Stopping the Cycle

Credit: PHOTOS COURTESY OF SEATTLE BIOMEDICAL RESEARCH INSTITUTE To get his start in malaria research, Stefan Kappe had to trick his university into letting him study biology. Although he'd majored in biology in high school, his grades weren't good enough to enter the competitive biology research track at the University of Bonn in Germany. "In high school, sometimes you have other interests than studying," Kappe says with a grin. After briefly trying out chemistry, history, and law, he found a loophole in the system: "You could enter the biology program if you went into the teaching track, not into the research track," says Kappe, who is now an assistant member of the Seattle Biomedical Research Institute (SBRI). "I went into the teaching track biology program and then switched quickly over without anybody noticing. And it worked. So I kind of avoided this whole GPA thing." Once in the program, he became fascinated by parasites, thanks to a parasitology professor, who "was very good at telling these exciting and exotic and rather, sometimes, gruesome stories about parasites," says Kappe. Since those undergraduate days, Kappe has studied malaria-causing Plasmodium parasites to determine how they infect and develop inside mammalian host cells. While the majority of malaria researchers focus on the blood stages of the parasitic life cycle, Kappe has researched how parasites develop first in mosquitoes and then in the host liver before they can infect the blood. He has identified components of the Plasmodium genome that are essential for infection and development inside the host, and he's now working to develop a live attenuated malaria vaccine by deleting some of these key infectivity genes. This approach could offer more than 90% protection against malaria, Kappe says - something that vaccines based on recombinant or synthetic technologies have not been able to do. Kappe's work has "broken open a field that's been of long-standing interest," says Patrick Duffy, director of SBRI's Malaria Antigen Discovery Program, "and created the possibility of this new type of vaccine: a genetically attenuated parasite vaccine." This possibility, Duffy says, "really has got a big buzz, a lot of excitement, in the malaria field." Invading a host Kappe grew up in Bad Honnef, a small town along the Rhine about 20 miles south of Bonn. At university, he began a thesis project focusing on how Plasmodium parasites enter mouse cells. His studies involved much microscopy and descriptive analysis, but eventually, "I decided to go to a lab that actually employed molecular tools to understand host-parasite interactions," he says. Kappe contacted John Adams at the University of Notre Dame, who had just moved from the National Institutes of Health, and asked about joining his lab. After gaining acceptance into Notre Dame's graduate program (a process he remembers most for the sweltering summer day he spent taking the Graduate Record Examination at the US Army barracks in Germany), Kappe became Adams' first PhD student in 1992. Adams had identified the Duffy binding protein that is essential for Plasmodium to dock to red blood cells and then enter them. This work was done in primate parasites, and Kappe's project was to find homologous molecules in rodent malaria parasites. First, though, he had to learn the necessary molecular techniques, "which are quite challenging in the beginning," he says. "I get very impatient now when people can't get their experiments to work the first time, but I remember that my experiments didn't work for the first two years." Eventually they did work, sort of. Kappe didn't find the homologs (those were identified later by another group), but he uncovered something else: a paralog of the Duffy protein that his group named MAEBL, for membrane antigen-1-erythrocyte binding-like protein. MAEBL is a chimera between two previously known molecules: AMA-1 (apical membrane antigen-1) and EBL (erythrocyte binding-like) protein. "It turned out to be a completely new molecule that we showed was also involved in red cell invasion," Kappe says. When they went back to the human parasites, they found MAEBL molecule there also. "MAEBL was a new malaria parasite molecule conserved in different parasite species," says Kappe. "It showed the smart ways the parasite uses to make new receptors for host tissue." Since Kappe's discovery of MAEBL, it's been shown that, besides helping merozoites invade red blood cells, the molecule is also important for mosquito salivary gland infection in the sporozoite stage. The work shows how Kappe is "creative at the same time as being very structured," Adams says. "The creativity comes through having really good ideas and then the structured part is being able to execute that, which may take years to do." How parasites move When it was time to look for a postdoc position, Kappe knew he wanted to stay in malaria research. He decided to examine a different part of the parasite life cycle, the sporozoite stage, which develops in the midgut and salivary glands of mosquitoes before infecting vertebrate liver cells. So he joined the lab of Victor Nussenzweig at New York University, whose group "did a lot of pioneering work on the sporozoite stages to find the first surface molecule of this transmission stage," Kappe says. Kappe focused his work on a protein called thrombospondin-related anonymous protein (TRAP). "That was a very fascinating molecule, because it was essential for the parasite to infect salivary glands of the mosquito and subsequently to infect the hepatocytes." Kappe's mutation analyses on TRAP showed that it underlies the unusual gliding motility that malaria parasites use to move into host cells. While many pathogenic microorganisms are taken into cells by passive phagocytosis, "malaria parasites actively move into a host cell, and they use their own actin-myosin system to do that," Kappe explains. He found that TRAP sits in the parasite membrane and links extracellular receptors on liver cells to the parasite's intracellular actin-myosin system. "It was a very significant finding," Kappe says, especially because all apicomplexan parasites (the phylum of protozoa that includes Plasmodium) use this actin-myosin apparatus to move. Identifying components of this molecular motor "opened up a completely new field," Kappe says. This new field now includes attempts to design drugs that could treat all apicomplexan human and animal parasite infections by inhibiting this motility machinery, says Nussenzweig. "It's a beautiful perspective." Expressing infectiousness Kappe next decided to take his research in a slightly different direction by analyzing gene expression in malaria sporozoites. Microarrays were becoming common around this time, he says, and researchers had published gene-expression analyses of malaria parasite blood stages. "But there was really no information for sporozoites," he says, until he published an expressed sequence tag analysis in 2001. "People were very excited about it because it was the first time we really understood what is expressed in that particular parasite stage," he says. He then took those gene expression data and teamed up with Kai Matuschewski, another postdoc in Nussenzweig's lab at the time, to try to solve a mystery of the malaria parasite life cycle: What differentiates the mosquito's midgut sporozoites, which cannot infect mammals, from the infectious sporozoites found in salivary glands. "Somehow, on their way from the midgut to the salivary glands, and subsequently in salivary glands, they gain the capacity to infect the mammalian liver," Kappe says. Kappe and Matuschewski compiled a cDNA subtraction library and found 30 genes that were silent in midgut sporozoites but highly induced in salivary glands. They knocked out two of these genes, which code for transmembrane proteins that sit in the host-derived membrane that surrounds the parasite, and found that disabling either of these genes resulted in parasites that couldn't develop normally in liver cells. "We took those knockout parasites and injected them into a mouse, and no blood stages came out on the other side," Kappe says. Further studies showed that these mutant parasites can infect liver cells normally but cannot develop inside them, presumably because the membrane proteins are key for the host-parasite communication that Plasmodium needs to enable its own growth. "Stefan is a really brilliant basic biologist," Duffy says. "He and Kai were really interested in ... the proteins that the parasite uses to establish itself in liver cells. I think it was only after studying them and seeing that they had identified some essential genes that they realized, 'Well, we have an attenuated parasite vaccine, don't we?'" Kappe and Matuschewski immunized mice with the mutant parasite, and one month later they found that the mice were completely protected from infection by the wild-type malaria parasite. The grand challenge Kappe and Matuschewski continued their collaboration as they both left Nussenzweig's lab. Matuschewski moved to the University of Heidelberg in Germany, and Kappe stayed on as a lecturer and then an assistant professor at NYU before moving to SBRI in 2003. Research technician Hilda Silva-Rivera with Kappe in the lab. Credit: PHOTOS COURTESY OF SEATTLE BIOMEDICAL RESEARCH INSTITUTE Using their rodent experiments as evidence for the feasibility of producing a genetically attenuated malaria vaccine, Kappe, Matuschewski, and their colleagues successfully applied for a $13.5 million, five-year grant from the Grand Challenges of Global Health Initiative, funded by the Bill and Melinda Gates Foundation. They plan to knock out membrane protein genes in human malaria parasites that are homologous to those that can disable parasite development in rodents. Once they've ensured that these parasites have the same defects in liver-stage development, they'll team up with the Walter Reed Army Institute of Research in Washington, DC, to take the attenuated parasites into human clinical trials. "There's no reason that it shouldn't be able to be done" in human malaria parasites, says Stephen Hoffman, CEO of Sanaria, which has no relationship with Kappe or SBRI. Sanaria is working to create a vaccine from sporozoites attenuated by gamma irradiation - a mechanism that was first shown to offer protection from malaria about 50 years ago but was later abandoned because irradiation damage to DNA is so unpredictable. That meant that each new batch would need to be tested for safety and potency, but Hoffman's group at Sanaria believes that it can overcome this problem. Whether parasites are attenuated by irradiation or by Kappe's method of genetic manipulation, it's the next steps that will be the true challenge, Hoffman says. They must produce sporozoites in sterile mosquitoes, extract and purify them into an injectable vaccine, and then bottle the vaccine in a form that can be transported and stored in the places it is needed most. "People say that when you look at the real situation in Africa, it will be impossible to deploy a live vaccine," Kappe says. "I don't think it's impossible. I honestly think that, since this is the only thing that works right now, we ought to really carefully look at it, and make sure that, if it's impossible, we have looked at every way of doing it." mphillips@the-scientist.com

Stopping the Cycle

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Melissa Lee Philips

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August 2006

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