Feature: A world without malaria

This feature appeared in the May/June 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Malaria is not only devastating to the individuals who suffer from it, but it also prevents many of the poorest countries around the world from emerging from poverty. A 2001 report by the Center for International Development found that countries with widespread malaria infection had income levels only one third that of similar countries without malaria - and that's adjusting for all other variables. So malaria is not just a health issue, it's a massive socio-political problem, the solving of which could benefit untold millions.

At its peak, malaria killed more than two million people each year, 90 per cent of them in sub-Saharan Africa, with at least 80 per cent of deaths being infants and children under five. Even in the wake of the World Health Organisation's concerted Global Malaria Action Plan of 2008, global deaths are still around 882,000.

The WHO's latest battle plan melds the tried and true with the high-tech new. Many homes in malaria-ridden regions are now protected by indoor residual insecticides sprayed on walls, while bed nets guard sleeping families. These simple, low-cost measures have cleared the air for a high-tech pincer movement against the five blood-borne Plasmodium parasites that cause human malaria. It employs rapid diagnostics and resistance-busting combinations of antimalarial drugs.

An earlier campaign to eradicate malaria in the 1950s an 1960s, was undone by the emergence of chloroquine-resistant strains of Plasmodium faiciparum in the 1960s, as well as an end to DDT manufacturing in industrialised nations, because of concerns about its environmental impacts DDT had shielded millions of African homes against mosquito invasion.

A crucial component to combating malaria is human treatments and vaccines. The last quarter of the 20th century saw a growing investment in malaria research, but only incremental progress towards a vaccine, and new antimalarial drugs. But it did deliver the tools for recent, spectacular advances, particularly in Australian laboratories. Several research teams around Australia are close to developing treatments or vaccines against malaria

Creating immunity

Dr Louis Schofield, a Howard Hughes Medical Institute International Scholar based at the Walter and Eliza Hall Medical Research Institute's Infection and Immunity Division in Parkville, Melbourne, says there is growing optimism in the malaria research community that malaria, like smallpox, can be consigned to history.

WEHI's Infection and Immunity Division is at the forefront of global malaria research and has been involved in many landmark developments since former Hall Institute Director, Sir Gustav Nossal, established its malaria research program with a Rockefeller Foundation grant in 1977.

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Five species of Plasmodium cause human malaria and, Schofield says, "wildly different" antigens have stymied the development of a universal malaria vaccine. P. falciparum accounts for most mortality but, according to Schofield, vivax has recently moved to centre stage after 50 years in the wings. "There was a misconceived view that vivax malaria was a relatively benign infection. Yet it's the second deadliest species."

Schofield says impressive recent advances in reducing malaria transmission have had limited impact on vivax. "It's much longer-lived than falciparum, and has a more robust transmission biology," he says. "It's immediately infectious to mosquitoes, where falciparum takes much longer. It relapses over many years, and this makes it harder to treat with standard drugs.

"You can go into an area where both falciparum and vivax are present, and treat people with drugs to eliminate their blood stages. Falciparum disappears, but vivax merozoites come straight out of the liver into the blood. Vivax will persist until we get better drugs to treat the liver stage."

P. ovale and P. malariae cause debilitating but non-lethal illness; P. knowlesi, whose primary host is the Southeast Asian long-tailed macaque, occasionally causes serious human malaria, but until recently, cases were misdiagnosed as vivax malaria.

All but P. knowlesi occur in Papua New Guinea, a conveniently adjacent natural laboratory for field work. The WEHI Institute works in partnership with the PNG Medical Research Institute, which has a long-standing epidemiology project, and advanced research capabilities.

"Our new ability to generate red blood cells from stem cells massively enables that work, because we can go to PNG, identify people carrying a mutations, and generate corresponding cell lines for study in vitro," says Schofield.

Schofield's team has a lead antigen for a vaccine in development: a highly conserved glycolipid toxin on the surface of the merozoites, and possibly sporozoites, of all five species. His team will target the toxin, glycosyl phosphatidyl inositol (GPI), hoping to see a strong, protective immune response.

GPI is an attractive target because it plays several roles during both the sporozoite and merozoite stages. In the merozoite (blood) stage, it serves as a toxin that inflames blood vessels and causes hyperactivation of the immune system in tissues like the brain and spleen.

"Red blood cells are filtered through the spleen every few minutes, and falciparum and vivax have different strategies for avoiding the spleen," Schofield says. "Falciparum enters erythrocytes and expresses PfEMP-1 [P. falciparum Erythrocyte Membrane Protein-1] on the surface of the infected cells, causing them to stick to the walls of the vasculature, in microcapillary beds in organs like the placenta, lung and brain."

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This specificity allows localised inflammation upon release of the GPI toxin, explaining the various clinical malaria syndromes, like cerebral malaria and placental malaria.

"We are pursuing two, complementary strategies: a vaccine based on whole attenuated parasites, and a vaccine targeting GPI," says Schofield. "Most malaria fatalities are due to the intersection between inflammation and organ-specific disease. They are the keys to pathogenesis, and our work is focused on preventing disease, as opposed to the brave new world of eradication.

"At WEHI we've have been fortunate in winning three Grand Challenge Exploration grants from the Bill and Melinda Gates Foundation. In the Infection and Immunity Division, Dr Krystal Evans and I got two of them. The Foundation issues a call for innovative ideas, and there is intense competition for the grants.

"Our other Gates-funded project headed by Dr Evans is a live attenuated vaccine. When I entered malaria research 30 years ago, people said there would never be a live attenuated vaccine. But returns from 30 years of research into recombinant antigens have been pretty disappointing, because single proteins are too polymorphic to be a good target.

"We know that people exposed to the whole organism in malaria regions eventually develop immunity. Regulatory and technical problems have precluded the live-organism vaccine approach until recently, but we have made some significant advances towards making a live attenuated vaccine."

The most significant, says Schofield, is a stunning technical achievement by Austrian postdoctoral researcher and erythropoiesis specialist, Sandra Pilat, whom he recruited after she produced fully and partially differentiated murine erythrocytes from immortal stem cells.

"Nobody had been able to crack vivax culture, and the field has been hugely neglected as a result. It's possible to infect chimps, but we couldn't study it in vitro. It has a very specific requirement for growing in reticulocytes, the earliest developmental stage of erythrocytes. In contrast, falciparum invades mature red blood cells that have lost all their RNA."

Pilat successfully adapted her protocols to grow human erythrocytes from stem cells from the peripheral blood supply. That capability could give blood banks an inexhaustible supply of virus-free erythrocytes. But, Schofield says, the more immediate significance of Pilat's feat is that researchers can now try to grow P. vivax in the laboratory - previously an impossibility because it specialises in colonising early-stage erythrocytes.

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Two vaccines are better than one

Alan Cowman's team is collaborating with Krystal Evans and Schofield to develop an attenuated blood-stage parasite using the same approach. Two vaccines, targeting different life-stages of the parasite, should be an effective barrier to infection. His group plans a collaborative project with the Centres for Disease Control in Atlanta, Georgia, which will perform the pre-clinical trials.

A prime target is PfEMP-1 (P. falciparum Erythrocyte Membrane Protein-1). Schofield says knocking out the gene for this potent virulence factor should significantly reduce the parasite's pathogenicity.

"Our attenuation project is targeting both virulence and pathogenicity," says Schofield. "We don't want to render the parasite completely unable to replicate, so we will attenuate the parasite to the point where it will have only a limited capacity to replicate before falling over, leaving the dead cells to stimulate a broad immunological response."

Cowman's research team is scheduled to begin clinical trials of its genetically attenuated sporozoite vaccine in the US May at the Walter Reed Army Hospital in Washington, which has a mosquito insectary.

"We're looking for proof of principle, and to confirm the vaccine's safety," Cowman says. "The live attenuated parasite is a genetically modified organism in which specific genes have been disrupted that are required for normal development of the malaria parasite in the liver.

"With Stefan Kappe's team in Seattle, we've identified the genes that the parasite requires to complete development of its liver stage. We've knocked out two of those genes so it can't complete its development and enter the blood. Stefan Kappe's group has already shown the approach works in mice, using Plasmodium yoelli as a model. It was 100 per cent protective.

"The phase I trial will move into a challenge trial if the vaccine proves safe. Volunteers who have had the attenuated parasite vaccine will be monitored after allowing themselves to be bitten by mosquitoes carrying a normal parasite strain known to be sensitive to chloroquine."

A subunit vaccine is a more distant goal, and contingent on identifying suitable antigens. "For 15 years we've been investigating how the merozoite enters red blood cells," Cowman says. "It has multiple invasion pathways, and we're trying to work out the best ones to target, and how many we need to block to lock it out of red blood cells.

"Clearly, a single protein won't work. If we apply strong selection pressure to one pathway, the parasite can switch to another. We've identified two candidate families, one sialic acid-dependent, the other sialic acid-independent. We have determined which of their proteins are most important with respect to invading the red cell.

"We have identified the critical regions of the ligands, and how we could block them with a combination vaccine, and confirmed that a particular combination vaccine approach does block invasion. So we know we can block this entry mechanism the parasite employs.

"The work is funded by the Malaria Vaccine Initiative. They've asked us to do a few more experiments, and we hope to move to a stage I clinical trial at the end of this year."

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Targeting the Achilles heel

Dr Jake Baum's group at WEHI is investigating parasite locomotion, another potential Achilles heel. "Malaria parasites use a basic actin-myosin motor to move, in which the actin filament acts like a clutch. We want to jam a spanner in the works with a drug that won't harm humans but would target the parasite motor, and observe what happens," he says.

"The problem is, the parasite is around a micron in diameter, the size of a bacterium. It's beyond the resolving power of light microscopy. To get around this we're using super-resolution methods like 3-D structured illumination microscopy, scanning confocal microscopy and rapid image capture to observe parasites in action. But its not easy, it's like trying to predict where a burglar might break into a high-rise tower whilst looking down from the top.

"The various Plasmodium species are quite different, but they all use this same actin-based motor to move. The next stage for us is to piece together actin regulation, understand what each individual regulator does, how they're switched on and off, and then find a drug that might target its function which would in turn block actin's key role."

Baum says there is a substantial research literature on genes likely to be involved in regulating actin. "Hundreds have been characterised in other organisms. We threw them against Plasmodium but remarkably only came up with six proteins. Either they have an incredibly pared down version of actin regulation in other organisms, or they're using a completely novel system. In either case, that makes actin control a good drug target."

Baum says he started out working solely on blood stage vaccines, but since movement happens across the lifecycle he's switched to more general drug targets. "The big push in malaria research now is for transmission-blocking strategies that cover the entire parasite life cycle."

His team collaborates with Dr James Beeson's group at the WEHI, and has achieved a quantum leap in their ability to image the tiny parasite breaking into giant erythrocytes.

"Last year my students tried 40 or more different assays to capture invasion, but detected invasion events less than one time in a thousand to a million. Now, thanks to work with James' group, we succeed every time," says Baum. "It takes the parasite only 30 seconds to gain access to the cell, but we can now break this event down and tell where each key protein localises. It's a powerful tool to work out what's going on. We couldn't do that before.

"We've watched parasite derived structures take shape that we know are absolutely essential to establishing a portal into the cell. One in particular - the tight junction - which the parasite limbs through like a window. Basically, whilst parasites have multiple keys the tight junction is completely conserved. It's the same for the liver stages.

"Using our new viewpoint we have identified one of the key molecules involved. The parasite uses it like a rock climber uses a piton to hang onto a cliff face. The parasite sticks in this protein peg anchoring itself to the erythrocyte membrane. Other structures are then built on this point of attachment, including the formation of a ring of actin. The motor can now engage, acquires traction against the junction and the parasite invades!"

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Inside immunity

James Beeson's research group is trying to understand how infants who survive falciparum malaria eventually develop protective immunity in their teenage years or in adulthood.

"It's critical to advancing vaccine development that we understand how immunity develops - or fails," he says. "We know antibodies are critical to acquired immunity to malaria, yet we know little about how they work, and what they target."

Beeson's group is investigating why the immune response is principally directed against erythrocyte-infecting merozoites, and infected erythrocytes expressing merozoite proteins on their surface.

"Antibodies eventually block the parasite's ability to infect red blood cells. We need to understand how people acquire those antibodies as they age, and how the parasite responds," he says. "It has multiple protein ligands that allow it to adhere to and invade the erythrocyte, and it can vary them. They're probably key targets of the immune response."

The challenge is to understand how the parasite adapts as antibodies progressively block its access points. "Immune individuals have effectively barred all the doors and windows against burglars."

To succeed, a vaccine must elicit antibodies against multiple proteins on the parasite's surface. "We've done all the human studies, and we're collaborating with Alan Cowman to determine which proteins we need to target.

"As for antibody function: do they trigger or facilitate responses from the immune system? It's obvious that if we could stimulate monocytes and macrophages to attack the parasites, it would speed their elimination. It wouldn't require antibodies.

"The other issue is complement. Antibodies can bind to the merozoite and bind complement to lyse the cell, but we don't know how these things work. Would a vaccine replicate that response?"

Beeson's team is looking at how merozoites induce their erythrocyte hosts to express particular forms of its suite of 100-odd PfEMP-1 proteins on its surface. PfEMP-1 proteins sequester parasites away from the immune system's reach, by mooring infected host cells to the walls of small blood vessel in safe-house organs like the brain and placenta. The clogged blood vessels explain malaria's tissue-specific symptomology.

PfEMP-1's variability makes it an unpromising target for a vaccine. Yet antibodies in individuals with acquired immunity appear to consistently target a subset of half a dozen PfEMP-1 variants that remains fairly consistent throughout malaria's global haunts.

"My lab works almost exclusively on P. falciparum. Despite substantial antigenic differences, proteins that differ markedly in sequence seem to serve identical functions in falciparum and vivax, so some our principles should be applicable to vivax."

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Breeding the buzz

Across Royal Parade, Professor Geoff McFadden's research group in Melbourne University's School of Botany has filled a gap in Australia's malaria research capabilities, by setting up the country's first mosquito insectarium.

"We lacked a facility for breeding parasites so, with some difficulty, we convinced the Australian Quarantine Inspection Service to allow us to build a secure facility to their specifications."

The new facility doesn't employ the real McCoy, but a Murinae model: Plasmodium berghei and its vector, Anopheles stevensii. "Telling AQUIS we would be using genetically modified parasites raised the bar even further, but we're up and running, and the mosquitoes are breeding well. We can produce heavy infections in mice, and track the parasites in the body by tagging them with green fluorescent protein," says McFadden.

"The main thrust of our work is to explore what metabolic pathways are essential to the parasite's life cycle, and how we can knock them out. We're excited because we've identified pathways specific to different stages of the parasite's life cycle. Some pathways are essential only during the liver phase, and we think we have found some that act only when the parasite is inside mosquitoes."

McFadden says there are no plans to prioritise such developments yet. They will inform the selection and design of drugs that could be combined to disrupt the parasite's complex life cycle at multiple points. McFadden developed the insectarium with collaborative projects with WEHI researchers in mind.

"Jake Baum is interested in how the parasite moves from the mosquito's gut to its salivary glands. Alan Cowman is interested in exploring whether the parasites put molecules on infected liver cells. We can make liver stages by using mosquitoes to reinfect the mice - we can't do that with the human parasites."

The WEHI is considering setting up another insectary to work directly on P. falciparum. "That's too complicated for us, and handling loaded mosquitoes involves risks that don't arise with P. berghei," says McFadden. "We hope to identify a drug target exclusive to the erythrocyte stage. Access to other life stages is more difficult, but there's a real need for liver-stage antimalarials.

"We need to think about our targets, and whether the need is for therapeutic or prophylactic drugs. A drug targeting the liver stage would be a useful prophylactic for young children who haven't developed immunity. In terms of prophylaxis, we have some exciting developments in the area of fatty acid synthesis pathways." says McFadden.

"The ultimate solution is a vaccine, but it's proving very elusive. There are technical obstacles to delivering an attenuated-parasite vaccine - for example, how do you generate enough genetically attenuated parasites to produce the vaccine in the volume required to prevent 250 million new cases of malaria annually?"

What is clear is that progress is being made. Far from being the incurable scourge of the developing world, malaria is now firmly in the sights of a number of research teams, all of whom are making tremendous progress towards treatment or vaccines. And Australia is playing a leading roll in bringing an end to this devastating disease.

This feature appeared in the May/June 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.