Charles Arntzen was in graduate school, in the 1960s, when agronomists introduced the techniques for high-yield agriculture that would dramatically increase food production in developing nations. But Arntzen, who was studying the development of chloroplasts in plant cells at Purdue University, had little interest in what would come to be known as the "green revolution."

"I was more interested in the revolution that was just starting to hit, on the molecular genetic manipulation of cells," he said. "Biology, at that time, was still very descriptive, and we were just developing the tools that allowed one to intervene and change a biological system and ask a whole new realm of more sophisticated questions. That's what captured me."

Now Arntzen, president and CEO of the Boyce Thompson Institute for Plant Research (BTI), a not-for-profit private research group affiliated with Cornell University, is at the heart of the current green revolution - this one aimed at transforming the world's food supply with genetically modified crops. With some of the same idealism that informed the first Revolution, Arntzen and Hugh Mason, a fellow BTI research scientist, are developing potato-, tomato-, and banana-based vaccines that they hope will provide safe, effective, and inexpensive protection against infectious diseases, still the primary cause of death in developing countries. Diarrheal diseases, alone, are estimated to kill four to six million people annually; most of them in developing countries, lacking the money, technology, or infrastructure to adequately immunize children against these illnesses.

In the July issue of the Journal of Infectious Diseases, Arntzen, Mason, and collaborators from the Center for Vaccine Development at the University of Maryland School of Medicine and Baylor College of Medicine will publish the results of a clinical trial showing that transgenic potatoes, expressing a Norwalk virus protein, can trigger an immune response in humans. Ninety-five percent of the participants who ate raw pieces of the vaccine-containing potato developed antibodies specific to the Norwalk virus, which is estimated to cause more than 90 percent of the nonbacterial gastroenteritis in the United States and other industrialized countries.

This trial follows on the heels of a 1998 proof-of-concept clinical study aimed at evaluating the efficacy and safety of a transgenic potato expressing the binding subunit of E. coli's heat-labile enterotoxin (LT-B), the cause of the dreaded "traveler's diarrhea." Ten out of the eleven subjects, who received three doses of raw potato pieces over the course of three weeks, developed a fourfold rise in serum antibodies to LT-B. Six of the eleven developed a fourfold rise in IgA (gut) antibodies.

The results of a third clinical trial, testing a potato-based vaccine against the hepatitis B virus, conducted in collaboration with Yasmin Thanavala of the Roswell Park Cancer Institute are still being analyzed.

Germinating an Idea

Arntzen started thinking about plant-based vaccines in 1990, when the Children's Vaccine Initiative, a consortium of global health and philanthropic organizations, called for the development of new, high-quality vaccines that would reduce the mortality of infectious diseases, especially in developing nations. "Two things hit me," Arntzen recalls, who had just relinquished his administrative responsibilities at Texas A & M University and was setting up a new research lab. "One was that they wanted more oral vaccines" - thus eliminating the health hazards associated with reusable needles - "and the second was that they wanted less expensive vaccines."

When the first recombinant subunit vaccine came on the market, the hepatitis B vaccine, a light bulb went off in Arntzen's head. "I thought, if yeast can do it, why can't a tomato do it?" Arntzen said. A plant-based vaccine, he reasoned, could be grown and processed locally for much less money than conventional recombinant vaccines. Ideally, it wouldn't even be necessary to purify the vaccine from the plant material.

He began looking for collaborators who, as he puts it, were also "interested in crazy ideas about making vaccines in plants." And crazy - or perhaps naïve - was how many scientists viewed the concept. "When we began doing the first clinical trials, you could line up 1,000 immunologists, ask them if the vaccine would work, and 999 of them would have said, 'No,'" said John Clements, one of the authors of the potato-LT-B study and a professor of microbiology and immunology at Tulane University School of Medicine. One of the major concerns was - and for some people, remains - the possibility that the vaccine would induce tolerance rather than protective immunity. Clements doesn't think this will be a problem. "I've seen a lot of mucosal vaccines fail for a lot of reasons," he said, "but never because of oral tolerance."

Cultivating the Idea

One of Arntzen's first recruits was Mason, then a postdoc in plant biochemistry at Texas A&M. Mason carried out much of the hands-on work of creating the transgenic plants, which he continues to do in his laboratory at BTI. The concept, in theory at least, is simple - insert the gene encoding the antigenic subunit under the control of plant-specific DNA regulatory sequences and integrate it into the nuclear chromosome of the plant cell. The antigen, once expressed, is retained within the cell wall and is, thereby, protected from stomach acids.

One of the challenges is inducing the plants to express high enough levels of the antigen to be effective, Mason said, without stunting the growth of the plant. They've gotten around that, to some extent, by creating a completely synthetic "plant-optimized" gene that produces a protein with the amino acid sequences specified by codons that are preferred by plants, and which lacks any problematic sequences that reduce the efficiency of expression. At the same time, they can limit high expression of the protein to mature plant tissues which can be harvested before any cell damage occurs.

Although the original concept was based on eating raw, unprocessed fruits or vegetables, Arntzen and Mason now believe that some degree of processing will be necessary to produce a more consistent and concentrated dose. So, for example, a banana-based vaccine might either be pureed and canned, like baby food, or made into banana chips. The coming tomato trials will use dehydrated tomatoes ground into a powder and then reconstituted with water. "We're very much focusing on the sort of technology that exists in developing countries today," Arntzen said.

Perhaps the greatest barrier to bringing plant-based vaccines to market will be the public's mistrust of genetically modified foods. Despite the promising initial results, cows, pigs, and chickens may be eating plant-based vaccines before we do. Their first financial backer, the British company, Axis Genetics, went belly-up last fall because protests over genetically modified food scared off investors, leading BTI to shift their focus to the livestock market. "Not that we've lost interest in the human vaccines," Arntzen said, "but it's turning out to be more complicated than we had anticipated. We believe that getting to a commercial market is going to be somewhat easier for animals." BTI has begun negotiations with companies that will back the research to develop plant-based vaccines for livestock.

Arntzen, who has spoken on behalf of plant biotechnology research before the Senate Committee on Agriculture, Nutrition, and Forestry, and other public forums, is well aware of the role that public perception will have in the success or failure of the vaccines. Although he believes that concerns about genetic escape or inadvertent overdosing on plant-based vaccines are unfounded, "we're trying to make sure that we've got all sorts of safeguards in place before we ever reach the next stage, such as transferring it on the to developing world. I just don't want any rumor to arise that would sidetrack a technology that we think has great value."