By David N. Leff
Food poisoning now comes in two pathogenic persuasions - bacterial and viral. Compared to the familiar scourges of E. coli and Salmonella infection, the viral version is a Johnny-come-lately perpetrator, known as the Norwalk virus (NV). The Centers for Disease Control recently estimated that 96 percent of non-bacterial diarrheal disease is due to NV.
Viruses - think Ebola, Lassa, Coxsackie and Hanta for example - generally are named for the places where they first showed their disease-wreaking prowess.
The Norwalk virus' name goes back to its first recorded outbreak, in the little town of Norwalk, Ohio (pop. 14,000), where half of the middle school student body came down - transiently - with extreme nausea, vomiting and diarrhea. Subsequent flare-ups worldwide clearly linked NV's spread by the same fecal-oral route on which bacterial gastroenteritis hitchhikes.
"Norwalk is a very simple, non-envelope virus," observed plant molecular biologist Charles Arntzen. "It just assembles in the cytoplasm of the intestinal epithelial cell. We get it routinely in contaminated food or water," he pointed out, "particularly in seafood - probably because a variety of seafood is filtering it out of the sewage effluent, concentrating it and delivering it back to us again. The virus infects and damages only the epithelial cells of the gut, and causes severe diarrhea - but only in humans. Normally the course of the disease lasts about 48 hours and then we get over it."
Why this fixation on the gastrointestinal tract? "Presumably, though it's not absolutely nailed down," he explained, "the viral surface protein has specific attachment mechanisms for the epithelial cells in the gut. So the protein is defining that, and if we can produce antibodies in the GI tract that recognize this protein, it can prevent that binding, and so stop the process of initiating the disease."
Arntzen has, in fact, led a multidisciplinary effort to create and test such a human vaccine against Norwalk virus infection. Last week, on Tuesday, June 20, he presented the team's first clinical results at a media backgrounder organized by the New York Academy of Sciences on the theme: "Agricultural biotechnology and the developing countries."
Veggie-Grown Vaccine Tries Out
Arntzen is president of the Boyce Thompson Institute for Plant Research Inc., which is affiliated with Cornell University in Ithaca, N.Y. Together with molecular virologist Mary Estes, at Baylor College of Medicine, in Houston, Texas, he designed and constructed an edible, vaccine expressed in the common potato (Solanum tuberosum). Raw, peeled, diced helpings of this enhanced vegetable were downed by 20 volunteers at the University of Maryland School of Medicine's Center for Vaccine Development, in Baltimore.
"I told the Academy press briefing," Arntzen recounted, "how we had come to create the Norwalk virus vaccine, and that we went through FDA approval for a human clinical trial. I laid out the nature of the trial," he added, "and simply reported that 19 out of 20 volunteers showed an immune response against the plant-delivered antigen."
But the very ubiquity of Norwalk virus in the human population limited the effectiveness of this experimental immunization. "In the case of NV," Arntzen pointed out, "our team couldn't find a naove patient. We have never found a volunteer who didn't have some level of his or her own antibodies against Norwalk virus. We've all seen NV, and some of us have had an inappropriate experience from it at some point. We'll need to do some more vaccine trials, and then challenge the individuals with the authentic, wild-type virus."
It took Baylor's Mary Estes two decades to clone the Norwalk genome and sequence the 530 amino acids of its protein. "It grows extremely well in people," she told BioWorld Today, "but we couldn't grow it in the laboratory."
Arntzen recalled, "There had always been a suspicion that there was a new virus causing diarrhea. Mary collected stool samples from diarrheal patients, and was able to purify Norwalk for the first time. Once she'd isolated it, she isolated enough of it so she could sequence its RNA viral genome. From that she found the gene encoding the surface coat - the capsid protein. She provided us with the gene, which we put into potatoes, and then began evaluating it as an oral vaccine."
The full story will appear in the Journal of Infectious Diseases, dated July 1, 2000.
From Potatoes To Tomatoes To Bananas
Meanwhile, Arntzen went on, "We're moving the Norwalk virus gene into tomatoes. We have to get a more concentrated form of the plant vaccine. The response that we saw in transgenic potatoes was inadequate to predict that we had a good immunization agent. We need to get a higher dosage of the capsid protein, which we can't seem to do in potatoes. We have higher expression in tomatoes. We're growing those plants up right now, and we've developed a very simple freeze-drying procedure that enables us to concentrate the tomato powder. We believe that will give us a 20- to 40-fold enrichment of the vaccine, just by drying the material down.
"So we can go to significantly higher dosage to test in our next human trials," he continued. "We're trying to get an amount of tomatoes that would yield at least a thousand doses of vaccine. We don't intend to test all that immediately, but we want to stockpile enough material so that if we get success in our next clinical trial, six or nine months from now, we could then go on to a challenge trial, using the same batch of material all the way."
In the Institute's greenhouse, Arntzen and his co-workers have their first transgenic banana fruit earmarked for eventual vaccines against Norwalk virus and hepatitis B virus. "These will never show up on supermarket produce shelves," Arntzen averred, but as he pointed out to his Academy audience, "in developing countries, the rapid and severe diarrhea caused by Norwalk virus infection is indicated as a prominent cause of infant mortality."