Science Editor

On July 6, 1885, a frantic mother brought her 9-year-old boy, Joseph Meister, to the door of Louis Pasteur (1822-1896). Two days before, the lad had been bitten 15 times by a dog thought to be rabid.

In an unprecedented effort, Pasteur had begun in 1884 injecting rabies-ridden spinal cord tissue into the brains of rabbits, and set up an experiment with 42 dogs. Half of them received 14 daily injections of the drying rabbit tissue. None of them came down with rabies, while all of the control canines died.

After 14 such prototype vaccinations, young Joseph Meister responded to the experimental tissue, and lived to a ripe old age. Rabies vaccine had arrived - ahead of time.

"A wide variety of vaccines are available today against rabies," observed Charles Rupprecht, chief of the Rabies Section at CDC, the Centers for Disease Control and Prevention in Atlanta. "When you consider," he pointed out, "some of the world is still using a technique mastered by Pasteur to produce vaccines all the way to continuous cell culture production in the industrial developed world . . . We've gone through multiple generations of rabies vaccines, from live attenuated to purified subunit antigens. And a key component to prophylaxis after being bitten is the simultaneous administration not only of vaccines but also antibodies.

"We've been using the same crude technique where the source of serum or immunoglobulin hasn't much changed for nearly a century. So we've made great progress in vaccine development, but usually we've been using as a source of serum either animals or people who have been vaccinated against rabies, and then merely taken their serum and purified it as a source of antibody.

"If you were bitten by a rabid animal," Rupprecht continued, "you would receive the first of five doses of vaccine on days 0, 3, 7, 14 and 28. At the same time, in and around the wound where the animal had bitten you, you would receive hyperimmune globulin, human or equine. That would neutralize virus in and around the local wound site while the patient started to make his or her own antibody. The average cost is $1,100 or 1,200 for five doses of vaccine and immune globulin. At such prices," he observed, "you won't get very far in coping with India's rabies pandemic. Of every five people in the world killed by rabies, four die in India."

Third World Can't Afford Anti-Rabies Weapons

"Current anti-rabies vaccines and antibodies are a luxury enjoyed by people in developed countries," Rupprecht commented. "At CDC, our primary interest is to make sure this global shortage of rabies immune globulin can some day be avoided by making inexpensive and infective products available to the developing world. The concept of using monoclonal antibodies - that is, antibodies produced in culture, or expressed via another vector, be it another expression vector system or in a plant - is as much of a breakthrough as it was when rabies virus was first produced in cell culture and grown to concentrations to make a vaccine.

"People in the late 1950s were among the first to adapt the rabies virus to primary cell culture," Rupprecht continued. "The late 1970s brought the gold standard to human-deployed vaccine. So you figure from Pasteur's time to the 1970s is 70 years to have cell-culture rabies vaccine available for humans. As of yet, we still have to rely upon animals or people as the first antibodies in postexposure prophylaxis.

"There are a variety of ways that one can use a plant to help produce antigens or antibodies or chemicals," he said. "One way is to make a transgenic plant that expresses in its genome a product of choice such as a vaccine. Another way is to find a plant expression vector or virus that grows in plants and that can express that material. And the utility of this technique is that you can produce relatively large concentrations."

Rupprecht is co-senior author of an online report dated June 3, 2003, in the Proceedings of the National Academy of Sciences (PNAS). Its title: "Function and glycosylation of plant-derived antiviral monoclonal antibody."

"The take-home finding of the paper," he told BioWorld Today, "is that you can express a monoclonal antibody in a plant and achieve not only neutralization against rabies virus but actual protection after exposure."

The paper's other senior author is Hilary Koprowski, professor of microbiology and immunology at Thomas Jefferson University in Philadelphia. He created the current rabies vaccine two decades ago. Koprowski deplores the present worldwide shortage of antibodies against rabies. "They are expensive to get from humans," he noted, "and antibodies derived from horse blood carry unwanted side effects."

Co-authors of the PNAS paper have inserted DNA coding for an antibody against the rabies virus into tobacco plants. Those in turn become botanical factories, churning out the much-needed antibody.

Rupprecht at CDC employed the transgenic plant antibody to prevent rabies in hamsters inoculated with a lethal dose of street virus.

Rabid Mexican Coyotes Supply Infectious Virus

"What we call a street virus [a coyote virus]," he explained, "is opposed to strains of rabies virus that are adapted to grow in the laboratory in cell culture, for example. We picked it because this coyote virus has killed people. Its reservoir runs free along our border with Mexico among dogs and coyotes.

"We chose that virus," he recounted, "to demonstrate that the antibodies not only neutralized it but will protect animals after they have been infected - as a surrogate for people. If a person has been infected with a virus of public health importance,' we asked, could this coyote antibody neutralize after the fact in concert with vaccine in a standard postexposure protective prophylaxis?' The answer is yes - comparable to commercial immune globulin.

"If a commercial partner who is serious about bringing it into the marketplace," Rupprecht surmised, "can be identified, one could then progress into Phase I clinical trials. I think this is a very exciting first step," he concluded, "and we look forward to additional trials in the future. Dr. Koprowski is looking for commercial partners to try and bring this potential to the marketplace. I know that there have been a few bites on this fishing expedition, and we're cautiously optimistic," he concluded, "that someone will be identified."