18 April 2003 17:00 GMT

by Hillary E. Sussman

Spinach could provide an answer to the quest for a safer, purer anthrax vaccine, suggest US researchers. The plant could readily be used as a vehicle for the production of an edible vaccine against the infection, say immunologists at Thomas Jefferson University in Philadelphia.

Efforts to develop a new vaccine have been stepped up since the current anthrax vaccine, which was licensed for human use in 1970, was recently deemed sub-optimal.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which exists as spores in the soil and, therefore, commonly affects grazing animals such as cattle and sheep. Human infection, although rare, occurs following direct skin contact with infected animals or their wool, hides or tissues, by ingestion of contaminated meat, or via inhalation of the spores. Left untreated, inhalation anthrax is almost always fatal and early intervention with antibiotics, such as ciprofloxacin, is essential.

Vaccination is recommended for persons at risk of exposure to anthrax spores. The current vaccine is based on cell-free culture supernatants of an attenuated strain of B. anthracis adsorbed on aluminium hydroxide (in the USA) or precipitated with aluminium phosphate (in the UK) - aluminium acts as an adjuvant. It is incompletely characterized and difficult to standardize and, therefore, exhibits inconsistency between lots. It is also relatively reactogenic, with side effects including a possible link to Gulf War syndrome (whose symptoms include chronic fatigue, depression, skin rashes and gastrointestinal disorders), and requires a lengthy dosing schedule, all of which suggest the need for an improved, alternative vaccine.

The main immunogenic component of the current vaccine has been determined to be the protective antigen (PA), and vaccination with PA alone can induce protective immunity to anthrax. PA binds mammalian cell surface receptors, where it is proteolytically cleaved and activated to form a heptameric pore-like structure that binds either edema factor (EF) or lethal factor (LF) to form edema toxin and lethal toxin, respectively. Following endocytosis of the toxin complex, PA facilitates the passage of the toxins into the host cell cytoplasm where they disrupt normal signaling pathways leading to cell lysis, toxic shock and, ultimately, death. However, "if you can block the very first stage - binding of the PA to the receptor - you block the mechanism-of-action of the toxin and essentially block the disease," said Alexander Karasev, assistant professor of microbiology and immunology at Thomas Jefferson University.

"The new vaccines will be based on recombinant PA and will be much purer than the current vaccine," said Meryl Nass, a Diplomat on the American Board of Internal Medicine, but "whether a pure PA vaccine will be safer is a big question."

Stephen Leppla, senior investigator in the Microbial Pathogenesis Section at the National Institute of Allergy and Infectious Diseases, (NIH) concurs. "The concerns about the existing vaccine may well apply to protective antigen-based vaccines regardless of whether they are made in plants, bacteria, yeast or other systems. There is no a priori reason to suggest that plant-derived vaccines will produce fewer side effects."

Nevertheless, many scientists believe that it is unlikely that the PA protein itself is associated with adverse reactions. Furthermore, plant-based vaccines are appealing. "One of the beauties of the plant system is that there are no pathogens that infect both plants and animals," said Karasev, and without having to continuously screen the production medium for contaminants, screening costs are significantly reduced.

Mohammed Azhar Aziz, senior research fellow at the Centre For Biotechnology, Jawaharlal Nehru University, India adds that the "production of subunit vaccines in plants offers the additional unique advantage of delivery in commonly consumed foodstuff, which may enhance the availability and ease of delivering immunizations." Aziz is part of a group of scientists that successfully integrated the PA gene into the nuclear genome of tobacco plants last year.

Because there is some public opposition to the genetic modification of plants, and tobacco is considered an experimental plant, the scientists at Thomas Jefferson University designed a system to transiently express PA within a normal spinach plant, moving yet another step closer to an edible anthrax vaccine.

Specifically, a fragment of PA that represents most of the receptor-binding domain was expressed as a translational fusion with a capsid protein on the outer surface of tobacco mosaic virus, and spinach was inoculated with the recombinant virus particles. "One of the rationales for using just a fragment of the protein is that basically you don't need the whole protein to elicit a protective immune response," said Karasev, whose data were presented last month at the American Society for Microbiology Biodefense Research Meeting, in Baltimore, USA.

The plant-expressed PA is highly immunogenic in laboratory animals, but producing antibodies that are specific to PA is not sufficient. "The key question is whether or not the antibodies are protective," remarked Sanford Kimmel, professor of family medicine at the Medical College of Ohio.

Karasev's group plans to first test whether these antibodies inactivate the anthrax toxin in vitro and then determine whether they protect laboratory animals against anthrax. Karasev also admits that evoking a good systemic immune response after the vaccine is delivered through the digestive system is "probably the biggest challenge today." The PA fragment was extracted and purified from the spinach to test it as an immunogen "but in theory if you solved the problem of eliciting a strong immune response you can probably just eat it as a vegetable salad," said Karasev.

The development of plant-based vaccines to protect against many other diseases, such as HIV-1, hepatitis B, rabies and non-Hodgkin's lymphoma, are ongoing.