The hard statistics about malaria are far from comforting. Killing over one million victims every year and infecting another 300 million, the disease continues to have a catastrophic effect in many developing countries, striking the most vulnerable members of society: pregnant women and children. The World Health Organization estimates that malaria costs many afflicted states 1.3% of their annual gross domestic product, cutting their economies by over 30% since 1960. Exacerbated by poverty, malaria exacts a high toll on populations that can least afford a cure. In the 1950s, many were optimistic that the disease could be eradicated with the development of powerful mosquitocides,environmental control and drug treatments. As a result there was a marked drop in the incidence of the disease in some regions, even complete eradication of the malaria carrier, Anopheles gambiae, in Jamaica and Taiwan. But by the end of the 20th century, concerns about the environmental safety of some mosquitocides and the parasite's increasing drug resistance saw the disease's incidence rise.
William Harvey explains that four major research strategies appear to dominate current efforts to combat the disease; two targeting the principal malaria parasite, Plasmodium falciparum, and two aimed at controlling mosquito populations, especially Anopheles gambiae. It seemed clear to him that each individual strategy was potentially powerful, although all had drawbacks. But instead of the four camps talking and working together,there seemed to be little discussion between proponents of the different approaches.
Suspecting that this isolation was hampering progress, Harvey felt it was time to bring all four fields together. With Andrew Spielman, he has assembled a unique collection of reviews, focusing on the diverse biological approaches to the malaria problem, entitled `The Experimental Biology of Malaria and its Vectors'. The reviews cover aspects of malaria, from disease control by drugs and vaccines, through transgenic approaches to mosquito control, to the development of modern mosquitocides.
Launching the collection with an editorial, the epidemiologist Andrew Spielman describes the history of research into vector-borne disease transmission (p. 3727). Sounding a note of caution that the present balance has led to an erosion of vector biology studies in favour of other approaches, Spielman discusses the conclusion drawn by a recent Institute of Medicine symposium that the `human resource capacity' capable of dealing with vector-related issues in health has been eroded and should be rebuilt to produce a `new generation of public health entomologists'.
DRUGS KEEP PACE WITH THE PARASITE
Anti-malarial drugs have always been a mainstay of defence against the malaria parasite. Kathryn Aultman of the National Institute of Allergy and Infectious Diseases explains that `there is no biological need for anyone to die of malaria in 2003'. It's not the lack of effective drugs that is the major problem facing infected populations, but the cost and complexity of modern treatments that makes them difficult to distribute. One of the main challenges facing drug developers is the need to find treatments that are not only simple to administer, but inexpensive. Philip Rosenthal explains that`even a price of $1 per treatment is probably unacceptable in many regions';he suspects that 10c is probably a more realistic goal(p. 3735). Describing vulnerable points in the parasite's physiology and a variety of drug identification strategies that might target them, Rosenthal summarises many promising new avenues of research in anti-malarial drug discovery.
However, if the current range of drugs are to remain effective, Christopher Plowe explains that it is essential to track drug resistance as it appears(p. 3745). Using a variety of pharmacological studies, and identifying molecular resistance markers, Plowe explains that knowledge of resistance patterns can be translated rapidly into novel treatment strategies. Plowe describes how monitoring resistance levels in the plasmodium population in Malawi showed that a significant decrease in chloroquine resistance occurred when treatment switched from chloroquine to sulfadoxine/pyrimethamine, suggesting that drug alternation could be an effective strategy to modulate drug resistance levels.
Although few new drugs made it into the anti-malarial pharmacopoeia during the last half of the 20th century, Piero Olliaro and Walter Taylor point out that modern multidrug therapies are having notable successes(p. 3753). Outlining the results from various drugs used in combination with the anti-malarial drug, artemesinin, Olliaro and Taylor describe many encouraging cases,especially when the drugs were administered in fixed doses from `blister packs'.
But it doesn't matter how effective the next generation of anti-malarial drugs are, if they aren't administered correctly. Writing with Patrick Kachur and Holly Ann Williams, Peter Bloland points out that as new therapies are costly and relatively complex to administer, it is essential that effective public health programs are established for the delivery of new therapies to reduce the risk of resistance developing later(p. 3761). One recent initiative proved successful in Ethiopia. Local mothers were trained to identify cases of childhood malaria in the community, and provide the parents with chloroquine accompanied with pictorial directions for correct usage.`This strategy led to a reduction of 41% in the under-five mortality rate over 2 years' says Bloland. But he emphasises that it will only be through research that alternative public health programs can be evaluated to produce effective strategies for the coming decades.
VANQUISHING VACCINATIONS
Although anti-malarial drugs have proved effective against the parasite,development of a vaccine is the Holy Grail of malaria research. For a vaccine to be effective, it must boost the immune system's response to eradicate the parasite from a patient's blood stream. Several creative approaches are now being taken to produce a vaccine that outwits the parasite.
Over the years, much work has focused on developing vaccine delivery systems for antigens, such as viral vectors, recombinant proteins and DNA vaccines. Unfortunately, few of these approaches were successful in isolation. However, Susanna Dunachie and Adrian Hill describe a promising new approach known as heterologous prime-boosting(p. 3771). Using this strategy, an antigen is presented in a series of different delivery systems that are administered sequentially. Establishing the best order of vaccine delivery and identifying candidate antigens are both key stages in the vaccine's development. According to Hill and Dunachie, clinical trials in both Europe and Africa show that heterologous prime-boost vaccines produce a significant increase in T-cell responses to an antigen expressed prior to red blood cell invasion. Although the `regimens currently under study may appear too complex for widespread use' Hill and Dunachie add that their goal is `to obtain good efficacy first and then develop ways to simplify the regimen' for use in the field.
Another target point for vaccine development is the blood-stage of the parasite's life cycle. Once inside the red blood cells, the parasite is protected from the body's immune response, and is only vulnerable when it prepares to invade a new cell. Therefore these vaccines must promote a significant antibody response in preparation for the rare occasions when the parasite is free. Louis Miller, Siddhartha Mahanty and Allan Saul describe recombinant protein vaccines that are based on merozoite surface proteins involved in red cell invasion. By raising an antibody response to these antigens, the team hope to block parasite invasion of red blood cells and inhibit parasite growth (p. 3781). Miller describes mixed results in clinical trials from vaccines based on blood cell stage antigens, and emphasises that there are not enough trials to evaluate an antigen's efficacy, but adds that these vaccines will eventually form `key ingredients of multi-component, multistage malaria vaccines'.
With the advent of whole organism genome sequencing, two of the creatures that were evidently high on the sequencer's wish list were the African malaria mosquito, Anopheles gambiae, and its malaria parasite, Plasmodium falciparum. The estimated 5300 proteins encoded by the parasite's genome offer rich picking grounds for the development of new vaccines based on novel antigens. Denise Doolan and her colleagues explain that `immunization with only one or a few parasite proteins will be unlikely to duplicate the broad and sustained immunity elicited by exposure to a parasite that has thousands of proteins' and contends that `a vaccine that mimics the complexity of the organism itself' should be a major focus of vaccine development(p. 3789). Doolan outlines a `genome-to vaccines' strategy that she believes will eventually prove to be a highly successful approach for combating the parasite.
But of all the vaccine approaches discussed in this collection, Stephen Hoffman's work with radiation attenuated malaria sporozoites is the most encouraging. Hoffman and Thomas Luke explain that the vast majority of disease vaccines currently in use are derived from `attenuated or inactivated whole pathogens', so they tested the levels of immunity conferred by irradiated sporozoites delivered by mosquitoes(p. 3803). Their results were spectacular, achieving protection against malaria in 13 out of 14 volunteers. According to Hoffman, his company Sanaria's goal is to develop a vaccine based on the irradiated sporozoite's success. Having listed some of the major hurdles that must be overcome to produce a viable sporozoite vaccine, Hoffman outlines the development strategy that he hopes will lead to the production and delivery of the vaccine to the population that needs it most: infants and children in Africa.
MALARIA'S TRANSGENIC MATCH
The next strategy moves from targeting the parasite, to targeting the parasite's vector, the infamous Anopheles mosquito, and blocking the parasite's progress through the insect's body. The mosquito's role in the parasite's lifecycle begins when it ingests the parasite's gametocytes during an infected human blood meal. The gametocytes mature rapidly, and fuse before producing an ookinete that invades the insect's gut wall. Developing in the gut epithelium, the oocyst eventually bursts, releasing sporozoites into the insect's haemolymph. But even though the sporozoites can pass throughout the insect's body, the only tissue that they invade is the salivary gland, ready to infect the next human victim. Of the many anti-malarial strategies that target the mosquito, surely the most ambitious is the development of transgenic mosquito populations that will block the parasite's progress through its insect host, terminating the deadly life cycle.
Marcelo Jacobs-Lorena and colleagues discuss some of the strategies designed to hinder the parasite's transition across the mosquito's midgut,including the development of transgenically modified mosquitoes(p. 3809). The main aim is to produce insects that prevent ookinete invasion of the midgut cells by expressing blocking peptides. But the real challenge is getting these modifications into the natural mosquito population. Jacobs-Lorena has identified two potential malaria-blocking genes, and describes several strategies that might drive the parasite-blocking genes into the wild type population, including transposable elements and selfish genes. Jacobs-Lorena is optimistic that combining this approach with knowledge of the parasite's genome could ultimately result in `[an engineered] mosquito that inhibits or kills the malaria parasite during multiple developmental stages'.
At the other end of the parasite's odyssey through the mosquito's body, the sporozoites invade the insect's salivary gland ready to infect the next human victim. Anthony James describes his work developing transgenic sporozoite-blocking mechanisms in mosquitoes. James suggests either inhibiting sporozoite development, or, destroying the sporozoites once they have invaded the salivary gland. But James believes that the most effective strategy is to develop transgenic insects that attack the sporozoites while they pass through the haemolymph to the salivary gland(p. 3817). Discussing a range of potential target sites and effector molecules that might destroy the parasite, James adds that the `use of multiple effector genes may be necessary to prevent the selection of resistance to any one mechanism'.
But all of these creative approaches are dependent on the technology to produce transgenic insects in the first place. David O'Brochta and a team of collaborators describe four of the current gene-vector systems that are being used to generate transgenic insects(p. 3823). All four systems are derived from Class II transposable elements, and although most seem successful in D. melanogaster, their behaviour in other insect species is less well understood. O'Brochta discusses each class's transposition and subsequent remobilisation in the yellow fever mosquito, Aedes aegypti, and concludes that `the stability of the transgenics being created with these insects will be very high even in the presence of homologous functional transposase'.
Although transgenic approaches will no doubt prove powerful, Alexander Raikhel points out that the `lack of a complete genetic tool box for mosquitoes remains a serious obstacle to our ability to study essential mosquito-specific mechanisms'(p. 3835). Optimistic that a combination of reverse genetics and RNA interference techniques could fill this vacuum, Raikhel has targeted the mosquito's immune system to respond to parasite infections in two new ways. In one case, he describes a transgenic mosquito that produces an endogenous anti-microbial agent that destroys the parasite. In another example, Raikhel has altered the insect's immune system so that it becomes susceptible to infection after a blood meal.
MOSQUITOCIDES TAKE OUT THE MIDDLEMAN
Historically, reducing the mosquito population has proved to be one of the most successful approaches to minimise, and even eliminate, malaria infections in human populations. Mosquitocides are still a mainstay of malaria control. Insecticide treated nets and indoor sprays protect humans from insect bites in their homes, and the development of modern environmentally safe mosquitocides will offer even more protection. Listing several aspects of the larvae's physiology, Harvey points out that `the mosquito larval midgut is a prime target for environmentally safe mosquitocides' because of its unusual trypsin regulating hormone, high pH, and voltage driven transporters.
Following this theme, Klaus Beyenbach describes his laboratory's research into transport mechanisms in the blood feasting Aedes aegypti(p. 3845). Outlining the role of diuretic peptides and their effects on various ion transporters,coupled with vacuolar H+-ATPases that energise epithelial ion transport, Beyenbach explains how the insect `deals with the enormous dietary load' exerted by its blood sucking diet. Sarjeet Gill and colleagues also focus on the role of ion transporters in the mosquito midgut and Malpighian tubules during ion regulation and fluid secretion(p. 3857). They discuss the roles of both sodium proton exchangers and cation-coupled chloride cotransporters in ion regulation with the aim of `identification and development of reagents that can potentially interfere with these crucial processes'.
Like most armies, mosquitoes `march' on their stomachs, so Dov Borovsky's approach, hitting the larvae in the stomach, seems very appropriate. Borovsky has identified a hormone, known as trypsin-modulating oostatic factor (TMOF),that prevents the larvae from producing trypsin, depriving the larvae of essential amino acids from brokendown proteins. Isolating the minimum peptide that is sufficient to obstruct the insect's digestion, Borovsky has cloned the peptide into yeast cells and fed them to mosquito larvae; 38-83% of the larvae died (p. 3869). Borovsky has also demonstrated the peptide's larvicidal properties in field tests. Harvey is very excited by Borovsky's approach and says that TMOF is the`first new class of mosquitocide in years'.
But humans aren't the only organisms intent on destroying insects. Several species of bacteria synthesise insecticides, and one of the most successful is Bacillus thuringiensis subsp. israelensis, known as Bti. Bti produces a series of proteins that combine with a receptor on the mosquito's midgut surface, destroying the midgut and killing the insect. Bacillus sphaericus also produces an inactive crystal toxin, which is activated in the insect's gut before destroying the tissue by lysis. Although both toxins are effective, Brian Federici and coworkers describe how together the combined toxins' effects are much more than the sum of their parts, increasing their potency ten fold, and limiting the insect's chance of developing resistance(p. 3877). And although he admits it sounds a little far fetched, Federici thinks that one day, it might even be possible to `design `smart' bacteria that will seek out and kill larvae of specific vector mosquitoes'.
THE FUTURE
Experimental biology clearly has a powerful role to play in combating the current malaria epidemic, but Harvey is keen to point out that epidemiology,ecology, and other disciplines are also essential for successful malaria control. Harvey hopes that by combining some of the innovative ideas discussed in this issue and working with organisations that have the political will to fund such large-scale projects, it will be possible to mount effective low-cost anti-malaria campaigns in the regions of the globe where they are needed most.
Clearly there is still a long way to go, but with the weight of creative might that is pitched against this appalling disease, there are plenty of reasons to hope that we will continue to see significant breakthroughs against an ancient parasite that still blights so many lives.
