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Seeking Immunity
by Paul Karr

In earnest, three-week stretches, Bolyn Hubby hunches over a microscope in a tiny lab above a medical clinic in Buenos Aires, Argentina. Downstairs, patients — some of them dying — offer samples of their blood.

Hubby diligently applies test vaccines developed at the University of Georgia to drop after drop of infected blood hoping to find a glimmer of acquired resistance — a clue in someone’s blood that might point the way to a successful vaccine.

“This is what I always wanted, to be involved directly with people suffering from the disease,” said Hubby, a UGA doctoral candidate in cellular biology from Savannah who also studied anthropology. “To combine what I do in the lab with actual contact with these people is a tremendous opportunity.”

It is also the motivating force behind the university’s new Center for Tropical and Emerging Global Diseases, directed by cellular biologist Rick Tarleton, which brings the work of scientists from a variety of disciplines to bear on some of the world’s most devastating diseases.

Infections and parasites contribute to one-third of all human deaths each year — a staggering 17 million, mostly in lesser-developed nations. Malaria alone kills 3 million people each year, many of whom are younger than age 10.

Yet tropical parasitic diseases historically haven’t received as much scientific attention as other diseases, in part because they usually occur in nations that have inadequate resources to find cures and vaccines. Scientists still have much to learn about the biology of these conditions.

That’s why center research concentrates on basic science, examining the underlying biology of infections — the building blocks of defense against disease. In the case of Hubby’s work in Argentina, she and her colleagues have found some promising signs that the vaccines that worked for mice in her Georgia lab may work for people too.

But it’s too early to tell. It may take many years of these sorts of confirmations — not just one person’s success — to make conclusions about a vaccine. There will be no dramatic ending to the quest, no single drop of blood that opens the floodgates and touches off celebration. That makes her efforts all the more impressive.

“Much of what we do, in research, is for the future,” she said. “It takes time and a lot of effort.”

Contributing to the effort, the National Institutes of Health awarded the UGA researchers a $3.3 million grant to establish a tropical disease research unit. The university is one of just four national recipients of the tropical disease awards, which are granted only once every five years.

The grant helps enable Tarleton’s team to arm itself with sophisticated equipment to help crack the case of tongue-twisting infections such as malaria, leishmaniasis, schistosomiasis, cryptosporidiosis and lymphatic filariasis.

Tarleton’s first target, however, is one he knows all too well — one he has been pursuing throughout his undergraduate and professional life for more than 20 years now.

Unraveling a pesky disease
Tarleton’s longtime quarry is a parasitic protozoan called Trypanosoma cruzi. The tiny microbe is transmitted to animals and humans through the feces of kissing bugs — a class of insects also known as reduviid.

These bugs, relatives of stinkbugs and wheelbugs, come into contact with people in the thatched huts of the tropical forests. The result is Chagas (pronounced SHA-gus) disease, a debilitating, often fatal illness most prevalent in South American countries — and one for which there is no known cure or vaccine.

“There are very few success stories with this tropical disease,” Tarleton said. “We’d like to see that change.”

The Centers for Disease Control and Prevention in Atlanta estimates that some 20 million people worldwide are infected with Chagas disease, also known as American trypanosomiasis, and that 50,000 more die each year. The symptoms may not show up for 10 or 20 years, but when they do, they can be serious. Heart enlargement and breathing problems are the most common.

Chagas has worked its way to the United States in recent years through immigrants, Tarleton said, and “is almost certainly being transmitted here through the blood supply.” The incurable disease even exists, in small numbers, in Georgia.

To combat it, scientists already have experimented with breeding techniques that neutralize the infectious pathogens while they still reside in the insects that transmit them. But integrating genetically altered insects into wider natural populations is a daunting — perhaps impractical — goal that might never be achieved.

And recent findings indicate that a Chagas sufferer is harmed by the cumulative effects of small, chronic amounts of the parasite that remain in the body over a period of years or decades. That’s why disease researchers like Tarleton keep working to find two vaccines: one that would arm the body against the parasite to ward off infection, and another that would provoke an immune response in sufferers and rid the body of the organism early enough to avoid cardiac trouble.

It’s tedious work.

“The conventional, old-fashioned way of developing a vaccine is simply to process the entire pathogenic organism and inject it into someone,” Tarleton said. “But that often doesn’t work very well, and doesn’t work at all for most parasites. An alternative approach is to find a component, a sub-unit, of that pathogen. One way to do that is to clone a gene and make a protein from the gene. But that doesn’t turn out to work very well, either, and it can take three or four years just to produce a single failure.”

One reason these approaches fail is because the T. cruzi parasite is a very complex organism, much more complex than, say, a virus.

“It contains tens of thousands of genes, and looking at them one at a time is not a very efficient process,” Tarleton said.

So his team decided to focus on different approaches. One of the most promising appears to be DNA gene delivery, a recent but simple technique whereby cocktails of specially selected DNA are injected directly into an infected person’s muscle or skin. If the mix is just right, the body’s immune system will be stimulated to produce the proper response and fight off the disease.

“It’s also nice because DNA vaccines are relatively easy to produce and inexpensive to store — an important consideration in developing nations,” Tarleton said.

However, finding the right mix is as difficult as it is crucial. Recall that T. cruzi contains at least 10,000 genes; experience has led Tarleton to believe the right combination would contain perhaps 20 to 30 pieces of those thousands. On the surface, it seems an impossible task.

Yet several new technologies are speeding the hunt. One, called expression library immunization, uses random cocktails of the T. cruzi’s DNA — “snapshots” of genetic material, taken at random from the entire “library” containing every possible bit of the protozoan DNA. These cocktails are then injected into mice and the mice are “challenged” by infecting them with T. cruzi.

It requires several painstaking months — looking for the presence of antibodies, immune responses and, ultimately, the survival of mice and the absence of disease — to be able to tell which mice have become infected and which have developed immunity. If a particular mixture seems to generate immunity more often than not in the mice, that mixture will then be examined in further and finer detail: Lab technicians subdivide it and run its pieces through more tests. Later, when very good candidates are uncovered through repeated vaccine trials, the researchers “sequence” the specific genetic code.

“Over the course of a year’s time, you can conceivably sift through tens of thousands of genes this way,” Tarleton said.

Another method, known as DNA microarray technology, is a bit different: Lab assistants prepare thousands of T. cruzi’s genes using a machine that automatically adds enzymes and produces large quantities of the DNA overnight. This DNA is laid out on glass slides. Then bits of mRNA taken from T. cruzi at different points during its life cycle are applied to the slides and allowed to hybridize — or react together with — the DNA.

“Doing this all by hand would be very time-consuming,” Tarleton said.

The researchers plot the results of these hybridizations on a graph. If a bit of T. cruzi “recognizes” its kin in the array, the pair will react and create a red or green spot on the slide. The color depends on which of two places — in the patient’s blood or inside another body cell — the parasite was located when that particular gene was expressed. If a particular spot of DNA doesn’t provoke either reaction, however, that gene may not evoke the best immune responses and may be discarded as a candidate for future study.

Most spots do turn yellow, but a few — on the outer edges of the graph — are bright red or green. These become potential diamonds in the rough.

Global reach
Tarleton’s lifelong pursuit to find a vaccine for Chagas disease is important, but it’s just one part of the center’s work around the globe. Liaisons with clinics and labs in Argentina, Ghana, Kenya, Guatemala, Mali and Brazil keep fresh data flowing to Athens, where the UGA researchers can focus an impressive array of technology on the problem.

“Like many ‘centers,’ this one involves a lot of people instead of a single location,” said Tarleton.

Indeed, the center has a small physical space but a large reach, stretching around East Campus to involve eight core investigators plus a cadre of research associates in the colleges of Arts and Sciences, Veterinary Medicine and Agricultural and Environmental Sciences.

“It’s more of a virtual center, at this point,” Tarleton chuckled.

Tarleton’s lab alone involves some 15 researchers working in several different areas of the labyrinthine Biological Sciences Building. Then there are various other researchers (see Bug Zappers on page 19) each with his or her own complement of assistants. You can’t walk into or phone a centralized CTEGD office, but collectively it has the resources of a powerful disease-research facility.

As computers on the sixth floor spit out, analyze and compare peptide sequences of T. cruzi, researchers in a basement lab — recently converted from what was once a loading dock — array its DNA or print images of brightly colored fluorescent cells and gels. Tarleton’s home lab is yet another complication of beakers, test tubes, machinery and a special room where live pathogens are grown in incubators; safety devices are mandatory here. Still another researcher works in borrowed space, waiting for a permanent home.

The temporary lack of a central facility hasn’t deterred the team from its research, though. Tarleton is excited about the recent addition of two new faculty members, bringing the total to eight core members.

Julie Moore is an expert in placental malaria — a lethal form of the disease that frequently strikes women in Africa whose immune systems are weakened by pregnancy. And Boris Striepen, a cellular biologist from Germany via the University of Pennsylvania and the other recent addition, studies the parasite Toxoplasma gondii. Striepen examines the infection process at a molecular level to try to understand how the parasite invades cells.

The CDC, the Medical College of Georgia in Augusta and the Emory Vaccine Center all have signed on to assist in various capacities. The most important of those partners is probably the CDC, which brings a wealth of experience in epidemiology — the long-term studies of disease factors and patterns. The CDC also has an extensive network of field sites in the tropics; several of their researchers are now UGA adjunct faculty.

The HIV connection
Tarleton’s next challenge may well involve increased collaborations. The center might join forces with animal disease specialists such as UGA ecology professor Peter Daszak, as well as institutions such as the World Health Organization and the NIH, to study the interaction among different diseases.

One of those is human immunodeficiency virus, or HIV.

“The impact of HIV on other pathogens is becoming an issue,” Tarleton said. “As HIV continues to gain in its impact on developing countries, it’s going to increase the problems that parasitic and opportunistic infections cause. We’ve seen people who have carried Chagas for 10 or 20 years, doing alright, and suddenly they get HIV and the next thing you know they are dead from an overwhelming parasitic infection.”

To do broader work like this, the center’s wish list doesn’t include high-tech equipment — the Georgia Research Alliance already has funded some equipment — but it does include a dedicated space or building that would centralize its scattered research operations, plus some additional staff to do the investigating.

“We hope to build expertise and have as many representatives of different areas and parasites as we can,” Tarleton said. “What we’ve tried to do with the center is not focus on any one parasite or set of approaches. We’re trying to get people who work in a variety of areas and cover as much of the field as we can.”

For more information about the CTEGD, access http://www.uga.edu/ctegd/.

Paul Karr is a prize-winning science journalist and writing coach who contributes regularly to Research Reporter.


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Chagas vitims (like the child to the right) become infected with Trypanosoma cruzi by kissing bugs, a class of bugs also known as reduviid. These insects, relatives of stinkbugs and wheelbugs, come into contact with people in the thatched huts of the tropical rainforests, such as those in Colombia. (All photos courtesy of Rick Tarleton and Boris Striepen)



















The growing of the heart.

A Chagas patient is harmed by the cumulative effects of small, chronic amounts of the parasite that remain in the body for decades. One of the most common symptoms of the disease is enlargement of the heart.