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3rd Millennium helps the U.S. Army enlist genomics and IT to fight an old foe: Plasmodium falciparum, also known as the malaria parasite.
 
By John Russell

July 15, 2003 | FOR MOST OF the developed world, malaria is someone else's problem, but if you live in sub-Saharan Africa, malaria — along with HIV — is a mass killer. Consider just a few scary statistics from the World Health Organization:

* There are 300 million to 500 million cases of malaria yearly, 90 percent of them in sub-Saharan Africa.

* More than 1 million malaria victims die each year, and the majority of deaths are among infants and pregnant women. Malaria is the leading cause of death for children under the age of five in Africa.

* Roughly 40 percent of public health expenditures in sub-Saharan Africa are to treat malaria.

Contrast those numbers with the roughly 1,200 cases reported annually in the United States, most of which arise in immigrants or travelers returning from regions of the world where malaria remains an epidemic. Most of these patients recover.

The truth is, the malaria epidemic grew worse, not better, during the past decade. Rising resistance to traditional drugs such as quinine, co-infection with HIV, and political and social turmoil in the regions most affected have all helped to stall headway against the disease. That's despite some high-profile international efforts to turn the tide.

What may be surprising, however, is that one of the staunchest combatants in the fight against malaria (and HIV) is the U.S. Department of Defense (DoD). Generals throughout history, from Hannibal to MacArthur, have had their progress stopped and their numbers diminished by the withering effects of malaria. Pack the ammo, but don't forget the quinine (or one of its modern counterparts), mosquito nets, and repellent!

Next month, DoD researcher

Taking SBIR Technology into the Mainstream 
As required by Small Business Innovation Research provisions, 3rd Millennium will bring the informatics technology developed for Sheila Peel's lab to a broader market.

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Sheila Peel and her colleagues at the Walter Reed Army Institute of Research (WRAIR) will tackle this old enemy with a new weapon — a state-of-the-art gene-expression lab and custom informatics platform delivered by consultant 3rd Millennium and paid for with a Small Business Innovation Research (SBIR) award (see "Taking SBIR Technology into the Mainstream").

Using the new system — comprising a large data warehouse, comprehensive LIMS, and a set of gene-expression analysis tools — Peel hopes to wring meaningful clues from a wealth of microarray data about the malaria parasite (primarily Plasmodium falciparum), its effects on mosquito and human hosts, and the results of co-infection with HIV.

"The Army has always been good at moving compounds through clinical development, and there are several drugs out now because of its efforts," says Malcolm Gardner, currently an associate investigator at The Institute for Genomic Research (TIGR), who has worked extensively on sequencing and analyzing the genome of the parasite. He was also a researcher at WRAIR from 1991 to 1993.

"What they didn't do well was basic research," Gardner says. "Funding was tight, and it was difficult to get approval to bring in the needed functional genomics technology." This new system is a good step forward, he says. "It sounds like they will now be able to examine biological pathways and the biochemistry."

That is certainly Peel's goal. She is one of a small army of civilian researchers working for DoD, and this spring received the 2002 Army Research and Development Award for her work in creating the new modern microarray facility to support basic research and drug and vaccine development to treat malaria and HIV.

"In terms of malaria, we're heavily engaged in drug development. We're looking at current and novel antimalarials," says Peel, who has a Ph.D. in molecular parasitology and an M.S. in public health, both from the University of North Carolina at Chapel Hill. Peel was also resident research associate for National Academy of Sciences' National Research Council from 1996 to 1999.

Several promising compounds are currently under investigation, Peel says as cagily as any private-sector pharmaceutical executive might.

The array-processing equipment and data warehouse for the new system are located in Peel's lab at the Department of Molecular Diagnostics/Pathogenesis in the Division of Retrovirology, WRAIR. However, access to the data and analysis tools will be provided to many malaria and HIV researchers throughout DoD over the Web, and the lab will supply needed chips to other Defense laboratories.


Do-It-Yourself Approach 
"The problem was that we didn't have the tools or capability in-house to manage this enormous glut of data for large-scale genomic experiments," says Peel, who joined the retrovirology division in August of 1999, moving there from the Division of Therapeutics, where she'd been working on functional genomics for antimalarial drug development. Researcher Maj. Karen Kopydlowski took over that effort. Peel wears two hats these days: She is chief of the de novo microarray laboratory that supports both divisions and chief of the flow cytometry resource laboratory.

"Karen and I have been developing this technology at WRAIR together," Peel says. "There's an enormous amount of synergy between our two laboratories, but neither of us had the money to develop it independently — there were only two of us in each lab. So I hit upon the solution of writing an SBIR."


On the firing line: Sheila Peel's new genomics lab supports DoD antimalarial research around the world. New drugs will follow shortly, she says. 
By law, DoD must set aside 2.5 percent of its R&D budget for SBIR awards. That amounted to $175 million in 2001. The idea is to bring innovative technology into DoD, support small companies, and spur commercialization of new technology. Landing an SBIR grant can be a breakthrough moment for small companies struggling to develop their ideas and a boon to DoD seeking access to the latest technology.

Peel was still in the early stages of ramping up her microarray laboratory at the Division of Retrovirology. She had brought the first instruments into the lab in October 2000. "We weren't really doing experiments. All through 2001, we were writing the Access database and running quality control to see how well we were printing [DNA] chips." She and her lab technician cobbled together a rudimentary system to store and analyze images using Microsoft's Access database, Excel, some Visual Basic programs for manipulating data, and an old program called CloneTracker. "Very quickly, we broke the Access database," Peel says.

It's worth remembering that these were the early days of industrializing the genomics revolution. Peel was using an array spotter from Engineering Services and an original Axon 4000A scanner from Axon Instruments. The equipment didn't talk well to each other, and technician Leon Simms ("an incredibly talented young man") was forced to write a bunch of macros to transfer data back and forth.

"As many labs have done, we ended up helping these companies develop their software because of the problems we encountered. We were really breaking new ground and had an impact on their software."

Sheila Peel, Walter Reed Army Institute of Research

"As many labs have done, we ended up helping these companies develop their software because of the problems we encountered. We were really breaking new ground and had an impact on their software," Peel says. Simms is now studying for a Ph.D. in computational biology at the University of Pennsylvania.

Peel was also working her way through the malaria genome. "We spent 2001 generating chips for the cDNA library and had about 200 open reading frames (ORFs), or about a third of the genome," she says. It was tedious work. The genome of P. falciparum is extraordinarily rich in A and T bases (82 percent), which often confounds sequencing and microarray analysis.

The complete malaria genome sequence was published in Nature last October, although much of the genome was known before. When Operon Technologies released a complete set of malaria genome oligonucleotides last year — based on sequences from The Wellcome Trust Sanger Institute, TIGR, and Stanford University — Peel and Kopydlowski purchased the library using the latter's funds. "We were interested in drug discovery and development," not making sequences, Peel says.

Still, that early work won't go to waste. Peel expects to publish a "techniques" paper later this year, "about developing a labeling mechanism for the very AT-rich parasite. The protocols almost always need to be modified from those for more GC-rich genomes, such as yeast and human. We have worked hard on this and are beta-testing a product," she says.

The work also informed Peel's thinking about what should be in the informatics platform as she wrote the SBIR proposal, which was finally posted in May 2001. As a rule it's rare for software projects, particularly research software, to win DoD funding. Peel shrugs and says this attitude is changing in the functional genomics era. Her project is a case in point.

SBIR awards are issued in phases. Companies compete vigorously for modest proof-of-concept Phase One grants, which can last six to nine months and be worth up to $70,000. If Phase One goes well, the project may advance to Phase Two, which can last up to 18 months and carry an award of $800,000. The contractor retains all intellectual property and is expected to commercialize the technology.


The New Millennium 
Several companies responded to the RFP, although Peel declines to name them. Each proposal was evaluated by several DoD reviewers. Calling 3rd Millennium's bid creative and flexible, Peel says the small Massachusetts-based company's proposal scored significantly higher than the others.

"We're a custom software company, and this project overlapped with other projects we'd done," explains Jack Pollard, one of two principal investigators from 3rd Millennium assigned to the project. "Sheila wanted an informatics platform that could help her store the data and analyze them in a statistically relevant way."

"We're a custom software company, and this project overlapped with other projects we'd done. Sheila wanted an informatics platform that could help her store the data and analyze them in a statistically relevant way." 

Jack Pollard, 3rd Millennium

Founded in 1996, 3rd Millennium has done similar projects for Ardais and Biogen. In 2000, it won a $1.8-million grant from the Advanced Technology Program (ATP) of the National Institute of Standards and Technology (NIST), which is under the auspices of the U.S. Department of Commerce to develop software for use in gene-based research. The company has since used this technology in other projects.

Pollard teamed with Roland Carel, senior systems architect at 3rd Millennium, on the project. "This is a good vignette of how we work," Pollard says. "We pair people like Roland and me who have a significant overlap in their understanding of biology and information systems but also have unique understanding in those areas." Pollard has a Ph.D. in biochemistry and molecular pharmacology; Carel's Ph.D. is in material sciences and engineering. The total staff at 3rd Millennium is 13, and all software development is done in-house.

Defining the data warehouse during Phase One was nontrivial. The requirement called for storing not only the spotted and Affymetrix expression data but also the biological context of the samples used, such as how the cells were collected, who harvested them, and what extraction procedures and instruments were used. When completed, the entire system will require about 200,000 lines of custom code.

"We were able to show that we could bring Sheila's spotted data into structures that were originally designed for Affymetrix kind of data and that we're also able to reuse the experimental design modules in the system for her data," Pollard says of Phase One.

3rd Millennium recommended moving to an Oracle database because of its flexibility and scalability. "It will scale easily into the terabyte range," Pollard says. That's good because Peel is particular about what information she wants captured.

"The LIMS will take all information associated with fabricating a chip and subsequent processing. The chips themselves have bar codes. Information about all of the processes, including labeling and hybridization, many of the chemicals and the concentrations involved, the position of the plate, concentration of the PCR product. All of that would be captured and get dumped into the archival database. You can get down to the level of each reagent," Peel says.

Peel's Lab at a Glance 
A look at the Lab's mission, IT systems and array capability.

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To provide needed computer power, two Sun Fire 280R servers were selected (see "Peel's Lab at a Glance"). "Truth be told, we actually spec'ed out a variety of different systems manufacturers and configurations that would have been acceptable. It was put out to competitive bid. I think the contracting office ultimately made the decision," Pollard says. The funds for purchasing the hardware are not part of the SBIR but paid directly by DoD. "We tend to be hardware agnostic," Carel agrees.

One area in which 3rd Millennium has gotten religion is open-source software. "We're not integrating any commercial software [on this project], but we are using lots of open-source software," Pollard says.

Cost is an obvious advantage: Open-source software is cheaper and can be distributed with fewer restrictions. This is a plus for Peel's project, which envisions providing access to a growing number of collaborators inside and outside of DoD. Initially, five sites will have access to the database and tools: Peel's lab in Silver Spring, Md.; Kopydlowski's lab nearby at WRAIR; another research center at Fort Detrick, Md.; and sites in Kenya and Thailand.

Commercial software availability was also an issue. When Peel began mulling LIMS requirements, the choices were limited. "I think PerkinElmer had a product, but it was very expensive," she says. Pollard suggested that Resolver from Rosetta Biosoftware could handle Peel's data analysis needs, but it too is expensive and would need to be customized.


Expert Coders 
Pollard and Carel drew heavily from the Bioconductor project for gene-expression analysis applications. The project's Web site (www.bioconductor.org) states: "The project was started in the fall of 2001. The Bioconductor core team is based primarily at the biostatistics unit of the Dana-Farber Cancer Institute at the Harvard Medical School/Harvard School of Public Health. Other members come from various U.S. and international institutions."

"This kind of software development is more suited to bioinformaticists than traditional software developers," Pollard says, adding cautiously, "Bioinformaticists are not necessarily known for the rigors of their development. Will they have tested all ranges and boundary conditions for given functions?"

Carel adds: "You're at the mercy of the person who originally coded it. On the other hand, people who are at the leading edge of microarray analysis contributed to this process and are the drivers of what's in the package. Realistically, there's very little chance that you can do as well as the people who dedicate their careers to analyzing these kinds of data."

The bottom line, Pollard says, is: "The bioconductor project is geared at providing open-source software for analyzing data that result from high-throughput data sets. When you think about commercial products, there's always a lag between something that gets published and then it appears in the product some six, eight, nine months later. But here, the person who wrote the paper publishing the routine is also contributing the [software]."

One key project decision was to limit its scope. "The analysis starts with the raw data that come off of the image analysis program, and it ends with [Peel] having a good understanding of which genes are differentially expressed in which samples and for what reason. That's the end point for us," Pollard says.

Peel was particularly interested in automating efforts to "trim" or normalize expression data for normal and so-called flip experiments (in which the indicator dyes are reversed) and to be able to search across the data. 3rd Millennium incorporated the ability to do this.

Not included in the system are tools for data mining and visualization. "We felt that the commercial packages that are out there do those things absolutely great, and it made no sense to try to reinvent the wheel," Pollard says, citing Spotfire's visualization software as an example. "We've provided an ability to integrate with for exporting data from the system to these packages."

Pollard says the project had few big surprises, but a fair number of changes. For example, the ability to store minimum information about a microarray experiment (MIAME)-compliant data wasn't part of the initial specification but was added as more scientific journals made it a condition for publishing.

Peel's small staff has also just expanded to three, with the addition of postdoc computer scientist Chining Wang, who brings considerable experience in Affymetrix data informatics.

"We know when you have malaria and HIV and there seems to be an effective acute infection, the [HIV] viral load increases by about a log. If you treat the parasitic infection and cure the malaria, the viral load declines."

Sheila Peel, Walter Reed Army Institute of Research

Mindful that drug and vaccine development is the endgame, Peel had built into the systems audit control and robust security provisions that can restrict which data and tools individual researchers have access to. "We'll be submitting data to the FDA and are ready to do that," she says.

Her own research currently focuses on parasite pathogenesis and the effects of co-infection with HIV and malaria. "We know when you have malaria and HIV and there seems to be an effective acute infection, the [HIV] viral load increases by about a log. If you treat the parasitic infection and cure the malaria, the viral load declines," she says, "but what the interaction is and how significant it is remains quite controversial."

Meanwhile, she has written another SBIR RFP, which was posted in May. This one seeks to develop a systems biology capability for her lab — a "product and interface package that will utilize recent advances in areas such as systems science, self-organizing processes, and/or adaptive cooperative computing ..."

No doubt a few 3rd Millennium types are out there right now, writing proposals to help her do just that. * 

Malaria Marches On - Growing resistance to antimalarial drugs, and political turmoil in the most affected areas, have enabled the worldwide pandemic to worsen. 90% of Deaths Are in Africa






ILLUSTRATION BY MARTIN O'NEILL; PHOTOGRAPH BY KATHERINE LAMBERT





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