It's back to the murine drawing board for a pioneer in thestubborn endeavor to make an anti-malaria vaccine.

For a quarter of a century, parasitologists Ruth and VictorNussenzweig have been leaders in the worldwide scientificmovement to foil the wily, deadly malarial parasite,Plasmodium falciparum -- an effort that has producedindifferent results. Now Ruth Nussenzweig and her colleagueshave turned back to the mouse model of malaria to develop anew approach.

Nussenzweig is lead author of a paper in the currentProceedings of the National Academy of Sciences (PNAS) titled"Priming with recombinant influenza virus followed byadministration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria." Thereport points the way to a detour from the endless dead endsthat have frustrated the vaccinologists.

What P. falciparum is to humans, P. yoelii is to mice: malaria'smost virulent, death-dealing parasitic species. Nussenzweig'simmunization target remains the same: Plasmodium'ssporozoite stage.

When an anopheles mosquito bites a human or a rodent, itssaliva squirts a culture of single-cell sporozites into thevictim's bloodstream. The needle-shaped, micron-sizeorganism swims swiftly to the liver, where it holes up for aweek while multiplying.

For years, malariologists have been cloning bits and pieces ofP. falciparum sporozoite protein and inserting them into anarray of bacterial and viral expression vectors, trying tocreate a recombinant vaccine. Now Ruth Nussenzweig at theNew York University School of Medicine, and Peter Palese,chairman of microbiology at Mount Sinai School of Medicine,have constructed and tested a pair of live-virus vectors --with a difference.

-- One is a vaccinia virus, long used for smallpox vaccination.The researchersL recombinant construct is a vector thatexpresses the entire 200- to 300-amino-acid P. yoeliisporozoite protein.

-- The other is an RNA influenza virus expressing P. yoelii'scytotoxic epitope. This is a 12-amino-acid, PCR-producedpeptide, replacing an eight-amino-acid sequence. Half of itssequence is an immunogenic target for CD8+ killer T cells.

Palese told BioWorld that his laboratory had altered theinfluenza RNA-virus genome "to make it amenable as a vectorfor genetic engineering." He made it from DNA, "and we cannow, for the first time on record, use this recombinant,chimeric influenza virus as a delivery vehicle for foreignepitopes," he said.

At the start of controlled trials of the one-two punch vectors,mice inhaled 500 units of the recombinant, epitope-bearinginfluenza virus, administered by aerosol. Three weeks later,they got booster shots of the recombinant vaccinia vector,packing the full-length sporozoite protein.

Then, on day 13 following this double-barreled vaccination, amosquito bite was simulated by injecting 100 P. yoeliisporozoite parasites from a hypodermic syringe into the tailveins of the mice.

The lethal challenge protected 60 percent of the immunizedmice, which never developed parasitemia because vaccinationprevented sporozoites from spilling out of the liver into thebloodstream.

This pre-erythrocyte killing off of parasites in the liver byCD8+ cytotoxic T lymphocytes (CTLs) also gave the other 40percent of the mice a longer lease on life before theyeventually succumbed.

Liver dissection among the immunized mice revealed a drop of96.3 percent of plasmodial rRNA, the PNAS paper reported,"revealing a synergistic effect of vaccinating with two live-virus vectors." It added, "Surprisingly, protective immunityfailed to be induced when the two viruses were administeredin reverse order."

Why? Nussenzweig theorized that "when the sequence ofimmunization is reversed, CTLs might be recruited away fromthe liver to the lung." Immunization with vaccinia, afterpriming with influenza anti-malaria vector, she suggested,"enhances the effectiveness of anti-parasite immunity,apparently by expanding the influenza virus-induced CD8+ Tcells."

She and Palese are now building other viral constructs,Nussenzweig told BioWorld, "trying to further improve theimmunogenicity." In anticipation of eventual human trials, theywill attenuate both viruses to traditional human-vaccinelevels of virulence.

Once they have optimized the two-vaccine strategy with P.yoelii, their plan is to turn back to P. falciparum and use it forrepeating and refining the mouse experiments, which shouldtake a year or so. "Once we are convinced we don't have anydeleterious side effects," Nussenzweig said, "then possibly, wewould do human trials."

David Kaslow is a research malariologist in the MolecularVaccine Section of the National Institute of Allergy andInfectious Diseases at the National Institutes of Health. Hetold BioWorld apropos the PNAS paper: "Dr. Nussenzweig, amongother things, continued to pursue this Plasmodial sporozoitething. And now she's gotten it to a point where I think there issome optimism in terms of this working in humans. Again, allthis work has been done in a mouse model, and it's completelyunknown as to whether it will extrapolate to humans."

Kaslow himself is developing a transmission-blocking vaccine.This, he explained, is the other end of the spectrum."Sporozoites are the forms of the parasite that go in, frommosquito to human. I'm working on the forms that go out,trying to prevent those forms from infecting mosquitoes. WhatI would envision is that what I'm working on would becombined with what Ruth is doing to make an effectivevaccine. We're hoping to get into human trials soon."

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-- David N. Leff Science Editor

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