The NIH last week awarded $350 million to eight institutions across the US to establish Regional Centers of Excellence for Biodefense and Emerging Infectious Diseases. The winning institutions were: Duke University, Harvard Medical School, The New York State Department of Health, University of Chicago, University of Maryland-Baltimore, University of Texas Medical Branch, University of Washington, and Washington University in St. Louis. Proteomics research will play an integral role in all eight of the winning institutions’ projects.
As ProteoMonitor reported last month (see PM 8-1-03), each RCE will bring together scientists from across the full spectrum of biological sciences and from several cooperating regional institutions, who will take a wide variety of approaches to producing diagnostics, therapeutics, and new vaccines for potential agents of bioterrorism — chief among them so-called “category A” pathogens like anthrax, botulism, smallpox, and viral hemorrhagic fevers — as well as diseases like SARS, about which there is little knowledge. The NIH will also announce soon the winners of two $120 million grants to build National Biocontainment Laboratories at the RCE sites.
Scientists from the proteomics cores of each of the RCEs spoke with ProteoMonitor this week about their projects.
Dhavalkumar Patel, chief of the division of rheumatology, allergy, and immunology at the University of North Carolina, will lead the proteomics core at the $45 million Southeastern RCE. The focus for the core will be to characterize the proteins involved in pox viruses. UNC will work with the University of Alabama at Birmingham, Vanderbilt University, Protometrix of Branford, Conn., and Molecular Staging of New Haven, Conn., to accomplish five aims: provide 3D crystals of proteins, antibody protein complexes and lead protein complexes involved in the relevant viruses; offer modeling and structural bioinformatics services for all RCE members; provide high-throughput tools — in particular, protein arrays — for the study of protein-protein interactions and protein profiling; provide high-throughput protein structural characterization of crystallization products to determine functional binding of prospective drug targets; and provide NMR tools to determine the structure of ligand binding conformations.
UAB will contribute most of the “advanced proteomics capabilities” for the group, such as 2D gel and mass spec analysis, and will use its X-ray crystallography capabilities to define 10 protein structures per year and 30 drug protein complexes per year. “It’s ambitious, but they have the capability to do it,” Patel said.
Protometrix, which is currently in the process of developing a whole human proteome chip, will provide protein chips for interaction analysis. Molecular Staging will provide chips for detecting cytokines and other immune response molecules in biological fluids, and will look at biomarkers for drug response and disease activity.
Harvard Medical School
Gerald Beltz, associate director for research at the $46 million New England RCE, said that Harvard’s proteomics core — one of four cores working on nine research projects on category A pathogens for the RCE —will build protein arrays based on cDNA arrays of pathogen genes. Other cores will use the arrays to analyze immune products such as antibodies or cells from challenged animals. The results will then be analyzed for vaccine and therapeutic development.
The proteomics core — which will use existing staff and equipment at the Harvard Institute of Proteomics — will start by developing “tools and reagents” that other cores could then use to screen for responses to proteins from Yersinia pestis (plague) and Francisella tularensis (tularemia). “The core would provide, say, an arrayed set of expressed proteins of all the genes expressed by Yersinia pestis to the investigator who is studying that as one of their objectives. Then they could analyze the immune response to all 4,000-odd genes of Yersinia pestis in one experiment,” Beltz explained. Beltz said that a typical information flow involving several cores might go from a host cell protein with unknown function, to the identification of proteins in the expressed library with which it interacts, to the screening of small molecules to identify potential drug candidates.
New York State Department of Health
Kenneth Williams at Yale University’s Keck Laboratory will be the lead project investigator for the proteomics core at the $45 million Northeastern RCE. Williams’ group will use traditional mass spec approaches to identify serum biomarkers from infected blood supplies that arise in the early phases of infection by category A pathogens, as well as in infections caused by more common viruses like tuberculosis. The biomarkers will be used to develop assays for early detection and diagnosis of disease.
A first stage in the project will be the development of methods to remove high abundance proteins — particularly albumin —from the serum. The group will then look at fractionating resulting samples, comparative testing of various mass spec platforms, and the development of statistical algorithms for identifying pathogen-specific biomarkers.
University of Chicago
Olaf Schneewind, chairman of microbiology at the University of Chicago and principal investigator on the $35 million Midwestern RCE, will lead a proteomics project using techniques including mass spec, phage display, NMR, and X-ray crystallography to look for gene products involved in the pathogenesis of a number of category A pathogens, initially concentrating on Bacillus anthracis (anthrax). The group will use automation for protein purification and gene expression techniques.
The RCE will look at protein products in relation to three areas: diagnostic, therapeutic, and vaccine production. Schneewind is particularly interested in discovering proteins that could induce vaccine protection in a patient without producing any virulent effects.
University of Texas Medical Branch
As ProteoMonitor reported last month (see PM 8-1-03), UTMB has already received $12 million in funding from the US Defense Advanced Research Projects Agency, the Centers for Disease Control, and the Defense Threat Reduction Agency over the past several years for proteomics research related to biodefense. The $48 million RCE award to UTMB will build upon this research.
Alex Kurosky will head the proteomics core, which will concentrate on providing “generic-type infrastructure” for proteomics, largely focused on improving mass spec methods. “The equipment is already there. [The proteomics core] would provide state of the art proteomics like we have already at UTMB as well as the technologies we have been developing [with other grants],” said David Gorenstein, a member of the core. Gorenstein has been working with Ciphergen to develop thioaptamers — aptamers that have chimeric part-sulfur backbones — for use on protein chips that would screen for immune response resulting from viral infection. This work will receive additional funding as part of the RCE grant.
UTMB also received a $15 million grant last year from the National Heart, Lung, and Blood Institute to establish one of 10 nationwide proteomics centers.
University of Washington
Tina Guina, research assistant professor in pediatrics at UW, and David Goodlett, proteomics facility director at the Institute of Systems Biology, will lead the proteomics core for the $50 million UW-led RCE. The core will use high-throughput micro-capillary LC tandem MS and ICAT-based differential expression profiling to analyze the proteomes of three gram-negative bacteria species: Yersinia pestis, Francisella tularensis, and Burkholderia pseudomallei, (melioidosis). The core will grow the bacteria in environmental conditions under which they produce virulence factors, although the ultimate goal will be to look at the bacteria’s protein production in vivo.
“Basically we’ll try to improve methodology to isolate these bacteria from infected macrophages and eventually maybe infected animal tissue and develop much higher sensitivity techniques to analyze bacteria in that environment,” Guida said. “Nobody has done it successfully yet, but I think we have a unique opportunity — we have access to the latest technology, the latest software development, and we can use microarrays [with] genome information too.”
Another emphasis of the core will be to develop better software to analyze mass spec data.
Washington University in St. Louis
Daved Fremont, associate professor of pathology and immunology at Washington University, will head the proteomics and structural genomics component of this $35 million RCE. Fremont’s group will focus on the ectromelia, or mousepox. The mousepox virus produces around 20 proteins that are expressed on the cell surface of virally infected cells, according to Fremont. Fremont’s group will study how these proteins mediate immune invasion and overcome immune protection.
In order to study this process, Fremont will take a somewhat unusual approach to proteomic analysis. “In contrast with a lot of other proteomic approaches that people take who are usually working with intracellular proteins, we can’t use the standard methods with yeast-two-hybrids and things of this nature because extracellular proteins don’t work in these assays,” Fremont said. Instead, the group will express the proteins in E. Coli in large quantities. Then, for proteins with unknown functions, the scientists will biotinylate the proteins, use streptavidin to make tetrameric versions, attach fluorescing tags, and look for protein interactions using FAC sorting, a common immunological method.
University of Maryland, Baltimore
Myron Levine, director of the University of Maryland Center for Vaccine Development, will lead the $42 million Mid-Atlantic RCE. The RCE will concentrate on the development of vaccines and diagnostics against anthrax and smallpox.
Although UMB representatives said there will likely be a proteomics component to the RCE, no involved scientist could be reached at press time for comment.