Molecular geneticist John Mekalanos thought he had it made _ "it"being an effective live attenuated vaccine against cholera.

After successful preclinical mouse testing, his vaccine was nowmidway through Phase I/II human trials.

Then he and his collaborator, post-doc Matthew Waldor, stumbled ona circumstance that looked as if it was the pathogen, Vibrio cholerae,that had it made. "It" being an unsuspected bacterial virus thatspreads the cholera toxin from one strain to another, and threatens totransmit its virulence to other forms of bacteria as well.

Their cautionary report on this sinister discovery appears in thecurrent issue of Science, dated June 28, 1996, titled: "Lysogenicconversion by a filamentous phage encoding cholera toxin."

Lysogenic refers to the process by which a bacteriophage infects abacterium, and integrates its DNA _ which encodes the choleratoxin _ into that target pathogen's genome. There the virus bides itstime, lying latent, until some event or agent triggers it to replicate,lyse the bacterial cell, and move on to its next cellular victim.

This sounds a little like the AIDS virus's life cycle, but V. choleraespreads from one human victim to another by ingestion of fecal-contaminated food or water, rather than a high-risk lifestyle.

Mekalanos, at the Harvard Medical School's Shipley Institute ofMedicine, is the paper's principal author. Its first author ismicrobiologist Waldor, now a faculty member at the Tufts School ofMedicine in Boston.

"The immediate effect of our work," Waldor observed half-humorously, "is probably to give a shot in the arm to our rivals, whoare developing oral killed vaccine, because it shows that our vaccine,as well as the other live, attenuated one being developed at theUniversity of Maryland, are both susceptible to infection with thephage that encodes the cholera toxin. So these vaccines couldpotentially revert to toxigenicity." He added, "Such reversion is atheoretical concern, but it's something I think we should work on.

"A direction for research now," Waldor told BioWorld Today, "is tofigure out how to prevent that phage from reinfecting our vaccine andmaking it toxic. We have a variety of new ideas how to accomplishthat," he added.

As Mekalanos and Waldor engineered the attenuated live vibriostrains for their vaccine, presumably they already have theappropriate gene deletions built into them to circumvent the phageattack.

Vaccine Shows In Vivo Immunity In Humans

So far, Phase I and II clinical trials of their live, attenuated vaccineare racking up results like toxin-busters.

"It's been given now to between 50 and 100 people at a minimum, onan outpatient basis," Waldor said, "without any side effects. And thevaccine has at least generated very good serum vibriocidal titers,which is a good surrogate for immunity."

An even better proof of efficacy is to challenge vaccinated subjectswith wild-type cholera. "A limited number of volunteers," Waldorsaid, "have gotten such a challenge, and they were completelyprotected. So it looks like a very promising vaccine."

A few years ago, Waldor said, "John Mekalanos co-founded acompany here in Cambridge called the Virus Research Institute[VRI], to which Harvard licensed some of the cholera vaccines."

He and Mekalanos discovered the hitherto unknown stealth phage"mostly by serendipity," while studying the horizontal transmissionof the cholera toxin genes. "We set out to investigate whether thesegenes were transmissible between V. cholerae strains," Waldorrecalled, "and found out the answer was `yes.' Then the mechanismof that transfer showed that there was in fact a bacterial virus, afilamentous phage, that encodes those cholera-toxin [CTX] genes."

In the human gut, a benign strain of the pathogen may occasion onlya mild abdominal discomfort, or none at all. But infection with thephage produces an outpouring of toxin, and a conversion tovirulence. This brings on severe diarrhea, dehydration, even death.(See BioWorld Today, Sept. 6, 1994, p. 1.)

"Virulence is dose-related to that toxin," Waldor observed."Vaccinologist Myron Levine at the University of Maryland has donesome classic studies, where he gave purified cholera toxin to humans,and found this could basically reproduce the disease symptoms in adose-response manner. So the vibrios become basically a vehicle todeliver the toxin to the intestines."

The Devil Is In the Pili

V. cholerae is a minute, curved bacillus, 1.5 to 2.0 microns long,shaped like a comma or a shelled cashew nut. When it enters the gut,its surface becomes covered by a forest of far tinier filaments, or pili,seven nanometers thick, which serve as receptors on which theinvasive bacteriophage docks.

"These hair-like filaments," Waldor observed, "the pili, turn out to bethe most important bacterial structure that we know of now thatallows colonization of the intestine."

The Harvard co-authors' new knowledge opened a new can ofignorance. "Can benign and virulent cholera strains co-exist in the gutor in the environment? We don't know," Waldor said. "Or was thishappening a long time ago in evolution, and it's not going on now?We don't know that either."

Seeking further insights, they co-inoculated two strains of mice, onethat was producing the phage, one that didn't have it. The latter, theyfound "could be infected at a remarkably high rate, suggesting in factthat it may be in nature where this kind of evolution has occurred."

Waldor observed that "this class of bacterial viruses has never beforebeen shown to mediate transfer of genes between bacterial species,certainly not genes that encode toxins. So we can look forfilamentous phages in other pathogens _ [such as Salmonella andYersinia, which causes bubonic plague] _ and see whether they infact might contain any genes important in pathogenesis."

Myron Levine, director of the Center for Vaccine Development at theUniversity of Maryland, Baltimore, put a less apprehensive spin onthe Harvard team's phage warnings.

"Their paper in Science," Levine told BioWorld Today, "is a veryelegant piece of basic research, and moves the field forward. "Interms of its relevance for live cholera vaccines," he observed, "So farit would appear to have no practical implications."

Levine continued: "Waldor spoke of 50 or 100 individuals gettingthe Harvard/VRI strain. About 6,500 subjects _ adults, school-agechildren, pre-schoolers, toddlers, even infants _ have received ourlive cholera vaccine in randomized, placebo-controlled, double-blindtrials, with no adverse reaction being observed.

"It's fair to raise theoretical concerns," he concluded. "but then wehave to sit back and see what happens in real life." n

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.

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