Work at the University of Maryland is suggesting that the TCP pilus is itself encoded by a phage, so perhaps as many as three organisms are involved in cholera virulence.

"The indication is that there is an ecological interplay of organisms here — some viruses, some segments of DNA that may be viruses but certainly move horizontally between strains. And they all come together to cause toxigenic strains — on occasion. But once those strains emerge, they are remain clonal for long periods after that, " says Mekalanos.

With the genome and the consequent chips "we'll really be able to look for fingerprints, like patterns, that will enable us to establish the evolutionary history of strains." And that might produce completely novel means of intervention? Such as attacking an organism completely separate from V. cholerae itself, in order to control the toxin genes? "Exactly."

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