Putting The Bite on Malaria

By any calculation, malaria's presence and impact are epic. Its domain covers the globe like a ragged shroud, reaching across Africa, India, Southeast Asia, and South America. It exacts a monstrous toll on humanity: 300 to 500 million infections per year and 1.5 to 3 million deaths (mostly of small children). Malaria remains a constant threat to more than 40 percent of the world's population.

A tireless migrant, stealthy invader, rapid reproducer, and constantly evolving organism, the Plasmodium parasite that causes malaria passes through multiple stages in its blood-borne journey from human host through the mosquito to the next human victim. In its earliest stage in the human body, only a handful of parasites are present. Within weeks, the parasite teems by the billions. At each stage, it leaves itself open to potential vaccines or drug treatments, almost taunting researchers with a multitude of options for them to strike. "It's a moving target," acknowledges David Sullivan, MD, an assistant professor in the W. Harry Feinstone Department of Molecular Microbiology and Immunology (MMI). But he is optimistic. In the parasite's constant shape-shifting from one life cycle stage to the next, Sullivan sees a "series of Achilles heels."

The School's announcement in May of an anonymous $100 million gift to fund the Johns Hopkins Malaria Research Institute (see sidebar) has refocused attention on the ancient scourge and infused the long battle against the parasite with new energy. The nascent Institute joins the front ranks of organizations devoted to stopping malaria, including the London School of Hygiene and Tropical Medicine, the National Institutes of Health, and the U.S. Army and Navy. The momentum added by the Institute comes at a fortuitous time. In recent decades malaria has roared back as the parasite developed resistance to once-powerful drugs like chloroquine, and the Anopheles mosquito similarly acquired resistance to insecticides in some areas.

"If there's no insecticide we can use safely and effectively, and there's no drug we can use safely and effectively, then what can we do? At this point we're helpless really," says Nirbhay Kumar, PhD, an MMI professor. 

A vaccine, the magic bullet of malaria research, seems to be the best answer, but remains fiendishly elusive for researchers. Summing up the situation that researchers face, a colleague of Kumar's said, "We have a problem of multiplicity." He meant that researchers must deal with multiple species of the parasite and multiple strains of each species, multiple parasite lifecycle stages, multiple strains of the mosquito, multiple epidemiologic areas, multiple im-mune responses, and so on. The parasite's complexity has ensured its survival for millennia and enshrined it as one of the most tenacious killers of human beings. 

The drama begins in the belly of the female Anopheles. 

When she takes her blood meal from a malarious person, she also sucks in male and female forms of the parasite (called gametocytes). As the blood arrives in the mosquito's midgut, the gametocytes sense the temperature and pH change and begin transforming almost instantly, as described in Bruce-Chwatt's Essential Malariology. The male gametocyte divides into four to eight smaller male cells called gametes. "This is quite a dramatic process. In less than 10 minutes, one parasite reproduces three times," says Kumar. Each female gametocyte matures into one female gamete. Similar to fertilization of a mammalian egg by the sperm, a male gamete presses itself into the female gamete and fertilizes it, forming a zygote. The gametes have only been in the mosquito midgut for 20 to 30 minutes. 

Within 24 hours, the zygote transforms into a banana-like shape, pierces the mosquito's midgut wall, and forms a cyst on its outer surface. Inside the cyst, its nucleus divides repeatedly over a week-long period, forming a thousand or so spaghetti-like shapes called sporozoites, which eventually burst through the cyst wall and spread through the mosquito's body cavity. The sporozoites that reach the mosquito's salivary glands will survive, ready to infect a human host when the mosquito takes her next blood meal. 

The malaria parasite has been infecting people for so long that one malaria expert argues that man's primate ancestors were "recognizably malarious before they were recognizably human." Indeed, humans have written about deadly fevers similar to malaria as long as they have been writing. More than 2,500 years ago, Hippocrates described the clinical nature of malaria and its complications. Throughout recorded history, the parasite has unleashed its power to conquer armies and humble civilizations. At times malaria has treated humanity more as a bug to crush than the other way around.

Before the age of scientific discovery, human imagination struggled to explain malaria's source, whether it be from a vengeful god or the mal'aria(bad air), as 17th-century Italians concluded. Fighting the disease, according to one modern newspaper account, was done with even greater imagination: eating a live spider on a butter pat; embracing a bald Brahmin widow at dawn; or resting the patient's head on the fourth book of the Iliad. Europeans only came upon a dependable treatment in the early 17th century, when Jesuit missionaries first learned about the fever-remedying properties of cinchona bark (whose active ingredient is quinine) from South American Indians.

However, separating the clinical source of the disease from millennia of superstition and ignorance really began less than 125 years ago at a French Army outpost in Algeria. On November 6, 1880, Charles Louis Alphonse Laveran placed blood from a malaria-infected patient on a microscope slide and observed malaria parasites for the first time. Within a few years, scientists around the world witnessed the parasites as well. (However, a doubtful William Osler, then at the University of Pennsylvania, reserved his acceptance of Laveran's discovery until 1887. Later, under Osler's direction, Johns Hopkins Hospital was the first in the world to do routine malaria blood smear examinations to diagnose febrile illness.)

That the malaria parasite existed in human blood was beyond doubt. But how could it move from one human to another? It wasn't until 1897 that a British Army doctor in India named Ronald Ross would positively link the malaria parasite to the mosquito. As malaria research developed, it was discovered that there was not one species of malaria parasite but four: Plasmodium falciparum (the deadly form), P. vivax (which causes almost half of all malaria cases), P. ovale, and P. malariae. 

The same year Ross made his discovery, W. G. MacCallum (then a student at Hopkins' School of Medicine) first described sexual reproduction of the malaria parasite in the blood of a crow, greatly advancing knowledge of how the parasite reproduces. Soon after the School was founded in 1916, its faculty members and researchers began key work in malaria. Robert Hegner, who founded the medical zoology program at the School, continued MacCallum's research in avian malaria, studying host-parasite relationships and testing quinine derivatives. In the 1920s, Francis Root became a world-renowned expert on mosquito taxonomy. Public health experts mailed him specimens from all over the world for identifi-cation. Lloyd Rozeboom, ScD '34, another medical entomologist, continued mosquito research in order to assist control efforts. 

Anopheles mosquitoes — the "brown mosquitoes" as Ross called them — were eventually linked with malaria. But they, like the parasite that rides within them, would prove to be a tricky, elusive target. 

A School alumnus scored a major success in malaria control by targeting the mosquito. During the 1930s, 20,000 people in Brazil died in one of the worst malaria outbreaks in the Americas. The Brazilians turned to Fred Soper, DrPH '25, MPH '23, then a Rockefeller Foundation regional director. The blunt, intimidating Kansas native wielded absolute power in Brazil, deploying an army of 4,000 men who used diesel oil and Paris green (an arsenic-based concoction) to put down the epidemic in less than two years, as recounted in a July 2, 2001, New Yorker magazine article. 

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.

The 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.

Hungry 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.

As 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.

"[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.

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." 

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."