By David N. Leff

"Eat your spinach, darling. It's good for you!"

This perennial plea from mother to child, pushing the virtues of Spinacia oleracea leaves, has just acquired a new dimension. Spinach is proving an efficacious vehicle as a vaccine against rabies.

"Rabies is a global problem," declared biomedical scientist Hilary Koprowski, "not just in the U.S. or the Western world. Of every five people in the world who die of rabies, four die in India."

The reason, he pointed out, is India's population of 80 million dogs, among which the rabies virus runs rampant. "To combat this situation," he continued, "to immunize those animals, you need an easily administered, inexpensive rabies vaccine. And the only way to fulfill these criteria is to use plants, with plant viruses as vaccine vectors."

A decade or more ago, Koprowski spearheaded a worldwide movement to eradicate rabid wild animals in Europe and the U.S. Foxes were a major carrier of the virus in France and Belgium, where he and his colleagues devised an edible, baited vaccine, that was distributed, largely by air, over French and Belgian forests. This led to control of rabies in those countries.

A similar campaign is targeting rabid raccoons along the Eastern U.S. seaboard, and Koprowski envisages deploying this strategy against India's canine population. "One advantage," he noted, "is that there is no wildlife transmission of rabies in India, only canine transmission. Because dogs don't eat spinach," he observed, "the bait for them will consist of tobacco-leaf extracts containing the viral vector."

Koprowski, who heads the Biotechnology Foundation Laboratories at Thomas Jefferson University, in Philadelphia, is the principal author of an article titled "Immunization against rabies with plant-derived antigen," in the current Proceedings of the National Academy of Sciences (PNAS), dated March 3, 1998.

"What my co-author, Vidadi Yusibov, did," he recounted, "was to insert in alfalfa mosaic virus (AIMV) the genes for antigenic rabies virus glycoproteins. Then he recombined that construct with tobacco mosaic virus (TMV), with which to infect leafy plants.

"That way," Koprowski went on, "he infected tobacco and spinach, by rubbing their leaves with the AIMV-TMV vector. He immunized mice with the leaves, which protected them against challenge by rabies virus."

Delivery System Safe, Cheap And Versatile

To deliver their plant-based vaccine, Koprowski and his co-authors fed mice spinach leaves infected with the AIMV-TMV construct carrying the genes that expressed the antigenic rabies virus particles.

Two weeks after this course of immunization, they exposed immune mice and non-immune control animals to the rabies virus challenge.

"We had to devise a method of challenging them based on mucosal exposure to rabies virus," Koprowski recalled, "rather than intracerebral infection. So we challenged them intranasally.

"Control mice, unimmunized, developed symptoms of murine rabies infection: Their coats roughened, they lost appetite and then 23 percent of their weight. Immunized animals developed mucosal immunity. They shed weight right after challenge, but then regained it."

Given this in vivo outcome, Koprowski said, "As our next step, we would like to get spinach vaccine to feed four or five us here in the lab, who have rabies antibodies. If we find that our antibody level is boosted, say from X to 10X, then essentially we could go to clinical trials of people who are not immunized against rabies, and find out if the spinach vaccine could be used in humans."

Incidentally, cooking destroys the antigen, so spinach salad it is. "And spinach is not the end," Koprowski pointed out. "You can put it in lettuce or herbs or many raw vegetables that vector will infect."

He counted the advantages of his plant delivery system.

"First of all, it's extremely safe to produce. You don't need animal products; you don't have the possibility of human exposure to something hazardous.

"Second, you can insert multivalent antigens in one plant. A particle of AIMV can carry three or four or five products. So, for instance, in the case of HIV, you can put in several different viral strains, or genes that express different proteins. We have some AIDS antigens in plants," he observed, "and we're pushing that work hard ahead.

"Third, and last, is the question of cost. Plants come very cheap."

Asked to assess the efficiency of the plant vaccine, Koprowski replied: "This we will tell you only after we have eaten our spinach, or when we experiment in dogs. The possibilities," he concluded, "are unlimited. You can take any gene you want and put it in plants." *

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