Hoping to develop a vaccine, a diagnostic, and a therapeutic for anthrax, scientists at the US Army Medical Research Institute of Infectious Diseases will begin performing animal experiments within the next few weeks to test the effects of selected Bacillus anthracis proteins on the bacterium's pathogenicity.
The proteins, selected by San Diego-based Diversa, are expressed more in vivo than in vitro by B. anthracis, according to Brian Green, a principal scientist at Diversa.
"The theory is that if you're seeing things going on in vivo that are not going on in vitro, those proteins may have an important key role in infection and disease," Green explained.
Diversa began working on finding anthrax target proteins in a limited fashion in 2002, then stepped up its efforts in 2003 and 2004 after it received $3.7 million from the National Institute of Allergy and Infectious Diseases to work on the project (see ProteoMonitor 4/16/2004). Recently, the company received a $1.4 million grant from the National Institutes of Health for the "characterization of proteomes of category A pathogens," which includes B. anthracis and Yersinia pestis, the bacteria that causes plague.
To analyze the B. anthracis proteins, Diversa scientists use multidimensional liquid chromatography, followed by mass spec analysis. Most of the mass spec work was done using ThermoFinnegan LCQs and LTQs, Green said. Diversa scientists also used software developed in house to compare levels of expression of proteins in a semi-quantitative manner.
To test the effects of the targeted proteins on the pathogenicity of B. anthracis, USAMRIID scientists have created about a dozen strains of the organism that lack genes for key proteins identified by Diversa. They plan to inject these modified strains into mice and observe if the modified strains still cause illness and death.
In theory, if a modified strain is not pathogenic, the protein that was deleted from that strain would make for a good therapeutic target and a good immunogenic basis for a vaccine.
In their search for target anthrax proteins, Diversa scientists originally selected 100 proteins that were expressed more in vivo than in vitro by B. anthracis. From that set, they selected proteins that are present in the membrane and unique to the organism because those properties make for better vaccine and therapeutic targets, the scientists said. In the end, they were left with about a dozen or so potential target proteins.
Some of the proteins that were selected were previously known B. anthracis virulence factors, Green said. The known factors include protective antigen (PA) and lethal factor, a lethal toxin produced by anthrax that disrupts enzymatic processes crucial for life.
PA, which acts as a binding intermediate between cells in the body and lethal factor, is the protein target used as the basis for the currently available anthrax vaccine. The problem with this vaccine, which was developed in the 1950s and 1960s, is that it contains not only PA but also a number of other contaminating proteins, Green said. People often develop a reaction to the contaminating proteins during the course of the vaccination regimen, which calls for six injections given within 12-18 months, followed by a booster shot every year.
"It's like they're using soup when you really want to inject the broth," said Martin Sabarsky, senior director of corporate development and investor relations at Diversa.
Diversa Also Looking to
Develop Vaccine, Dx,
Tx for Plague Bacterium
In addition to its work on anthrax, Diversa is also trying to develop a vaccine, a diagnostic, and a therapeutic for Yersinia pestis, the bacterium that causes plague. For this disease, Diversa is collaborating with the University of North Dakota and the US Army Medical Research Institute of Infectious Diseases.
The USAMRIID facility in Fort Deitrick, Md. has one of the few laboratories in the US equipped to deal with lethal pathogens like anthrax and plague, according to Brian Green, a principal scientist at Diversa. The US Army facility was featured in the 1995 Dustin Hoffman movie Outbreak.
Aside from working on terrorism-related pathogens, Diversa also works on many non-terrorism related projects, Green said, including developing industrial and agricultural enzymes and pharmaceuticals for other types of infectious diseases.
Green said that scientists are hoping that with the next anthrax vaccine that is developed, people will get a good immune response to it, so that only one shot would be needed for protection. He said it isn't clear when the new vaccine would be broadly available. He also said that a vaccine would most likely be available exclusively for the US armed forces but that the diagnostic and therapeutic would be broadly available to the general population. It is also not clear when a diagnostic or therapeutic would be available, Green said.
The current treatment for anthrax
is the antibiotic Cipro. Cipro is effective in killing B. anthracis, but it is not effective in disabling toxins such as lethal factor that are released by the bacterium. Consequently, if an anthrax infection is detected too late, the infected person may still die from toxins even after being treated with Cipro.
"Lethal factor does all the damage," said Green. "It killed a lot of the people that came down with anthrax in 2001. They started taking the antibiotic, but by that time they had already been subjected to too much of the toxin."
The Diversa anthrax group comprises about 15 people, including three bioinformaticists. The group is equipped with about 10 Thermo Finnegan mass specs, in addition to a Micromass Q-TOF, an ABI Q-STAR and a MALDI-TOF.
Financial terms of the alliance between Diversa and the US Army have not been disclosed.