In the spring of 1987, ripley ballou, then a major in the U.S. Army and a specialist in infectious diseases, entertained the delusion that he had beaten malaria. Ballou was part of a research team from the National Institutes of Health and the Walter Reed Army Institute of Research (known as wrair) that had developed a vaccine against Plasmodium falciparum, the parasite that causes the most virulent strain of malaria. The perception was,’’ says Ballou, this is going to be a slam dunk.’’ His teammate Steve Hoffman, a commander in the Navy, shared the delusion, as did Ruth and Victor Nussenzweig, the elder statesmen of the field, who worked out of New York University and had developed a competitive vaccine.
Of the four, the Nussenzweigs were the lucky ones. Although their discoveries had led directly to the development of the two vaccines, they were both pushing sixty at the time, which put them over the age limit for testing the nyu vaccine on themselves. Ballou and Hoffman, however, were then in their thirties and looked like the stars of a television medical drama or perhaps a sequel to M.A.S.H. Fifteen volunteers, Hoffman and Ballou among them, were injected with the vaccine. Hoffman, Ballou, and four other volunteers were then bitten repeatedly by malaria-infected mosquitoes. (Two more volunteers who had not received the vaccine also allowed malaria-infected mosquitoes to bite them, to give the researchers a basis for comparison.) In the parlance of vaccinology, the volunteers were challenged’’ with malaria. They were to be tested regularly for Plasmodium, and the minute the parasite showed up in anyone’s blood, of course, they would be treated with antimalarial drugs.
A couple of weeks passed, which is the time it takes Plasmodium to be fruitful and multiply in the human body. Three of the six vaccinated volunteers fell ill. Not so Ballou and Hoffman, who believed they were protected. It was a heady feeling. Hoffman flew off to San Diego, where he was scheduled to speak at a conference, while Ballou ran six miles, after which he attended a party. One beer in, Ballou was sweating, feverish, and, as he describes it, in trouble.’’
I had chills and then a high fever—a 104-degree temperature—and a bad headache. I’ve never been so sick, he says. The next morning I went into the hospital and, sure enough, the parasites were in my blood. They gave me antimalaria medication, and I was still sick for another day and a half. I wasn’t really well for six weeks after that. In San Diego, Hoffman was going through a public version of the same suffering. I was in the middle of my presentation, Hoffman says, and I developed the malaria rigor, the shaking chills. That was it.
Hoffman and Ballou had painfully relearned the primary lesson in the battle against Plasmodium. As Ballou puts it, Malaria is a hell of a disease. It is very difficult to protect against it.
By World Health Organization tally, malaria kills some 2.7 million people each year, most of them children under five years of age. It infects 300 to 500 million others, more than 90 percent of them in Africa, the rest mostly in south and southeast Asia and Central and South America. The mosquito-borne parasite may have killed one out of every two human beings who ever lived on the planet. To make matters worse, after years in modest abeyance, the disease is on the rise. The mosquitoes that carry it have become resistant to pesticides, the parasite itself to antimalarial drugs. The World Bank is talking about launching a 30-year effort to stem this rising parasitic tide, while the nih and who are collaborating to combat the problem as well. The news is not all bleak, however. Vaccinologists, including Ballou and Hoffman, are once again permitting themselves a glimmer of optimism.
In retrospect, those painful 1987 trials may have been a turning point in the battle against malaria. The lessons learned and the techniques employed have led to a number of promising vaccines. In January 1997 a Ballou-led collaboration between wrair researchers and SmithKline Beecham Biologicals in Belgium published the results of a study in which a vaccine protected six of seven volunteers. wrair and the Nussenzweigs both began testing new vaccines last fall, while Hoffman and his colleagues at the Naval Medical Research Institute have begun clinical trials of a radical new type of technology known as a dna vaccine. Researchers at the nih even have a benevolent vaccine in the works that will not actually prevent an individual from coming down with malaria but will inhibit his transmitting the parasite back to mosquitoes. If it works, it should prevent small malaria outbreaks from ever reaching epidemic proportions.
Despite the hopeful news, however, it’s worth mentioning a couple of important qualifications right away. First, even if these vaccines work, none is likely to reach the population at large for at least a decade. And then, researchers have learned to lower their standards for what constitutes victory over malaria. We’re not talking about polio-type vaccines, where you get immunized and you never get the disease, explains Bob Gwadz, an entomologist who helps run the malaria research program at the nih. We’re probably talking about vaccines that eliminate mortality and significantly reduce the level of sickness associated with the disease.
The perniciousness of the parasite lies at the heart of the vaccine problem. Plasmodium falciparum is considerably more complex than a virus, or even a bacterium. A virus is nothing more than a small strand of nucleic acid, either rna or dna, wrapped in a protein coat. To replicate itself, it has to infiltrate your cells and co-opt their machinery. A bacterium, such as salmonella, is a simple single-celled organism. The malaria parasite is also single-celled, but unlike a bacterium it takes many forms over the course of its lifetime, ingeniously escaping the body’s defenses. After perhaps a million years of evolutionary struggle with the human immune system, it has evolved ways to prosper, making it seem almost consciously malicious. These parasites have an extraordinary capacity to evade our immune system, says Gwadz, and they have been doing it for a long time. Every time the human immune system has found a way to get around the parasite, the parasite has found a way around the immune system.
The life cycle of Plasmodium circles from mosquito to man and back to mosquito (or, depending on your point of view, from man to mosquito and back to man). It begins, for sake of discussion, when a mosquito dribbles the malaria parasite into your skin with its saliva while taking a blood meal. At this stage the parasites are in a form known as sporozoites. They enter your bloodstream and stay there for all of half an hour before burrowing into your liver cells, where they remain sequestered for a week or so, multiplying furiously. By the time they reemerge, destroying the cells as they burst out, the parasites are no longer sporozoites but a rounder, more compact form known as merozoites. There are now tens of thousands of the latter for every one of the former.
The merozoites spend only seconds floating in your bloodstream before they invade your red blood cells and colonize them. Comfortably ensconced, the merozoites multiply for 48 hours, increasing their numbers twentyfold before bursting out to invade virgin red blood cells. Sometimes, though, they take on a new form within the red blood cells, becoming what are known as gametocytes. This is the sexual stage, the only time the parasites exist as males and females. After a couple of cycles in and out of the red blood cells, millions if not billions of parasites are likely to have made your blood their home. At this point, any uninfected mosquito that bites you will suck up red blood cells. The gametocytes emerge from the infected red blood cells only after a drop in temperature convinces them they’re in the mosquito’s relatively chilly gut. Now called gamete/zygotes, they breed and produce oocysts—think of a cross between a cyst and an egg sack. From the oocysts emerge sporozoites, which is the stage we started with. The sporozoites take two weeks to develop in the mosquito, spread through its gut into its blood, and finally infect its salivary glands. Now they can be dribbled back into a human and the show opens all over again.
Plasmodium has adapted itself to its human host with ghastly efficiency. You can think of your immune system as having two tiers of defense: antibodies, which identify alien invaders in your bloodstream and flag them for other immune cells to attack; and cellular defenses, such as killer T cells, which hunt down and kill your own cells once an invader has made its way into them. Malaria eludes both.
For starters, once injected by the mosquito, the parasites are in your blood for only a half hour before hiding in liver cells. Thus they are susceptible to your antibody defense system for only that short time—which is often not long enough for the antibodies to kill all the parasites. Once they colonize your liver cells, your killer T cells will go to work, but this takes time. The immune system develops this response probably over a 10- to 12-day period to kill its infected cells, says nih research physician David Kaslow. So what does the parasite do? It jumps out of the liver cell just about the time this would kick in, and jumps into a red blood cell. Red blood cells are not susceptible to this particular T cell response. If you were a parasite and you wanted to pick the one cell type in the body that is most abundant and has no response, you’d go right to red blood cells. But the parasites are not yet done protecting themselves from your immune defenses. As they develop inside your red blood cells, they extrude minute knobs onto the surface of the cells. The knobs cause these infected red blood cells to stick to the lining of your capillaries and the very small vessels in your brain and other organs. The knobs serve a protective purpose: to keep the infected red blood cells out of your spleen, where they would be recognized as bad blood and cleansed from the system. Stuck to the vessel walls, the infected cells never get swept through the spleen. Clever? says Kaslow. Well, the parasite’s not, but evolution is clever. The clinical symptoms of the disease appear only when the parasite is in this red-blood-cell stage. With the infected red blood cells clogging up your small blood vessels, oxygen flow to your tissues decreases. And when the blood cells rupture, releasing merozoites, they further cripple your body’s ability to transport oxygen. Plasmodium falciparum can colonize up to 70 percent of your blood cells. When those burst, you lose 70 percent of your ability to transport oxygen. The immediate result is anemia, followed by cerebral malaria as your brain begins to suffer the effects of severe oxygen deprivation.
Malaria infections are not permanent. Falciparum, which imparts the most serious of the malarial strains, will kill up to half the people it infects if left untreated. Those who don’t die and don’t get antimalarial treatment will eventually rid themselves of the disease in three to five years. The catch is that if they live in a malaria-infected area, they are likely to get reinfected, and 2 billion individuals—one-third of the world’s population—live in malarial areas. In Africa, says Gwadz, one-third of the deaths are from severe malaria, one-third are from chronic anemia. In some people, the parasite goes so high you get cerebral malaria and die. In others, it goes up, it goes down, it percolates along, but every 48 hours it’s destroying red cells. So you have chronic anemia. Where does chronic anemia leave you? It basically reduces your resistance to everything else, and you may die of measles or the common cold. A third of the deaths are caused by reduced oxygen and nutrient flow to the developing fetus—low-birth-weight babies, stillbirths, and miscarriages.
Notwithstanding the havoc wreaked by malaria parasites throughout the long history of mankind, the human immune system has evolved to defend us from disease-causing agents, and for the most part it does a hell of a job. It has to work quickly enough to stop irreversible assaults, however, and sometimes by the time it mounts its counterattack, the virus or bacteria or parasite has ensconced itself permanently or fatally in your body.
In preparation for the real thing, vaccines work by stimulating the immune system to produce antibodies to an apparent threat. When the real thing comes along, the immune response is vastly accelerated; the defenses mobilize quickly enough to take out the infectious agent before it settles in for a debilitating or fatal long haul.
The most effective vaccines rely on attenuated or, simply, dead versions of the real agent to stimulate the immune system. These will induce an immune response while lacking whatever it takes to cause actual disease. The classic example was Edward Jenner’s use, in 1796, of cowpox—a disease related to smallpox that causes serious illness in cattle but not in humans—to vaccinate against its deadly human cousin. Should you ever get a yellow-fever vaccine before traveling to the tropics, you will be getting a live virus rendered harmless, although still capable of inducing an immune response against the real thing. One dose of yellow-fever vaccine and you’re protected for at least ten years.
Malaria, however, brings a host of unique problems to immunologists trying to concoct a vaccine. Foremost is that the viruses or bacteria we can beat do not metamorphose in our bodies; they don’t exhibit the bewildering variety of stages that a malaria parasite does. Malaria has its four stages in a human. A vaccine aimed at the sporozoites won’t necessarily go after the liver stages, or the blood stages, or the gametocytes, nor will a vaccine aimed at the later stages necessarily stop a sporozoite. And if your sporozoite vaccine prompts an antibody response that kills off 99 percent of the sporozoites the mosquito has dribbled into your blood, that might still leave one alive to spawn its 30,000 merozoites in your liver. If you make a vaccine that induces the killer T cells to attack the parasite in the liver cells, but they miss one or two, you will still have thousands of merozoites running rampant in the blood. When you’re creating a malaria vaccine, 99.9 percent efficiency doesn’t seem to keep a person disease-free. It’s a complex multistage organism, Hoffman says simply, and we don’t have vaccines against complex multistage organisms. We’re trying to do something that has never been done before.
One possible solution is to create a vaccine of attenuated parasites, but that comes with its own difficulties. For starters, researchers can’t grow the parasite in the laboratory. Or to be precise, they can grow the red-blood-cell parasites, but, says Ballou, your parasite is inside a red cell. How do you mass-produce red cells? It can’t be done. Meanwhile the sporozoites grow only in mosquitoes, and the liver stages of the parasite will grow only in human liver cells, which just a few labs in the world can grow, and those only in small numbers.
Not that a successful malaria vaccine can’t be made. It’s been known for 30 years now that if you take a mosquito infected with the sporozoites and zap it (and thus them) with radiation, the parasites inside the mosquito will become suitably attenuated, just like an attenuated yellow-fever virus. You can then let the mosquito dribble them into your blood, adding them to your body’s natural flora and fauna. These irradiated parasites will travel to your liver but they will not mature, produce merozoites, and colonize your red blood cells. The sporozoites simply remain in your liver and do nothing but generate an impressive response from your immune system. Now if an errant mosquito delivers a healthy malaria parasite into your blood, your body will kill it off, at least for the next few months.
In the 1940s, researchers demonstrated that irradiated sporozoites would protect chickens, which are susceptible to a form of bird malaria. In 1968, Ruth Nussenzweig and Jerome Vanderberg at nyu showed that this method of vaccination worked for mice and monkeys, and they and others showed that it worked in humans a few years later. But while some researchers in the field have taken to immunizing themselves with irradiated sporozoites—Hoffman, for instance—the bottom line is it’s simply not practical for the billions exposed to the disease in the wild. It can take a thousand bites from an irradiated mosquito to induce immunity, and, as Hoffman says, you can’t go and expose billions of people to thousands of infected mosquitoes each. It’s not cost-effective.
It is the irradiated sporozoites, however, that have held the key to most of the progress that’s been made since. As Ruth Nussenzweig describes it, when researchers put sporozoites into blood serum from someone who had been bitten by irradiated mosquitoes—which meant he or she now had antibodies against malaria—and examined the sporozoites with an electron microscope, they could see a thick coat covering each sporozoite, and a tail trailing behind that was maybe five times as long as the parasite itself. Victor Nussenzweig describes it as looking like a snake shedding its skin. Hoffman says, Basically the surface coat falls off and you look at it and you think, ‘Hey, that sporozoite is probably not viable any longer.’ The obvious implication was that this reaction was the result of an antibody attack. The hope, says Victor, was that if you identified the molecule that gives this reaction, it could be used as a vaccine.
It took them a while, but by 1979 the Nussenzweigs and their collaborators had identified the target of the antibodies as a protein that made up the protective coat of the sporozoite; they called it the circumsporozoite (or cs) protein. Pausing only for the technology of molecular biology to catch up with them, they synthesized important pieces of the protein in the lab, injected them into mice, watched the mice develop antibodies against them, and watched those antibodies go after the living sporozoite when it was introduced. Not only did the antibodies protect against malaria but they did so impressively. Not a single sporozoite survived to colonize the liver cells of the mice.
This led to the 1987 trials and their feverish denouement. Thanks to the Nussenzweigs’ work, malaria researchers could viably attack the parasite. Next, a research team at nyu and one at nih and wrair cloned the gene that encoded for the cs protein. This is when Ballou and Hoffman got involved as junior members of a team led by Wayne Hockmeyer of wrair and Lou Miller of NIH.
NIH had this technology to pull out the gene, says Ballou. wrair had this technology to recognize when we had the gene. We put the two together and within a month had the damned thing cloned. The nih/wrair team then looked for a drug company to help generate a vaccine and found SmithKline, one of the biggest. The Nussenzweigs and their collaborators at NYU found Hoffman–La Roche, also one of the biggest, and the race was on. The nih/wrair team put their gene in bacteria and let the bacteria generate copies of the cs protein, which they then injected into mice and eventually six human volunteers, including Ballou and Hoffman. The Nussenzweigs and their colleague Fidel Zavala used what’s called a synthetic protein, which means they created the crucial portion of the protein piece by piece in the laboratory. Then their collaborators at the University of Maryland injected large doses of the vaccine into three human volunteers, none of whom was a Nussenzweig.
And then, says Victor Nussenzweig, there was big publicity, which was impossible to control. It created a false, optimistic perspective, which really was deadly afterwards.
The trials, however, were not a total loss. Of the six volunteers immunized by the nih/wrair researchers, one remained malaria-free. And in two of the five who did get the disease, Hoffman and Ballou, its progress was slower than it would have been with no vaccine. The vaccine was doing something, says Ballou. As for the Nussenzweigs, of their three immunized volunteers, one remained free of malaria and the other two reached the blood stage only after a delay, because most of their sporozoites had been eliminated. The trials both failed and didn’t fail, says Victor. We didn’t consider [the trial] a failure, because it established that it was possible to protect against malaria infection by vaccination with the cs protein. Others considered it a failure because their expectations were not reasonable. They expected, says Ruth, that with one shot, one immunologic bullet, we would protect against malaria. No vaccine has ever been developed like that.
In the decade between the first cs protein vaccine in 1987 and the wrair success reported last year, the researchers went from protecting one out of six volunteers to six out of seven, but it took them a score of clinical trials. The essence of their approach has been to do what Ballou calls molecular biology tricks to get the cs protein to induce the greatest possible response from the immune system. These tricks involve combining pieces of the cs protein with chemical additives known as adjuvants.
They’ve also looked at different ways to express the protein in humans—for instance, by cloning a gene that would produce the protein in combination with a piece of the hepatitis B virus. The result was a protein that balled up like a particle rather than a long string. Particles are more like real viruses, says Ballou, which means the immune system takes a more vigorous approach to counterattacking.
The result so far is that six-out-of-seven number, which is impressive, although it’s not clear how impressive, because it’s not known how long the protection will last. For whatever reason, the immune system doesn’t seem to do a good job of remembering that it had the malaria antibodies. We’ve made tremendous progress, says Ballou. The message we’ve learned is that we have to go to extraordinary lengths to get those kinds of immune responses, but it is doable.
Now Ballou and his colleagues at wrair have begun testing yet another variation on his vaccine, hoping for added protection, while the Nussenzweigs are gearing up to test three new vaccines. Their colleague Elizabeth Nardin has spent the last few years identifying yet more pieces of the cs protein that will raise an immune response in humans, and she has created vaccines using synthetic versions of these pieces that, says Ruth, have produced astronomical amounts of antibodies, at least in mice. Moreover, these pieces of the cs protein seem to raise a variety of immune responses. The immune system is full of defense systems, including killer T cells and molecules such as gamma interferon that help excite the immune system. These pieces of the parasite seem to stimulate many different responses. The Nussenzweigs are optimistic, but they also know that optimism may be a delusional state of mind when dealing with malaria.
Whatever happens with these vaccines, very few people in the field believe they can beat Plasmodium by going after only the sporozoites. That’s why these researchers, while still plenty competitive, talk about the pursuit of a vaccine as a team effort in which the end product is likely to be a cocktail of multiple vaccines aimed at different stages of the parasite’s life cycle.
Hoffman, for instance, has spent the last five years with his colleagues working on a vaccine that will attack the parasite when it’s breeding in the liver. They’ve managed to identify half a dozen genes that these liver stages express when they’re turning from sporozoites to merozoites, and this past summer they began testing the safety of a new dna vaccine. The idea is to shoot the cs-protein gene into your muscles, which will take up the gene and begin generating the same protein the parasite generates when it’s in your liver. If all goes well, your immune system will then recognize the protein as an invader and be suitably prepared with an army of killer T cells. By this summer, Hoffman and his colleagues hope to learn whether the vaccine works in humans as well as it has worked in mice.
In England, Tony Holder of the National Institutes for Medical Research in London has been going after the blood-stage parasites, the merozoites. Back in 1981, Holder and his colleagues identified a protein on the merozoites that seems to be an essential tool for breaking down the walls of the red blood cells and getting the merozoites inside successfully. Holder and his colleagues immunized mice with this protein, the mice developed the proper antibodies, and when the mice were later injected with infected blood, the antibodies made a beeline for the protein, and the merozoites found themselves locked out of the red blood cells.
If they can’t get into the red blood cells, the cycle is interrupted, Holder explains. If we can interrupt the cycle completely, not only would we prevent clinical disease but we’d largely prevent transmission to the next person, because it’s the blood stage that forms the sexual stage that’s then transmitted to the mosquito. Three years ago, Holder showed that immunizing rodents with just part of the blood-stage protein stopped malaria dead. Fantastic, he says. You’d see no parasites at all. Now he and his colleagues have been working on protecting humans, and he, too, feels quite optimistic, although with the usual malaria-related cautions. Until they prove they can protect humans, their immunizing mice against malaria is meaningful only to mice.
Immunizing mosquitoes against the parasite, however, can help not just mosquitoes, which brings us to the benevolent vaccine. This is the vaccine that doesn’t stop you from getting sick but stops you from passing on the malaria to the next uninfected mosquito that takes a sip of your blood. The vaccine is the work of nih’s David Kaslow, whose idea is to actively vaccinate humans so they passively immunize mosquitoes. So when the mosquito takes the blood meal, he explains, along with the infected blood, she’ll take up the antibodies, which will protect her from becoming infected.
Kaslow’s strategy is to attack the parasite at its weakest point. While the bloodstream of an infected human contains billions of merozoites, very few go on to develop into the gametocytes, the sexual stage. And clearly, he explains, mosquitoes don’t suck people dry when they feed, so of the circulating gametocytes, only a fraction are taken up by the mosquitoes. A mosquito might imbibe only a couple of thousand gametocytes, of which maybe ten will survive in its gut to produce an oocyst, out of which will come the tens of thousands of sporozoites that will reinfect a human. So we’re talking about blocking a couple of oocysts from forming, says Kaslow, not blocking billions of parasites. Moreover, in the midgut of the mosquito, the parasite deploys proteins that have never had to deal with a human immune system, so they have never developed evolutionary strategies to evade them. This adds to their vulnerability.
Now what we’re doing is creating an artificial situation by making an immune response to something that’s never been under immune selection, says Kaslow. He plans to vaccinate humans with gamete/zygote proteins usually expressed only in the mosquito’s midgut. The vaccinated people will produce antibodies, which will be sucked into the mosquito with the blood meal. Thus, when gamete/zygotes emerge from the gametocyte-infected red blood cells in the mosquito’s gut, the antibodies will be there waiting for them.
Back in 1988, Kaslow and his colleagues identified a protein that was generated by the parasite’s genes only during its stay in the mosquito’s gut. Since then Kaslow has been working on turning it into a vaccine. It’s been a decade of one annoying problem after another. First the researchers had to make their protein fold properly so the antibodies would recognize it. That took a year. Then they had to abandon the virus used to produce and deliver the protein, because it might hurt the many sub-Saharan Africans whose immune systems are crippled by hiv. So they spent two years generating the vaccine from yeast. It looked great, says Kaslow, really good. Then they tried to scale up production, and it failed. It was degraded, says Kaslow. There were all kinds of problems when we tried to go from a bench-top to a huge fermenter. By that time, around 1993, their pharmaceutical company of choice lost interest, so they spent a year finding a new company, and so it went.
Two years ago Kaslow and his colleagues finally started human trials. The humans made antibodies to the protein, but the antibodies did not block transmission to the mosquito. On the other hand, Kaslow had shown that the vaccine was relatively safe, and the formulation he was using wasn’t particularly optimized to elicit a good immune response. So now Kaslow is starting the routine that Ballou and his wrair colleagues have been following for a decade—playing with adjuvants and delivery systems to get the best possible immune response. It looks really quite promising, he says.
If it’s safe, and if it blocks transmission in controlled settings here, then they’ll go to Africa and try it in the field. And this will assuredly bring a whole new host of obstacles. You’re going to have to vaccinate anything with two legs, he says, because you want the whole population to be vaccinated. It doesn’t really do you much good if you vaccinate only adults, because children can transmit it. And then, once we have something that looks good, we want to start combining it with other protective vaccines, or other transmission-blocking vaccines, or other interventions.
Kaslow is optimistic, as is virtually everyone when talking about their own vaccines. They have to be, as Hoffman points out—otherwise they’d give up. But when they talk about taking the vaccines to the field, the researchers sound less hopeful. The problem is immense. Two to three million people die every year from malaria. Several billion are at risk. Children are the most threatened, but vaccinating infants under six months, which will have to be done, comes with its own unique problems. So the vaccinologists and tropical medicine experts are talking freely about what they’re willing to consider a victory. The military, for instance, which funds much of the vaccine testing, would like to develop a vaccine that will protect troops against the parasite for six months, long enough to go into combat in a tropical zone. Such a vaccine would also be good for travelers. For the hundreds of millions in sub-Saharan Africa, the researchers might settle for anything that reduces the burden of suffering and death from the disease. A vaccine that makes a child’s immune system act like that of a 20-year-old, for instance, would keep children alive and healthier, because a 20-year-old will live through a bout of malaria that might kill a child.
Many of the researchers confess that in their bad dreams they think of the parasite as unbeatable. Even if they come up with something to stop the parasite, they fear, the parasite will evolve a way around the vaccine. But that’s just in their bad dreams. I am well aware of how difficult a problem this is, Hoffman says. I realize that the odds of success are minimal to slim in the short term, but we have made phenomenal progress over the years, and we are putting together the pieces of the puzzle that eventually will lead to the capacity to prevent malaria with vaccines. Whether that’s this year, next year, or 10 or 15 years from now, is what remains to be seen.