Armed with a two-year, $1.5 million grant from the US Department of Energy, researchers at George Mason University, including Lance Liotta and Emanuel Petricoin, are using proteomic technology to identify protein biomarkers for early exposure to infectious diseases.
The effort is a joint collaboration between the university’s National Center for Biodefense and Infectious Diseases and its Center for Applied Proteomics and Molecular Medicine, and addresses one of the major concerns of the post-Sept. 11 world: the threat of bioterrorism.
Since the Sept. 11, 2001, attacks, the threat of biological weapons has been a looming specter, heightened by anthrax attacks in New York, Washington, DC, and Florida, as well as a ricin attack directed at then-Senate Majority Leader Bill Frist in 2004.
With that as a backdrop, the GMU researchers are aiming to develop a diagnostic with higher specificity and sensitivity that can tell within hours whether someone has been infected with a bioterrorism agent.
“The goal is to be able to detect the infection at a point where it potentially can make a difference in treatment,” Liotta, co-director of CAPMM, told ProteoMonitor this week.
While any test developed from their work would have immediate application within military scenarios, it would also have applications elsewhere, said Charles Bailey, director of the NCBID, which is responsible for culling the samples for analysis and exposing the cell lines to the infectious agents.
“It also could be applied toward vaccine evaluations as well,” he said. “[For] people who have been vaccinated for some of these pathogens, or maybe biomarkers that become circulating within the blood following vaccination, [a test] would indicate whether or not a person is going to be protected or not, whether or not the vaccine was effective, or whether they need to have a booster dose.”
The two centers have received the funding and begun their work, which initially is focusing on anthrax, because Bailey’s center has samples for the bacteria responsible for the infection, Bacillus anthracis, readily available. The researchers are in negotiations with federal labs for samples for other kinds of biopathogens, including pox viruses, and may conduct research in those areas later on.
A test for smallpox will not be explored, however, because the NCBID lab is not equipped to study it, Bailey said.
Terror in a Protein
Much of the proteomics work will be mass spectrometry based. Researchers will use a Thermo Fisher Scientific LTQ Orbitrap mass spectrometer to identify candidate biomarkers from serum. Mark Ross in Liotta’s lab will then employ multiple reaction monitoring and electron transfer dissociation technology to sequence and validate the proteins.
Liotta and Petricoin’s team will also draw upon a nanotechnology-based harvesting method they developed for pulling down low-abundance markers from serum.
“We’re particularly interested in biomarkers that are in the lower molecular weight range, that are fragments, that are low-abundance,” Liotta said. “And we’re looking at biomarkers that are related to the host response because in many of these diseases, the host response is what kills the infected individual.”
The harvesting technology is based on nanoparticles that Liotta said will be introduced to the complex protein mixtures. The nanoparticles will interact and remove from the solution the low-abundance biomarkers and purify them based on their size and affinity.
The technology eliminates the need for chromatography for separation, Liotta said. Rather, candidate markers are eluted from the nanoparticles, then tryptically digested and analyzed by ETD or MRM. Eventually, antibodies are used to validate the biomarkers.
While work is being conducted elsewhere in the area of biological weapons testing, Liotta said that most of the separation work involves 2D gel analysis, which requires a large sample size and doesn’t work well in the low molecular-weight range — a “rich source” of biomarkers, according to Liotta.
“We think we can just get rich candidate markers that are just not possible by standard proteomic methods,” he said.
Another differentiating characteristic of the GMU work is the expertise that Bailey’s group offers in infectious diseases, Liotta said. A pathologist by training, he said he understands infectious disease from that point of view, but not “from the epidemiology point of view, or the mechanistic effects of toxin” that Bailey and his group has been studying.
“Since they’re experts in infectious disease, they can say, ‘Hey, this is a really interesting marker that makes sense [to further study] because it may relate back to some host reaction or some effect of some toxin,’ and we can follow up on that,” Liotta said.
Playing Catch-Up With Speed
How much of an advantage a GMU-derived test will confer is unclear, however. Indeed, the GMU team is coming into the game relatively late.
“Right now, people look at antibody development and that usually doesn’t occur for three, four, five days following infection, and we feel that through this technology we may be able to find biomarkers circulating in the bloodstream in a matter of hours following the initial infection.”
Among the tests already available is one for anthrax based on Cepheid’s GeneXpert technology that has been on the market for more than four years. A protein-based test for the infection developed by Tetracore, called Redline Alert, was approved by the US Food and Drug Administration in late 2003.
In addition, last fall the National Institutes of Health awarded Genomics USA $2.8 million to develop a microarray product to analyze the natural genetic variation that modifies vaccination response to biosecurity agents such as bird flu, small pox, and B. anthracis.
And last summer, the US Department of Defense awarded Invitrogen a $1.2 million contract extension to use its ProtoArray protein microarray technology to detect and analyze biothreat agents such as hemorrhagic fever viruses, pox viruses, and B. anthracis.
According to Bailey, what separates the GMU effort from others is that he and his colleagues are concentrating on a test that can detect disease in the very early stages of infection.
“Right now people look at antibody development, and that usually doesn’t occur for three, four, five days following infection, and we feel that through this technology we may be able to find biomarkers circulating in the bloodstream in a matter of hours following the initial infection,” he said.
But because biomarker discovery has, to date, moved at a glacial pace, it is not certain that the GMU researchers will be successful in finding and validating any biomarkers within the two-year period covered by the DOE grant. Bailey said he is confident they will be.
The DOE project represents a new frontier for Liotta and Petricoin, perhaps best known for their work applying proteomics — in particular the surface enhancement laser desorption ionization platform — to cancer research. Most recently, they formed a company, Theranostics, employing an assay-based technology they say will allow researchers to look at the activity state of proteins in tissue samples [See PM 03/29/07].
While they have done smaller-scale proteomics work in infectious diseases, the DOE project is their first large, funded work in the area, Liotta said. A total of 14 people will be working on the project. He said that his cancer work will continue to be the main priority for his center, but he anticipates hiring additional staff for the DOE project.
Officials at the DOE were traveling and could not be reached for comment, a spokeswoman for the department said.