Tobacco Plants Yield First Vaccine For Dreaded 'Cruise Ship Virus'

In his laboratory, Charles Arntzen holds a modified tobacco plant able to produce vaccines to fight diseases like the "cruise ship virus," a dreaded cause of diarrhea and vomiting. (Credit: Charles Arntzen)

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Scientists have used a new vaccine production technology to develop a vaccine for norovirus, a dreaded cause of diarrhea and vomiting that may be the second most common viral infection in the United States after the flu. Sometimes called the "cruise ship virus," this microbe can spread like wildfire through passenger liners, schools, offices and military bases.

The new vaccine is unique in its origin — it was "manufactured" in a tobacco plant using an engineered plant virus. Researchers are enlisting plants in the battle against norovirus, swine flu, bird flu, and other leading infectious diseases. This plant biotechnology opens the door to more efficient, inexpensive ways to bring vaccines quickly to the public, especially critical in times when viruses mutate into unpredictable new strains, said Charles Arntzen, Ph.D., who reported on the topic today at the 238th National Meeting of the American Chemical Society (ACS).

"The recent outbreak of H1N1 influenza virus has once again reminded us of the ability of disease-causing agents to mutate into new and dangerous forms," Arntzen points out. "It will be at least six months until a vaccine for this new strain will be available, and it will take even longer to create large stock piles of vaccine. For a case like the H1N1 influenza virus, you want to be able to move very rapidly and introduce a commercial vaccine in the shortest possible time. We think we have a major advantage in using engineered plant viruses to scale-up vaccine manufacture within weeks instead of months."

Noroviruses are always mutating, making it a moving target for vaccine developers. Arntzen says this has presented an obstacle for big pharmaceutical companies who might have considered developing a vaccine. Production costs can skyrocket when a single disease may frequently require new vaccines that must be developed and tested for safety and effectiveness. As a result, vaccines do not exist for many diseases that sicken enormous numbers of people each year. Arntzen notes that plant biotechnology could create a cheaper, quicker vaccine manufacturing technique uniquely suited to combat mutating viruses like norovirus and the flu.

Norovirus temporarily disables its victims, giving them severe diarrhea or nausea for up to three days. While not as life-threatening as the flu, Arntzen says it is equally important.

"It essentially closes down wings of hospitals, schools, day care centers and homes for the elderly. In the case of the military, it can shut down an entire ship and delay military operations while there is a cleanup in process. Because the disease spreads so rapidly, the major economic consequences are caused by the disruption of normal daily life and commerce," says Arntzen.

Norovirus will continue to evolve new strains, so Arntzen's team designed a vaccine manufacturing process quick enough to keep up with it and other shape-shifting viruses.

"With plant-based vaccines, we can generate the first gram quantities of the drug and do clinical tests within eight to 10 weeks… We could easily scale that up for commercial use in a two to four month period," explains Arntzen.

Plant-based vaccine production also offers cost advantages. Building greenhouses is more cost effective than the sterilized facilities, expensive manufacturing technology and stainless steel tanks required for the insect or mammalian cell cultures used in most traditional vaccines.

"The other cost advantages relate to vaccine purification and formulation. Purification from plant extracts is simpler because there are no infectious agents to clean up. There are no viruses in plants which can infect humans, so you don't have to worry about viral removal," notes Arntzen.

The team re-engineered plant viruses to produce high levels of specially designed "virus-like" nanoparticles in tobacco plants. At about 25 nanometers in diameter, the particles are about the same size as the norovirus, but they consist only of the outer surface protein — the portion of the virus recognized by the human immune system. The particles contain none of the infectious material of the original virus, but they stimulate a robust immune response to fight off an actual infection.

To battle each new strain of the norovirus and to keep full resistance to older strains, Arntzen says the vaccine could be administered as a booster every 12 to 18 months. After successful experiments in mice, his team is developing a nasal delivery system for the virus-like particles. Arntzen expects to start clinical trials in late 2009 or early 2010.

Several companies, most notably pharmaceutical heavyweight Bayer, are investing in new facilities to create plant-based vaccines for cancer, as well as other pharmaceutical proteins. He suggests the first plant-based vaccines should be publically available within four to five years.

"Mammalian and insect-based vaccines are tried and true — some have barely changed in nearly 60 years," says Arntzen. But that doesn't necessarily mean they are the best in terms of manufacturing costs or flexibility. It simply means that the industry is not accustomed to using plant biotechnology.

"Among other factors, the uncertainty on how such products would be viewed in the FDA approval process has created uncertainty in big pharma companies, and uncertainty is often a 'kiss of death' in product development that can involve hundreds of millions of development cost." But, he adds, "the current pipeline of new products now working their way to FDA approval is sure to change these opinions in coming years."
Adapted from materials provided by American Chemical Society.

Frozen Assets: Decades-old Frozen Infant Stool Samples Provide Clues To Norovirus Evolution

A search through decades-old frozen infant stool samples has yielded rich dividends for scientists from the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health. The team customized a laboratory technique to screen thousands of samples for norovirus, a major cause of acute gastroenteritis outbreaks in people of all ages. What they discovered about the rate of evolution of a specific group of noroviruses could help researchers develop specific antiviral drugs and, potentially, a vaccine against a disease that is very unpleasant and sometimes deadly.

The research, led by Kim Y. Green, Ph.D., and Karin Bok, Ph.D., of NIAID's Laboratory of Infectious Diseases, will appear in a future issue of the Journal of Virology, and is now available online. NIAID scientist Albert Z. Kapikian, M.D., is a co-author on the paper. In 1972, Dr. Kapikian and colleagues identified and characterized the virus, now known as norovirus, responsible for an outbreak of acute gastroenteritis in Norwalk, Ohio, in 1968.

"Thanks to the foresight of Dr. Kapikian and others at NIAID and the Children's National Medical Center who established and have maintained these clinical samples since 1974, our researchers have a unique resource that represents one of the oldest sets of norovirus samples in the world," says NIAID Director Anthony S. Fauci, M.D. "This is the first study to look at samples that date back almost to the first recorded cases of norovirus outbreaks, more than 40 years ago."

Highly contagious, noroviruses are responsible for an abrupt onset intestinal ailment also called winter vomiting disease or cruise-ship disease. The Centers for Disease Control and Prevention (CDC) estimates that 23 million cases of acute gastroenteritis each year are due to norovirus infection and that noroviruses are the cause of more than half of all foodborne gastroenteritis outbreaks. In elderly people, infants and people with compromised immune system function, dehydration resulting from vomiting and diarrhea following norovirus infection can be life-threatening. In developing countries, according to a 2008 estimate by CDC researchers, up to 200,000 children under 5 years old die of norovirus infection each year. There is no vaccine against norovirus and no specific antiviral drugs to treat infections.

A key question for norovirus researchers is determining when a dominant variant, called genotype II.4 (or GII.4), first emerged, notes Dr. Green. "This genotype has been associated with the majority of global outbreaks of acute norovirus gastroenteritis since the mid-1990s," says Dr. Green. "The GII.4 genotype was first described around 1987, but no one knew for sure whether that genotype emerged then or if it existed earlier."

To answer the question, Dr. Bok customized a new technique—real-time reverse transcriptase-polymerase chain reaction (RT-PCR)—and applied it to stool samples originally collected from infants and young children hospitalized at the Children's National Medical Center in Washington, D.C., between 1974 and 1991. Samples were taken from infants and children with gastroenteritis and from others (controls) who did not have gastroenteritis. Essentially, Dr. Bok crafted genetic hooks capable of fishing out matching genetic sequences of any norovirus present in the samples. Fifty out of 5,424 samples tested contained norovirus. The most commonly seen genotype was GII.3 (48 percent), but the second most common genotype was GII.4 (16 percent). Some GII.4-containing specimens dated back to 1974, allowing the researchers to conclude that this now-dominant genotype had been circulating for years before its more recent identification as the cause of severe global outbreaks of norovirus disease.

Next, using a strategy developed by NIAID scientist Stanislav Sosnovtsev, Ph.D., the researchers determined the complete genetic sequences of five older GII.4 viruses and compared those sequences to gene sequences of contemporary GII.4 noroviruses. The comparison allowed the investigators to determine how much the archival viruses differed from the most recent representatives of the same genotype and, thus, to calculate how quickly the GII.4 genotype is evolving.

Currently, there are no antiviral drugs specifically targeted to noroviruses, but the new knowledge about which segments of the norovirus genome change the least could aid in the development of novel drugs that could be targeted at those more genetically static portions of the virus, say the researchers. Noroviruses, like influenza viruses, mutate readily and evolve rapidly, explains Dr. Green. If vaccines against noroviruses become possible in the future, researchers would need to take into account shifts in the virus's genetic make-up and reformulate the vaccines to match the virus, she adds. However, unlike influenza viruses, noroviruses cannot be grown in the lab, raising an additional hurdle to vaccine development.

"By examining the history of norovirus evolution contained within these archival samples, we can see how the virus has changed during this time, and we also can better predict how the virus is likely to change in the future," says Dr. Bok. If scientists one day crack the problem of growing norovirus in the lab, information about the rate of evolution will be invaluable to developing vaccines, adds Dr. Green.

"This research is the first to reveal the speed at which the molecular clock of norovirus runs," says Dr. Green. Dr. Green and her colleagues are now looking at stool samples from the 1960s in Dr. Kapikian's collection. If norovirus can be detected in those samples, knowledge about the ancestry and rate of evolution of this virus will be further expanded.

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Journal reference:

1. Bok et al. Evolutionary Dynamics of GII.4 Noroviruses over a Thirty-four Year Period. Journal of Virology, 2009; DOI: 10.1128/JVI.00864-09

Adapted from materials provided by NIH/National Institute of Allergy and Infectious Diseases.