By David N. Leff

Catch 22 should really be written Catch 22-2 or 22-3, when it comes to vaccination against infectious diseases. To wit:

* Successful vaccination depends on user compliance.

* Such user-friendly immunization calls for single-dose vaccination, not multiple booster shots, nor invasive needle injection.

* Oral vaccines fill the no-needle part of this bill, but their live-pathogen antigens require that the microorganisms they employ be sufficiently attenuated (weakened) to avoid infection.

* However — and here's the catch — the higher the attenuation, the lower the immunogenic efficiency of the vaccine.

This litany of the hang-ups that bedevil current vaccination programs leads off a paper in the May 1998 issue of Nature Biotechnology. Its title: "Homologous and heterologous protection after single intranasal administration of live attenuated recombinant Bordetella pertussis."

"I think we have showed, for the first time," said the article's senior author, molecular biologist Camille Locht, "that genetic attenuation actually may increase the immunogenicity of the vaccine-targeted antigen." Locht directs a bacterial-pathogenesis laboratory at the Pasteur Institute, in Lille, France.

Pertussis — whooping cough — is a severe childhood infection, with a 1 to 2 percent mortality rate in infants under 12 months of age. Immunization requires five shots between birth and school age, and these can sometimes cause severe side effects.

In an infected youngster, the spasmodic coughing spells, each ending in a high-pitched, rooster-crow-like whoop, are not only agonizing to its victim, but a menace to any person within breathing distance who is not immunized.

Bordetella pertussis invades the body via the nostrils, and moves down the airways into the lungs, colonizing the mucus membranes on its way.

"A single whooping-cough infection," Locht told BioWorld Today, "induces strong mucosal and systemic humoral and cellular immune responses, as well as long-lasting protection, in humans."

Alien Antigen Was 'Right Next Door'

He and his co-authors set out to evade or avoid the immunological and compliance handicaps that beset present-day whooping-cough vaccination. Their genetic-engineering strategy resorted to two tactics: ridding B. pertussis of its virulent primary toxin, and equipping the resulting attenuated strain with a high-profile foreign-antigenic immunogen as the vaccine target.

That alien antigen was "right next door," Locht recalled. "We used the cercariae [worms] and eggs of the Schistosoma mansoni parasite, with which our institute has a long-standing research experience." S. mansoni infests the water, and infects the populations, over much of Africa and Latin America.

"We had cloned the toxin genes about 12 years ago," Locht recalled, "while I was at the U.S. National Institute of Allergy and Infectious Diseases. And now we used a system of homologous recombination to cleanly take those sequences out of the bacterium's chromosome. That is, we didn't add any resistance markers, for instance.

"Thereby," he continued, "we had a B. pertussis strain exactly the same as the wild-type parent strain, except that it didn't have any of the primary toxin genes. It expressed all the secondary toxins and protective antigens in mice."

On the toxin-free Bordetella organisms, Locht's team devised a way of expressing, or presenting, the S. mansoni antigen to best advantage. They found one molecule of particular interest, namely, filamentous hemagglutinin (FHA). "This B. pertussis antigen," Locht pointed out, "is extremely well secreted, and very well exposed at the surface of the organism.

"People or mice infected by B. pertussis," he observed, "make high levels of both mucosal and serum antibodies against FHA, and we had mapped important epitopes recognized by these human and murine antibodies on the FHA molecule."

Mice Infected With New Strain Survived

Next, they chose an FHA region that seemed immunologically dominant, into which they inserted the S. mansoni antigen gene. "Our idea," Locht explained, "was to present this target antigen on this FHA carrier molecule, in a domain that is particularly immunogenic. We found that using this parasite's antigen actually increased the genetically attenuated B. pertussis bacterium's immunogenicity."

A single drop of this toxin-deleted B. pertussis strain suspension, introduced into the nostrils of mice, Locht's article reported, "protected against subsequent challenge as well as did the parent strain, and better than immunization with commercial vaccine."

Locht said all mice infected with the new strain survived. "There were no fatalities," he said. "And none of the animals seemed sick; they all looked very happy."

Had they been human subjects, their happiness might have been even greater.

"We were able to show — again, I think, for the first time — that the single intranasal dose protected the mice simultaneously against the schistosomiasis parasite as well as against whooping cough," Locht said.

Besides the poetic justice that the vaccine went in intranasally — the same route taken by the contagious B. pertussis bacterium — the Lille researchers noted a pulmonary circumstance.

"When we did pathology on the mouse lungs," Locht said, "we did not see major infiltration, major inflammation. There was a big difference between the wild-type and our attenuated strains. Yet, these strains colonized the lungs very well, and multiplied. But they were able to boost the protective antigens within the lungs.

"Of course," he added, "what we really want to do now is see how these kinds of constructions work in humans. But there's still a lot of work to be done before we can think of initiating clinical trials. We need to further attenuate the Bordatella. So far, we've taken out only the one main toxin, and we know that there are other, secondary, ones. Work in my lab is presently focused on trying to eliminate all of these. But clearly, then we would like to go into people, and see how we can induce their immune responses by nasal infections."

Locht estimated the elapsed time to that goal as "about one to two years for the final genetic engineering. And then, of course, we'll need ethical clearance in Europe, for the different countries where we want to conduct these clinical trials, so we have to set all this up.

"My wish would be," he concluded, "that within the next three or four years, maybe we can go into clinical trials. Perhaps even before, because some people are very interested in this approach, which may accelerate the process." *

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