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Putting the Bite On Malaria continued...


"You sleep under a mosquito net. You close the windows. You spray the walls… Then you are praying that mosquitoes don't bite."
— Taha Taha, Associate Professor

The advent of the insecticide DDT during World War II emboldened Soper to help initiate in 1955 the Global Malaria Eradication Programme. Initial results were encouraging, but as the years passed mosquitoes became resistant to DDT, funding for the program dried up, and Rachel Carson's 1962 publication of Silent Spring awakened people to the toxic environmental effects of widespread use of chemicals such as DDT. The Anopheles mosquito surged back, bringing with it new malaria outbreaks. The program was abandoned in 1969. (The current, more modest effort by the World Health Organization Roll Back Malaria is trying to "halve the world's malaria burden" by 2010.)

Current research at the School involves more subtle techniques than spraying tons of chemicals on a given area in hopes of killing the Anopheles.

From Nirbhay Kumar's perspective, a parasite of multiplicity calls for multiple lines of attack. His research is attempting to stop malaria transmission from three different angles. His most encouraging results have come from the transmission-blocking vaccine he's been working on since 1982. Rather than immunizing an individual against the disease, Kumar's vaccine has the goal of preventing the individual from transmitting the disease to others. "If we can contain and stop transmission, then everything stops then and there," he says.

imgThe vaccine aims to stop development of sexual forms of the parasite (male and female gametocytes) in the mosquito midgut. Kumar has already demonstrated that the vaccine can produce enough antibodies in mice to stop the parasite's sexual development. He is currently testing the vaccine with rhesus monkeys. If that research is similarly successful, a decision to pursue human trials can be made in two to three years, says Kumar, who himself suffered a bout of malaria as a PhD student in India in 1976.

Kumar and his colleagues have also shown that by removing a key gene from the Plasmodium falciparum parasite they can suppress a protein it needs for sexual development.

And a third research interest of Kumar's aims to stop the sporozoite in its journey from the cyst on the mosquito's midgut to the salivary glands. "No more sporozoites in the salivary gland, no more transmission again," Kumar says.

* * *

imgHungry again, the female mosquito searches for another blood meal. Imagine she lands on you. The stylets of her proboscis saw into your skin. She injects saliva into the wound to speed the process. Dozens of Plasmodium sporozoites in her salivary glands spurt into the tiny hole in your skin. A single sporozoite is enough to initiate a full-blown infection.

If you live in an area where malaria is endemic, a bite by an infected Anopheles is nothing new. You are probably permanently infected. For those who survive childhood, the constant re-infection leads to a partial immunity. If you're otherwise healthy, your immune system can hold off the parasite. But if you are weakened by malnutrition or other infectious disease, malaria can suddenly overwhelm you with debilitating illness or death. If the steady re-infection stops for an extended time, for example if you move to North America, you lose whatever immunity you had.

On the other hand, if you're not from a malarious area, you are considered immunologically naïve. But it takes just one bite of an infected
Anopheles to rid you of your naïveté.

It starts with the sporozoites carried in the mosquito's saliva into the bite on your skin. Once in the bloodstream, a sporozoite can reach the liver within minutes. There, it invades a liver cell and stealthily starts reproducing. Your body, with its powerful immune system, stands by dumbly. Symptomless, you walk about completely unaware of the firestorm about to erupt in your body.

imgAs one of the 2 billion people of the world living in an area threatened by malaria, Taha Taha, MD, PhD '92, MPH '86, associate professor in Epidemiology, took regular precautions to stave off malaria infection when he was working in Malawi in the mid-1990s. "You do the little ritual things," says Taha, who grew up in Sudan and battled malaria there as well. "You sleep under a mosquito net. You close the windows, check to make sure the screens aren't damaged. You spray the walls [regularly with insecticides]. Then you are praying that mosquitoes don't bite."

At some level, the likelihood of getting malaria from an Anopheles mosquito becomes a dance of numbers. There are more than 3,000 species of mosquitoes. Of the 420 species of Anopheles, about 70 are capable of transmitting malaria. And of those 70, only about 30 to 40 are considered "good transmitters," according to Douglas Norris, PhD, an assistant professor in MMI. "For any one of those species, only 5 to 10 percent of the population are capable of transmitting malaria," Norris says. These odds seem favorable to humans until one factors in the countless numbers of Anopheles mosquitoes that range over the globe.

While they may be found all over the world, Anopheles mosquitoes naturally cause the greatest concern in malarious regions. Suzanne Maman, PhD, an assistant scientist in International Health, has worked in Kenya and Tanzania, where malaria is a daily fact of life. "When somebody says, 'I have malaria,' it's not like everybody is shocked," Maman says. "They just live with it. They describe malaria like a flu.

img"[But] having said that, it is the number one killer for children under five and pregnant women," who may succumb to severe anemia, cerebral malaria, or renal dysfunction, she says.

Clive Shiff, PhD, an associate professor in MMI and a veteran of more than 30 years of malaria control in Africa, says he has found that some Tanzanians suffered 300 to 1,000 infectious bites per person per year — one to three infections per day. Among schoolchildren ages 12 and 13, some 60 percent were infected with malaria. "This is debilitating. These kids couldn't concentrate on their work," he says. "But people survive. You see people doing work, and women having children."

* * *

Inside your body, the killer is multiplying.

Within a week of its invasion, a single Plasmodium parasite can multiply into tens of thousands of parasites. The frantic reproductive pace is interrupted when the parasites (now called merozoites) burst out of the liver cell. Each merozoite invades the nearest red blood cell, where it feeds on the hemoglobin. One merozoite will yield a dozen or more merozoites every 48 to 72 hours (depending on the type of malaria). The cycle of reproduction, bursting free, and invading new red blood cells continues over and over until literally billions swarm in the blood.

LAUNCHING THE JOHNS HOPKINS MALARIA RESEARCH INSTITUTE

Since the euphoric days in May when the School announced a $100 million gift to found the Johns Hopkins Malaria Research Institute, celebration has given way to rounds of organizational meetings, search committee meetings, and faculty recruitment efforts.
      "We're getting things off the ground," says Diane Griffin, MD, PhD, chair of the W. Harry Feinstone Department of Molecular Micro-biology and Immunology (MMI). With new projects getting under way in the next few months, Griffin and others are designing an infrastructure of people, facilities, and equipment for the Institute.
      Over the next five years, 10 to 12 new faculty members will be hired. They will have appointments in MMI, Biochemistry and Molecular Biology, Biostatistics, and International Health. Piles of CVs from experts in entomology, structural biology, bioinformatics, molecular biology, parasitology, immunology, and other areas have already arrived.
      Faculty interviews will begin this fall. A minimum of 50 faculty, students, postdocs, and support team members will eventually staff the Institute, according to Griffin.
      Space will be cramped until 2004 when the Teaching and Research buildings (TR) 5 and 6 will be completed. Until then, some lab space will open up early next year as some researchers move to TR3, the latest addition to the School's main building. Despite the space restrictions, the Institute has already begun to acquire the necessary state-of-the-art equipment it needs. The Institute recently purchased, in partnership with the departments of Environmental Health Sciences and Biochemistry, a $200,000 microarray facility used in analyzing changes in gene expression.
      Current researchers at the School are already fanning out across the globe to initiate collaborative research. Douglas Norris, PhD, assistant professor in MMI, and Clive Shiff, PhD, associate professor in MMI, spent part of their summer in Africa meeting with colleagues there and working out the logistics of potential collaborations. Additionally, malaria researchers from the U.S. Navy and Army and the World Health Organization's Roll Back Malaria program visited this summer.
      Griffin, MMI professor Nirbhay Kumar, and others are already preparing for the Institute's first conference: a January 2002 meeting on malaria in the post-genomic era.
      Furthering the Institute's "basic science" initiative, researchers will be working to better understand the parasite, the disease-host response, what makes the mosquito such a good transmitter of malaria, and other issues. The ultimate goal is development of a vaccine and new drugs.
      David Sullivan, MD, assistant professor in MMI, says he believes the basic science acquired at the Institute will go beyond the specific results of new drugs and vaccine candidates. "It's like the space program. A lot of technology used to monitor astronauts in space is now used to take care of patients here," he says. "A lot of research knowledge gained in tackling malaria can be used in other research."

- BWS

The merozoite invades the red blood cell primarily to dine on hemoglobin to fuel its astounding reproductive capacity. Even in mild infections, the merozoite consumption of hemoglobin can be debilitating, leaving the infected person feeling exhausted, as if he or she has just run a marathon or performed a couple days of hard labor, says MMI's Sullivan.

Sullivan researches the parasite's vulnerable iron metabolism in the red blood cell. There, the parasite encounters a large amount of what is known as "heme iron" in the hemoglobin. "Red blood cells have 20,000 times the iron concentration of other mammalian cells," says Sullivan. "The parasite has to detoxify the iron through a unique crystallization process."

Ever adaptable, the parasite makes crystal polymers of the heme iron, metabolizes the unwanted iron, and sequesters it. Chloroquine, the powerful antimalarial drug that successfully treated the disease for decades, inhibits the parasite's crystal-making detoxification efforts. The parasite essentially overdoses on the excess iron (which makes toxic oxygen radicals). However, the parasite has acquired resistance in recent years to chloroquine in Africa, Southeast Asia, and South America. Sullivan's hope is that a better understanding of the parasite's iron metabolism will lead to more effective drugs.

Gary Posner, PhD, Scowe Professor of Chemistry at Homewood with a joint appointment at the School, is also targeting the parasite's iron metabolism. Using "molecular architecture," Posner, Theresa Shapiro, MD, PhD, professor of Clinical Pharmacology in the School of Medicine, and their students have created a synthetic antimalarial. It is based on the artemisia plant, long used by the Chinese as an herbal remedy for malaria. The drug interacts with iron to generate oxygen and carbon radicals that destroy the parasite. In late August, Posner announced that preclinical testing in mice and rats had shown the carboxyphenyl trioxane compound to be safe and effective. Human testing of the new antimalarial drug is still two to five years away.

Other researchers at the National Institutes of Health and in Britain are trying, like Posner and Sullivan, to stop the parasite's merozoite stage in the blood. Meanwhile, the U.S. Navy and the Army have projects under way to develop vaccines that target the sporozoites in humans just after the mosquito bites.

Ultimately, even if scientists develop a vaccine that successfully knocks out the parasite during one of its life stages, that may not be enough. The vaccine would not only have to be 100 percent effective, but would also have to stand up to Plasmodium's expert ability to develop resistance through mutations. Explains Kumar: "Given the complexity and smartness of the parasite, I think that is expecting too much. In the end, we may have to make a cocktail vaccine, a vaccine that targets the parasite at different stages in the life cycle."

* * *

Too late, your body recognizes it has been invaded.

Depending on the species of Plasmodium parasite, you may feel nothing for 9 to 30 days after the mosquito bite. Symptoms usually begin with malaise and fatigue. You may become dizzy and nauseated. You might think it is a cold or flu. Soon, however, malaria is unmistakable. The horrors of the malaria rigor (pronounced RYE-gor) begin. You start to shiver, mildly at first and then violently. Your entire body shakes and your teeth chatter uncontrollably. You grab for more blankets, desperate for warmth. Finally, the warmth comes, but you keep getting warmer. Hot. Your temperature increases. You kick off any covers. Your heart races. For two to six hours, your skin burns. Then, you sweat profusely, drenching the sheets. Your fever peaks at 106 degrees, before declining slowly. The rigor lasts 8 to 12 hours. If your case of malaria follows the classical model, you will suffer this every 48 or 72 hours, a cycle that continues until drug therapy or your body's immune response subdues the parasite. If, however, you are an infant or pregnant woman infected with Plasmodium falciparum, you risk death.

* * *

Some researchers are bypassing the parasite altogether and focusing on the other host in the malaria cycle: the Anopheles mosquito.

Norris, a vector biologist in MMI, studies the basic science of mosquito populations in order to control them more effectively. He tackles difficult questions: What genetically defines a mosquito population? How big is a given population? Is its range the size of a village or 60 kilometers square?

"We're applying really high-end molecular tools to field populations," Norris says. "We are at the point where we can do essentially paternity testing on mosquitoes to find out how much transfer of genetic material is going on."


"We are at the point where we can do essentially paternity testing on mosquitoes to find out how much transfer of genetic material is going on."
— Douglas Norris, Assistant Professor

Conventional thinking had been that female mosquitoes mate once in their lifetimes, store the male's sperm, and fertilize eggs as they are deposited. Recent studies have shown, however, that female mosquitoes can mate more than once, and on rare occasions with a male outside of her population. "By doing the paternity test, we can identify more than one mate's DNA," Norris says. "We can sample the mom pretty easily — we have the whole mosquito — and we can also sample the dad - from the [stored] sperm."

By studying mosquito populations and how genetic material passes through them, researchers can better predict the likelihood of a mosquito population acquiring insecticide resistance.

* * *

As the Plasmodium parasite grinds along in its seemingly endless life cycle in mosquito and man, researchers are divining knowledge from the parasite's multiplicities, seeking out its weak points and learning where the odds favor a successful attack. The scientists rely on multiplicities of their own, drawing on genetic, social, biological, statistical, chemotherapeutic, and other knowledge that will eventually enable them to extract the parasite from its eternal reservoir, mankind.

"Basically, the parasite has developed a biological mechanism that has allowed it to survive forever, as long as human beings have existed," says Kumar. "We are talking about breaking a symbiotic relationship between the parasite and host. It's not going to be easy. But the fight is on."

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