DARKNESS has fallen in the swampy Wosera river plain of Papua New Guinea. George Maruke, a lean, dark-skinned villager in his thirties, sits alone on a log staring intently at his bare legs. He is waiting for hungry female mosquitoes to come and drink his blood. After a while, one alights on his shin and begins feeding. Unflinching, Maruke slips a plastic tube over her and deposits her in a vial so she can be tested for malaria parasites. Then he settles down and waits for the next bite. He will do this until dawn.
Maruke鈥檚 work is part of an ongoing effort to meticulously document the complex relationships between the malaria parasite, mosquitoes and people. What proportion of mosquitoes carry the disease, for example, and how many infected bites do people suffer each year? How does this affect their level of immunity to the parasite? Researchers desperately need answers if they are to realise the dream of a vaccine that will outwit malaria. For many are now cautiously hopeful, after decades of disappointment, that a useful vaccine is at last within our grasp.
New research is beginning to reveal why our efforts to create an effective vaccine have failed so far. The malaria parasite is a much more formidable and devious opponent than we ever imagined. As well as playing a devilish game of hide-and-seek inside the human body, it seems that the parasite can turn our immune system against itself and even force immune cells that should be fighting it to commit suicide instead, making it hard for people to develop long-lasting and effective resistance to the disease.
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As well as providing explanations, these discoveries are opening up new lines of attack. 快猫短视频s are developing a battery of counter-technologies, from vaccines made of DNA to proteins that can prime our blood with weapons to attack the parasite while it is inside the mosquito鈥檚 gut. Some vaccines in development could mimic the partial immunity that develops naturally in people who are exposed to malaria for decades, while others will employ tricks to produce types of immunity that never develop naturally (see Table). In any case, a vaccine is urgently needed: the parasite is becoming increasingly resistant to drugs, and threatens to blight the lives of over 40 per cent of the world鈥檚 population (see Map).
I have come to Papua New Guinea because it is here, in one of the worst affected areas in the world, that researchers eventually hope to trial some of these new approaches. Although malaria is endemic to all of lowland Papua New Guinea, its miasma hangs especially heavily here in Wosera. In some wet months, villagers experience up to 300 mosquito bites a night 鈥� a staggering 20,000 per year, 200 of which will carry the malaria parasite. At any given time, at least 60 per cent of the locals, young and old alike, harbour the parasites in their blood
What鈥檚 more, Wosera is one of the few places on Earth to harbour all four species of the Plasmodium parasites that cause malaria in humans. 鈥淭his place is about as complicated as it gets,鈥� admits John Reeder, Director of the Institute of Medical Research in Goroka, Papua New Guinea, which runs the public health surveys in Wosera. 鈥淏ut it鈥檚 also realistic. If you can get a vaccine to work here, it is going to work in most other places.鈥� For now, however, all eyes are on the latest game plans for tackling this foe.
The main drive is to understand every nuance of the malaria parasite, and that is a tough problem. The parasite is a single-celled creature with a staggeringly complex life cycle. When an infected mosquito bites her victim, she transfers the parasite in the form of long slender cells called sporozoites. These surf through the blood to the liver in a mere 30 minutes, where they hide from the immune radar by slipping inside liver cells. There they lie low, multiplying rapidly and developing.
After about a week, tens of thousands of parasites burst into the bloodstream and infect red blood cells. There they multiply again until the cells rupture, releasing more parasites that in turn infect more cells, making the victim anaemic. Worse, the parasite produces proteins and a toxin that make infected red blood cells stick to the insides of blood vessels. This leads to blockages that can cause kidney and heart failure, coma and death.
Of course, this carnage does not go unnoticed by the immune system. But the parasite is armed with several ingenious countermeasures against our defences. For starters, it spends most of its time holed up inside cells. This shields it from direct attack from one of our major weapons 鈥� antibodies, the mainstay of traditional vaccines such as those for polio or measles. Unfortunately, antibodies can鈥檛 鈥渟ee鈥� or access the insides of cells. Although infected red blood cells do have proteins made by the parasite on their surfaces, which antibodies can attack, the parasite constantly changes the shapes of these proteins, always leaving the immune system one step behind. For example, one called PfEMP1 has about 50 different versions.
So spurring the immune system to chug out antibodies is useful, but it is not enough to stop malaria. A malaria vaccine must also stimulate the other arm of the immune system, the T-cells that attack the cells where the parasite hides, causing them to implode. And priming that mechanism means far more than just inoculating people with a bunch of poorly understood malaria proteins and hoping for the best. New research has revealed that getting the right sort of immune response is absolutely crucial.
Several proteins used by the parasite, including MSP1, MSP2, MSP4, and MSP5, and AMA1, are being investigated as potential vaccine targets. The parasite uses these proteins as 鈥渃laws鈥� to pry its way into red blood cells, and antibodies against some of them can halt the cell invasion. Yet there is something fishy about some of 鈥� perhaps many of 鈥� the parasite鈥檚 proteins. It seems that they can act as decoys, drawing the immune fire away from the parasite鈥檚 vulnerable points. People regularly exposed to malaria have twice as many antibodies in their blood as those who aren鈥檛, yet most of those antibodies are completely useless.
Worse, the parasite somehow makes the immune system turn against itself by tricking it into making rogue antibodies that actually block some of the useful antibodies. So a vaccine containing the wrong proteins, or parts of proteins, will be self-defeating.
In 1998, Michael Good, Director of the Queensland Institute of Medical Research in Brisbane, Australia, discovered that the parasite uses an even nastier trick: it actually assassinates T-cells that recognise it (Proceedings of the National Academy of Sciences, vol 95, p 1715). These cells, called CD4 T-cells, are essential not only for killing the parasite as it hides inside red blood cells, but also for helping the immune system remember which invaders it has encountered before. This immune 鈥渕emory鈥� allows the body to unleash a rapid response when that invader attacks again.
No one knows how the parasite kills these cells. One idea is that it hijacks a natural mechanism the body uses to cull superfluous CD4 cells. Another, more sinister possibility is that when the parasite infects a pregnant woman, even though it can鈥檛 cross the protective placenta itself, it sends protein messages to the fetus, tricking the developing immune system into recognising it as part of the baby鈥檚 own tissues rather than as a foe. If parasites then enter the baby once it is born, any CD4 cell that attacks them is destroyed by the body as a rogue agent, leaving the baby wide open to attack.
Whatever the mechanism, it is bad news. Without the benefit of malaria-targeting CD4 cells, you are in trouble. 鈥淥ne of the impediments to developing immunity is that every time you鈥檙e exposed to the infection, your responding T-cells simply die off,鈥� explains Good. 鈥淪o every time you are re-exposed, your immune system has to virtually start again.鈥�
But Good has a plan to turn the tables on the parasite. He has previously found that if you inoculate people with ultra-low doses of infected red blood cells 鈥� no more than 3 dozen or so 鈥� and then kill the parasites a few days later with drugs, you can stop this CD4 massacre altogether. What鈥檚 more, this also induces an immune response that helps protect people from infection (快猫短视频, 31 August 2002, p 14).
Good has now isolated the protein responsible for inducing this immune response. It turns out to be the same one he suspects of sweet-talking the fetal immune system. He now plans to test whether ultra-low doses of this protein will also protect CD4 cells. But it remains to be seen whether this form of immunity, which doesn鈥檛 occur naturally in infected people, will be the whole answer.
The challenges presented by malaria have prompted many researchers to develop a new, more pragmatic approach to vaccination. The search for a vaccine has already seen several high-profile disappointments (快猫短视频, 21 September 1996, p 8), prompting some researchers to speculate that we may never find a vaccine that provides complete protection against infection. 鈥淎 malaria vaccine has got to do something that鈥檚 pretty amazing,鈥� admits Louis Miller, who is involved in malaria vaccine development at the National Institutes of Health in Bethesda, Maryland.
People like Miller are not necessarily expecting to create a vaccine that gives near-perfect protection, as the polio vaccine does. Rather, they envisage something that would speed up the development of the partial immunity seen in adults after long-term exposure 鈥� enough to blunt the most severe attacks and hopefully see infants and children through their vulnerable years. Ross Coppel, a malariologist and vaccine researcher at Monash University in Melbourne, agrees. 鈥淚f you can reduce blood parasite levels by 20 per cent,鈥� he says, 鈥渢hat will translate into reductions in mortality and improved health. Even an imperfect vaccine can do good things.鈥�
Furthest along the development conveyer belt is a vaccine called RTS,S created by GlaxoSmithKline Biologicals in Rixensart, Belgium, and the Walter Reed Army Institute of Research in Washington DC. It is intended to target the parasite while it is still in the liver. RTS,S consists of a hollow hepatitis B virus shell with about 100 copies of a parasite protein fused to its surface. A recent trial produced mixed results. RTS,S gave 71 per cent of recipients protection from infection for up to nine weeks. Although it failed to completely prevent infection after this time, the vaccine still delayed the onset of symptoms. Future trials will show whether this will help prevent severe disease.
Attack on all fronts
One way of defeating the parasite鈥檚 habit of changing its spots would be to design a vaccine containing many different protein targets that appear at different stages of the parasite鈥檚 life cycle. Our knowledge of the malaria genome could make it easier to design a many-protein vaccine. But there are problems. 鈥淲hat will you do if you find 30 good proteins?鈥� asks Stephen Hoffman, who used to direct the US naval malaria vaccine programme. 鈥淵ou have no capacity to deliver them.鈥� Vaccines containing one or more synthetic malaria proteins are expensive to produce, and become uneconomical once you include more than four or five proteins.
DNA vaccines may be the answer. These consist of rings of DNA containing parasite genes, which are injected into muscle or fired into the skin on microscopic gold beads. Cells absorb the DNA and manufacture malarial proteins that prime the immune system. Even better, DNA vaccines are dirt cheap, and you can deliver multiple proteins at very little extra cost. And there is no need to refrigerate them 鈥� a boon to isolated bush communities.
Unfortunately, researchers have trouble getting DNA vaccines to produce a robust immune response. Some researchers are trying to improve this with 鈥減rime-boost鈥� vaccines, where an initial dose of DNA rings is followed by a booster dose of an engineered virus, such as cowpox, containing the same malaria genes. The viral proteins in the booster intensify the immune response. This has produced good results in monkeys, completely protecting 20 per cent against infection, and allowing 60 per cent to beat the infection after a few days of suffering only mild symptoms.
Yet despite all these rather sophisticated techniques, there is a much simpler approach that has long been something of an ignored elephant in the room. In 1973 scientists found that exposing people to weakened malaria parasites produced excellent immune responses. It was again brilliantly shown in 2001. Fourteen people were immunised with radiation-weakened sporozoites and then repeatedly exposed to the parasite. All but one person gained protection from infection 鈥� even a mild one 鈥� for at least 42 weeks. Such vaccines are great for delivering lots of different immune-stimulating proteins at once.
However, malariologists rejected this approach because sporozoites only grow in mosquito salivary glands, making it almost impossible to get enough of them together to make a vaccine. But frustration with other vaccine strategies is leading some to reconsider. 鈥淲e keep saying it鈥檚 not practical to make this vaccine,鈥� says Hoffman, 鈥渂ut we are staring at a situation where we have nothing that is in any way, shape or form comparable to this degree of protection. Shouldn鈥檛 we re-explore the practicality of this?鈥� Hoffman has founded a company called Sanaria, based in Bethesda, Maryland, to do just that, but is keeping details of the technology under wraps for now.
Meanwhile, there are other ways of seeing people through the dangerous period before they develop partial immunity. Louis Schofield, an immunoparasitologist at the Walter and Eliza Hall Institute of Medical Research in Melbourne, is developing a vaccine against the parasite鈥檚 toxin. A recent study found that anti-toxin antibodies protect mice against coma and death even when their parasite levels skyrocket.
But the wiliest ploy of all may be to use antibodies to attack the parasite while it is inside the mosquito. Joseph Vinetz, a tropical disease specialist at the University of Texas Medical Branch at Galveston, is studying an enzyme that the parasite uses to find its way from the mosquito鈥檚 stomach into its salivary glands. Prime the blood with antibodies against this protein and the parasite can鈥檛 use it to escape the mosquito鈥檚 stomach. Even if such a vaccine were only 70 per cent effective, it could still cause malaria rates to spiral downwards over several years, says Vinetz.
Back in Wosera, I have come to the Kunjigini village clinic, a no-frills, cinder-block shell with two rooms, which mothers with feverish children walk four hours to reach. The line of waiting patients stretches out of the door. It is a depressing scene, and it is repeated over a third of the world, where 400 million people are infected with malaria and 2 million die every year, most of them children under five. And although there are some brilliant new ideas that could lead to a vaccine breakthrough, there is only one sure thing 鈥� it can鈥檛 come too soon.
