Nature outlook malaria by Javier Ortega

OUTLOOK

MALARIA

26 April 2012 / Vol 484 / Issue No. 7395

OUTLOOK MALARIA

Produced with support from: Medicines for Malaria Venture (MMV), Sigma-Tau and Vestergaard Frandsen New York

The struggle to gain ground

Cover art: Nik Spencer

Editorial Herb Brody, Michelle Grayson, Tony Scully Art & Design Wes Fernandes, Alisdair Macdonald, Andrea Duffy Production Donald McDonald, Susan Gray, Leonora Dawson-Bowling Sponsorship Reya Silao, Yvette Smith, Gerard Preston Marketing Elena Woodstock, Hannah Phipps Project Manager Christian Manco Art Director Kelly Buckheit Krause Magazine Editor Tim Appenzeller Editor-in-Chief Philip Campbell Editorial Advisors Sheilagh Molloy, Claudia Lupp

omo versus Plasmodium. It’s a war that has raged for millennia and that still claims hundreds of thousands of lives each year. The struggle against this mosquitoborne parasite has shaped the genomes of people in endemic regions. Past attempts to eradicate malaria have failed. What will it take to finally beat it into submission? It is predominantly the richer countries with temperate climates that have eliminated malaria so far. The disease is still endemic in hotter, poorer countries; in some parts of western Africa, the burden is so high that elimination would require more than a 99% reduction in transmission rate (page S14). At least in Africa, where 90% of malaria-related deaths occur, the standard artemisinincombination therapy (ACT) is still an effective cure. Parts of Southeast Asia are not so lucky. There is evidence that resistance to ACT is emerging there but, despite huge international drug development efforts, there are no new treatments to replace it (S16). A vaccine for malaria has been a big research goal for more than a hundred years. Plasmodium is a master of disguise, and researchers have to try a diverse range of tactics to target the parasite — in both its human and mosquito hosts (S24). Indeed, understanding human immunity to malaria and identifying parasite antigens are two of the top research priorities identified by our panel of experts (S22). And any successful strategy to eradicate malaria will have to include controlling mosquitoes — such ideal vectors (S26). But by far the greatest challenge is not in the lab; it is on the ground in endemic countries. It concerns access to care, encompassing education about malaria, availability of ACTs, infrastructure, presence of healthcare workers and government support. And in a country like Uganda, as our reporter Amy Maxmen found when she visited — these are in short supply (S19). We acknowledge the financial support of Medicines for Malaria Venture (MMV), Sigma-Tau and Vestergaard Frandsen New York in producing this Outlook. As always, Nature has full responsibility for all editorial content. Michelle Grayson Senior Editor, Nature Outlook.

Nature Outlooks are sponsored supplements that aim to stimulate interest and debate around a subject of interest to the sponsor, while satisfying the editorial values of Nature and our readers’ expectations. The boundaries of sponsor involvement are clearly delineated in the Nature Outlook Editorial guidelines available at http://www. nature.com/advertising/resources/pdf/outlook_guidelines.pdf CITING THE OUTLOOK Cite as a supplement to Nature, for example, Nature Vol XXX, No. XXXX Suppl, Sxx–Sxx (2012). To cite previously published articles from the collection, please use the original citation, which VISIT THE OUTLOOK ONLINE can be found at the start of each article. The Nature Outlook Malaria supplement can be found at VISIT THE OUTLOOK ONLINE http://www.nature.com/nature/outlook/malaria_2012. It features all newly The Nature commissioned Outlook Malaria content supplement as wellcan as abe selection found atof relevant previously published material. http://www.nature.com/nature/outlook/malaria_2012

CONTENTS S14 MALARIA

The numbers game Charting the major challenges

S16 DRUG DEVELOPMENT

Holding out for reinforcements Danger signs in Southeast Asia

S19 PUBLIC HEALTH

Death at the doorstep Reality bites in Uganda

S22 PERSPECTIVES

The missing pieces Nine experts identify the major unknowns

S24 VACCINES

The take-home lesson Complex lifecycle frustrates researchers

S26 VECTOR CONTROL

The last bite How many ways to kill a mosquito?

COLLECTION S28 Global health hits crisis point

L. Garrett

S30 A world without mosquitoes

J. Fang

S33 Moving in and renovating: exporting

proteins from Plasmodium into host erythrocytes D. E. Goldberg and A. F. Cowman

S38 Artemisinin resistance: current status

and scenarios for containment A. M. Dondorp et al.

S47 Two-pronged tactics for malaria control

P. Kirkpatrick

S48 New neglected disease research scheme pools IP and expertise S. Frantz

All featured articles will be freely available for 6 months.

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S49 Experimental human challenge

infections can accelerate clinical malaria vaccine development R. W. Sauerwein, M. Roestenberg and V. S. Moorthy

S57 Mosquitoes score in chemical war

D. Butler

S58 Malaria vaccine results face scrutiny

D. Butler

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OUTLOOK MALARIA

THE NUMBERS GAME

Nature Outlook maps the challenges in tackling the malaria epidemic. By Priya Shetty.

MAPPING MALARIA Malaria is still one of the ‘big three’ diseases, along with HIV and tuberculosis, affecting the developing world. While it has been eliminated in many regions, it remains a scourge of poorer countries, especially those in sub-Saharan Africa, where Plasmodium falciparum is the leading cause of malaria. Elsewhere in the world, malaria is caused by a mix of P. falciparum and P. vivax, as well as a few less common sub-species.

Malaria-free Eliminating malaria Endemic

90% Africa 6% Southeast Asia 2% Eastern Mediterranean 1% The Americas 1% Western Pacific

DEATHS BY REGION

Required reduction in transmission to eliminate malaria

(WHO)

90–99% >99%

Every 60 seconds a child dies of malaria

90%

MALARIA DEATH DISPARITY

90% of malaria deaths are in Africa

The Lancet numbers differ because the authors used verbal autopsy, in which they interview the relatives of a person who has recently died to determine a cause of death. Interviews can identify people who died of malaria but went undiagnosed or treated.

1.24M deaths worldwide, 714,000 under the age of 5

655,000 deaths worldwide, 563,300 under the age of 5

COTE D’IVOIRE

BENIN

UGANDA

WHO: 1,023

WHO: 964

WHO: 8,431

Lancet: 31,664

Lancet: 14,415

Lancet: 41,648

THE MONETARY GAP The good news is that if current efforts to control malaria are strengthened, the costs of the epidemic (including prevention through bed nets and insecticides, treatment with antimalarials, diagnosis, and R&D) are set to fall over the next few decades. However funding has not matched these ambitions. Costs of malaria control

Projected costs

Funds raised

6.0

US$ billion

5.0 4.0 3.0 2.0 Lancet = 50,000 people

WHO Deaths Deaths under 5yrs

US$1.7 billion

US$2 billion

1.0 0

2009

2010

2011

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2021

2031

2041

MALARIA OUTLOOK

Pregnant women: A pregnant woman is 4-times more likely to get malaria, and twice as likely to die from it, than another adult. This is because her immune system is partially suppressed during pregnancy. Malaria in pregnancy has dangerous consequences for the baby too.

Children: Most malaria deaths happen in children under 5 years old because their immune systems are not developed enough to fend off the parasite’s attack.

People living with HIV: HIV infection weakens the immune system, making people more vulnerable to malaria. Malaria infection causes HIV viral loads to shoot up, which could increase its transmission. The diseases are linked in other ways too – the DARC gene that protects against vivax malaria might increase susceptibility to HIV.

TIMELINE OF RESISTANCE Malaria has defeated many drugs in the past, often less than a decade after they were introduced.

1920

1930

1940

1950

1960

1970

1980

1990

2000

2010

Chloroquine resistance emerges 1957 Sulfadoxine-pyrimethamine resistance emerges 1953 Mefloquine resistance emerges 1982 Artemisinin resistance emerges 2009

DANGER ZONE: DRUG RESISTANCE THAILAND, CAMBODIA Artemisinin-combination therapies (ACTs) were the great hope of malaria control, as the parasite has steadily become resistant to older drugs. Just a couple of years after ACTs were introduced to this part of Southeast Asia in 2005, drug resistance emerged. Why this area is such a hotspot for drug

INDIA

MYANMAR

WHO: 1,023

WHO: 788

Lancet: 46,970

Lancet: 21,995

resistance is not entirely clear, there seems to be many factors: counterfeit medicines are rife, drugs are available too easily over the counter, and the drugs are sold on their own rather than in combination (which makes it easier for the parasite to develop resistance).

LOFTY AMBITIONS In 2007, Bill Gates’s rallying call for eradication took some malaria experts by surprise. New drugs and insecticide-treated bed nets were helping control the spread of the infection, yet the world was some way off beating the parasite for good. Opinion is divided, especially as many of the goals set by the WHO so far haven’t been achieved.

ZERO

| | | |

| | | | |

| | | |

| | | | |

75 %

| | | | |

Achieved 25% (from 26.6 to 19.8 deaths per 100,000)

| | | |

Achieved 16 million

50 %

| | | | |

1.5 Bn

| | | |

Reduction in cases

| | | |

| | | | |

Achieved 66 million

| | | |

Number of deaths

| | | | |

730 M

| | | | |

Achieved 100 million

| | | |

Reduction in death rate

Diagnostic tests

247 M

| | | | |

Bed nets needed

Drugs distributed

TARGET BY 2015:

TARGET BY 2010:

Can these targets be achieved?

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Feachem, R. G. A. et al. Lancet 6736 (10), 61270–61276 (2010). /Roll Back Malaria Progress & Impact Series: Report 8 (WHO, 2011). /www.vivazmalaria.com/Gates Foundation/WHO.

VULNERABLE GROUPS

Cambodia is cracking down on counterfeit malaria drugs and has outlawed artemisinin monotherapies.

DRUG DEVELO PMENT

Holding out for reinforcements Signs of emerging drug resistance are turning the hunt for new malaria treatments into a race against the clock. BY MICHAEL EISENSTEIN

he first signs of trouble were subtle. Several patients living near the ThaiCambodia border had received first-line malaria treatment of artemisinincombination therapy (ACT), but failed to clear the Plasmodium parasites from their blood. Their doctors were flummoxed. “There were these sentinel sites reporting reduced efficacy of ACTs,” recalls Arjen Dondorp, deputy director of the Mahidol-Oxford Tropical Research Unit in Bangkok. Dondorp and his colleagues suspected drug resistance. “But it was always uncertain, and could potentially be ascribed to the drugs not being taken properly or underdosed.” In 2006, they started to investigate the likelihood of ACT resistance more thoroughly. The stakes are high. The emergence of drug resistance has already rendered once-effective malaria treatments — chloroquine and sulfadoxine-pyrimethamine — less reliable. Today, ACTs are the weapon of choice against malaria, and the possibility of losing them has the research community scrambling to

understand the situation and to develop new drugs as reinforcements or even replacements.

CRACKS IN THE ARMOUR

Artemisinin is a molecule extracted from the sweet wormwood plant Artemisia annua. In its natural form, it is degraded in a matter of hours within the body. Treatment requires a weeklong course — a demanding regimen that can fail if patients do not complete it. Clinical researchers overcame the limitations of natural artemisinin by developing more robust derivatives, such as artesunate or artemether, and coupling them with partner drugs such as mefloquine or lumefantrine. The World Health Organization (WHO) recommends five such combinations for distribution to endemic malaria regions. They have, in general, proven successful, ridding most patients of the immature blood-stage parasites after only 3 days. Resistance has only recently become the biggest concern with ACT. For years, the primary issue was uncertainty with supply. Production depended on A. annua agriculture, and drug

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prices were prone to dramatic fluctuations. “The average price for 1 kilogram of highquality artemisinin spiked to US$1,000 in 2005 due to a shortage, but dropped to around US$195 in 2007 due to overproduction,” says Tue Nguyen, vice president for research and preclinical development at OneWorld Health, a non-profit organization in South San Francisco, California. “The current price is around US$450/kg and increasing.” Nguyen’s organization is leading efforts to counter this volatility, partnering with Amyris — a synthetic-biology company in Emeryville, California — and French pharmaceutical giant Sanofi to produce bulk quantities of artemisinic acid, an artemisinin precursor, using geneticallyengineered yeast. In 2012, Nguyen anticipates making 9,000 kg of artemisinin — enough for millions of doses — at an initial price of US$400/kg; in 2013 they expect to quadruple production. Stabilizing supply of artemisinin should help reduce some of the volatility in the cost of ACTs and smooth the availability of drugs. Nevertheless, a stable supply will be of little comfort to patients in Southeast Asia facing potential treatment failure. Several independent investigations by Dondorp and Harald Noedl of the Medical University of Vienna have confirmed the emergence of ACT resistance in the region1, and the problem appears to be spreading. “There was a dramatic slowing of parasite clearance,” says Dondorp. “ACTs are starting to fail in western Cambodia, but what may be even worse is that this phenotype has also arrived in western Thailand, at the Thai–Myanmar border.” In some communities, a standard ACT course no longer stops malaria in up to half of the patient population. This resistance is specific to artemisinin, but it could eventually lead to resistance to partner drugs too. Southeast Asia has incubated treatmentresistant malaria in the past (see The numbers Game, page S14). With both chloroquine and sulfadoxine-pyrimethamine, resistance spread to the Indian subcontinent and eventually Africa. With ACT, “So far, we have not seen any signs of resistance outside of Southeast Asia,” says Pascal Ringwald, coordinator of the Drug Resistance and Containment Unit within the WHO’s Global Malaria Programme, which is closely monitoring reports of potential malaria resistance. Unfortunately, the basis of this resistance remains unclear. “We have screened 185 different P. falciparum isolates — mostly from Southeast Asia — and they are all extremely sensitive to the artemisinins in vitro,” says Xinzhuan Su, chief of the Malaria Functional Genomics section at the National Institute of NATURE.COM Allergy and Infectious The latest research Diseases in Bethesda, on artimisinin Maryland. Nevertheless, resistance: many of these strains go.nature.com/cteys2

©WHO/SONNY KRISHNAN

OUTLOOK MALARIA

MALARIA OUTLOOK show resistance to artemisinin in malaria patients. A study published in April 2012 identified a segment of the P. falciparum genome that appears to contribute to reduced parasite clearance in patients from Cambodia and Thailand, although further analysis will be needed to identify the relevant genetic variants2. David Fidock, a microbiologist at Columbia University, New York, suspects that the resistance seen in Plasmodium is only a transient survival mechanism, and that genetic mutations that confer robust artemisinin resistance without weakening the parasite’s fitness are yet to emerge. “My personal sense is that it’s very difficult for the parasite to gain stable resistance to artemisinin,” says Fidock. “Lab studies indicate that the initial gain of drug tolerance is readily lost by the parasite after the removal of drug pressure.” Furthermore, many scientists think that artemisinin works in a nonspecific manner: as Plasmodium feeds on haemoglobin, iron is released that activates artemisinin, causing extensive chemical damage to the parasite (see ‘One parasite — many hiding places’).“There’s probably not a specific target on the parasite that the drug is attacking that could mutate to prevent inhibition,” says Fidock. Even without a clear cause, the problem is undeniable, and the WHO has produced the Global Plan for Artemisinin Resistance Containment (GPARC), now being implemented across Southeast Asia. GPARC pairs surveillance with improved clinical practice: catching malaria early and ensuring complete elimination of Plasmodium. “We’re focusing on the success of diagnostics and treatment,” says Ringwald, “and using very effective first-line drugs, mosquito control, lots of advocacy and research into new operational strategies to kill off reservoirs of the parasite.” New drugs would also help. One of the most promising is a compound known as OZ439, which differs structurally from the artemisinin drugs, but retains the endoperoxide chemical group that is crucial to their success. OZ439 outperforms artemisinin in an important regard. “It has a half-life that has never been seen for an artemisinin derivative,” says biochemist Sergio Wittlin of the Swiss Tropical and Public Health Institute (Swiss TPH), who helped discover the compound. “Natural products have half-lives in the range of 1 hour, but this has a half-life of 20 hours in orally dosed rats, which also held true in a phase I trial.” The drug is currently in phase II trials, and hopes are high that OZ439’s prolonged existence in the bloodstream, paired with its novel structure, will make it effective where ACTs are failing.

NOT SO NEGLECTED

The past decade has seen a surge in investment in antimalarial drug discovery. According to a report by the Bill & Melinda Gates Foundation’s Global Funding of Innovation for Neglected Diseases (G-FINDER) programme, 2010

ONE PARASITE — MANY HIDING PLACES Plasmodium falciparum and P. vivax, the parasite species primarily responsible for malaria in humans, have complicated lifecycles. Both parasites have multiple developmental stages, each with its preferred target cell type in humans or mosquitoes, making it a challenge to eliminate infection — especially for the hibernation-prone P. vivax. However, scientists are ﬁghting back, by expanding the arsenal of antimalarial drugs (in white boxes).

• Chloroquine

3 2

Sporozoites infect liver cells

1 Female mosquito injects sporozoites into human blood while feeding

Liver cell ruptures, releasing thousands of merozoite-stage parasites into bloodstream

Liver cell

Merozoites infect red blood cells

Red blood cell

• Artemisinins/ 0Z439 • NITD609/ Spiroindolones

Methylene blue

For P. vivax, parasite remains dormant as hypnozoite for weeks or even years

• Primaquine • Tafenoquine

Parasites divide and infect new red blood cells

Some merozoites mature into gametocytes for sexual reproduction

Mosquito takes in gametocytes in infected red blood cells

Human Mosquito

Salivary gland

Gut Midgut wall

8 9

• Sulfadoxinepyrimethamine

Ookinetes become oocysts, yielding sporozoites that migrate to the salivary gland

global spending on malaria research and development (R&D) totalled US$547.2 million, with drug research accounting for the largest component (42%). Nearly a quarter of this R&D investment came from the private sector, an important contribution to what was previously considered a neglected tropical disease. Alongside the Gates Foundation as a driver of this funding boom is the Medicines for Malaria Venture (MMV). With pharmaceutical and biotechnology partners, MMV coordinates distribution of public-sector and philanthropic funds to ensure that money can be rapidly allocated to scientists making progress in malaria drug development at any institution, anywhere in the world. “MMV is so knowledgeable and focused and professional in its efforts — it’s like a virtual drug discovery organization,” says Nick Cammack, who heads medicinal development for GlaxoSmithKline (GSK) in Tres Cantos, Spain. The Tres Cantos campus is devoted to tropical-disease research and a nexus for public–private sector collaboration. “We fund research on a 50:50 basis, with GSK and MMV each contributing

Sexual reproduction occurs in mosquito gut, creating ookinetes, which migrate to the midgut

half, and we have joint objectives to find new molecules for clinical development,” he says. As well as investment and expertise, drug companies engaged in the drive for antimalarial research are also contributing libraries of chemicals — resources once kept under lock and key. Wittlin’s Swiss TPH colleague Matthias Rottmann is part of a multi-institutional collaboration with the Novartis Institute for Tropical Diseases that has worked with one such library. “It was almost 2 million products, basically all the chemicals Novartis has,” says Rottmann. In 2010, GSK surprised many observers by making public the chemical structures and screening data of more than 13,000 molecules with apparent antimalarial activity, identified from its library of roughly 2 million compounds3. MMV has since worked with GSK, Novartis and St Jude Children’s Research Hospital in Memphis, Tennessee, to compile a ‘greatest hits’ collection called the Malaria Box, a freely available sampler of 400 chemicals with demonstrated activity against P. falciparum. According to MMV’s chief scientific officer Tim Wells, this curated collection 2 6 A P R I L 2 0 1 2 | VO L 4 8 4 | NAT U R E | S 1 7

OUTLOOK MALARIA is proving popular. “It became available in the last week of December 2011, and it has already ‘sold out’ its first batch,” he says. Compounds that show promise in these screens are only starting points. “I saw one article that described our findings as ‘13,000 new malaria drugs’, which is an exaggeration to say the least,” says GSK’s Cammack. Each promising lead will need significant optimization work, he adds. Accordingly, although scientists are free to use the Malaria Box as they like, Wells anticipates that most will seek further assistance through MMV or private sector partners to shepherd promising molecules into clinical testing.

MMV is assisting in early stage development of dozens of potential drug candidates. Among the most promising is the synthetic molecule NITD609, one of several spiroindolones isolated in 2010 from a Novartis library4. “They were fairly potent in cellular assays right away, and they were attractive with regard to how long they stay in the bloodstream and their chemical tractability,” says cell biologist Elizabeth Winzeler at The Scripps Research Institute in La Jolla, California, and a corresponding author on the study. NITD609 is now poised for a phase II trial in Thailand. “It has a very good safety profile, and looks very promising,” says

DA N G E R B E LOW T H E R ADAR Targeting the ‘other’ Plasmodium species When people talk about malaria, they’re generally talking about Plasmodium falciparum, the predominant species in Africa and the cause of hundreds of thousands of deaths each year. Plasmodium vivax, more of a problem in Asia and South America, tends to get overlooked, largely because of its reputation for being less lethal. Epidemiologist J. Kevin Baird, of the Eijkman-Oxford Clinical Research Unit in Jakarta, Indonesia, sees this as a serious mistake. “You can still read in current medical textbooks that P. vivax very rarely kills,” says Baird. “But that’s just not so, and there’s very good evidence that it kills patients in the same variety of ways as P. falciparum.” He cites recent unpublished data from his team indicating that P. vivax was the sole parasite found in 28% of malaria deaths in one Indonesian hospital. Plasmodium vivax’s mild reputation is only part of the problem — it’s also hard to study. It infects only blood-cell precursors known as reticulocytes, which are difficult to obtain and vanish quickly in culture. “Vivax can only be cultured on a short-term basis,” says Matthias Rottmann, a biochemist at the Swiss Tropical and Public Health Institute. “A reticulocyte ages very fast: after a few days it’s not a reticulocyte any more — it’s an erythrocyte.” This means drug studies must be performed at sites where P. vivax is readily available, and makes genetic manipulation of the parasite virtually impossible. Furthermore, it has a nasty tendency to hide. P. vivax enters a dormant stage — a hypnozoite — in the liver, lingering for months before re-awakening. “We know nothing about hypnozoite metabolism,” says Elizabeth Winzeler, a cell biologist at The Scripps Research Institute in La Jolla, California. “We don’t know what targets would be useful, and we have no way of validating them.”

Antimalarial drugs that kill blood parasites leave hypnozoites unscathed. A drug called primaquine eliminates P. vivax hypnozoites, but can be profoundly toxic for people with a mutated form of the G6PD gene. In a cruel irony, such mutations are widespread in malarial regions — and detecting them requires clinical resources that can be scarce in the developing world. “Even here in Jakarta, I would have a hard time finding somebody who could do a G6PD test,” says Baird. A related drug, tafenoquine, now in phase II trials, allows a sharply reduced dosing regimen, lowering the risk of harm for G6PD-deficient patients. Nevertheless, toxicity may still prove an issue, in which case effective use would require improved G6PD diagnostics or better-designed dosing strategies. Lack of access to good experimental models is another limiting factor. Winzeler’s team developed an assay to screen compounds against liver cells infected with a rodent-specific strain of malarial parasite6. This yielded at least one lead, although it has not yet been tested against human hypnozoites. Otherwise, the best models available are rhesus macaques — far from ideal, as monkey models are logistically complicated, expensive and ill-suited to large-scale drug discovery. Fortunately, this once-neglected problem is getting a lot more attention. “The Gates Foundation put quite a lot of money in two years ago, and there are five or six programmes looking at how to improve cell culture,” says Tim Wells, chief scientific officer at Medicines for Malaria Venture. But Baird believes that priority should go to bolstering research efforts in regions where the crisis is most acute. “As difficult and expensive as it might be,” he says, “we have to invest in research sites near where malaria patients live.” -ME

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Rottmann, who has also worked on NITD609. Unfortunately, patient recruitment has stalled owing to events such as catastrophic flooding — problems that have also dogged trials of OZ439. “Now the malaria season is past its peak, researchers at the trial sites will have to wait until next season to finish the phase II trial,” said Wittlin, in January 2012. The onset of the rains in June should enable these trials to get back on track.

SEEKING THE ULTIMATE WEAPON

Although the early data for both OZ439 and NITD609 are encouraging, their efficacy has been demonstrated only against blood-stage parasites. Yet Plasmodium has a complex, multi-stage lifecycle — especially P. vivax, which remains an especially tricky target (see ‘Danger below the radar’). Any therapeutic strategy intended to eradicate malaria must wipe out gametocytes — the stage of the parasite lifecycle that moves from humans to mosquitoes. The ultimate goal for researchers is to design treatments that render a person inhospitable to parasitic infection after one dose: a strategy termed ‘single-encounter radical cure and prophylaxis’. Efforts are underway to pin point vulnerabilities in non-blood-stage parasites, and although no new drugs have unambiguously passed this test, Fidock’s team has obtained surprising data on a very old drug — methylene blue5. “This was the first synthetic compound ever used in humans — in 1891 — and it has very potent activity against both early and late-stage gametocytes, including the ability to block transmission to mosquitoes,” says Fidock. Methylene blue fell into disuse because of its disconcerting tendency to turn urine green and the whites of the eyes blue, but these might be acceptable side effects should the compound prove valuable for parasite eradication. The motivation and resources are clearly present for antimalarial drug discovery and development, but time is a looming problem. “When you talk about next-generation drugs, launch dates are projected for 2018 or 2019,” says MMV’s Wells, “but people are worried about what’s going to happen next year.” For the time being, surveillance and containment strategies are the best hope. “It all comes back to whether emerging ACT resistance might soon leave us with essentially no effective treatments for a period of some years,” says Fidock, “and that’s an unresolved question right now.” ■ Michael Eisenstein is a freelance science journalist based in Philadelphia, Pennsylvania. 1. Dondorp, A.M. et al. N. Engl. J. Med. 365, 1073– 1075 (2011). 2. Cheeseman, I. H. et al. Science 336, 79–82 (2012). 3. Gamo, F.-J. et al. Nature 465, 305–310 (2010). 4. Rottmann, M. et al. Science 329, 1175–1180 (2010). 5. Adjalley, S.H. et al. Proc. Natl. Acad. Sci. USA 108, E1214–1223 (2011). 6. Meister, S. et al. Science 334, 1372–1377 (2011).

MALARIA OUTLOOK AMY MAXMEN

malaria — as a supernatural phenomenon, most mothers nevertheless recognize the progression of joint pain, fevers, vomiting and dehydration that comes with the onset of the disease. That malaria has an inexpensive cure makes these deaths even more devastating, says Grant Dorsey, an infectious disease researcher at the University of California, San Francisco, who works in Tororo for a couple of months each year. “In our clinical trials we’ve monitored well over 5,000 patients with malaria and they all respond to the therapy within a day or two. So my own feeling is that — at least in Africa where malaria is caused by Plasmodium falciparum — no one dies if treated quickly. The obstacle really just becomes about access to care.” Rates of malaria incidence and mortality are falling around the world, thanks in part to the widespread distribution of artemisinin-based combination therapies (ACTs), along with insecticide-treated bed nets. Although such measures have come to Uganda, this country of 33 million people is yet to witness much success in reducing the ravages of malaria. In fact, a study published in 2011 claims that the incidence of malaria in Uganda has, if anything, risen since 2005 (ref. 1). In neighbouring Rwanda, by comparison, malaria incidence dropped by 60% between 2005 and 2010 (ref. 2). Uganda’s tragic failure to abate malaria has numerous political, geographic, economic and social factors — and illustrates the reality that it takes more than scientific breakthroughs and cheap drugs to solve this persistent menace. A child’s grave lies near a hut in Tororo — an all too familiar sight in Uganda.

P UBLIC HEALTH

Death at the doorstep Even a cure is not preventing deaths from malaria in Uganda. Poor education and limited access to healthcare are among the reasons why. BY AMY MAXMEN

ary Nyaburu recalls how, several years ago, one of her nine children convulsed in her arms while she waited at the district hospital in Tororo, a rural area in eastern Uganda. The other women waiting at the hospital urged her to go at once and see a traditional healer who could exorcise the evil spirits in possession of her one-yearold baby. Worried, Nyaburu fled the clinic and found a healer who sold her a concoction as useless in the fight against malaria as the

anti-inflammatory paracetamol she had tried earlier that week. The following day, her child could no longer swallow water. Death followed soon after. Many mothers in Tororo have similarly heart wrenching stories. In small villages across Uganda, a child-size grave lies within a few metres of many of the huts. Young children are particularly vulnerable to malaria. And in Uganda, where 1 in 7 children die before their fifth birthday, this mosquito-delivered disease is the biggest killer. Although some villagers interpret seizures — one symptom of severe

BLOOD-THIRSTY SWARMS

Uganda is a victim of its own lush lands. The moist soil, wetlands and great lakes for which the country is celebrated also provide a yearround refuge for the mosquitoes that transmit the malaria parasite. The World Health Organization (WHO) estimates that, in 2010, the country had more than 11 million cases of malaria — the most in Africa — and ranked it fifth in the number of deaths from the disease across the continent. What’s more, Uganda has the world’s highest recorded rate of malaria transmission: reaching 1,586 infective bites per person in 2001 in the swampy Apac district near the Nile River. On average, Tororo’s rate is less than half this level, but still higher than rates recorded within Rwanda (81 bites per person per year), Kenya (120) and Sierra Leone (541). Children in Tororo can expect to catch malaria several times each year. Compounding this problem is a feeble healthcare infrastructure that cannot manage malaria’s toll on the poor. And Uganda is very poor: 81% of its population live in rural areas, where 96% of households lack electricity and 91% do not have access to flushing toilets. If Tororo wasn’t so plagued by Plamodiuminfected mosquitoes, bed NATURE.COM nets might do more for What is the state of villagers, says Abel Kakscience in Africa? uru, a doctor who colgo.nature.com/ylnyfw laborates with Dorsey in 2 6 A P R I L 2 0 1 2 | VO L 4 8 4 | NAT U R E | S 1 9

Tororo. When their study site was in the capital city of Kampala, Kakuru says he saw about one case of malaria every two weeks. “And then we moved to Tororo and although the cohort was half the size, we began to see at least five cases in our study cohort every day,” he says. “The burden here is so heavy that we need multiple interventions, like indoor residual spraying.” But this control measure is too costly to roll out across the entire country — especially in poor places like Tororo. A steady stream of women clutching infants and small children flows through the gates of Tororo district hospital. Some ride in on the back of bicycles or motorcycle taxis called boda-bodas. Those who can’t afford the US$3 ride arrive on foot. Patients will often wait for most of the day to see a doctor, meaning that mothers who accompany sick children also miss a day’s work. Waiting in the hospital is Florence Aketch, a mother with a shy 4-year old boy on her lap. Every few weeks, Aketch travels the 7km to the hospital with either her son, who gets ill with malaria monthly, or his twin sister, who becomes feverish slightly less often. Both twins sleep under a mosquito net, but it doesn’t seem to matter. She reckons that her children get bitten while they eat dinner just after dusk. Even if they dine inside the house, mosquito-proof sealing or window screens would be of little use: in Aketch’s village, straw thatch covers clay or cement huts about the size of a one-car garage. If there’s a door, it is a flimsy piece of wood.

with international and local groups to control malaria in Africa and Asia. The African branch of the consortium is based in Kampala. “We essentially had no ACTs in the country in 2006 and 2007, and we were delayed with deploying insecticide-treated nets. We still have not recovered.” The suspension of international funding can trigger tumultuous cascades in countries with frail infrastructure. When funds froze in 2005, for example, Uganda was gearing up to switch from cheaper, less effective antimalarials to ACTs. At the highest levels of the Ministry of Health, this switch meant that administrators had to draft lots of different documents — contracts with drug suppliers, reports to aid organizations, procurement plans and bidding documents — all to be discussed and signed. At the other end of the chain, vendors in cramped makeshift drug stores on dirt roads needed to be taught about the usage, price and storage of the new antimalarials. Each step requires manpower and money. And when logistics don’t flow — as when the staff at large pharmacies have neither the time nor the training to predict demand and

ABANDONED INFIRMARIES

IN SHORT SUPPLY

Public hospitals in Uganda offer ACTs free of charge. The private sector dropped the price of drugs to about US$1.30 for a 3-day course, thanks to the Affordable Medicines Facility for malaria (AMFm), a programme run by one of the largest malaria-control funders, The Global Fund to Fight AIDS, Tuberculosis and Malaria. The AMFm subsidies have helped slash the cost of malaria treatment in Uganda and five other African countries. However, cost is incidental when the drugs aren’t available. So-called ‘stock-outs’ have been the rule rather than the exception during the past five years, especially at small public clinics, says David Okumu, a doctor and an administrator at the Ugandan Ministry of Health who coordinates health services in Tororo. Stock-outs began abruptly in 2005 when The Global Fund froze grants totalling US$201 million over allegations that some members of Uganda’s Ministry of Health had misused the money. Although the funds were partially restored within a few months, and despite the fact that The Global Fund and the Ugandan government made arrangements to continue to supply vital drugs, many lives were lost. The supply chain for medicines like ACT “came to a halt”, says James Tibenderana, the African technical director of the Malaria Consortium, a non-profit organization that partners

a steady supply chain and train hospital staff nationwide. In the meantime, doctors continue to treat severe malaria with intravenous quinine — a harsh substance that causes tinnitus, vomiting and vertigo, as well as increasing the risk of cardiac arrest. When Nature Outlook visited public and privately owned pharmacies in Tororo and Kampala in January 2012, ACTs were available. And according to an administrator at Tororo district hospital, they had been for a while. But Moses Kamya, head of the Department of Medicine at Makerere University College of Health Sciences in Kampala, predicts that stock-outs will happen again. As prices continue to drop, sales will rise and could exceed capacity. A big reason for this supply instability is that in Uganda, as in many African countries, privately owned pharmacies dispense ACTs to anyone who can pay for them, even without a prescription. One taxi driver in Kampala told Nature Outlook he swallows ACTs as a prophylaxis whenever he feels slightly sick. “Globally, we are in a precarious situation for ACTs,” says Sonali Korde, an advisor at the US President’s Malaria Initiative based in Washington, DC. If countries mismanage their supplies or sell too many to people without the disease, she warns, stock-outs will happen and people will perish.

Even in the dry season, Uganda’s lush lands provide plentiful ground for mosquitoes to breed.

place appropriate orders — stock-outs occur. And because the drugs expire in a matter of months, they cannot be stockpiled. There are stories of ACTs going to waste in the relatively malaria-free southwestern regions of Uganda, while children in northern places like Tororo die for lack of medicines. Funding bottlenecks, inefficient procurement processes, transportation problems and inadequate stock keeping share the blame for the delay in introduction of ACTs for routine use — which did not happen in Uganda until 2008 (an advancement which regressed in 2009 with more nationwide stock-outs). Likewise, the latest firstline treatment for severe malaria recommended by the WHO is yet to reach the country. Before the Ugandan government can endorse this new intravenous therapy, which consists of the artemisinin derivative artesunate, it must ensure

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Boosting the number and quality of staff in public healthcare would undoubtedly improve Uganda’s situation. A shortage of doctors plagues much of sub-Saharan Africa. Uganda, like its neighbours Rwanda, Kenya, Ethiopia, the Democratic Republic of Congo (DRC) and Tanzania, has at most 1 doctor per 10,000 people, compared to 8 per 10,000 people in South Africa, 27 in both the US and UK, and 64 in Cuba. As a result, long waits for overwhelmed doctors often deter people from seeking medical help until it’s too late. And village health workers, who deliver health education and occasionally malaria medicines, are unpaid volunteers in Uganda — as a result, Kamya says, these positions are often vacant. Some say the issue boils down to money. The son of peasants in western Uganda, Kakuru dreamed of being a doctor from a young age. He says he never tires of watching sick children recover. But he worries that the US$300 per month salary paid to public sector doctors will not be enough for him to provide a better quality of life for his young daughter than he himself experienced. “When we talk about a pay raise, the government just tells us that we have to love our nation,” Kakuru says. Many of his colleagues from medical school have left the country or joined non-governmental organizations (NGOs) that offer US$1,200 per month or more. “My friends have even taken NGO jobs in dangerous places like South Sudan if it means earning a better living,” he says. It is staff retention, not recruitment, that is Uganda’s problem. “There are enough health

AMY MAXMEN

OUTLOOK MALARIA

AMY MAXMEN

MALARIA OUTLOOK

Villagers travel to Tororo public hospital on foot, bicycle or boda-boda (left) where mothers and children wait in long queues to see a doctor.

workers trained in this country, but few stay in the public sector,” says Seraphine Adibaku, the malaria programme manager at the Ministry of Health. “You find healthcare facilities that are closed or struggling with very few staff,” he says. “The President has said that health is a priority area, but usually when it comes to sharing the annual budget, you find it falls short of what well-meaning leaders want to achieve.”

SEEKING SUSTAINABLE ANSWERS

Aid from international organizations is vital to malaria control. The Global Fund, which assembles donations from 54 governments, the Bill & Melinda Gates Foundation and other donors, has disbursed nearly US$162 million to Uganda to fight malaria since 2003. In addition, the US President’s Malaria Initiative has given US$144 million since 2006. Aid has helped buy and distribute insecticide-treated bed nets and ACTs, educate villagers about malaria, train health workers, and spray homes in northern districts with insecticide. However, the projects don’t include supplementing the salaries of doctors as an incentive for them to remain in the public sector. In other African countries, including Rwanda, The Global Fund provides salary ‘top-ups’ for doctors who meet performance criteria determined by its Ministry of Health. Yet the Ugandan government has not asked for this type of support, and The Global Fund has not provided it. Grants from international research institutions can enhance health infrastructure, although only indirectly. Fourteen years ago, Kamya collaborated with Phil Rosenthal, from the University of California, San Francisco, on a study funded by the WHO and the US National

Institutes of Health (NIH); their efforts led to the formation of a non-profit organization called the Infectious Diseases Research Collaboration (IDRC). The IDRC now employs about 200 people, who help run clinical trials, studies and surveys in Uganda. Kamya says that patients who enrol in the trials receive quality care as well as health education, and the hospitals that host the research teams also benefit. “In Tororo, our doctors work as surgeons when they can, and we let the hospital use our “Some people generator when the say that $6 lights go out during billion is a lot operations,” he says. of money, but I Unfortunately, don’t think it’s like the programmes an outrageous operated by internathing to ask for.” tional organizations, grants for research projects must eventually end, often abandoning their local staff and patients. Still, past collaborations have created a cadre of trained investigators: a local resource that did not exist in Uganda when Kamya authored his first scientific paper in 1995. Kamya and other investigators now lead their own studies and train students year round. Nurturing local talent is important from more than simply a resource point of view. “We regularly share data with the Ministry of Health,” he says, “and hearing about the country’s needs from a Ugandan is different than hearing about it from an American.”

GLOBAL PROBLEM; LOCAL SOLUTIONS

The global death toll from malaria is anywhere from 655,000 to 1.2 million (ref. 3). The reasons people die from this preventable and curable disease vary from region to region.

Mountainous terrain in northeast India complicates the distribution of ACTs, whereas conflict in Burma, South Sudan and DRC has destroyed clinics where people might have gone for help. What is common to all the countries with a high incidence of malaria is that their people are poor and have inadequate access to education, drugs, diagnostic tests and doctors. Infrastructure improvements will be essential for malaria elimination, says Rob Newman, director of the Global Malaria Programme at the WHO — so if international aid lapses, health workers and hospitals will still be there. These systems aren’t impossible to build, Newman says, but they do take long-term investments in human resources, logistics, regulation and surveillance. According to the 2011 WHO World Malaria Report, malaria elimination will cost nearly US$4 billion more than the US$2 billion pledged (see ‘The numbers game’, page S14’). However, there are no new sources of funding on the horizon, so meeting this target before 2015 seems unlikely. “Some people say that $6 billion is a lot of money, but I don’t think it’s an outrageous thing to ask for,” Newman says. “Ask someone if they think a person should die because they can’t afford a $5 bed net, a 50-cent diagnostic test and a $1 drug.” Although sophisticated in their simplicity, these things mean nothing without practical ways to get them into the hands of mothers in distress. ■ Amy Maxmen is a freelance science journalist in New York. 1. Okiro, E. A. et al. BMC Medicine 9, 37 (2011). 2. US President’s Malaria Initiative, Malaria Operational Plan, Rwanda Fiscal Year 2011. 3. Murray, C. J. et al. Lancet 379, 413–431(2012). 2 6 A P R I L 2 0 1 2 | VO L 4 8 4 | NAT U R E | S 2 1

OUTLOOK MALARIA P E RS P ECTIVES

The missing pieces Nine experts give their opinion on the ‘known unknowns’ in malaria research.

Burnet Institute, Melbourne, Australia. Parasite development goes through several distinct stages in both the human host and mosquito (see ‘One parasite — many hiding places’, page S17). After repeated infections with Plasmodium falciparum or P. vivax, the two main causes of malaria, people do eventually develop effective immunity that prevents symptomatic and severe illness and controls the blood-stage infection. This observation has long provided a strong rationale for malaria vaccine development, yet we know remarkably little of how malaria immunity works in naturally exposed individuals. Our limited knowledge of both the key molecular targets and the specific immunological mechanisms has severely constrained vaccine development. What we do know is that protective immune responses predominantly act against the blood stages of Plasmodium parasites and have multiple targets and effector mechanisms. Although immune responses also develop against liverstage parasites and transmissible forms of the parasite (gametocytes), even less is known about their nature and relevance. Malaria research needs to establish the relative significance of the many known or predicted antigens. It should focus on

A malaria parasite in the liver stage of its complex lifecycle.

ROGERIO AMINO & ROBERT MÉNARD Identify the critical antigens Unité de Biologie et Génétique du Paludisme, Institut Pasteur, Paris. The symptoms of malaria arise only after Plasmodium has left the hepatocytes (liver cells) and infected red blood cells — or erythrocytes. Therefore, an important goal is to develop an efficient vaccine against the pre-erythrocytic (PE) stages of the parasite — the sporozoites injected into the skin by the mosquito and the parasite forms that develop inside hepatocytes. Live PE parasites attenuated by irradiation or gene inactivation are known to provide solid protection against infection; however, their use as vaccines for humans in endemic areas faces major technical and logistical limitations (see ‘The take-home lesson’, page S24). The subunit vaccines now in clinical trials, which are based on sporozoite antigens, have shown limited efficacy. Clearly, other vaccine candidates must be identified. The biggest research need is to identify the right antigens among the thousands expressed by PE parasite stages. Studies of the attenuated parasite vaccine in rodents indicate the crucial protective role of a certain type of immune cell, CD8+

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T cells, which detect and destroy hepatocytes infected by the malaria parasite. A functional assay that exploits this ability of CD8+ T cells would help identify such antigens. Recent technical developments, such as transcriptomics and proteomics to identify PE antigens, expression cloning procedures to catalogue the many antigens found, and new imaging technologies, make possible systematic screens to find antigens capable of eliciting protective immunity.

ANDY WATERS Focus on the ookinete Institute of Infection, Immunity and Inflammation, University of Glasgow, United Kingdom. Female Anopheles mosquitoes transmit the malaria parasite. In the mosquito gut, male and female gametocytes fuse to create a zygote, which develops into an ookinete. The ookinete crosses the mosquito midgut wall and implants, where it continues to develop. A successful ookinete has overcome tremendous odds to survive, and it exists in a hostile environment, surrounded by human blood cells that are being enzymatically digested. It is here in the mosquito gut that the Plasmodium parasite suffers the greatest proportional loss of numbers of any stage of its lifecycle, making it an interesting target for blocking transmission. In 1911, Ronald Ross (who won a Nobel prize for his work linking malaria transmission to the Anopheles mosquito) recognized that, in many endemic settings, malaria dies out if transmission rates drop below a certain level. Although transmission is dropping in Africa, mainly thanks to insecticide-treated bed nets, we need additional measures to accelerate that trend. An increased understanding of ookinete biology would help this effort, including the identification of virulence factors critical to its survival, and the discovery of elements that could stimulate an innate immune response in the mosquito. Logically, targeting the weakest link in the Plasmodium lifecycle will be the best way to develop new vaccines, drugs

SEATTLE BIOMED

BRENDAN S. CRABB & JAMES G. BEESON Unravel natural immunity

defining the mechanisms that clear or prevent infection, and should be complemented by studies into how malaria immunity is acquired and maintained as well as how the parasite evades the immune response. To help achieve these goals, government and private funding agencies must build research capacity in malaria-endemic countries, which will help promote greater linkages between immunology research, population studies and clinical trials. Understanding the basis of human immunity will be key to developing long-lasting malaria vaccines, and will also enable us to identify populations at highest risk and to monitor those populations in malaria elimination programmes over time.

MALARIA OUTLOOK or vector-control methods that will further reduce the passage of the parasite through the mosquito and hasten its extinction.

ELIZABETH A. WINZELER Don’t ignore vivax University of California, San Diego, School of Medicine, La Jolla. Malaria caused by P. vivax is more widespread than that caused by P. falciparum — it endangers up to 40% of the world’s population. Indeed, of the two parasites, most scientists agree that the malaria caused by P. vivax will be the more difficult to control and eliminate: infections are trickier to detect; the parasite can hide as a hypnozoite (a dormant form) in the liver and cause relapses years after initial infection; and there are fewer tools available to study P. vivax. The only licensed drug that is able to eliminate P. vivax hypnozoites is primaquine. This drug needs to be given at low doses over 2 weeks because of its potentially lethal toxicity to individuals who are deficient in the metabolic enzyme glucose-6-phosphate dehydrogenase. Parasite resistance to primaquine is suspected but, within an endemic region, there is currently no accepted method to distinguish relapses caused by drug failure from those caused by reinfection. Furthermore, ex-vivo assays cannot accurately assess resistance because primaquine becomes active only after the liver metabolizes it. Although it is widely agreed that we need a new drug to replace primaquine, there are no established anti-hypnozoite targets, nor are there accepted screening methods that do not involve non-human primates. These are crucial gaps for researchers to address.

MATS WAHLGREN Tackle severe malaria Department of Microbiology, Tumour and Cell Biology Karolinska Institutet, Stockholm. Improvements in supportive care, including adjunct drugs, could lower the fatality rate for the 10 million or so individuals who develop severe malaria each year. The critical factor in malaria pathogenesis is the obstruction of blood-flow that results when parasitized red blood cells block the

human microvasculature and cause inflammation. Indeed, in patients with cerebral malaria, the level of vascular obstruction correlates directly with the depth of coma. For severe P. falciparum malaria cases, death rates remain high in spite of the availability of anti-parasitic drugs (artesunate — derived from artemisinin), intravenous fluids and state-of-the-art intensive care. No adjunctive treatment has been shown to be of benefit in severe malaria and there are few clinical data regarding the optimal supportive care of patients in the early stages of their hospitalization. In patients with severe malaria, the majority of deaths occur within the first 48 hours of hospitalization. It is therefore crucial for the physician to be able to ‘unstick’ the infected cells and restore blood-flow without delay. Research that helps us understand the molecular details of this process is vital because it will allow for the development of novel anti-adhesive and anti-inflammatory strategies. Questions such as ‘how does the parasitized red blood cell bind in the humanmicrovasculature’ and ‘how do these cells block the capillaries and cause inflammation’ need to be answered.

DAVID A. FIDOCK Define resistance Departments of Microbiology & Immunology and of Medicine, Columbia University Medical Center, New York. Recent evidence for the emergence of P. falciparum parasite resistance to derivatives of the antimalarial artemisinin suggests that in a few short years we may be faced with the loss of this vital drug (see ‘Holding out for reinforcements’, page S16). Despite intense efforts to discover and develop new antimalarial agents, no suitable alternative is ready to replace artemisinin. How can the research community best help, at a time of shrinking science budgets? Understanding the biological features of artemisinin tolerance or resistance is key to defining molecular markers to monitor its spread, and to developing therapeutic strategies that effectively treat drug-resistant strains. This will require an exceptional level of sharing of reagents, technologies and knowledge, as the genetic and molecular basis of decreased parasite susceptibility to this drug is likely to be particularly complex. More research is also required to define how artemisinins work, how this translates into parasite death, and what metabolic pathways enable parasites to withstand drug action. Such investigations will involve the

application of next-generation sequencing, genetic association studies to define candidate loci, faster methods of genetic manipulation of P. falciparum, and metabolomics. Research into mechanisms of resistance also needs to extend to the partner drugs used in artemisinin-based combination therapies and to the new chemical entities that are entering clinical trials. The recent reductions in malaria deaths are very encouraging, but these gains are fragile. We cannot allow malaria to resurge.

SOLOMON NWAKA Harness local knowledge African Network for Drugs and Diagnostics Innovation (ANDI); and Special Programme for Research and Training in Tropical Diseases, World Health Organization, Geneva. About 80% of the populations of Africa, Asia and Latin America rely on local traditional medicines to meet their primary healthcare needs. In some instances, these local solutions have spurred innovation that has transcended national borders to save millions of lives around the world. Take artemisinin, for example. Now the mainstay of malaria control, this drug was derived from centuries-old traditional Chinese medicine. But there is a significant knowledge gap on how best to tap into local knowledge. Research and guidelines are needed to: inform and develop sample collection methods; generate and evaluate data on the efficacy, safety and quality of traditional medicines; and to help us understand how these approaches work. Several discoveries based on traditional African medicines are also in our hands, but these are in desperate need of translation into usable products. This is true not only for treatment and control of malaria, but for a variety of other diseases. Our work with the African Network for Drugs and Diagnostics Innovation (ANDI) and by other groups suggest that what is needed are coordinated mechanisms and investment to support the translation of local knowledge into affordable, safe and accessible products. Sustainable solutions can be realized only if appropriate regulatory and policy frameworks are established to guide research, development, production and use of such medicines. ■ Elizabeth A. Winzeler and Mats Wahlgren declare conflicts of interest: go.nature.com/spwwfg 2 6 A P R I L 2 0 1 2 | VO L 4 8 4 | NAT U R E | S 2 3

JOHN STANMEYER/VII/CORBIS

OUTLOOK MALARIA

Mosquitoes bred for vaccine research are reared in cardboard cups.

VAC C INES

The take-home lesson The nearly century-long search for a malaria vaccine might end in the bottom of a cup. BY SARAH DEWEERDT

akeout containers — cardboard cups of the sort used to bring home soup from the deli — are the surprisingly low-tech housing in which Seattle BioMed rears 20,000 to 30,000 malaria-carrying mosquitoes each week. At the nonprofit research institute in Washington, dozens of containers line the shelves of its humid walk-in incubator, mosquitoes clinging upside-down to mesh inserts. There are similar set ups in labs around the world: mosquitoes biding time, before being encouraged to gorge on human volunteers, or have their parasite-laden salivary glands dissected by a deft lab technician. Although the first partially effective malaria vaccine might be only a few years away from clinical use, the search for a truly effective vaccine is far from over. The problem is the complexity of malaria parasites — single-celled organisms belonging to the genus Plasmodium. The parasite has more than 5,000 genes — 50 times more than some viruses, for example — and has a multiphase lifecycle that means it shows a different side to the immune system at different stages of its development. “It’s difficult to see which of the tsunami of immune responses that are provoked by the parasite are associated with protection,” says Robert Sauerwein, head of the Centre for

Clinical Malaria Studies at Radboud University in Nijmegen, the Netherlands. Without knowing which immune response a vaccine needs to stimulate, researchers can only guess. Immunology is not the only uncertainty: producing vaccines practically and in large volumes is another hurdle. Malaria researchers “know how to make a protective malaria vaccine, but don’t know how to manufacture it,” says Stefan Kappe, malaria programme director at Seattle BioMed. “And the vaccines we know how to manufacture don’t protect that well.”

BITS AND PIECES

A number of malaria vaccine candidates have entered clinical trials. The furthest along, known as RTS,S, is about 50% effective against the disease. It’s a subunit vaccine which presents to the immune system the circumsporozoite protein (CSP), a molecule that studs the surface of the sporozoite stage of Plasmodium falciparum. It’s the sporozoite that passes from the mosquito to the human host (see ‘One parasite — many hiding places’, page S17). Investigators in seven African countries have enrolled more than 15,000 infants in a phase III clinical trial of RTS,S. In late 2011, they reported the first set of results from 6,000 of these infants. Those who received RTS,S had 56% fewer malaria episodes and 47% fewer cases of severe malaria over the course of a year

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compared to controls1. “This is the first vaccine that has proved substantial efficacy,” says Didier Leboulleux, the Ferney, France-based director of the Clinical Unit at the PATH Malaria Vaccine Initiative (MVI), a nonprofit that is developing RTS,S in partnership with pharmaceutical giant GlaxoSmithKline. If initial results hold up, RTS,S could be rolled out for clinical use in 2015. But, Leboulleux acknowledges, “It’s clearly a first-generation vaccine.” Most investigators agree that eradication of malaria will require a vaccine that can prevent the disease 80–90% of the time. One approach to developing a more effective vaccine is to try different vaccination strategies using CSP. “We’re trying to milk that antigen for as much as we can with things like prime–boost approaches,” says Ashley Birkett, director of research and development at PATH MVI. Path’s prime-boost strategy delivers the same antigen in two different ways in order to improve the immune response. Another possibility might be to add more antigens to the vaccine. For example, researchers have tested vaccines based on merozoite surface protein 1 (MSP1) and apical membrane antigen 1 (AMA1), each of which is expressed by the parasite during the blood stage of its lifecycle. These antigens have shown poor effectiveness on their own, but adding them to RTS,S might

SEATTLE BIOMED/KAPPE LAB

MALARIA OUTLOOK improve the chances of generating a protective immune response, reckons Christopher Plowe, professor of medicine at the University of Maryland in Baltimore. Other researchers are not as confident. “It will be very hard to reach that 80–90 percent efficacy with a subunit vaccine,” says Sauerwein. He argues that a whole-organism vaccine, which presents the immune system with hundreds of parasite antigens, is a better bet. “Everybody makes his or her own cocktail of immune responses, eventually leading to protection,” he adds. There is precedent for a whole-organism approach. In the 1970s, researchers showed that people bitten by irradiated malaria-carrying mosquitoes gained protection from the disease. The radiation-weakened sporozoites couldn’t cause malaria, yet were up to 90% effective at inducing immunity. But it took up to 1,000 mosquito bites to generate this protection — hardly a practical approach for a mass-vaccination campaign. “Nobody thought it would be possible to make a whole-sporozoite vaccine,” says Stephen Hoffman, chief scientific officer of the Rockville, Maryland-based biotech firm Sanaria. Hoffman, formerly director of the malaria programme at the US Naval Medical Research Center, founded Sanaria in 2003 to try to counteract that pessimism within the malaria community. The company developed a method that involves freezing sporozoites painstakingly dissected by hand from the salivary glands of mosquitoes. But standard intradermal or subcutaneous injection of irradiated sporozoites has failed to protect humans against malaria2. Intravenous injection might work better. Monkeys inoculated intravenously generated high levels of CD8+ T cells in their livers specific to the malaria parasite — a key component of immunity to the disease, Hoffman says. Sanaria, in collaboration with the Vaccine Research Center, part of the US National Institutes of Health, has entered the vaccine into clinical trials, with results expected in late 2012.

IN THE GENES

“Whoa,” says Seattle Biomed’s Kappe, sucking in his breath, “giving intravenous vaccinations to children in Africa is difficult to envision.” Seattle BioMed has a different strategy for a wholeorganism vaccine. Instead of using radiation to weaken the parasites, Kappe favours a genetic engineering approach: knocking out selected genes so that sporozoites can infect liver cells and trigger the immune system, but are unable to progress to the blood stage of their lifecycle and cause disease3. Kappe and others say that genetic engineering will weaken the parasites in a more predictable, uniform way to enable vaccination — eventually using a needle and syringe — at a much lower dose than irradiation. Kappe describes an unpublished study, where five of six volunteers bitten by mosquitoes infected with sporozoites lacking the p36 and p52 genes developed robust

T H E ALT R UI ST I C AP P R OAC H Transmission-blocking vaccines Most efforts at developing a malaria vaccine target the parasite in the liver or blood of the human host. But what if you could give a person a vaccine that would affect parasites inside a mosquito? “It’s odd, yes. I know,” says Rhoel Dinglasan, a professor of molecular microbiology and immunology at Johns Hopkins University in Baltimore, Maryland. And yet, despite the oddness of the idea, there’s growing interest in ‘transmissionblocking vaccines’ as this approach is called. Such a vaccine would be like altruism in a syringe: it wouldn’t prevent vaccine recipients from contracting malaria, but it would prevent any mosquito that bites them from passing the parasite to someone else. Researchers have tried vaccines based on a variety of proteins — P25, P28, P230, to list a few — that are produced by the parasite inside the mosquito. But it has often been difficult to manufacture these proteins in sufficient quantities and, in a few cases, the vaccines have caused serious side effects4. Dinglasan is taking a different approach. The vaccine he is working on is based on a mosquito antigen — a molecule called AnAPN1, found on the surface of cells lining the mosquito’s gut that serves as a kind of docking station for malaria parasites5. The theory is that a person injected with an AnAPN1 vaccine will make antibodies to the molecule, which a mosquito then imbibes along with the parasites in its blood meal. Once in the mosquito gut, the antibodies

immune responses. But the sixth got malaria from the vaccine — unacceptable odds when it comes to safety. Kappe plans to test lower doses of this genetically weakened parasite, and is developing another strain of sporozoite with other genes missing. He hopes that additional safety mechanisms could be engineered in, such as a suicide gene that would cause the parasite to selfdestruct if it did reach the blood stage. Sauerwein is also preparing genetically engineered parasites and, in parallel, is investigating an even more radical approach: immunizing with live Plasmodium parasites that aren’t weakened at all. Ten people exposed to malariacarrying mosquito bites and dosed with the antimalaria drug chloroquine to protect them from getting sick, all developed immunity to the disease; some remained protected for as long as 28 months (ref. 3). Sauerwein’s radical approach “gives the best protective immunity of any malaria intervention ever” — even better than those experiments with irradiated parasites that were the benchmark for the field for decades, says Hoffman, who is

block AnAPN1 and prevent the malaria parasites from latching on and entering the cells. The vaccine is at least a year or two away from being tested in humans. So far, Dinglasan’s team has had encouraging results in rabbits. Interestingly, as well as inhibiting Plasmodium falciparum, the antiAnAPN1 antibodies can block P. vivax, a species of malaria parasite that is common outside Africa. Very little vaccine research targets P. vivax, so the possibility of a double-strike is especially tantalizing. “This is something that evolution has given us as a gift,” Dinglasan says, “and we’re lucky enough to have found it.” SD

Target: the parasite (green) in the mosquito gut.

collaborating with Sauerwein on related work. He suggests Sanaria’s method for manufacturing sporozoites could make this type of controlled infection a feasible approach to vaccination, at least for military personnel and travellers to malaria-endemic regions. Sauerwein and Hoffman are not alone in trying multiple approaches — many researchers in this field are cooking several leads for a malaria vaccine at the same time. Kappe, for example, muses on the possibility that, rather than make it all the way to an approved vaccine, his engineered parasites will simply reveal promising antigens to include in a next-generation subunit vaccine. For now, nothing is off the menu. ■ Sarah DeWeerdt is a freelance science writer in Seattle, Washington. 1.The RTS,S Clinical Trials Partnership. N. Engl. J. Med. 365,1863–1875 (2011). 2. Epstein, J. E. et al. Science 334, 475 (2011). 3. Roestenberg, M. et al. Lancet 377, 1770–1776 (2011). 4. Wu, Y. et al. PLoS ONE 3, e2636 (2008). 5. Dinglasan, R. R. & Jacobs-Lorena, M. Trends Parasitol. 24, 364–370 (2008). 2 6 A P R I L 2 0 1 2 | VO L 4 8 4 | NAT U R E | S 2 5

V ECTOR CO NTRO L

The last bite Preventing mosquitoes from transmitting the malaria parasite is a crucial piece of the eradication puzzle. B Y L A U R E N G R AV I T Z

osquitoes are the ideal vector — they provide a safe haven for a parasite to reproduce, then inject it directly into the human bloodstream. What’s more, mosquitoes are a moving target. The females of nearly 70 species of Anopheles transmit malaria, biting indoors and out. They have evolved resistance to once highly effective pesticides1, and some species might have even changed their behaviour to bite earlier in the evening when people are less likely to be shielded by bed nets. To achieve a definitive end to malaria transmission, mosquito populations must be controlled. Some countries with low transmission rates, such as Tunisia and the United Arab Emirates, got rid of malaria using vector control, aggressive treatment and stiff monitoring, but in parts of Africa transmission rates are so high that even reducing bite frequency by 99% would still result in around 10 infectious bites per person per year. In these circumstances, elimination means extraordinary levels of control. “You’re talking 99.99% control, which is different from any other pathogen on the planet,” says Gerry Killeen, a mosquito biologist and malaria expert who splits his time between the Liverpool School of Tropical Medicine in the United Kingdom and the Ifakara Health Institute in Tanzania. “It makes flu look difficult to transmit.”

The last big international push to eradicate malaria, spearheaded by the World Health Organization (WHO), ran from 1955 to 1969 and involved spraying insecticides — primarily the newly developed DDT — on the inside walls of houses2. But coverage was inconsistent, high in some countries while low or non-existent in others, and some mosquito populations, such as those that lived in tropical forests, went untouched. This effort managed to eliminate the disease in North America, Europe and parts of Asia, and tamped it down in other regions. Yet by the early 1970s malaria was resurgent on several continents, often reaching epidemic proportions. The WHO campaign was a massive disappointment — and eradication became known as ‘the E word’ in some circles. It wasn’t until after the successful roll out of insecticide-treated bed nets in the 1990s that governments across the world thought it worth trying again. At first glance, the effort appears to be working: the WHO estimates that it has saved more than a million lives since 2000. But more is needed in order to extinguish the last hotspots of malaria transmission.

HISTORY REPEATING

Many researchers in the field believe that eradicating malaria is possible, but it would require a global strategy that employs a variety of insectcontrol techniques. “We’ve seen a vast resurgence

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of interest and investment in malaria, which is attributable to the advent of the treated net,” says Jo Lines, an entomological epidemiologist at the London School of Hygiene and Tropical medicine. As with the 1955 campaign, “The whole business again rests on the effectiveness of the massive scale-up of a single technology.” And once again there are signs of weakness. Lines and others point to insecticide-resistant mosquitoes as a particularly disturbing trend. Pyrethroid insecticides are, by far, the most commonly used pesticide in endemic regions: they’re cheap, long-lasting, effective at both repelling and killing mosquitoes, and safe enough that a baby can suck on a pyrethroidtreated bed net without harm. That optimal profile, however, also means that the family of chemicals has been massively overused and is in danger of becoming ineffective. Similar concerns apply to chemicals besides the pyrethroids. All of the insecticides currently in use for malaria control have come from agriculture; decades of use against bollworms and beetles have had the unintended side effect of exposing mosquitoes to the same chemicals. Now that the mosquitoes are encountering them in people’s homes, resistance is spreading. “The frequency with which we hear new reports of insecticide resistance is increasing,” says Tom McLean, chief operating officer of the Innovative Vector Control Consortium (IVCC), a UKbased non-profit that partners with companies to pursue novel insecticides. It seems that almost wherever an entomologist looks, he adds, “they find insecticide resistance.” The global health community is working on solutions. In the short term, for example, one aim is to repurpose agricultural pesticides: fastdegrading chemicals for crop use are being reformulated to create longer-lasting insecticides for indoor spraying. Yet these are only stop-gap measures. Many malariologists contend that, in order to maintain progress, they need a larger toolbox.

SPRAY AWAY

Vector control should combine a diverse mix of insecticides for indoor spraying and treating bed nets, a range of repellents — both new and old — and novel ways to decrease population sizes of the worst-offending mosquito species3. Researchers are pursuing all strategies. McLean is optimistic about the outcome of IVCC’s projects. “The rate at which we’re finding new chemicals suitable for development suggests we’ll reach our endpoint by around 2020: a whole new set of insecticides with which we can implement mosaics of vector control,” he says. Matthew Thomas, an entomologist at Pennsylvania State University in University Park, has been working on a different type of insecticide. Rather than synthetic pesticides, he’s developing NATURE.COM one based on a fungus, Efforts to control with the aim of making it the mosquito resistance-proof. The fun- continue: gus, Beauveria bassiana, go.nature.com/zu1djn

STEPHEN MORRISON /EPA/CORBIS

OUTLOOK MALARIA

MALARIA OUTLOOK TO KILL A MOSQUITO

Various technologies to control malarial mosquitoes are available or are in development Strategy

Advantages

Disadvantages

Current research aims

Indoor residual spraying

• Deters mosquitoes from entering buildings • Kills mosquitoes that land on walls

• Lasts only 6–12 months • Growing issues of resistance • Needs trained workers • Useless outdoors

• Develop inexpensive and long-lasting insecticides • Develop novel insecticides that act on resistant mosquitoes

Insecticide-treated bed nets

• Deters mosquitoes • Kills mosquitoes that land • Relatively inexpensive

• Last up to 3 years • Growing issues of resistance • Need to be under them indoors

• Investigate long-term • Develop alternative, safe pesticides

Odour-baited insecticide traps

• Reduce outdoor biting

• Can be expensive • Not as useful in rural settings

• Determine most effective structure • Perform cost–benefit analyses

Pesticide applied to water where mosquitoes breed

• Targets species that breed in large, stagnant bodies of water • Hits a different stage of lifecycle, prolonging time to resistance

• Useless against breeding sites in puddles or streams • Must monitor breeding sites • Very expensive and energy intensive

• Develop baited stations and install near breeding pools

Fungus that hobbles and eventually kills mosquitoes

• Works in a different way from current insecticides, so can target resistant mosquitoes

• Not yet field-tested • Only effective in enclosed spaces

• Determine if slow-acting formulations better than fast-acting

Bacterial infection of mosquito to prevent parasite transmission

• Potential long-term control of mosquito populations • Potential to slow transmission of infection

• Requires species-specific Wolbachia strains, introduced separately • Anopheles proven difficult to infect

• Develop ways to infect the common species • Prove transmission of bacteria through mosquito generations

Genetically engineered mosquitoes

• Long-term control of mosquito populations • Slows transmission of disease

• Sheer number of different species • Widespread fear of genetically modified organisms (GMOs)

• Find most effective mutations • Assess whether GMO would integrate into native populations

starts to work upon contact — the spores attach to a mosquito, penetrate its exoskeleton and burrow inside where they grow, multiply, and produce toxins to fend off an immune attack. The mosquitoes can’t fly, eat or smell properly. “It’s like a head cold,” Thomas says. “Ultimately the insect dies, and how quickly that happens, whether 5 days or 2 weeks, depends on the isolate [fungal strain] we choose.” A faster-acting isolate kills a mosquito before she’s old enough to transmit the malaria parasite, which takes 2 weeks to mature. However, the risk is that only the most vulnerable mosquitoes will die whereas more resilient ones will manage to breed and spread resistance. A slower-acting isolate could act as a late-life insecticide: hampering a mosquito’s ability to feed and to transmit the parasite yet allowing her to lay some eggs before she dies, thus limiting selective pressure. “We should rethink how we use insecticides,” says Thomas. “Not that killing quickly is a bad thing, but it increases speed to resistance.” He adds that using the fungal pathogen in combination with other pesticides could further deter resistance by reducing reliance on a single technology.

STEALTH ATTACK

Perhaps the more ambitious of the long-term projects are the ones that aim to change the mosquitoes themselves. Researchers are investigating an assortment of techniques: male sterilization, genetic engineering, even the introduction of bacteria that reduce a mosquito’s ability to transmit the malaria parasite. It’s an uphill battle — because dozens of different species transmit malaria, scientists will need to separately engineer, breed and distribute each kind of modified mosquito. The uphill battle, at least for genetic modification, has an army of industry and academic

researchers on the case. “There are people doing genomics, bioinformatics, population genetics, protein engineering, computational protein design, germline transformation of mosquitoes, modelling, and working with potential field sites,” says Mark Benedict, a molecular biologist and visiting fellow at the University of Perugia in Italy. Benedict is part of an international team that is starting with some conservative modifications — sexually sterile males — and working up to more ambitious plans, such as males that have mostly male offspring. He is developing a strategy to move some of the more promising mosquito strains out of the lab to see if the experimental insects can integrate themselves into natural populations. “The lab experiments are moving very well, very fast. If we had the most powerful, most effective strains in the field in 10 years, I’d be quite happy,” he says. But genetically engineered insects have another hurdle to get over: the widespread fear of genetically modified organisms. Some scientists are trying to circumnavigate that fear by working another angle. Bacteria from the Wolbachia genus can infect mosquitoes and decrease parasite transmission. Moreover, the bacteria are transmitted from one generation of mosquitoes to the next, making the infection self-sustaining. Wolbachia has been successfully introduced into one mosquito species that transmits another problem disease: dengue. Small trials of the infected vectors in Australia look promising, and show that the bacteria-laden insects quickly integrate into native populations. Within 14 weeks of the release of 250,000 Wolbachia-infected mosquitoes, 90% of the populations in the test areas were positive for the bacterium4. Such an approach could work for malaria, too. The trick is finding the appropriate Wolbachia

species — something that has proved very difficult. But entomologist Zhiyong Xi, at Michigan State University in East Lansing, might have found one. He says so far he’s managed to introduce the bacterium into one species that’s prominent in India and the Middle East and has begun promising efforts with three African species. Xi says he is just beginning talks with India’s National Institute of Malaria Research about testing the relevant mosquitoes. Of course, by the time modified mosquitoes are ready for release, any number of advances could be available, including an anxiously awaited vaccine (see ‘The take-home lesson’, page S24). Environmental change will also affect the malaria burden. Land development has removed the forests that shelter large mosquito populations. More modern-style houses, with tin roofs instead of mud — ideally with screens on the windows and doors — to prevent mosquitoes from entering in the first place. The future of mosquito control must ultimately consist of a mixture of current technologies combined with both concentrated science and social development (see ‘To kill a mosquito’). Insecticide-treated nets and indoor spraying have helped enormously, “but there’s a limit to what they can do,” says Killeen. In Tanzania, thanks to these measures, “malaria has crashed down to levels that are normal in other areas. Even at those levels, it’s still a major public health problem.” ■ Lauren Gravitz is a freelance science journalist in Los Angeles, California. 1. Greenwood, B. et al. J Clin Invest. 118, 1266 (2008). 2. Nájera, J. A., González-Silva, M. & Alonso, P. L. PLoS Med. 8, e1000412 (2011). 3. Ferguson, H. M. et al. PLoS Med. 7, e1000303 (2010) 4. Hoffman, A. A. et al. Nature 476, 454–457 (2011). 2 6 A P R I L 2 0 1 2 | VO L 4 8 4 | NAT U R E | S 2 7

WORLD VIEW

A personal take on events

V. FLORESCU

Global health hits crisis point

The Global Fund’s drive to ensure sustainability and efficiency means that it may not be able to meet its commitments to combat disease, says Laurie Garrett.

ast week, Michel Kazatchkine tendered his resignation as executive director of the Global Fund to Fight AIDS, Tuberculosis and Malaria. Regardless of whether you’ve heard of the French AIDS scientist, or even of the fund, you should keep reading. This is a crucial, dangerous moment for global health. Kazatchkine made clear the political struggle that forced his resignation. “The Global Fund has helped to spearhead an entirely new framework of international development partnership,” he wrote in his resignation letter. But under stress during the world economic crisis, with radically declining support from donors, a battle developed. “Today, the Global Fund stands at a cross-road. In the international political economy, power-balances are shifting and new alignments of countries and decision-making institutions are emerging or will have to be developed to achieve global goals. Within the area of global health, the emergency approaches of the past decade are giving way to concerns about how to ensure long-term sustainability, while at the same time, efficiency is becoming a dominant measure of success,” he wrote. It is almost possible to hear Kazatchkine spitting out the words ‘sustainability’ and ‘efficiency’. Since the financial crisis of November 2008, a storm has been brewing over these concepts, one that affects everything from humanitarian responses to projects that distribute malaria bed nets. It is a fight, and on one side are those who believe that crises in general, and the AIDS pandemic and allied diseases in particular, constitute global ‘emergencies’ that must be tackled with full force, mistakes be damned. On the other are those who feel that AIDS is now a chronic disease that can be managed with medication and therefore requires investment in permanent infrastructure of care and treatment that can eventually be operated and funded by the countries themselves. It is a classic battle of titans, pitting urgency against long-term sustainability. In his resignation letter, Kazatchkine essentially conceded victory to the forces for sustainability. Charitable urgency didn’t stand a chance once the donor states started cinching their domestic budget belts so tightly that they had to punch new buckle holes. The fund was established ten years ago as a unique mechanism to move billions of dollars from rich countries to poorer ones, to combat and treat three infectious diseases: HIV, malaria and tuberculosis. It acts as a granting agency, accepting applications from governments and health organizations, and convenes regular replenishment meetings to tell donors — mostly the governments of the United States, United Kingdom, France and Germany — how much money is needed for the next round. NATURE.COM By the end of 2009, the fund was disbursing Discuss this article US$2.7 billion a year, and was underwrit- online at: ing almost half of all HIV treatment in poor go.nature.com/ge9til

countries, about two-thirds of all malaria prevention and treatment in the world and about 65% of all tuberculosis efforts. The fund’s most marked impact has been on malaria. At the end of 2011, the World Health Organization estimated that the number of malaria deaths had fallen by one-quarter between 2000 and 2010. But Global-Fund cash has spawned dependency and expectation among its recipients. Should it disappear, or radically diminish, countries would be hard-pressed to finance malaria and tuberculosis efforts. Indeed, the great diminishment has commenced. In October 2010, the fund asked donors for $20 billion for five years’ worth of disbursements. The donors were indignant and committed just over half that. In response, the fund’s flabbergasted leadership cancelled the next grant round, and it will now not distribute new grants until 2014. Donor scrutiny increased and a high-level independent review panel set up by the fund’s governing board, which includes representatives of United Nations agencies and the World Bank, released a scathing report, citing a litany of problems, including fraud, theft and inconsistent decision-making by grant reviewers. At a meeting in Accra, Ghana, on 21 November, the board members expressed shock at the problems identified by the high-level panel, and by reports commissioned on the situation on the ground in some countries. Some African leaders described riots and demonstrations at the lack of vital medicines, especially for HIV. The board’s own investigation showed that the fund had committed assets of $10 billion for 2011–13, but had only about $4 billion in its bank accounts. The board called for ways to stretch available resources and eliminate inefficiencies. Key to that would be the appointment of a general manager to oversee all spending, pushing Kazatchkine aside. Stepping into that position is Colombian banker Gabriel Jaramillo. To try to give Jaramillo a running start, in Davos, Switzerland, last week, Bill Gates handed over some $750 million, redeemable by the fund in full during 2012, or spread out over time. And the Saudi Arabian government announced a $25-million donation. As generous as these millions may be, the fund needs billions just to stay alive and fulfil country grants, let alone to grow. Right now we have no idea where that money will come from. Should the fund collapse, the consequences will be severe. Progress against tuberculosis and malaria will stall, and more than a million people living with HIV could be left without treatment. ■

GLOBAL-FUND CASH HAS SPAWNED

DEPENDENCY AND EXPECTATION AMONG ITS

RECIPIENTS.

Laurie Garrett is senior fellow for global health at the Council on Foreign Relations in New York, recipient of the 1996 Pulitzer Prize in Journalism and author most recently of I Heard The Sirens Scream: How Americans Responded to the 9/11 and Anthrax Attacks. e-mail: lgarrett@cfr.org

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A WORLD WITHOUT

MOSQUITOES

wIld wonders of europe/sMIt/nAturepl.coM

very day, Jittawadee Murphy unlocks a hot, padlocked room at the Walter Reed Army Institute of Research in Silver Spring, Maryland, to a swarm of malaria-carrying mosquitoes (Anopheles stephensi). She gives millions of larvae a diet of ground-up fish food, and offers the gravid females blood to suck from the bellies of unconscious mice — they drain 24 of the rodents a month. Murphy has been studying mosquitoes for 20 years, working on ways to limit the spread of the parasites they carry. Still, she says, she would rather they were wiped off the Earth. That sentiment is widely shared. Malaria infects some 247 million people worldwide each year, and kills nearly one million. Mosquitoes cause a huge further medical and financial burden by spreading yellow fever, dengue fever, Japanese encephalitis, Rift Valley fever, Chikungunya virus and West Nile virus. Then there’s the pest factor: they form swarms thick enough to asphyxiate caribou in Alaska and now, as their numbers reach a seasonal peak, their proboscises are plunged into human flesh across the Northern Hemisphere. So what would happen if there were none? Would anyone or anything miss them? Nature put this question to scientists who explore aspects of mosquito biology and ecology, and unearthed some surprising answers. There are 3,500 named species of mosquito, of which only

a couple of hundred bite or bother humans. They live on almost every continent and habitat, and serve important functions in numerous ecosystems. “Mosquitoes have been on Earth for more than 100 million years,” says Murphy, “and they have co-evolved with so many species along the way.” Wiping out a species of mosquito could leave a predator without prey, or a plant without a pollinator. And exploring a world without mosquitoes is more than an exercise in imagination: intense efforts are under way to develop methods that might rid the world of the most pernicious, disease-carrying species (see ‘War against the winged’). Yet in many cases, scientists acknowledge that the ecological scar left by a missing mosquito would heal quickly as the niche was filled by other organisms. Life would continue as before — or even better. When it comes to the major disease vectors, “it’s difficult to see what the downside would be to removal, except for collateral damage”, says insect ecologist Steven Juliano, of Illinois State University in Normal. A world without mosquitoes would be “more secure for us”, says medical entomologist Carlos Brisola Marcondes from the Federal University of Santa Catarina in Brazil. “The elimination of Anopheles would be very significant for mankind.”

Arctic pests

Wish you were gone: mosquito clouds can be thick enough to choke caribou in the Arctic. 432

Elimination of mosquitoes might make the biggest ecological difference in the Arctic tundra, home to mosquito species including Aedes impiger and Aedes nigripes. Eggs laid by the insects hatch the next year after the snow melts, and development to adults takes only 3–4 weeks. From northern Canada to Russia, there is a brief period in which they are extraordinarily abundant, in some areas forming thick clouds. “That’s an exceptionally rare situation worldwide,” says entomologist Daniel Strickman, programme leader for medical and urban entomology at the US Department of Agriculture in Beltsville, Maryland. “There is no other place in the world where they are that much biomass.” Views differ on what would happen if that biomass vanished. Bruce Harrison, an entomologist at the North Carolina Department of Environment and Natural Resources in Winston-Salem estimates that the number of migratory birds that nest in the tundra could drop by more than 50% without mosquitoes to eat. Other researchers disagree. Cathy Curby, a wildlife biologist at the US Fish and Wildlife Service

M. Bowler AMAzon-IMAges/AlAMy

Eradicating any organism would have serious consequences for ecosystems — wouldn’t it? Not when it comes to mosquitoes, finds Janet Fang.

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in Fairbanks, Alaska, says that Arctic mosquitoes don’t show up in bird stomach samples in high numbers, and that midges are a more important source of food. “We (as humans) may overestimate the number of mosquitoes in the Arctic because they are selectively attracted to us,” she says. Mosquitoes consume up to 300 millilitres of blood a day from each animal in a caribou herd, which are thought to select paths facing into the wind to escape the swarm. A small change in path can have major consequences in an Arctic valley through which thousands of caribou migrate, trampling the ground, eating lichens, transporting nutrients, feeding wolves, and generally altering the ecology. Taken all together, then, mosquitoes would be missed in the Arctic — but is the same true elsewhere?

Food on the wing

At your service

As larvae, mosquitoes make up substantial biomass in aquatic ecosystems globally. They abound in bodies of water ranging from ephemeral ponds to tree holes2 to old tyres, and the density of larvae on flooded plains can be so high that their writhing sends out ripples across the surface. They feed on decaying leaves, organic detritus and microorganisms. The question is whether, without mosquitoes, other filter feeders would step in. “Lots of organisms process detritus. Mosquitoes aren’t the only ones involved or the most important,” says Juliano. “If you pop one rivet out of an airplane’s wing, it’s unlikely that the plane will cease to fly.”

Mosquito larvae form a substantial part of the biomass in water pools worldwide.

“If there was a benefit to having them around, we would have found a way to exploit them. We haven’t wanted anything from mosquitoes except for them to go away.”

The effects might depend on the body of water in question. Mosquito larvae are important members of the tight-knit communities in the 25–100-millilitre pools inside pitcher plants3,4 (Sarracenia purpurea) on the east coast of North America. Species of mosquito (Wyeomyia smithii) and midge (Metriocnemus knabi) are the only insects that live there, along with microorganisms such as rotifers, bacteria and protozoa. When other insects drown in the water, the midges chew up their carcasses and the mosquito larvae feed on the waste products, making nutrients such as nitrogen available for the plant. In this case, eliminating mosquitoes might affect plant growth. In 1974, ecologist John Addicott, now at the University of Calgary in Alberta, Canada, published findings on the predator and prey structure within pitcher plants, noting more protozoan diversity in the presence of mosquito larvae5. He proposed that as the larvae feed, they keep down the numbers of the dominant species of protozoa, letting others persist. The broader consequences for the plant are not known. A stronger argument for keeping mosquitoes might be found if they provide ‘ecosystem services’ — the benefits that humans derive from nature. Evolutionary ecologist Dina Fonseca at Rutgers University in New Brunswick, New Jersey, points as a comparison to the biting midges of the family Ceratopogonidae, sometimes known as no-see-ums. “People being bitten by no-see-ums or being infected through them with viruses, protozoa and filarial worms would love to eradicate them,” she says. But because some ceratopogonids are pollinators of tropical crops such as cacao, “that would result in a world without chocolate”. Without mosquitoes, thousands of plant species would lose a group of pollinators. Adults depend on nectar for energy (only females of some species need a meal of blood to get the proteins necessary to lay eggs). Yet McAllister says that their pollination isn’t crucial for crops on which humans depend. “If there was a benefit to having them around, we would have found a way to exploit them,” she says. “We haven’t wanted anything from mosquitoes except for them to go away.”

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“Mosquitoes are delectable things to eat and they’re easy to catch,” says aquatic entomologist Richard Merritt, at Michigan State University in East Lansing. In the absence of their larvae, hundreds of species of fish would have to change their diet to survive. “This may sound simple, but traits such as feeding behaviour are deeply imprinted, genetically, in those fish,” says Harrison. The mosquitofish (Gambusia affinis), for example, is a specialized predator — so effective at killing mosquitoes that it is stocked in rice fields and swimming pools as pest control — that could go extinct. And the loss of these or other fish could have major effects up and down the food chain. Many species of insect, spider, salamander, lizard and frog would also lose a primary food source. In one study published last month, researchers tracked insect-eating house martins at a park in Camargue, France, after the area was sprayed with a microbial mosquito-control agent1. They found that the birds produced on average two chicks per nest after spraying, compared with three for birds at control sites. Most mosquito-eating birds would probably switch to other insects that, post-mosquitoes, might emerge in large numbers to take their place. Other insectivores might not miss them at all: bats feed mostly on moths, and less than 2% of their gut content is mosquitoes. “If you’re expending energy,” says medical entomologist Janet McAllister of the Centers for Disease Control and Prevention in Fort Collins, Colorado, “are you going to eat the 22-ounce filet-mignon moth or the 6-ounce hamburger mosquito?” With many options on the menu, it seems that most insecteaters would not go hungry in a mosquito-free world. There is not enough evidence of ecosystem disruption here to give the eradicators pause for thought.

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d. Allen/IstocKpHoto.coM

Humans have made many concerted, if not always effective, efforts to eliminate mosquitoes. the more successful attempts include the eradication campaign against Aedes aegypti in the early 1900s, which relieved yellow fever enough to allow the completion of the panama canal; and the use of the larvicide paris green to rid Brazil of the malaria vector Anopheles gambiae by 1940. Application of the adulticide ddt allowed the united states to be declared free of malaria in 1949. But the chemicals sprayed then are banned in many countries now. “we can’t mount those top-down, military-style efforts today,” says roger nasci, an entomologist at the centers for disease control and prevention in fort collins, colorado. “And we don’t have ddt any more. It came with a lot of baggage but was an outstanding product for mosquito reduction.” Mosquito control using lesstoxic chemicals is key to keeping the insects in florida and parts of

southeast Asia and latin America at tolerable levels. worldwide malaria control in 2010 requires about us$1,880 million for indoor residual spraying and $2,090 million for insecticidal nets. “It’s a complicated business, and that’s why we still have mosquitoes,” nasci says. “they’re not going anywhere.” researchers are developing alternative mosquito-control methods; some are outlined below. RNA interference ● rnA-based insecticides kill female A. aegypti by promoting cell suicide6. “It basically tells the mosquito to go kill itself,” says stanton cope, director of the us Armed forces pest Management Board, washington dc. ● formulation not yet developed to spray it in large quantities. Male sterilization ● Introduced in large-enough numbers, sterile males can slow reproduction. screw worms were

Ultimately, there seem to be few things that mosquitoes do that other organisms can’t do just as well — except perhaps for one. They are lethally efficient at sucking blood from one individual and mainlining it into another, providing an ideal route for the spread of pathogenic microbes. “The ecological effect of eliminating harmful mosquitoes is that you have more people. That’s the consequence,” says Strickman. Many lives would be saved; many more would no longer be sapped by disease. Countries freed of their high malaria burden, for example in sub-Saharan Africa, might recover the 1.3% of growth in gross domestic product that the World Health Organization estimates they are cost by the disease each year, potentially accelerating their development. There would be “less burden on the health system and hospitals, redirection of public-health expenditure for vector-borne diseases control to other priority health issues, less absenteeism from schools”, says Jeffrey Hii, malaria scientist for the World Health Organization in Manila. Phil Lounibos, an ecologist at the Florida Medical Entomology Laboratory in Vero Beach says that “eliminating mosquitoes would temporarily relieve human suffering”. His work suggests that efforts to eradicate one vector species would be futile, as its niche would quickly be filled by another. His team collected female yellow-fever mosquitoes (Aedes aegypti) from scrap yards in Florida, and found that some had been inseminated by Asian tiger mosquitoes (Aedes 434

d. wylIe/MAgnuM pHotos

war against the winged

Battle to the death: fumigating the streets of Calcutta, India.

eradicated in the united states in the early 1980s in this way: irradiated pupae grew into sterile males that were released until the species bred itself out of existence. ● Hasn’t been widely field tested for mosquitoes. Improved chemicals ● Mosquitoes are becoming resistant to current adulticides, which target the nervous system. researchers are seeking agents with new mechanisms, including

natural products such as cedar oil. ● Basic research to be done to find compounds and modes of action. Mosquito traps ● In 2003, Aedes taeniorhynchus was mostly eliminated from an island in florida by researchers at the us department of Agriculture, using traps that generate carbon dioxide to lure mosquitoes. ● good for gardens or small islands, but probably not feasible on a larger scale. J.F.

albopictus), which carry multiple human diseases. The insemination sterilizes the female yellow-fever mosquitoes — showing how one insect can overtake another. Given the huge humanitarian and economic consequences of mosquito-spread disease, few scientists would suggest that the costs of an increased human population would outweigh the benefits of a healthier one. And the ‘collateral damage’ felt elsewhere in ecosystems doesn’t buy much sympathy either. The romantic notion of every creature having a vital place in nature may not be enough to plead the mosquito’s case. It is the limitations of mosquito-killing methods, not the limitations of intent, that make a world without mosquitoes unlikely. And so, while humans inadvertently drive beneficial species, from tuna to corals, to the edge of extinction, their best efforts can’t seriously threaten an insect with few redeeming features. “They don’t occupy an unassailable niche in the environment,” says entomologist Joe Conlon, of the American Mosquito Control Association in Jacksonville, Florida. “If we eradicated them tomorrow, the ecosystems where they are active will hiccup and then get on with life. Something better or worse would take over.” ■ Janet Fang is an intern in Nature’s Washington DC office. 1. poulin, B., lefebvre, g. & paz, l. J. Appl. Ecol. 47, 884–889 (2010). 2. daugherty, M. p. & Juliano, s. A. Am. Midl. Nat. 150, 181–184 (2003). 3. daugherty, M. p., Alto, B. w. & Juliano, s. A. J. Med. Entomol. 37, 364–372 (2000). 4. Heard, s. B. Ecology 75, 1647–1660 (1994). 5. Addicott, J. f. Ecology 55, 475–492 (1974). 6. pridgeon, J. w., zhao, l., Becnel, J. J., strickman, d. A., clark, g. g. & linthicum, K. J. J. Med. Entomol. 45, 414–420 (2008).

Progress Moving in and renovating: exporting proteins from Plasmodium into host erythrocytes Daniel E. Goldberg and Alan F. Cowman

Abstract | Malaria parasites live within erythrocytes in the host bloodstream and induce crucial changes to these cells. By so doing, they can obtain the nutrients that they require for growth and can effect the evasion and perturbation of host defences. In order to accomplish this extensive host cell remodelling, the intracellular parasite exports hundreds of proteins to commandeer the erythrocyte for its own purposes. An export motif, a processing enzyme that specifies protein targeting and a translocon that mediates the export of proteins from the parasite into the host erythrocyte have been identified. However, important questions remain regarding the secretory pathway and the function of the translocon. In addition, this export pathway provides potentially useful targets for the development of inhibitors to interfere with functions that are vital for the virulence and survival programmes of the parasite. Parasites of the genus Plasmodium are obligate intracellular organisms with a complex life cycle (FIG. 1), switching between a mosquito vector and a vertebrate host, in which they grow and reproduce inside erythrocytes. In humans, Plasmodium falciparum causes the most severe form of malaria and results in over one million deaths each year, although other species such as Plasmodium vivax also account for considerable morbidity 1. After these parasites reach the bloodstream they undergo continuous asexual multiplication, and it is this stage of the life cycle that is responsible for the clinical symptoms of malaria2. Erythrocytes — the takeover In order to survive inside an erythrocyte in the bloodstream, the parasite must undertake major host cell remodelling. This is mediated by the export of hundreds of parasite proteins from the parasite into the erythrocyte and, in some cases, under and within the host cell membrane3,4 (FIG. 1). These proteins include a key virulence protein, P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is exported to

the red blood cell surface and is responsible for antigenic variation5–7. PfEMP1 also mediates the adherence of P. falciparuminfected erythrocytes to receptors on endothelial cells, which helps the parasitized red blood cells to avoid destruction by the reticuloendothelial system and allows the microaerophilic parasite to mature in a hypoxic environment 8. Knob-associated histidine-rich protein (KAHRP) reorganizes the erythrocyte membrane into knob structures that provide a raised platform of clustered PfEMP1 molecules linked to the cytoskeleton (FIG. 1); this enhances receptor binding under the flow conditions that the parasite faces in this hostile environment 9,10. Adherence of the P. falciparum-infected erythrocytes allows their sequestration in organs such as the brain so that they are not passed through the spleen, where they can be readily destroyed. In addition, the intracellular parasite exports an array of proteins that modify the erythrocyte cytoskeleton through phosphorylation, binding and cleavage of host cytoskeletal proteins11–13. This leads to an alteration in the mechanical properties of the infected erythrocyte and

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may also be important in parasite egress14. The parasite establishes a new permeation pathway on the erythrocyte membrane for nutrient acquisition from the bloodstream, and it even changes the major erythrocytic cation from K+ to Na+ (REF. 15). This wholesale takeover converts a terminally differentiated host cell lacking such basic functions as protein trafficking and transcription into a niche in which the parasite can flourish. Topological problems for protein export During invasion of a red blood cell, the malaria parasite is encased in a second membrane known as the parasitophorous vacuolar membrane (PVM). The plasma membrane of the parasite is close to the PVM; the space between the membranes is referred to as the parasitophorous vacuole. Parasite proteins exported into the host cell must therefore cross three membranes. They are first inserted into the endoplasmic reticulum (ER) of the parasite and are subsequently transported through the secretory pathway and released into the parasitophorous vacuole, from where they must cross the PVM to reach their final localization in the erythrocyte cytoplasm (for reviews, see REFS 3,4). For erythrocyte surface-located proteins, such as PfEMP1 and other transmembrane proteins, the challenge is even greater, as these membrane-associated proteins must traverse the erythrocyte cytosol (see below). The parasite has developed specialized and specific mechanisms for achieving the desired topological localization of both soluble and membrane-associated exported proteins. Entry into the secretory pathway In P. falciparum, fusion of a signal sequence to a reporter is sufficient for the reporter to gain access to the ER lumen for subsequent secretion into the parasitophorous vacuole (reviewed in REF. 16). Integral membrane proteins can also access this pathway 17. Signal sequences that are substantially recessed from the amino terminus can still be functional18. Indeed, most exported proteins have a hydrophobic signal sequence that is 20 to 60 amino acids downstream of the N terminus. The functional VoluME 8 | SEPTEMbER 2010 | 617

Progress relevance of this interesting feature is currently unknown. A conserved export signal Entry into the host cell from the parasitophorous vacuole requires an additional sequence downstream of the signal a

sequence19,20. by aligning the N termini of known exported proteins, two groups identified a pentapeptide motif — RXlX(E/Q/D), where X is any amino acid — that is able to target a protein for export. This motif, called the protein export element (PEXEl) or the vacuolar targeting signal21,22, is located

b Merozoite 0 h 1–24 h

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Figure 1 | Plasmodium falciparum life cycle. a | Infection starts when an infected mosquito releases sporozoites into the skin of a human during a blood meal. The sporozoites travel to the liver, where Nature | Microbiology they enter hepatocytes and subsequently divide for approximately 10 days, afterReviews which they differentiate into merozoites. growth in hepatocytes requires export of proteins into the host cell. The merozoites are released into the bloodstream, where they infect erythrocytes. b | Inside erythrocytes, the parasite undergoes a 48-hour-long developmental process that starts with the ring stage (0–24 hours), followed by DNA replication and parasite growth during the trophozoite stage (24–36 hours) and, ultimately, the schizont stage (36–48 hours), during which infectious merozoites are formed. A small percentage of the parasites undergo differentiation into gametocytes (not shown), which can be taken up by a mosquito during a blood meal and can then mate inside the mosquito gut. export of parasite proteins also occurs at this stage. c | During the intracellular stages in the vertebrate host, the parasite actively remodels the host erythrocyte. An outline of the export pathway of a protein is shown, starting with secretion through the secretory pathway (initiating in the endoplasmic reticulum) into the parasitophorous vacuole (PV). exported proteins are then transported past the parasitophorous vacuolar membrane (PVM) by an export complex. Transmembrane domain-containing proteins may be transported through vesicle-like structures. exported proteins, including Plasmodium falciparum erythrocyte membrane protein 1 (PfeMP1), are part of the knobs and the Maurer’s clefts. 618 | SEPTEMbER 2010 | VoluME 8

25–30 amino acids downstream of the signal sequence. using bioinformatics these groups generated lists of 200–400 proteins carrying this sequence, and this ‘exportome’ was subsequently refined through bioinformatics and cell biology approaches23,24. Most genes encoding predicted exported proteins are clustered in the subtelomeric regions of the Plasmodium spp. chromosomes and, particularly in P. falciparum, an expansion and radiation of some genes led to the generation of large families of predicted exported proteins, including the FIKK kinases, the PHIST (Plasmodium helical, interspersed, subtelomeric) proteins and the DNAJ proteins11. Although these analyses provided clues about the functions of some exported proteins, most showed no similarity to any known proteins. During the liver stage, proteins are probably exported into the host hepatocyte using a PEXEl-based mechanism. Circumsporozoite protein (CS) has a functional PEXEl motif 25 and is exported into the hepatocyte, where it then translocates to the nucleus to alter the expression of genes involved in host defence. Early-stage gametocytes also use the PEXEl pathway for protein export 26. Although the PEXEl motif, and therefore this general mode of protein export, is conserved in all Plasmodium species, it is not present in related apicomplexan parasites23. PEXEL is a protease cleavage motif The signal sequence in exported proteins is essential for targeting these proteins to the ER, and the PEXEl motif is recognized and cleaved by a protease27. Analysis of the biosynthesis of two exported proteins from P. falciparum, histidine-rich protein II (HRPII) and PfEMP2, showed that the PEXEl motif is a protease recognition sequence that results in removal of the N terminus of the protein, including the signal sequence. both proteins were cleaved after the conserved leucine, with subsequent acetylation of the N terminus28. Mutational studies revealed that the arginine and leucine residues of the motif are required for cleavage, and that the final residue (which can be glutamic acid, glutamine or aspartic acid) is necessary for subsequent export but not for cleavage27. N-acetylation of the exported proteins after cleavage is not sufficient for their correct export to the host cell, and whether it is necessary for export has not yet been established. Two observations suggest that the ER is the site of processing. First, brefeldin A, an agent that prevents trafficking from the ER to the Golgi29, does www.nature.com/reviews/micro

Progress not block cleavage of proteins destined for export 27,28. Second, a PEXEl-containing chimaera possessing an ER retention signal (SDEl) is fully processed30. Recently the resident ER aspartic protease, plasmepsin V, was identified as the PEXEl-processing protease31,32. It is an integral membrane protein and is expressed in the erythrocytic and hepatic stages of the P. falciparum life cycle33. Plasmepsin V derived from P. falciparum or produced as a recombinant protein in Escherichia coli was able to cleave the PEXEl sequences of two recombinant proteins and several synthetic PEXEl motif-containing peptides from different exported proteins after the conserved leucine31,32. This cleavage was found to require the arginine and leucine but not the fifth, polar residue, similar to the cleavage properties of the PEXEl motif in vivo27. Plasmepsin V is an essential enzyme, as gene knockouts in P. falciparum and Plasmodium berghei were unsuccessful and allelic replacement of a catalytic aspartate codon was possible only when a synonymous mutation was introduced31,32. Expression of a catalytically inactive plasmepsin V had a dominant-negative effect, impairing PEXEl motif cleavage, protein export and parasite growth31. A carboxyterminal truncation of the endogenous plasmepsin V gene was viable, and the protein was targeted correctly as long as the truncation did not extend to the membranespanning region. The transmembrane domain is necessary and sufficient for ER targeting, and ER localization is clearly crucial for protein function31. Recognition of the PEXEl motif by plasmepsin V is more than just a cleavage event; it seems to be the necessary first step in a pathway for export, as formation of an identical mature N terminus by a different protease does not result in export of the protein. A transgenic PfEMP3 protein lacking a PEXEl motif but containing a signal sequence was engineered such that cleavage by the signal peptidase would result in the same protein as the native, processed PfEMP332. both the engineered and wild-type proteins contained the mature, acetylated N terminus (AcXQ)27 but, in contrast to the wild-type protein, the engineered PfEMP3 accumulated in the parasitophorous vacuole and was not exported. Therefore, plasmepsin V probably designates processed proteins for export through the secretory system. Interestingly, chaperones that are thought to be involved in the transport of exported proteins across the PVM (see below) are also found in association with

plasmepsin V, leading to the hypothesis that these chaperones are loaded with cleaved proteins by plasmepsin V during or after cleavage and then transport their cargo to the PVM31. Protein unfolding has been shown to be required for translocation across the PVM34, and chaperones are likely to be involved in this step as well35. A translocon at the PVM? It was previously proposed that a translocon at the PVM would be required for export across this membrane and for insertion of the exported proteins into Maurer’s clefts for trafficking through the parasite-infected erythrocyte21. Known PVM proteins are highly represented in detergent-resistant membrane fractions, so it was reasoned that the set of peptides identified in these fractions by proteomic analysis was likely to contain components of the putative translocon36. These contenders were analysed using carefully thought out criteria, including the presence of an ATPase, conservation among Plasmodium spp., expression during the erythrocytic phase of the parasite life cycle and interaction with PEXEl-containing proteins; in this way, a complex termed the Plasmodium translocon of exported proteins (PTEX) was identified. This complex localizes at discrete foci in the PVM and resides at the apical end of merozoites, from where it could be released and injected into the nascent PVM during invasion. At least five proteins were detected in the PTEX complex 36 (FIG. 2). Heat shock protein 101 (HSP101), an AAA+ ATPase that was detected in this complex, might function as the power source for the translocation process. Another protein in the complex, PTEX150, has an expression profile similar to that of HSP101 and is present in Plasmodium spp. but not in apicomplexans that lack the PEXEl motif export system. The three other proteins are EXP2 (a known PVM protein), a thioredoxin-like protein (TRX2) and an uncharacterized protein called PTEX88. EXP2 was shown to be partially resistant to carbonate extraction from membranes and was predicted, from structural modelling, to be a pore-forming protein. Therefore, EXP2 could form the translocon channel, and TRX2 could assist in protein oxidation and reduction. HSP101 was found to interact with plasmepsin V and may therefore form the chaperone that delivers the exported proteins to the translocon complex 31, although a second interaction study did not find HSP101 to be associated with plasmepsin V, and the primary location of HSP101 is in the PVM. The PTEX is a strong

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candidate for the translocation apparatus, as it has all of the properties that would be expected for a translocation machine, but further functional data are required to characterize this complex definitively. Exported proteins without a PEXEL A small group of exported P. falciparum proteins, including the early ring proteins ring-exported protein 1 (REX1), REX2, membrane-associated histidine-rich protein (MAHRP1) and skeleton-binding protein 1 (PfSbP1), lack a canonical PEXEl motif (reviewed in REF. 37). REX2 also lacks a classical signal sequence and instead requires the ten N-terminal residues and a downstream transmembrane sequence for export 38. The N terminus of REX2 is processed just before, rather than after, a leucine in the N-terminal decapeptide, generating a mature N-terminal sequence of lXE. The leucine is not important for export, whereas the glutamic acid residue is essential. Currently there is no evidence that REX2 processing is required for export 39. The N termini of REX2, MAHRP1 and PfSbP1 are all interchangeable for export, suggesting that they are exported by the a

b HSP101 EXP2

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PTEX88

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Figure 2 | Proposed components of the export translocon complex. a | The Plasmodium transNature Reviews | Microbiology locon of exported proteins (PTeX) complex has been detected in a punctate pattern in the parasitophorous vacuolar membrane. eXP2 has been postulated to form the pore through which the proteins traverse the membrane. Heat shock protein 101 (HsP101) and possibly the thioredoxinlike protein TrX2 are thought to be involved in guiding the exported proteins to the pore. The functions of PTeX150 and PTeX88 are unknown. b | The distribution of HsP101 and PTeX150 in ring-stage parasites is shown. Part b image is reproduced, with permission, from Nature REF. 36 © (2010) Macmillan Publishers Ltd. All rights reserved.

Progress same mechanism. All three proteins, as well as REX1, localize to the Maurer’s clefts, which are membranous structures that bud from the PVM and move out to the underside of the erythrocyte membrane, to which they attach through tethers40,41. It is possible that these proteins flow from the PVM to the contiguous clefts and therefore do not require translocation through the PVM, although the putative translocon may still have a role in sorting these proteins and moving them to the nascent Maurer’s clefts. Export of PfEMP1 one of the most important exported proteins in P. falciparum is PfEMP1, which is a variant surface antigen and adhesin that is encoded by approximately 60 var genes5–7. Monoallelic transcription of var genes results in the expression of one variant type of PfEMP1 on the P. falciparum-infected erythrocyte. PfEMP1 proteins are large (over 300 kDa) and do not have a signal sequence but do have a C-terminal transmembrane domain that seems to be essential for targeting to the ER42. There is a conserved N-terminal sequence, a portion of which has some homology to the PEXEl motif, with the consensus sequence (R/K)X(V/F/M/l)X(E/D). PfEMP1–reporter chimaeras have been shown to be exported to the erythrocyte, and this requires the N-terminal region of PfEMP1 (REFS 21,42). It is not clear whether the conserved sequence in the N terminus of PfEMP1 is a PEXEl motif; it is not cleaved by plasmepsin V (J. A. boddey, A.F.C. and D. Marapena, unpublished observations), suggesting that it is functionally distinct. PfEMP1 reaches the erythrocyte surface through Maurer’s clefts, and delivery from these membranous structures to the erythrocyte surface occurs by an unknown mechanism that may involve vesicle formation43. Not surprisingly, movement of PfEMP1 onto the erythrocyte surface requires a remarkable network of proteins that function at different steps of its trafficking through the host cell44. PfSbP1, a Maurer’s cleft protein, is required for the localization of PfEMP1 at the erythrocyte surface45,46. A large-scale knockout screen has identified eight additional proteins that are involved in the display or function of PfEMP1 on the surface of the infected erythrocyte44. Analysis of the loss-of-function mutants showed that these proteins could be divided into two functional groups. The first group is involved in loading PfEMP1 into Maurer’s clefts at the PVM, as disruption of the corresponding genes results in blockage of trafficking at

this point. The second group is involved in transferring PfEMP1 from Maurer’s clefts to the erythrocyte surface, as disruption of these corresponding genes blocks transfer but does not block loading of the protein into Maurer’s clefts. Conclusions Export of Plasmodium spp. proteins into the host cell is a process that we are only just beginning to understand. A PEXEl sequence is required for most exported proteins, and this bifunctional motif comprises a recognition and cleavage site for the protease plasmepsin V31,32, the action of which generates a new N terminus that is necessary for subsequent steps in the export pathway 27,28. It has been proposed that a translocon located at the PVM is required for export21, and the PTEX complex that has been identified has all of the features that would be expected for such a machine36 (FIG. 2). Many key questions with respect to protein export in Plasmodium spp. are yet to be answered. First, how do proteins that are destined for export and that have been processed by plasmepsin V get from the ER to the translocon? Most of the PEXEl sequence that confers export specificity is cleaved in the ER, leaving AcX(E/Q/D) at

the processed N terminus27. However, it is clear that plasmepsin V-mediated cleavage of PEXEl is required not only for processing but also for ensuring that the mature protein gets to the right place31,32. Perhaps plasmepsin V passes its product to chaperones that are required to escort the proteins to the translocon31,47,48 (FIG. 3a). Alternatively, plasmepsin V could be located in a region of the ER that determines cargo destination49, and the export signal could be recognized locally by a receptor for packaging into specialized vesicles that dock with specific regions of the parasite membrane, with the translocon located nearby in PVM subdomains36 (FIG. 3b). This model was suggested in an early form by lingelbach and Przyborski50 and has recently been reviewed48. Although a PVM complex has been identified that has all the properties expected of a translocon machine, direct evidence for its function in export has yet to be obtained36. EXP2 is the best candidate for the pore through which exported proteins pass, as it has structural homology with haemolysin E and it is strongly associated with the membrane. Nevertheless, further proof is required to demonstrate that it functions as a channel and to determine the roles of associated proteins. little

Erythrocyte cytoplasm PVM PV PM

Secretion pathway

Export pathway

ER Nucleus Exported protein Protein export translocon

PEXEL motif

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Figure 3 | Two possible models for protein export specificity.Nature a | The chaperone model. Reviews | Microbiology Plasmepsin V recognizes the protein export element (PeXeL) motif in a protein in the endoplasmic reticulum (er), cleaves it and transfers the processed protein to the chaperone heat shock protein 101 (HsP101). HsP101 shepherds the protein through the general secretory pathway and docks to the translocon (of which HsP101 is a component) at the parasitophorous vacuolar membrane (PVM). b | The er subdomain model. Plasmepsin V recognizes the PeXeL motif, cleaves it and transfers the processed protein to a PeXeL-cargo-sorting receptor in an er subregion. Vesicles then deliver the protein to a special region of the parasitophorous vacuole (PV) (delimited by the pink ovals), where the translocon can export it. These vesicles would be distinct from those involved in the general secretory pathway. PM, plasma membrane of the parasite.

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Progress is understood about the export of proteins that do not have a canonical PEXEl motif; it is unclear whether they use the same machinery as proteins that have a PEXEl motif or whether there is an alternative transport machinery. Similarly, the pathway used for the export of PfEMP1 remains to be determined. The discovery of components of the Plasmodium spp. protein export pathway opens a new area for the development of inhibitors that may have potential for use in antimalarial therapy. Plasmepsin V and the putative translocon components seem to be essential for intra-erythrocytic parasites31,32,36. blocking the export function of these proteins would therefore prevent parasite survival as well as parasite virulence. As we understand more about the individual export components, we might be able to develop inhibitors to thwart the takeover of the host cell by the parasite. Daniel E. Goldberg is at the Howard Hughes Medical Institute, Washington University School of Medicine, Departments of Medicine and Molecular Microbiology, St Louis, Missouri 63110, USA. Alan F. Cowman is at The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne 3050, Australia, and at the Department of Medical Biology, University of Melbourne, Melbourne 3050, Australia.

12.

13. 14.

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e-mails: goldberg@borcim.wustl.edu; cowman@wehi.edu.au

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doi:10.1038/nrmicro2420 Corrected 13 August 2010

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members in Plasmodium falciparum, all with an export signature. BMC Genomics 6, 30 (2005). Waller, K. L. et al. Interactions of Plasmodium falciparum erythrocyte membrane protein 3 with the red blood cell membrane skeleton. Biochim. Biophys. Acta 1768, 2145–2156 (2007). Glenister, F. K. et al. Functional alteration of red blood cells by a megadalton protein of Plasmodium falciparum. Blood 113, 919–928 (2009). Glenister, F. K., Coppel, R. L., Cowman, A. F., Mohandas, N. & Cooke, B. M. Contribution of parasite proteins to altered mechanical properties of malariainfected red blood cells. Blood 99, 1060–1063 (2002). Kutner, S., Breuer, W. V., Ginsburg, H., Aley, S. B. & Cabantchik, Z. I. Characterization of permeation pathways in the plasma membrane of human erythrocytes infected with early stages of Plasmodium falciparum: association with parasite development. J. Cell Physiol. 125, 521–527 (1985). Tonkin, C. J., Pearce, J. A., McFadden, G. I. & Cowman, A. F. Protein targeting to destinations of the secretory pathway in the malaria parasite Plasmodium falciparum. Curr. Opin. Microbiol. 9, 381–387 (2006). Klemba, M., Beatty, W., Gluzman, I. & Goldberg, D. E. Trafficking of plasmepsin II to the food vacuole of the malaria parasite Plasmodium falciparum. J. Cell Biol. 164, 47–56 (2004). Lingelbach, K. R. Plasmodium falciparum: a molecular view of protein transport from the parasite into the host erythrocyte. Exp. Parasitol. 76, 318–327 (1993). Wickham, M. E. et al. Trafficking and assembly of the cytoadherence complex in Plasmodium falciparuminfected human erythrocytes. EMBO J. 20, 5636–5649 (2001). Lopez-Estraño, C., Bhattacharjee, S., Harrison, T. & Haldar, K. Cooperative domains define a unique host cell-targeting signal in Plasmodium falciparuminfected erythrocytes. Proc. Natl Acad. Sci. USA 100, 12402–12407 (2003). Marti, M., Good, R. T., Rug, M., Knuepfer, E. & Cowman, A. F. Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306, 1930–1933 (2004). Hiller, N. L. et al. A host-targeting signal in virulence proteins reveals a secretome in malarial infection. Science 306, 1934–1937 (2004). Sargeant, T. J. et al. Lineage-specific expansion of proteins exported to erythrocytes in malaria parasites. Genome Biol. 7, R12 (2006). van Ooij, C. et al. The malaria secretome: from algorithms to essential function in blood stage infection. PLoS Pathog. 4, e1000084 (2008). Singh, A. P. et al. Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite. Cell 131, 492–504 (2007). Silvestrini, F. et al. Protein export marks the early phase of gametocytogenesis of the human malaria parasite Plasmodium falciparum. Mol. Cell. Proteomics 9, 1437–1448 (2010). Boddey, J. A., Moritz, R. L., Simpson, R. J. & Cowman, A. F. Role of the Plasmodium export element in trafficking parasite proteins to the infected erythrocyte. Traffic 10, 285–299 (2009). Chang, H. H. et al. N-terminal processing of proteins exported by malaria parasites. Mol. Biochem. Parasitol. 160, 107–115 (2008). Wiek, S., Cowman, A. F. & Lingelbach, K. Double cross-over gene replacement within the sec 7 domain of a GDP-GTP exchange factor from Plasmodium falciparum allows the generation of a transgenic brefeldin A-resistant parasite line. Mol. Biochem. Parasitol. 138, 51–55 (2004). Osborne, A. R. et al. The host targeting motif in exported Plasmodium proteins is cleaved in the parasite endoplasmic reticulum. Mol. Biochem. Parasitol. 171, 25–31 (2010). Russo, I. et al. Plasmepsin V licenses Plasmodium proteins for export into the host erythrocyte. Nature 463, 632–636 (2010). Boddey, J. A. et al. An aspartyl protease directs malaria effector proteins to the host cell. Nature 463, 627–631 (2010). Klemba, M. & Goldberg, D. E. Characterization of plasmepsin V, a membrane-bound aspartic protease homolog in the endoplasmic reticulum of Plasmodium falciparum. Mol. Biochem. Parasitol. 143, 183–191 (2005).

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34. Gehde, N. et al. Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum. Mol. Microbiol. 71, 613–628 (2009). 35. Banumathy, G., Singh, V. & Tatu, U. Host chaperones are recruited in membrane-bound complexes by Plasmodium falciparum. J. Biol. Chem. 277, 3902–3912 (2002). 36. de Koning-Ward, T. F. et al. A newly discovered protein export machine in malaria parasites. Nature 459, 945–949 (2009). 37. Spielmann, T. & Gilberger, T.-W. Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways? Trends Parasitol. 26, 6–10 (2010). 38. Haase, S. et al. Sequence requirements for the export of the Plasmodium falciparum Maurer’s clefts protein REX2. Mol. Microbiol. 71, 1003–1017 (2009). 39. Brown, G. V. et al. Localization of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum in merozoites and ring-infected erythrocytes. J. Exp. Med. 162, 774–779 (1985). 40. Waterkeyn, J. G. et al. Targeted mutagenesis of Plasmodium falciparum erythrocyte membrane protein 3 (PfEMP3) disrupts cytoadherence of malariainfected red blood cells. EMBO J. 19, 2813–2823 (2000). 41. Hanssen, E. et al. Targeted mutagenesis of the ringexported protein-1 of Plasmodium falciparum disrupts the architecture of Maurer’s cleft organelles. Mol. Microbiol. 69, 938–953 (2008). 42. Knuepfer, E., Rug, M., Klonis, N., Tilley, L. & Cowman, A. F. Trafficking of the major virulence factor to the surface of transfected P. falciparum-infected erythrocytes. Blood 105, 4078–4087 (2005). 43. Kriek, N. et al. Characterization of the pathway for transport of the cytoadherence-mediating protein, PfEMP1, to the host cell surface in malaria parasiteinfected erythrocytes. Mol. Microbiol. 50, 1215–1227 (2003). 44. Maier, A. G. et al. Exported proteins required for virulence and rigidity of Plasmodium falciparuminfected human erythrocytes. Cell 134, 48–61 (2008). 45. Cooke, B. M. et al. A Maurer’s cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells. J. Cell Biol. 172, 899–908 (2006). 46. Maier, A. G. et al. Skeleton-binding protein 1 functions at the parasitophorous vacuole membrane to traffic PfEMP1 to the Plasmodium falciparum-infected erythrocyte surface. Blood 109, 1289–1297 (2007). 47. Charpian, S. & Przyborski, J. M. Protein transport across the parasitophorous vacuole of Plasmodium falciparum: into the great wide open. Traffic 9, 157–165 (2008). 48. Crabb, B. S., de Koning-Ward, T. F. & Gilson, P. R. Protein export in Plasmodium parasites: from the endoplasmic reticulum to the vacuolar export machine. Int. J. Parasitol. 40, 509–513 (2010). 49. Struck, N. S. et al. Spatial dissection of the cis- and trans-Golgi compartments in the malaria parasite Plasmodium falciparum. Mol. Microbiol. 67, 1320–1330 (2008). 50. Lingelbach, K. & Przyborski, J. M. The long and winding road: protein trafficking mechanisms in the Plasmodium falciparum infected erythrocyte. Mol. Biochem. Parasitol. 147, 1–8 (2006).

Acknowledgements

The authors are supported, in part, by a grant to D.E.G. from the US National Institutes of Health (grant AI047798) and by grants to A.F.C. from the Australian National Health and Medical Research Council.

Competing interests statement

The authors declare no competing financial interests.

FURTHER INFORMATION Daniel e. goldberg’s homepage: http://dbbs.wustl.edu/ DBBs/website.nsf/FA/3ACB8200C2999B1586256D4e005B2 D13?openDocument All links Are AcTive in The online Pdf

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Artemisinin resistance: current status and scenarios for containment Arjen M. Dondorp*‡, Shunmay Yeung*§, Lisa White*‡, Chea Nguon§, Nicholas P.J. Day*‡, Duong Socheat§|| and Lorenz von Seidlein*¶

Abstract | Artemisinin combination therapies are the first-line treatments for uncomplicated Plasmodium falciparum malaria in most malaria-endemic countries. Recently, partial artemisinin-resistant P. falciparum malaria has emerged on the Cambodia–Thailand border. Exposure of the parasite population to artemisinin monotherapies in subtherapeutic doses for over 30 years, and the availability of substandard artemisinins, have probably been the main driving force in the selection of the resistant phenotype in the region. A multifaceted containment programme has recently been launched, including early diagnosis and appropriate treatment, decreasing drug pressure, optimising vector control, targeting the mobile population, strengthening management and surveillance systems, and operational research. Mathematical modelling can be a useful tool to evaluate possible strategies for containment. Parenteral Administered by injection.

*Mahidol Oxford Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand. ‡ Centre for Tropical Medicine, Churchill Hospital, University of Oxford, Oxford OX3 7LJ, UK. § London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK. || The National Center for Parasitology Entomology and Malaria Control, Phnom Penh, Cambodia. ¶ Joint Malaria Project, Tanga, Tanzania. Correspondence to A.M.D. e-mail: arjen@tropmedres.ac doi:10.1038/nrmicro2331 Published online 8 March 2010

In nearly all countries in which malaria is endemic, artemisinin combination therapies (ACT) are now the recommended first-line therapy for uncomplicated Plasmodium falciparum malaria, a policy endorsed by the WHO1. This change in policy followed a period of increasing failure rates with chloroquine and later sulphadoxine–pyrimethamine treatment, which arose from the development of resistant P. falciparum strains. The spread and increased levels of resistance to the generally available and affordable drugs resulted in an increase in the number of deaths caused by malaria in children under 5 years of age in sub-Saharan Africa during a period when overall childhood mortality was decreasing 2–4. This trend has now been reversed with the introduction of ACTs and other control measures, specifically the widespread use of insecticide-treated bed nets. A marked decrease in malaria burden has been observed in several Asian and African regions, where these effective control measures have been deployed actively 5–8. In addition, parenteral artesunate, an artemisin derivative, became the treatment of choice for severe malaria in adults after it was shown to reduce mortality by 35% compared with quinine9. Artemisinins extracted from the ubiquitous annual wormwood Artemisia annua have been used in traditional Chinese medicine for more than 2,000 years for the treatment of febrile illnesses10. In the 1970s the chemical structure of a sesquiterpene peroxide with powerful

antimalarial properties (artemisinin) was identified, and several more potent derivatives were synthesized, including artesunate, artemether and dihydroartemisinin11 (FIG. 1). Artemisinin derivatives have an excellent safety profile in the treatment of malaria, a rapid onset of action and are active against the broadest range of stages in the life cycle of Plasmodium spp. compared with other antimalarials11,12 (FIG. 2). Artemisinins also kill immature and developing gametocytes, the sexual stages that are essential for transmission13,14, thereby reducing gametocyte carriage and infectivity. In the blood, the dihydroartemisinin derivatives artesunate, artemether and artemotil are quickly and completely hydrolysed back to dihydroartemisinin, which has a short plasma half-life of ~ 1 hour 10. A once or twice a day dosing regimen with artemisinin derivatives results in a reduction of four orders of magnitude of the asexual parasite biomass per 48-hour treatment cycle (FIG. 2). Despite this remarkable antimalarial activity, artemisin derivative monotherapy for 7 days covering 3 cycles of the asexual life cycle of the parasite is needed to completely eliminate a biomass of 10 12 parasites, which corresponds to a parasitaemia of ~2% in an adult 11. The short half-life of artemisinin derivatives minimizes the period available for the selection of resistant strains (known as the selective window)15. However, there is still the potential for the emergence of resistant strains when artemisinin derivatives are deployed

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f o c u S o n a n t I m I c R o b I a l R E SRI SE ta cE V I EnW S emergence of resistant P. falciparum strains The first reports of higher recrudescence rates of P. falciparum malaria after treatment with ACTs emerged O O from observational data collected in Cambodia since O O 2004 (ReFs 21,22 ). It was not clear initially whether these high failure rates resulted from resistance to artemisiO O CH3 nins, their partner drugs or unusual host or pharmacokiArtemisinin Artemether netic factors23,24. A study carried out in 2006 and 2007 in Battambang province, Cambodia, showed that a minority of patients with uncomplicated P. falciparum malaria O O harboured parasites with decreased in vitro sensitivity to O O artesunate and showed delayed parasite clearance times O O in the presence of apparently adequate plasma drug conO O centrations after treatment with artesunate in a dose of O O 4 mg per kg per day for 7 days25. COO– O Conclusive evidence came from a recent study comArtemotil Artesunate paring the therapeutic responses to artesunate in patients Figure 1 | Chemical structure of artemisinins. Artemisinin with uncomplicated P. falciparum malaria in pailin, is the compound that is produced by the plant Artemisia western Cambodia, and Wang pha, western Thailand, annua. The derivatives arthemether and artesunate have where artemisinin derivatives remain effective26 (FIG. 3). Natureand Reviews | Microbiology Clearance rates were much slower in western Cambodia better bioavailability than artemisinin are used clinically in artemisinin combination therapy. Artemotil and showed little heterogeneity (FIG. 4). Specifically, after (also known as arteether) is infrequently used. artesunate monotherapy in a dose of 2 mg per kg per day for 7 days or artenusate in a dose of 4 mg per kg as monotherapies in areas of increasing drug pressure, per day for 3 days followed by mefloquine in a dose of so researchers suggested more than a decade ago that 25 mg per kg per day, the median parasite clearance artemisinin derivatives should be used only in combi- time was 84 hours (interquartile range (IQr) = 60 to nation with partner drugs in ACTs16. The artemisinin 96) in pailin compared with 48 hours (IQr = 36 to 66) component of ACTs rapidly kills the bulk of the organ- in Wang pha (p=0.001), with similar drug concentraisms and a partner drug with a longer plasma half-life tion profiles in both sites. The difference in clearance eliminates the remaining parasites. This not only opti- rates was not explained by genetic polymorphisms in mizes the therapeutic benefit, but also mutually protects the P. falciparum genes pfcrt (chloroquine resistance both components of the ACT and minimizes the risk transporter gene), pfmdr1 (multidrug resistance gene 1) of resistant parasites emerging and spreading 17. In the or pfserca (a sacroplasmic reticulum Ca2+ ATpase) — absence of an efficient partner drug, repeated exposure which had been suggested previously as the targets of to artemisinin monotherapies at subtherapeutic doses, artemisinins — or amplification in pfmdr1. Heritability especially in hyperparasitaemic patients and over studies suggest that the observed artemisinin resistprolonged periods of time, will provide a risk for the ance phenotype of the parasites has a genetic basis and emergence of resistance18. thus is expected to spread within parasite populations After a lengthy delay before general acceptance, that live where artemisinins are deployed unless assoACTs have now been implemented as first-line treat- ciated fitness costs of the putative resistance mutation ment in the national malaria control programmes of or mutations outweigh selective benefits27. The study most malaria-endemic countries19. Deployment in the shows that in patients treated with artesunate, genetiprivate sector, however, lags far behind. To ensure the cally identical parasite strains (defined by microsatellite use of the combination, rather than the individual com- typing) strongly cluster in patients with slow versus fast ponents, fixed-dose ACTs have been developed, includ- parasite clearance rates. To date, the molecular basis for ing artemether–lumefantrine, artesunate–mefloquine the resistance mechanism remains unknown, although and artesunater–amodiaquine. Dihydroartemisinin– intensive molecular and phenotypical characterization piperaquine and artesunate–pyronaridine are in is under way. advanced stages of clinical testing and drug registration. The short and mid-term pipeline for antimalarial drug Development of resistance development depends on artemisinin derivatives20; los- Several factors may have contributed to the emering the artemisinin derivatives because of P. falciparum gence of reduced artemisinin sensitivity in Cambodia. drug resistance would be a disaster for malaria control Cambodia was one of the first countries to adopt ACTs and treatment and would seriously threaten current as first-line treatment in 2001, but unregulated artemmalaria elimination efforts. isinin or artesunate monotherapy has been available This review presents the evidence that resistance to since the mid-1970s. A recent survey showed that 78% artemisinins has emerged in western Cambodia and dis- of patients obtain their antimalarial treatment through cusses alternative therapies and treatment regimens that the private sector, mostly as artesunate monotherapy 28. can decrease the spread and independent occurrence of The problem is compounded by the unavailability of the drug resistance. fixed dose combination of the current first-line ACT O O

Recrudescence Reoccurence of a disease after treatment. This can be caused by parasites that were not completely eliminated during the treatment.

O O

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REVIEWS artesunate–mefloquine. Although it has been developed as a fixed dose combination, it is currently available only as separate tablets, facilitating the continued use of artemisin or artesunate monotherapy. Counterfeited or substandard tablets that contain less active ingredients than stated are additional sources of subtherapeutic dosing of artemisinins, which may also have contributed to the selection of resistant parasite strains29. moreover, it is possible that the different pharmacokinetic properties of artemisinins in subgroups of the population, such as pregnant women and children, have resulted in underdosing. Why has this problem emerged in western Cambodia and not in other parts of Southeast Asia with similar conditions? We think two factors have been vital. First, there has been uniquely massive drug pressure. Second, the low malaria transmission in the area was probably essential to allow resistant parasite populations to establish themselves31,32. In addition, it is possible that parasite factors, such as a unique P. falciparum phenotype30, or host factors have played a part18,31,32. Western Cambodia has previously been a focal point for the emergence of chloroquine resistance33 and for sulphadoxin–pyrimethamine resistance34 (box 1). The relative affluence generated by the mining of precious stones in the area has attracted a highly mobile,

Mosquito Sporozoite

Sporozoite Liver Oocyst Merozoite

Zygote Erythrocyte

Erythrocytic stage

Ookinete

Gametocytes

Figure 2 | The life cycle of Plasmodium falciparum. The infection in humans is initiated by the bite of a mosquito, which releases sporozoites in theReviews bloodstream. Nature | Microbiology The sporozoites infect hepatocytes and undergo many rounds of replication, ultimately releasing merozoites. These invade erthrocytes, leading to the symptoms of malaria. Infection of erythrocytes results in the formation of additional merozoites, allowing the infection to expand. A small percentage of merozoites differentiates to form male or female gametocytes. After these are taken up by a mosquito during a bloodmeal, they convert to male and female gametes and fuse to form a zygote. Subsequently development through the ookinete and oocyst stages leads to the formation of sporozoites, which migrate to the salivary glands of the mosquito, from which they can be injected into the host. Artemesinin targets the parasites in the erythocytic stages, the merozoites and the gametocytes, preventing both their growth and spread.

susceptible population into an area where malaria is transmitted and has supported informal sales of antimalarials. migrants working in the area are an added concern as they could carry and spread artemisinin-resistant strains to other countries. However, 5 years after the first indications of reduced susceptibility to artemisinins, the clearly resistant P. falciparum phenotype is still confined to the Cambodia–Thailand border, although parasite clearance times after treatment with artemisinin derivatives have also, but to a much lesser extent, increased on the border of Thailand and myanmar (formerly known as Burma)35. It should also be noted that ACTs are still effective in western Cambodia, with cure rates usually exceeding 90%, as resistance is not complete and the parasites are still killed by artemisinins, albeit at much lower rates. It will be of utmost importance to continue monitoring the spread of the artemisinin-resistant phenotype through the region. Because a sensitive in vitro test and a molecular marker for artemisinin resistance is not currently available, clinical monitoring of the affected population is necessary.

treatment during artemisinin failure There is currently no group of drugs that can replace artemisinins in the way that sulphadoxine–pyrimethamine replaced chloroquine and ACTs replaced sulphadoxine–pyrimethamine. Semisynthetic artemisinins and synthetic endoperoxides have the same advantages as the artemisinin derivatives used in ACTs (that is, rapid parasite killing and broad stage specificity) and will undergo clinical testing in the near future, but it is uncertain whether these new drugs will be more effective against artesunate-resistant parasites than the currently available ones. none of the currently licensed antimalarial drugs has a similar safety and efficacy profile to the artemisinin derivatives. The non-artemisinin-based compounds that are in the early stages of development are likely to take at least a decade until they become available for clinical use20. Quinine remains useful for cerebral malaria and other forms of severe malaria, but a 7-day course is needed for the complete treatment of uncomplicated malaria. Furthermore, patients are unlikely to adhere to a full course of quinine because of its frequent adverse events, three times per day dosing and an unpalatable taste surprisingly. not surprisingly, recrudescence is observed frequently 36. One of the few remaining effective drugs without an artemisinin derivative component is the combination of atovaquone–proguanil. The use of atovaquone– proguanil has been limited to prevention and treatment in travellers, not because of a lack of efficacy or safety concerns, but because of its prohibitively high price. The lack of availability has so far minimized the drug pressure and prevented the appearance of resistance, but a single point mutation in codon 268 in the cytochrome b gene of P. falciparum confers a high level of atovaquone resistance. This rapid emergence of resistance to atovaquone despite optimal dosing suggests that the drug would have a short lifespan if it were widely used for malaria treatment 37.

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f o c u S o n a n t I m I c R o b I a l R E SRI SE ta cE V I EnW S

Vietnam Laos

Thailand Bangkok

Pailin Cambodia

Phnom Penh Ho Chi Min City

Nature Reviews Cambodia. | Microbiology Figure 3 | The study site in Pailin, western Decreased artemisinin sensitivity was first detected in Pailin, western Cambodia, near the border with Thailand.

Hypnozoite A dormant form of the liver stage parasites found in several Plasmodium spp., including the human parasites Plasmodium vivax and Plasmodium ovale.

Treatment of artemisinin-resistant malaria will rely on optimizing the combinations of existing drugs. Four antimalarials (lumefantrine, mefloquine, amodiaquine and sulphadoxine–pyrimethamine) are widely used in the combination with artemisinin derivatives. However, amodiaquine38 and sulphadoxine–pyrimethamine 39 resistance is already widespread, and although lumefantrine and mefloquine can still be used everywhere40, resistance could be readily selected, particular if monotherapies were deployed41. Drugs that have been more recently included in ACTs include piperaquine and pyronaridine. piperaquine was used extensively as a treatment in China42, which led to the rapid selection of resistance locally; however, it has retained efficacy elsewhere and has proved to be a valuable partner drug in combination with dihydroartemisinin43. The dihydroartemisinin–piperaquine combination still awaits pre-qualification by the WHO, but it is already widely used in Southeast Asia. Indeed, the ministry of Health in Cambodia has recently changed its first-line treatment of uncomplicated P. falciparum malaria to dihydroartemisinin–piperaquine in the affected regions in western Cambodia. pyronaridine is a newer drug that is likely to be licensed next year as a co-formulation with artenusate44,45. pyronaridine on its own is an effective and apparently safe antimalarial46. As there has been minimal use of the pyronaridine components as monotherapy, the combination with artenusate holds considerable promise. Another possibility is the simultaneous administration of three or more drugs, which is recommended and widely accepted for other diseases, including tuberculosis, multibacillary leprosy, Helicobacter pylori gastric ulcers and AIDS47,48. Triple therapy, including an artemisinin derivative and two partner drugs with matching plasma terminal half-lives, has yet to be fully explored for the treatment of uncomplicated P. falciparum malaria. A practical difficulty in the development of triple therapies is the increased potential for drug interactions and

a longer time frame for approval from regulatory institutions. Adding a gametocytocidal drug to existing ACTs would be ideal to block or at least minimize the transmission and hence the spread of artemisinin-resistant malaria. The only current gametocytocidal drugs are 8-aminoquinolones, such as primaquine. primaquine is effective against mature-stage gametocytes, but has low activity against the erythrocytic stages in the lifecycle of P. falciparum. When given as a 7-day course as an adjunct to an ACT, primaquine accelerates gametocyte clearance14. Whether a single dose of primaquine given on the last day of a course of an ACT has a similar effect has not been well studied. nevertheless, single dose primaquine has been widely recommended by malaria control programmes for many years (although often not actually used). modelling suggests that primaquine, which has a short half-life of approximately 8 hours, should ideally be given as a follow-up treatment to ACT 8 days after the start of ACT49. This is because gametocytaemia tends to peak about a week after the initial acute attack. The same model also suggests that the intervention should be deployed only on top of an effective antimalarial drug combination that kills the erythrocytic stages of the parasite. Another problem for deployment is that the safety profile of primaquine, which has not been well studied, has bedeviled the treatment of hypnozoites50. primaquine and 8-aminiquinolones have oxidative properties, and these cause intravascular haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6pD) deficiency 2. G6pD deficiency is an X-linked, hereditary genetic defect that results from different mutations in G6PD and has many biochemical and clinical phenotypes51. At least 140 mutations have been described, which differ extensively with regard to the severity of the corresponding reduction in G6pD activity. The risk–benefit assessment for the use of primaquine as a transmission blocking agent in settings in which parasite transmission is low and elimination might be possible52 is currently being assessed. The benefit to the community of reduced malaria transmission should be balanced with the risk to the individual patient who derives no personal direct benefit from blocking transmissibility. These safety concerns currently present a barrier for the acceptance of 8-aminoquinolones in triple therapy combinations.

Delaying the spread of artemisinin resistance Several public health strategies to interrupt the spread and prevent further emergence of artemisinin resistance are under consideration, and recently a programme was launched for the containment of partial artemisinin resistance in western Cambodia and eastern Thailand53. The proposed programme involves a multifaceted approach, including early diagnosis and appropriate treatment of malaria, decreasing the drug pressure, optimizing insect vector control, targeting the mobile population, strengthening disease management and surveillance systems, and operations research. The technical, operational and financial feasibility of interventions depends on the epidemiology of malaria, quality of

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REVIEWS existing health infrastructures, skilled manpower, funding, health-seeking behaviour, free-market forces, and regulatory and ultimately political forces. each of these parameters varies between regions. For example, interventions that are suitable for western Cambodia could be inappropriate for parts of Thailand. As the empirical testing of some strategies would be prohibitively slow and expensive, choosing the optimal approach requires reliance on observational data, expert opinion and predictive modelling. Increasing coverage with effective antimalarial treatment. To ensure cure and reduce further transmission of resistant infections, it is essential that symptomatic patients are diagnosed promptly and treated effectively 16. Therefore, patients need to have easy access to affordable, high-quality and effective treatment. Cambodia has applied to pilot the Affordable medicine Facility — malaria (AmFm; see AmFm website), an innovative financing mechanism that aims to lower the price of good-quality ACTs sold in the public and private sector and to not-for-profit buyers. This effort will be funded by the Global Fund for HIV, Tuberculosis and malaria,

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Hours after admission Artesunate (2 mg per kg) monotherapy in Pailin, Cambodia Artesunate (4 mg per kg) –mefloquine (25 mg per kg) therapy in Pailin, Cambodia Artesunate (2 mg per kg) monotherapy in Wang Pha, Thailand Artesunate (4 mg per kg) –mefloquine (25 mg per kg) therapy in Wang Pha, Thailand

Figure 4 | Parasite clearance rates. Parasite clearance rates in patients with uncomplicated P. falciparum malaria in Pailin (western Cambodia) Wang Pha Nature and Reviews | Microbiology (western Thailand). These study results clearly show that the parasites in patients from the Pailin region in Cambodia require longer treatment with either artesunate (shown in red) or artesunate–mefloquine (shown in purple) therapy than the parasites from the Wang Pha region in Thailand (shown in yellow and blue) to be cleared. Figure is reproduced, with permission, from ReF. 26 © (2009) Massachusetts Medical Society.

which will pay a large proportion of a negotiated low price directly to manufacturers on behalf of the buyers. This means that buyers will only pay approximately uS$0.05 for each course of an ACT. However, it will only be appropriate to subsidize a fixed-dose ACT and not the current combination of two individual pills of artesunate and mefloquine. efforts are being made in western Cambodia to strengthen the capacity for malaria diagnosis and treatment in public primary healthcare facilities to ensure early diagnosis and adequate treatment. lay village malaria workers are also being trained to provide free malaria diagnosis using rapid diagnostic tests and treatment with a good-quality ACT. This will also help to encourage patients to take the full course of the treatment, thereby avoiding the selection for antimalarial drug resistance. Reduction of drug pressure. A strategic priority is to reduce the drug pressure exerted on parasites. The limited and controlled use of atovaquone–proguanil is being explored, but the artemisinin-based drugs will continue to be the basis of malaria treatment. This is particularly problematic in Cambodia, where the use of artemisinin monotherapies, substandard drugs and subtherapeutic doses of treatment courses in the private sector is common. The sale of oral artemisinin derivative monotherapies in the private sector has recently been banned in Cambodia, and there are ongoing efforts to strengthen the capacity for drug quality monitoring, regulation of the private sector and law enforcement. However, this remains a challenging area, and until a cheap, effective and high quality fixed-dose ACT becomes available in sufficient quantities, the problem is likely to continue. The AmFm initiative described above could prove to be an effective mechanism to push substandard and monotherapy antimalarials out of the market. Deployment of multiple first-line therapies (mFTs) is another strategy to reduce drug pressure on the parasite pool (discussed below). Mass drug administrations. One approach to eliminate resistant malaria from a limited area is to administer a complete course of effective therapy to the whole population, irrespective of disease status. Several mass drug administrations (mDAs) to control malaria have been reported over the past 75 years42. However, the logistic challenges are formidable. A fraction of the population invariably refuses to participate, and adherence to multidose regimens is likely to be incomplete. To interrupt transmission a gametocytocidal drug has to be added, and to assure high coverage the therapy has to be free of side effects. perhaps most importantly, mDAs result in a massive increase in drug pressure, which, as discussed above, should be minimized. past mDAs have succeeded in reducing parasite prevalence and the incidence of clinical malaria but have failed to interrupt malaria transmission. models predict that one round of mDA will not have an impact because the acceptable drug regimens fail to remove all gametocytes and cannot therefore break the transmission cycle54.

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f o c u S o n a n t I m I c R o b I a l R E SRI SE ta cE V I EnW S Box 1 | chloroquine resistance Chloroquine was discovered a by Andersag and co-workers in 1934 and patented 1939 by I.G Farbenindustrie AG60. Following several pitfalls and errors the drug became 1960 1980 available for the treatment of malaria only from 1946 1980 onwards61. Chloroquine had 1959 1957 1970 an enormous impact on the 1974 treatment of malaria and has probably saved hundreds of millions of lives61. It was hoped that chloroquine would have a pivotal role in the WHO malaria eradication programme, which begun b in 1955 (ReF. 62). However, chloroquine resistance was first reported in 1957 — the first documented cases of chloroquine resistance were observed in the 1982 Cambodia–Thailand border. Independently, chloroquine resistance emerged in South 1997 America in 1959, and was reported in Africa 17 years after the first cases in Asia. However, once choloroquineresistant Plasmodium falciparum strains had gained a foothold in Africa, they dispersed rapidly from country to country63. By the early 1990s chloroquine had become useless for the treatment of P. falciparum malaria in east Africa and gradually became useless in west Africa as well over the rest of the decade61. The figure part a shows the global spread of chloroquine resistance; represent the Naturearrows Reviews | Microbiology routes of spread and dates indicate the year of arrival of clinically significant antimalarial drug resistance in that region. Chloroquine resistance is caused by multiple mutations in pfcrt (chloroquine resistance transporter gene), which encodes a transmembrane protein that is present in the digestive vacuole of Plasmodium parasites. The Lys76Thr mutation in this gene confers chloroquine resistance, but only in a genetic a background with several additional mutations in the same gene64,65. Molecular epidemiological analysis of pfcrt mutations and surrounding microsatellites have shown that the lineages from South American and Southeast Asia with the mutation in pfcrt are distinct and that the pfcrt that spread through Africa originated from Southeast Asia66. A recovery in chloroquine sensitivity following the halt of chloroquine use over a period of 10 years has been observed in Malawi, and this coincided with a decrease in the prevalence of the Lys76Thr mutation, suggesting a fitness disadvantage owing to the presence of the mutation in the absence of drug pressure67,68. As Malawi is surrounded by countries with extensive chloroquine resistance, reintroduction of chloroquine in Malawi is currently no option, as resistant parasites would probably return rapidly, imported in people from neighbouring areas. When the utility of chloroquine decreased, sulphadoxine–pyrimethamine became an important treatment. However, high level resistance to pyrimethamine spread rapidly (see the figure, part b). Interestingly, resistance to the drug emerged in the same areas as chloroquine resistance (the Amazon basin and Southeast Asia). Artemisinin resistance has now emerged in one of these regions, Southeast Asia. Figure modified, with permission, from ReF. 69 © (2009) Elsevier.

In settings such as the Cambodia–Thailand border, where parasite prevalence is low, most of the population receiving mDAs will not harbour malaria parasites. In this case it has been suggested that the population should be screened, and parasitaemic individuals should be targeted for treatment. mass screening and treatment (mSAT) has not been empirically tested, and there are concerns that it will not be possible to detect lowdensity parasitaemias and achieve the coverage rates

that are needed to affect transmission. However, the effect of mSAT on the spread of resistant malaria has been modelled. In combination with the successful replacement of artemisinin monotherapies with ACTs for presumptive treatment, the model predicts that the absolute number of infections could be reduced by mSAT but the proportion of resistant infections may increase54. Furthermore, mSAT would have to be repeated regularly for almost a decade, as terminating

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REVIEWS mSAT campaigns early would result in resurgence of infections with an increased proportion of artemisinin resistance. modelling suggests that to eliminate artemisinin resistance using ACT, all P. falciparum malaria has to be eliminated, as the last surviving parasite strains will be the most resistant ones54. It is therefore important to decrease the parasite burden in the community as low as possible and then treat the remaining artemisinin resistant strains with different drugs. However, there are only limited options to use non-ACT antimalarials for mDA or mSAT. resistance levels to chloroquine and sulphadoxin–pyrimethamine are high and fixed in Southeast Asia. Atovaquone–proguanil could be used instead but, as discussed above, the parasites can become resistant through a single point mutation in the cytochrome b gene, which confers an up to 10,000-fold reduction in sensitivity to atovaquone55. Surveillance, active case investigation and focal control. Instead of broad regional mass population approaches, which can be used in the end phase of an malaria elimination programme, interventions that target foci of infection are likely to be a more effective and sustainable method to reduce the parasite burden in the region. For active case investigation and focal control, a well-functioning surveillance system is essential as are dedicated and motivated teams to follow up patients and carry out local intensive malaria control measures such as screening and treating nearby households and controlling the insect vector population. Although logistically still difficult, the routine screening and treatment of populations at risk of spreading resistant malaria, such as miners, loggers and military families, is likely to be effective. Multiple first-line therapies. It has been proposed that varying first-line antimalarial therapies by area or age group could be an effective strategy to treat malaria56. Temporal cycling of insecticides is currently used to minimize the emergence of resistance 57 and could also be applied to first-line therapies. In this case, a population that harbours parasites that have developed resistance to the first drug in the cycling sequence would be treated with another drug after a set amount of time, decreasing the level of the resistant parasites. An evolutionary–epidemiological model based on clinical data from eight endemic regions in sub-Saharan Africa suggests that population-wide use of mFTs using three different drugs simultaneously in a population can reduce the emergence and spread of resistance in almost all transmission settings compared with a single first-line therapy or temporal cycling policies58. A population level combination of partner drugs is likely to be less effective than the application of triple therapies but it can be applied immediately and will provide some protection of the partner drugs while triple therapies are being developed and licensed. The logistical difficulties in adding and changing antimalarial policy should not be underestimated59. In reality mFT is already the prevailing practice in many countries such as Cambodia,

where the private sector has an important role in providing malaria treatment and where there is a range of antimalarial products. Thus, in practice it might not be necessary to promote mFT actively but instead to encourage the availability of multiple effective drugs in the private sector. Other malaria control measures. Other malaria control measures at the population level include vector control and, in the future, vaccines, although the most advanced malaria vaccine candidate is at least 5 years away from being made available to the public. In the meantime a goal of 100% coverage with long-lasting insecticide-treated nets (llIns) is being vigorously pursued on the Cambodia–Thailand border and includes the use of insecticide-treated hammock nets for forest workers. unfortunately, the main malaria vector species in this area, Anopheles minimus, Anopheles maculatus and Anopheles dirus, start feeding early in the evening, making the use of bed nets less effective than in sub-Saharan Africa. However, modelling work indicates that high coverage with llIns as part of an elimination programme in the region could result in an estimated reduction in transmission of 30%, which could halve the time it takes to eliminate malaria completely 54. Similarly, although indoor residual spraying has been shown to be effective in southern Africa5, it is likely to be much less effective in the Cambodia– Thailand border because the vectors are exophilic (that is, they have a preference for resting outdoors rather than indoors). However, they might have an effect when being used in selected regions. There is also a need to explore alternative methods of vector control, including protection for individuals through the use of insect repellents and insect repellent clothing.

conclusions partial artemisinin resistance has emerged in western Cambodia that is characterized by much slower parasite clearance rates after artemisinin treatment. The resistant strains have the potential to spread to different parts of the region and to subsequently become a global threat for malaria control and treatment. There are currently no alternative drugs to replace artemisinin derivatives. Atovaquone–proguanil is too expensive to deploy on a large scale and is prone to resistance development. Treatment of resistant P. falciparum strains therefore has to rely on the use of ACTs containing a potent partner drug, such as piperaquine or pyronaridine, which have not yet been compromised by resistance. Affordable high-quality, fixed combination ACTs should be made universally available to completely displace the artemisinin monotherapies and substandard drugs. Triple therapy has not yet been formally evaluated but should probably be developed and assessed before its use becomes necessary because of the development of resistance to artemisinins and multiple partner drugs. The addition of an 8-aminoquinolone as a gametocytocidal drug would be important to reduce transmission, but its safety needs to be further assessed in regions with a high prevalence of G6pD deficiency, such as western Cambodia.

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f o c u S o n a n t I m I c R o b I a l R E SRI SE ta cE V I EnW S Containing the problem in western Cambodia requires a multifaceted approach. mathematical models are useful tools to evaluate possible strategies, and these suggest that mFTs hold greater promise to contain the spread of artemisinin resistance than a single first-line treatment. ultimately, regional malaria elimination seems the only way to solve the problem, as the proportion of resistant parasites will increase with continued artemisinin exposure in a shrinking parasite pool. The WHO has launched

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a containment programme for western Cambodia and Thailand: this includes strengthening of early diagnosis and treatment programmes, banning artemisinin monotherapies in the private sector, increasing impregnated bed net coverage and documenting the migration of workers. Achieving effective control of malaria will crucially depend on the successful implementation of these strategies. The world cannot afford to lose artemisinin and artemisinin derivatives in the fight against malaria.

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32. Maude, R. J. et al. The role of mathematical modelling in malaria elimination and eradication. Trans. R. Soc. Trop. Med. Hyg. 103, 643–644 (2009). 33. Verdrager, J. Epidemiology of the emergence and spread of drug-resistant falciparum malaria in South-East Asia and Australasia. J. Trop. Med. Hyg. 89, 277–289 (1986). 34. Verdrager, J. Localized permanent epidemics: the genesis of chloroquine resistance in Plasmodium falciparum. Southeast Asian J. Trop.Med. Public Health 26, 23–28 (1995). 35. Carrara, V. I. et al. Changes in the treatment responses to artesunate-mefloquine on the northwestern border of Thailand during 13 years of continuous deployment. PLoS ONE 4, e4551 (2009). First study reporting a slight decrease in treatment responses to an ACT for uncomplicated P. falciparum malaria in an area outside the Cambodia–Thailand border region. 36. Achan, J. et al. Effectiveness of quinine versus artemether-lumefantrine for treating uncomplicated falciparum malaria in Ugandan children: randomised trial. BMJ 339, b2763 (2009). 37. Gebru, T., Hailu, A., Kremsner, P. G., Kun, J. F. & Grobusch, M. P. Molecular surveillance of mutations in the cytochrome b gene of Plasmodium falciparum in Gabon and Ethiopia. Malar. J. 5, 112 (2006). 38. Olliaro, P. & Mussano, P. Amodiaquine for treating malaria. Cochrane Database Syst. Rev. 2003, CD000016 (2003). 39. Gesase, S. et al. High resistance of Plasmodium falciparum to sulphadoxine/pyrimethamine in northern Tanzania and the emergence of dhps resistance mutation at codon 581. PLoS ONE 4, e4569 (2009). 40. Pilz, J. B. et al. In vitro sensitivity of Plasmodium falciparum to lumefantrine in north-western Thailand. Wien. Klin. Wochenschr. 116 (Suppl. 4), 41–46 (2004). 41. Wongsrichanalai, C., Pickard, A. L., Wernsdorfer, W. H. & Meshnick, S. R. Epidemiology of drug-resistant malaria. Lancet Infect. Dis. 2, 209–218 (2002). 42. von Seidlein, L. & Greenwood, B. M. Mass administrations of antimalarial drugs. Trends Parasitol. 19, 452–460 (2003). 43. Tran, T. H. et al. Dihydroartemisinin-piperaquine against multidrug-resistant Plasmodium falciparum malaria in Vietnam: randomised clinical trial. Lancet 363, 18–22 (2004). 44. Ramharter, M. et al. Fixed-dose pyronaridineartesunate combination for treatment of uncomplicated falciparum malaria in pediatric patients in Gabon. J. Infect. Dis. 198, 911–919 (2008). 45. Vivas, L. et al. Anti-malarial efficacy of pyronaridine and artesunate in combination in vitro and in vivo. Acta Trop. 105, 222–228 (2008). 46. Ringwald, P., Bickii, J. & Basco, L. Randomised trial of pyronaridine versus chloroquine for acute uncomplicated falciparum malaria in Africa. Lancet 347, 24–28 (1996). 47. Carr, A. & Amin, J. Efficacy and tolerability of initial antiretroviral therapy: a systematic review. AIDS 23, 343–353 (2009). 48. Grant, A., Gothard, P. & Thwaites, G. Managing drug resistant tuberculosis. BMJ 337, a1110 (2008). 49. Lawpoolsri, S. et al. Optimally timing primaquine treatment to reduce Plasmodium falciparum transmission in low endemicity Thai-Myanmar border populations. Malar. J. 8, 159 (2009). 50. Vale, N., Moreira, R. & Gomes, P. Primaquine revisited six decades after its discovery. Eur. J. Med. Chem. 44, 937–953 (2009). 51. Cappellini, M. D. & Fiorelli, G. Glucose-6-phosphate dehydrogenase deficiency. Lancet 371, 64–74 (2008).

REVIEWS 52. [No authors listed]. Roll Back Malaria Partnership. Global Malaria Action Plan for a malaria-free world [online] (2009). 53. Samarasekera, U. Countries race to contain resistance to key antimalarial. Lancet 374, 277–280 (2009). 54. Maude, R. J. et al. The last man standing is the most resistant: eliminating artemisinin-resistant malaria in Cambodia. Malar. J. 8, 31 (2009). Modelling paper showing that the absolute number of artemisinin-resistant malaria cases will decrease, but the proportion of artemisinin-resistant malaria cases will increase over time when elimination of resistant malaria is attempted. 55. Korsinczky, M. et al. Mutations in Plasmodium falciparum cytochrome b that are associated with atovaquone resistance are located at a putative drug-binding site. Antimicrob. Agents Chemother. 44, 2100–2108 (2000). 56. Okell, L. C., Drakeley, C. J., Bousema, T., Whitty, C. J. & Ghani, A. C. Modelling the impact of artemisinin combination therapy and long-acting treatments on malaria transmission intensity. PLoS Med. 5, e226 (2008). 57. Castle, S. J., Toscano, N. C., Prabhaker, N., Henneberry, T. J. & Palumbo, J. C. Field evaluation of different insecticide use strategies as resistance management and control tactics for Bemisia tabaci (Hemiptera: Aleyrodidae). Bull. Entomol. Res. 92, 449–460 (2002). 58. Boni, M. F., Smith, D. L. & Laxminarayan, R. Benefits of using multiple first-line therapies against malaria.

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Proc. Natl Acad. Sci. USA 105, 14216–14221 (2008). Modelling paper showing the delay in development of antimalarial drug resistance with regional deployment of MFTs. Shretta, R., Omumbo, J., Rapuoda, B. & Snow, R. W. Using evidence to change antimalarial drug policy in Kenya. Trop. Med. Int. Health 5, 755–764 (2000). Coatney, G. R. Pitfalls in a discovery: the chronicle of chloroquine. Am. J. Trop. Med. Hyg. 12, 121–128 (1963). Jensen, M. & Mehlhorn, H. Seventy-five years of Resochin in the fight against malaria. Parasitol. Res. 105, 609–627 (2009). Greenwood, B. M. et al. Malaria: progress, perils, and prospects for eradication. J. Clin. Invest. 118, 1266–1276 (2008). Payne, D. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol.Today 3, 241–246 (1987). Djimde, A. et al. A molecular marker for chloroquineresistant falciparum malaria. N. Engl. J. Med. 344, 257–263 (2001). Fidock, D. A. et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol. Cell 6, 861–871 (2000). Wootton, J. C. et al. Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum. Nature 418, 320–323 (2002).

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67. Kublin, J. G. et al. Reemergence of chloroquinesensitive Plasmodium falciparum malaria after cessation of chloroquine use in Malawi. J. Infect. Dis. 187, 1870–1875 (2003). 68. Laufer, M. K. et al. Return of chloroquine antimalarial efficacy in Malawi. N. Engl. J. Med. 355, 1959–1966 (2006). 69. Plowe, C. V. The evolution of drug-resistant malaria. Trans. R. Soc. Trop. Med. Hyg.103, S11–S14 (2009).

Acknowledgements

We thank N. J. White for his critical review of the paper. This work was supported by the Wellcome Trust.

Competing interests statement

The authors declare no competing financial interests.

Databases Entrez Genome Project: http://www.ncbi.nlm.nih.gov/sites/entrez?db=genomeprj Plasmodium falciparum Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez pfcrt | pfmdr1

furtHer information arjen m. Dondorp’s homepage: http://www.tropmedres.ac amfm website: http://www.theglobalfund.org/en/amfm All links Are ACTive in The online Pdf

RESEARCH HIGHLIGHTS

A N T I PA R A S I T I C D R U G S

Two-pronged tactics for malaria control Most existing and investigational drugs for malaria are targeted at the stages of the Plasmodium spp. parasite life cycle in red blood cells, which underlie the disease manifestations. Targeting the earlierstage exo-erythocytic forms, which multiply in the host liver following infection before emerging into the bloodstream, could be prophylactic but developing high-throughput assays for such activity has been more technically challenging than for blood-stage activity. Writing in Science, an academia–industry consortium describes the use of a high-content imaging assay of liverstage activity to identify a new class of compounds that confer complete protection against parasite challenge in rodent models of malaria, as well as showing potent in vivo activity against blood-stage parasites. In vitro, only ~1% of liver cells become infected by malaria parasites at the sporozoite stage of the life cycle, and so to provide the throughput necessary for screening thousands of compounds the authors refined an in vitro assay with Plasmodium yoelii sporozoites to identify infected cells through high-content imaging analysis. They then used this assay to screen a set of >4,000 commercially available compounds that had previously been shown to have blood-stage activity. Based on an analysis of the structures of compounds that possessed

both liver-stage and blood-stage activity, a cluster of compounds containing an imidazolopiperazine scaffold was selected for further investigation, which was considered to be attractive because it is structurally unrelated to existing drugs and is chemically tractable. By synthesizing >1,200 imidazolopiperazine derivatives and optimizing their potency against blood-stage parasites, a compound — known as GNF179 — was identified that also possessed suitable pharmacokinetic properties for oral testing in animal models of malaria. In mice infected with Plasmodium berghei, GNF179 lowered parasitaemia levels by 99.7% after the administration of a single 100 mg per kg oral dose, and prolonged the survival of mice by an average of 19 days, compared with 12.5 days and 7 days for the existing antimalarial drugs chloroquine and artesunate, respectively. Furthermore, GNF179 provided complete protection against a P. berghei sporozoite challenge after a single 15 mg per kg oral dose. Initial studies of the mechanism of action of GNF179 indicated that it was distinct from existing drugs, and also that its target might be a protein (encoded by a parasite gene known as pfcar1) that has been tentatively linked to protein folding

NATURE REVIEWS | DRUG DISCOVERY

in the endoplasmic reticulum, which would presumably be important in both the liver and blood stages of parasite infection. Given that antimalarials with novel mechanisms of action are desirable because they could be less likely to show crossresistance with existing drugs, this study provides a valuable starting point for developing more effective drugs that might be used not only to treat infected patients but also to provide protection against infection and reduce transmission.

Peter Kirkpatrick

ORIGINAL RESEARCH PAPER Meister, S. et al. Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery. Science 334, 1372–1377 (2011) FURTHER READING Wu, T. et al. Imidazolo piperazines: hit to lead optimization of new antimalarial agents. J. Med. Chem. 54, 5116–5130 (2011) | Mazier, D. et al. A pre-emptive strike against malaria’s stealthy hepatic forms. Nature Rev. Drug Discov. 8, 854–864 (2009)

NEWS & ANALYSIS New neglected disease research scheme pools IP and expertise WIPO Re:Search aims to encourage drug discovery for neglected diseases by broadening the scope of the assets members are willing to share. Simon Frantz A consortium of private and public organizations launched in October 2011 by the World Intellectual Property Organization (WIPO) aims to accelerate the discovery and development of new drugs, vaccines and diagnostics for neglected tropical diseases, malaria and tuberculosis. Because much of the knowledge and data essential for efficient drug discovery are not patented, patentable or publicly available, the new initiative — called WIPO Re:Search — intends to pool not just intellectual property (IP) but also intellectual capital, including screening hits, expertise and know-how. “What we’re trying to do is to take out some of the error from the trial-and-error process of drug discovery, so that neglected tropical disease researchers, many of whom may be coming from the developing world, can have the same level of expertise or resources that big companies have at their disposal,” says Donald Joseph, COO of BIO Ventures for Global Health (BVGH), the non-profit organization that is administering the initiative. “Knowing what’s worked before, what hasn’t worked, that’s the kind of access we’re trying to provide and make available.” The initiative reflects a growing trend towards openness in tackling global health challenges. The Medicines Patent Pool, launched last year, focuses on making products that are already approved for HIV/ AIDS available on favourable terms to developing world markets. WIPO Re:Search, however, has grown out

of GlaxoSmithKline (GSK)’s Pool for Open Innovation for Neglected Tropical Diseases (POINT), which was launched in 2009 to bolster the early-stage neglected disease pipeline by providing a platform to share IP. POINT, which is now absorbed into the new initiative, was to some extent limited in that it was perceived to be a GSK-only initiative, says Joseph. It led to only one publicly disclosed partnership, in which GSK, iThemba Pharmaceuticals and the Emory Institute of Drug Discovery started working together to develop inhibitors of malate synthase and isocitrate lyase as targets for latent-stage tuberculosis. “It wasn’t as though [POINT] was doing anything wrong or ineffectively, but simply that [WIPO Re:Search] turbo-charges the effort, and gets more scale, more depth, more scope,” says Joseph. WIPO Re:Search has already signed up major pharmaceutical companies — including AstraZeneca, GSK, Novartis, Pfizer and Sanofi — academic institutes and non-profit organizations like the Drugs for Neglected Diseases initiative (DNDi). One lesson Joseph says they learned from administering POINT is the constraints of restricting assets to IP. “Scientists don’t typically think in terms of IP, they think in terms of the knowledge that they have, and the knowledge that they need for the experiments that they want to generate and the clinical trials that they want to run,” he says. “I’m not saying patent pools are a bad model. It’s more the idea of broadening the scope beyond pure IP to reach what actually happens in the labs and what happens in relationships, and so it was

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a conscious part of WIPO Re:Search to engage expertise and services.” Some companies, for instance, intend to host neglected disease researchers in their facilities. “We’ll be opening up access to AstraZeneca labs and will consider requests from neglected disease scientists wishing to work on their targets,” says Manos Perros, Vice President and Head of Infection Innovative Medicines at AstraZeneca. “Guest scientists will benefit from scientific mentoring and access to innovative technologies including cheminformatics support, and it’s also an opportunity for us to learn from other researchers how they are thinking about diseases and mechanisms of actions for treating those diseases.” For Bernard Pécoul, Executive Director of DNDi, the scheme is “clearly a step in the right direction”. Yet he remains critical of its narrow scope. Whereas it currently aims to improve access to neglected disease medicines in just the 49 least-developed countries, he thinks it should aim to increase access for all developing countries. Access is important, says Joseph, but the aim in initial stages is to get as much engagement as possible. Although the initiative is not about making money, profit is nevertheless always a powerful motivating factor, says Jeremy Phillips, Editor of Journal of Intellectual Property Law & Practice. He therefore proposes that one way to increase the scheme’s odds of success could be to bolt on financial incentives like tax breaks and entitlement to investment grants. “After all, one of the reasons why neglected tropical diseases are neglected is that there’s no material incentive to un-neglect them.” VOLUME 11 | JANUARY 2012 | 5

PerSPecTIveS Science and Society

Experimental human challenge infections can accelerate clinical malaria vaccine development Robert W. Sauerwein, Meta Roestenberg and Vasee S. Moorthy

Abstract | Malaria is one of the most frequently occurring infectious diseases worldwide, with almost 1 million deaths and an estimated 243 million clinical cases annually. Several candidate malaria vaccines have reached Phase IIb clinical trials, but results have often been disappointing. As an alternative to these Phase IIb field trials, the efficacy of candidate malaria vaccines can first be assessed through the deliberate exposure of participants to the bites of infectious mosquitoes (sporozoite challenge) or to an inoculum of blood-stage parasites (blood-stage challenge). With an increasing number of malaria vaccine candidates being developed, should human malaria challenge models be more widely used to reduce cost and time investments? This article reviews previous experience with both the sporozoite and blood-stage human malaria challenge models and provides future perspectives for these models in malaria vaccine development. Half of the world’s population (more than 3 billion people) live in malaria-endemic areas, and an estimated 243 million cases of malaria led to nearly 863,000 deaths in 2008 according to the World Health Organization (WHO) World Malaria Report 2009. There are five species of human malaria parasite: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi. Recent evidence indicates that P. ovale is composed of two subspecies1. Most infections are caused by P. falciparum, which is particularly dominant in sub-Saharan Africa. P. vivax is the most widely spread cause of malaria, being responsible for an estimated 80 million to 300 million cases every year, and thus it accounts for a major burden of disease2. Plasmodium parasites are highly prevalent in Asia and South America, where individuals can be infected with more than one malaria parasite species simultaneously. Infective foci of P. knowlesi have been identified in the past decade in Malaysia, where P. knowlesi is transmitted from simian hosts to humans.

Plasmodia are transmitted by the bites of infected Anopheles mosquitoes. Control strategies are based on the early diagnosis and treatment of uncomplicated infections with artemisinin-based combination therapies, thereby also decreasing transmission3, combined with preventive measures aimed at vector (mosquito) control. Artemisinin-based combination therapies — such as artemether plus lumefantrine, artesunate plus amodiaquine, artesunate plus mefloquine or artesunate plus sulphadoxine–pyrimethamine — have been the WHO-recommended treatment for uncomplicated P. falciparum malaria since the development of widespread resistance to chloroquine and sulphadoxine–pyrimethamine. Unfortunately, in P. falciparum, resistance has been observed to all current antimalarial drugs (amodiaquine, chloroquine, mefloquine, quinine and sulphadoxine-pyrimethamine) and, more recently, also to artemisinin derivatives. For uncomplicated P. vivax infection, treatment with chloroquine is recommended in

NATURE REVIEWS | Immunology

those areas without chloroquine resistance. Artemisinin-based combination therapies can be used as an alternative treatment for chloroquine-resistant P. vivax. In these cases, artemether plus sulphadoxine–pyrimethamine is not recommended because P. vivax can acquire resistance to sulphadoxine– pyrimethamine. To fully eradicate P. vivax infection, primaquine must be administered to prevent relapses. P. ovale and P. malariae infections are treated similarly to P. vivax infections, although there is no need for primaquine treatment in patients who are infected with P. malariae, as this species does not form dormant or latent hypnozoites in hepatocytes (see the WHO Guidelines for the Treatment of Malaria). Vector control is the primary intervention for decreasing malaria transmission at the community level. When universal vector control coverage is achieved by impregnating bed nets and spraying indoor surfaces of houses with insecticides, malaria transmission can be decreased to close to zero. Unfortunately, the increasing resistance of mosquitoes to insecticides such as dichlorodiphenyltrichloroethane (DDT) and pyrethroids, particularly in Africa, poses challenges to current prevention policies (see the WHO World Malaria Report 2009). In this context, the development of an effective vaccine could make a significant contribution to the fight against malaria. Ambitious goals in this regard have been set by the Malaria Vaccine Technology Roadmap Process, which aims to achieve a licensed first-generation P. falciparum malaria vaccine with more than 50% protective efficacy against severe disease and death, lasting for at least 1 year, by the year 2015. Malaria vaccine development has been fuelled by new technology enabling the sequencing of the P. falciparum, P. vivax and Anopheles gambiae genomes and the development of experimentally relevant animal models, combined with significant increases in financial resources from funders such as the Bill & Melinda Gates Foundation, the European Union, the US National Institutes of Allergy and Infectious Diseases and the US Agency for International Development. Currently, there are 38 P. falciparum and two P. vivax candidate malaria vaccines or VOlUME 11 | jANUARy 2011 | 57

PersPectives

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Figure 1 | Plasmodium falciparum life cycle showing the three developmental stages of the parasite that are targeted by malaria vaccine candidates. Parasites (sporozoite stage) are injected into the skin by a female Anopheles spp. mosquito. From the skin, a proportion of sporozoites will travel 0CVWTG 4GXKGYU ^ +OOWPQNQI[ through the bloodstream to the liver. Some sporozoites will be trapped in regional lymph nodes. In hepatocytes, parasites develop and multiply for 6â&#x20AC;&#x201C;7 days before merosomes are budded from the cell and enter the hepatic sinusoids. Merosomes eventually rupture, releasing merozoite-stage parasites that invade erythrocytes for further reproduction. clinical malaria is caused by the 48-hour cyclical proliferation of asexual-stage parasites in erythrocytes. Malaria mortality is primarily due to organ dysfunction, in particular of the brain, following sequestration of infected erythrocytes in the microvasculature. The development of sexual forms of the parasite (gametocytes) in the blood allows the transmission of parasites to mosquitoes with subsequent bites. Once ingested, the parasite gametocytes taken up in the blood further differentiate into male or female gametes and then fuse in the mosquito gut. This produces an ookinete that penetrates the gut lining and produces an oocyst in the gut wall. When the oocyst ruptures, it releases sporozoites that migrate through the mosquitoâ&#x20AC;&#x2122;s body to the salivary glands, where they are then ready to infect a new human host. Image is modified, with permission, from REF. 59 ÂŠ (2004) Macmillan Publishers Ltd. All rights reserved.

vaccine components in advanced preclinical or clinical development as listed by the WHO Malaria Vaccine Rainbow Tables. Malaria vaccine candidates are categorized according to the Plasmodium life cycle stage at which the targeted antigen is expressed (FIG. 1). Pre-erythrocytic stage vaccines aim to prevent the passage of parasites through the human liver and subsequent blood-stage infection, leading to

the induction of sterile immunity. Asexual erythrocytic stage vaccines focus on delaying or decreasing parasite multiplication in red blood cells, thereby decreasing morbidity and preventing mortality. Transmissionblocking vaccines consist of sexual- or mosquito-stage antigens that prevent infection passing from humans to mosquitoes, thereby decreasing the spread of malaria in the population.

58 | jANUARy 2011 | VOlUME 11

Generally, less than 10% of preclinical vaccine projects progress to Phase III clinical evaluation4. Clinical development is time consuming and costly. Candidate malaria vaccines are selected downstream of clinical testing on the basis of safety, immunogenicity and, eventually, efficacy profiles. Whereas the first two criteria can generally be assessed in a small initial Phase I trial, field vaccine efficacy can only be assessed in Phase II trials, which require larger study groups in malaria-endemic areas. The sample size of Phase II trials depends on the prevalence of malaria infections in that area and the expected efficacy of the candidate vaccine. Ideally, immunological assays carried out in initial clinical trials should predict potential efficacy in subsequent trials, by analogy with, for example, hepatitis B virus surface antigen (HBsAg)specific antibody titres for the hepatitis B vaccine. However, with the current lack of unequivocal correlates of immune protection against malaria in either animal models or in vitro assays on human samples, there is a continuous need to test field efficacy in time-consuming and costly Phase II trials in malaria-endemic areas. There are only a limited number of competent field trial sites for malaria that can adhere to the good clinical practice guidelines established by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH), which describe the monitoring, reporting and archiving responsibilities of all participants in the conduct of clinical trials (see The Malaria Product Pipeline: Planning for the Future). Finally, there seems to be a downward trend in malaria incidence in several endemic areas, most probably as a result of improved policy and adherence to malaria control measures, and this will further increase the size and costs of Phase II field trials5. Human experimental sporozoite infections carried out under strictly controlled laboratory and clinical conditions, in which volunteers are exposed to the bites of laboratory-reared Plasmodium-infected mosquitoes, are an intermediate step between Phase I and Phase II trials, providing information on preliminary vaccine efficacy. It is common practice to test the efficacy of pre-erythrocytic stage malaria vaccine candidates by experimental sporozoite infection before going into the field. In such cases, a distinction is thus made between Phase IIa trials using experimental infection of volunteers in non-endemic areas and Phase IIb field trials in endemic areas. www.nature.com/reviews/immunol

PersPectives Poor preliminary efficacy in Phase IIa trials may subsequently halt progression of the vaccine candidate to Phase IIb trials. By contrast, the efficacy of asexual erythrocytic stage vaccine candidates is generally assessed in field trials only, although blood-stage challenge models have been used. Here, we present the history of artificial malaria challenge infections, the clinical aspects of P. falciparum challenges using sporozoites or blood-stage parasites and experience with P. vivax challenges. We discuss the strengths and limitations of both models and provide future perspectives. Historical perspective Deliberate infection of humans with malaria was first carried out in 1917 by Wagner von jauregg6 as a therapy primarily for patients with neurosyphilis, and he was awarded the Nobel Prize in Medicine for his work in 1927. Thousands of patients have undergone this treatment, which was administered by the bites of infectious mosquitoes or by intravenous or subcutaneous inoculation of dissected Plasmodium sporozoites suspended in media. Historically, P. vivax was used most frequently, but infections were also carried out with P. falciparum, P. malariae and P. ovale. The objective was to induce a febrile illness that was thought to be beneficial for the prognosis of the disease. This practice stopped with the advent of antibiotics for the treatment of the Treponema pallidum infection that causes syphilis. In the 1960s, experimental human malaria infections were used to assess the effects of anti-malaria treatments on healthy non-immune male inmates in the United States7. Following the discovery of protocols for the continuous culture of P. falciparum in 1976 (REF. 8) and protocols for the generation of mature P. falciparum gametocytes in vitro in 1981 (REF. 9), laboratory-reared infectious mosquitoes could be produced10 and human malaria sporozoite infections could be carried out more routinely. The first well-documented study of human experimental malaria infection with these laboratory-reared infectious mosquitoes was carried out in 1986 at the US Walter Reed Army Institute of Research (WRAIR), the US Naval Medical Research Institute (NMRI) and the US National Institutes of Health (NIH). Six volunteers were infected with P. falciparum sporozoites by the bites of infectious Anopheles freeborni and Anopheles stephensi mosquitoes11. The following year, the efficacies of the first recombinant protein and synthetic peptide

P. falciparum vaccines were tested in experimentally infected volunteers12,13. Since the late 1980s, the number of institutions carrying out experimental infections with P. falciparum sporozoites has been growing. In 2007, data were published from a total of 532 volunteers14. So far, unpublished analysis shows that a total of 1,343 volunteers have been experimentally infected with P. falciparum between 1985 and 2009 (REF. 15); 526 of these volunteers took part in vaccine trials (TABLE 1), and of these, 118 volunteers were protected against infection by the vaccine candidate. The most successful immunogens were RTS,S (a pre-erythrocytic stage vaccine consisting of the P. falciparum circumsporozoite protein combined with HBsAg; developed by GlaxoSmithKline in partnership with PATH Malaria Vaccine Initiative) and irradiated whole parasites delivered by mosquito bite. comparison with field trials Differences between natural and experimental infections mean that it is important to validate the results of Phase IIa challenge trials with data from Phase IIb field trials in malaria-endemic areas. Only three candidate vaccines have been assessed by both types of trial, allowing a comparison of the protective outcomes. The best studied candidate vaccine, RTS,S, which is currently in Phase III trials, has repeatedly demonstrated a protective efficacy of ~30–50% in Phase IIa trials with sterile protection as the study end point 16–18. Interestingly, a similar ~30–50% efficacy of RTS,S was found in Phase IIb trials in the field using time to first clinical malaria episode as the primary study end point19. A similar association between the results of Phase IIa and Phase IIb trials was found when testing long-term protection in adults19,20. A second pre-erythrocytic stage candidate vaccine, ME-TRAP (a multi-epitope string fused to thrombospondin-related adhesion protein), delivered by a DNA prime and attenuated poxvirus boost, induced complete protection in only a few volunteers (three out of 74) in Phase IIa trials, and no protection was found in adult Phase IIb field studies in the Gambia21,22. Artificial blood-stage challenge has been used in a Phase II trial after immunization with Combination B (a combination of merozoite surface protein 1 (MSP1), MSP2 and ring-infected erythrocyte surface antigen (RESA)) in 17 volunteers, which resulted in no decrease in parasite growth rates23,24; this is in line with results from a Phase IIb trial of Combination B conducted in Papua New

NATURE REVIEWS | Immunology

Guinea25. These limited data indicate that results obtained in experimental challenges are generally in line with results in the field, but more comparisons are required before definite conclusions can be drawn. experimental sporozoite infection The delivery of sporozoite-stage malaria parasites by mosquito bites has traditionally been used as a model to test pre-erythrocytic stage vaccines. Since the late 1980s, standardization of experimental sporozoite infections has improved and efforts to further increase harmonization are ongoing. Such infections are currently routinely carried out at: the US Military Malaria Vaccine Program; the University of Maryland, USA; Radboud University Nijmegen Medical Centre (RUNMC), the Netherlands; the University of Oxford, UK; and, more recently, Seattle Biomed, USA15. All centres use A. stephensi mosquitoes that feed on either the chloroquine-sensitive NF54 strain of P. falciparum or the 3D7 clone of NF54. In addition, limited numbers of volunteers have been challenged with the 7G8 strain of P. falciparum14. Approximately 14–21 days after feeding, mosquitoes are checked for infection by microscopic examination of salivary glands. Healthy human volunteers are subsequently exposed to the bites of five infectious mosquitoes for either 5 or 10 minutes (FIG. 2). Almost 100% of volunteers bitten by five infected mosquitoes develop patent parasitaemia, with very rare exceptions26,27. Infection rates drop significantly when volunteers are exposed to fewer than five infected mosquitoes27,28. After infection, subjects are monitored closely on an outpatient basis. Signs and symptoms such as headache, myalgia and fever are noted, and a physical examination and thick blood smears (a drop of blood on a glass slide) are carried out once, twice or thrice daily. The period before blood-stage parasites can be detected in thick smears by microscopy (the prepatent period) ranges from 7 to 20 days, with a mean of approximately 11 days7,14,26. As soon as parasites are microscopically detected, volunteers are treated with a curative regimen of chloroquine, artemether plus lumefantrine, or atovaquone plus proguanil. Nearly all volunteers will develop symptoms of clinical malaria infection; approximately one-fifth of volunteers temporarily develop symptoms graded as severe (symptoms that prevent daily activities), but severe or life-threatening malaria has never occurred26. The most common symptoms are fatigue and headache, VOlUME 11 | jANUARy 2011 | 59

PersPectives Table 1 | Summary of published Phase iia sporozoite challenge trials with Plasmodium falciparum candidate vaccines Vaccine

Plasmodium protein

Category

number of number of volunteers volunteers challenged protected

year of publication

Institution or company

Irradiated sporozoites

Whole parasite

Pre-erythrocytic 37

Several products

cSP

Several products AMA1 with AS02A or AS01B

20 (54.05%)

1970s–1993

NMrI* and WrAIr*; University of Maryland, USA; University of Sydney, Australia

Pre-erythrocytic 317

94 (29.65%)

1987–2009

University of Maryland; WrAIr*; University of Oxford, UK; Johns Hopkins University School of Hygiene and Public Health, Maryland, USA; NMrI*; University of Lausanne, Switzerland

12,13,16, 18,20,22, 40,45,62, 66–73

TrAP

Pre-erythrocytic 74

3 (4.05%)

2003–2006

University of Oxford

22,74,75

AMA1

Asexual erythrocytic

0 (0%)

2009

US Military Malaria vaccine Program

LSA1-Nrc with AS01 or AS02

LSA1

Pre-erythrocytic 22

0 (0%)

2010

WrAIr*

NYvAc-Pf7

cSP, SSP2, LSA1, MSP1, SerA, AMA1, Pfs25

All stages

1 (2.86%)

1998

WrAIr*

FFM Me-TrAP plus Pev3A

cSP, TrAP and AMA1

Pre-erythrocytic 24 and asexual erythrocytic

0 (0%)

2008

University of Oxford

SPf(66)30 or SPf(105)20 with Alum

MSP

Asexual erythrocytic

0 (0%)‡

1988

Universidad Nacional de colombia

MuStDO 5

cSP, eXP1, SSP2, LSA1, LSA3

Pre-erythrocytic 31

0 (0%)

2005

Naval Medical research center*

FMP1 with AS02A

MSP1

Asexual erythrocytic

0 (0%)

2005

WrAIr*

Unknown

Refs

60–65

Alum, aluminium hydroxide adjuvant (Alhydrogel; Brenntag biosector); AMA1, apical membrane antigen 1; AS01, GlaxoSmithKline adjuvant system 01; cSP, circumsporozoite protein; eXP1, exported protein 1; FFM Me-TrAP, multi-epitope string fused to TrAP that is delivered in fowlpox virus strain FP9 and modified vaccinia virus Ankara vectors in prime–boost combinations; FMP1, carboxy-terminal region of MSP1; LSA, liver-stage antigen; LSA1-Nrc, full-length carboxy- and amino-terminal flanking domains and two of the 17 amino acid repeats from the central repeat region of LSA1; MSP, merozoite surface protein; MuStDO 5, multi-stage DNA vaccine operation 5 antigens; NMrI, Naval Medical research Institute, USA; NYvAc-Pf7, a highly attenuated vaccinia virus with seven P. falciparum genes inserted into its genome; Pev3A, virosomal formulation of cSP and AMA1; Pfs25, 25kDa ookinete surface antigen; SerA, serine-repeat antigen protein; SSP2, sporozoite surface protein 2; SPf, synthetic P. falciparum peptides of MSP; TrAP, thrombospondin-related adhesion protein; WrAIr, Walter reed Army Institute of research, USA. *currently the US Military Malaria vaccine Program. ‡Three of five volunteers immunized with SPf(66)30 eventually cleared parasitaemia after they experienced asexual parasitaemia that was detectable by microscopy.

and severe symptoms can include headache, fatigue, malaise, chills, myalgia, rigors, nausea and vomiting. Clinical symptoms generally coincide with the detection of blood-stage parasites at densities of 10–20 parasites per μl of blood by microscopy of thick blood smears26. This corresponds to a parasitaemia of approximately 0.0004%. Severe malaria is generally diagnosed when parasitaemia is 3 to 4 logs greater than the peak parasitaemia in challenge trials. After the start of malaria treatment, symptoms can temporarily increase in severity but subside quickly with an average duration of approximately 2–3 days. Routine laboratory checks generally show a moderate decrease in leukocyte and platelet numbers during infection, with no change in haemoglobin concentration27. Bleeding or thrombogenic complications have never been described26,27.

Abnormalities of liver enzymes have been observed, but these abnormalities did not result in clinical manifestations and they resolved after a few days26,27. Immediate treatment of volunteers at the earliest phase of microscopically detectable blood-stage infection ensures that the potential risks of complications associated with severe malaria are minimized to the greatest extent possible. Indeed, human malaria challenge infections have been shown to be safe in the 1,343 volunteers challenged so far 14,26,27. Recently, safety concerns were raised because of a cardiac event in a young volunteer shortly after treatment for diagnosed malaria, although a definite relationship between the cardiac event and the experimental malaria infection was not established29. Nevertheless, it has been generally agreed that volunteers with an increased risk of cardiac disease should be excluded from such trials.

60 | jANUARy 2011 | VOlUME 11

In addition to the clinical manifestations, participation in an experimental sporozoite infection trial has a major impact on the daily life of volunteers, particularly because of the intense follow-up with blood sampling several times daily. Volunteers’ perception of their participation in such a trial depends mainly on whether they have realistic expectations of trial procedures and the severity of symptoms, indicating the importance of providing accurate and sufficient information to volunteers before the onset of the trial. Measurement of parasitaemia A real-time quantitative PCR assay based on 18S ribosomal RNA gene transcripts has been developed for tracking the kinetics of developing parasitaemia before a positive diagnosis of infection can be made from a thick blood smear using microscopy30. This www.nature.com/reviews/immunol

PersPectives assay is becoming increasingly important for assessing very low parasite densities and incremental changes in density in smallscale Phase IIa trials31. The detection of parasites below microscopy thresholds by PCR allows for a detailed analysis of cyclical parasite growth in the blood, albeit for a short time window of 2â&#x20AC;&#x201C;3 days between liver-stage infection and microscopic detection30. Several statistical models have been developed to further analyse profiles of parasitaemia and partial protection in vaccine trials32â&#x20AC;&#x201C;34. For example, these models allow a separate estimation of liver-stage and blood-stage parasite development. From the first wave of parasite DNA that is detected in the blood, an estimation can be made of the number of merozoites released from the liver, making it possible to approximate the extent of pre-erythrocytic stage inhibition resulting from a vaccine (simulated in FIG. 3a). Similarly, the ratio of parasite DNA between the second and first replication cycles in the blood reflects the growth rate of blood-stage parasites. Comparing these ratios between test subjects and controls can indicate inhibitory effects of a vaccine candidate on the growth of blood-stage parasites (simulated in FIG. 3b). Such analyses could be of particular interest in trials of multi-stage vaccines (combining both liver- and blood-stage antigens) to assess stage-specific protective immunity. For example, statistical modelling of parasitaemia from a recent Phase IIa trial with the vaccine candidate apical membrane antigen 1 (AMA1), a protein that is mainly expressed by blood-stage parasites, indicates inhibition of pre-erythrocytic parasite stages35, which highlights the possible role of sporozoite-expressed AMA1 in disease progression36. experimental blood-stage infection The evaluation of asexual erythrocytic stage vaccine candidates requires follow-up of blood-stage parasitaemia over a sufficiently lengthy period of time to determine parasite growth rates. This requirement could compromise the safety of volunteers, as blood-stage parasitaemia is responsible for malaria morbidity and even mortality. In currently accepted protocols, the appearance of Plasmodium-infected erythrocytes in thick blood smears examined microscopically leads to immediate initiation of treatment with curative anti-malarial drugs. Harbouring higher numbers of parasites in the bloodstream increases the risks to volunteer safety, so the length of the observation period for parasite multiplication in erythrocytes is limited.

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Figure 2 | Timeline of Plasmodium falciparum sporozoite challenge infection in humans. Gametocytes are derived from in vitro parasite culture in donor blood and are fed to laboratory-reared 0CVWTG 4GXKGYU ^ +OOWPQNQI[ Anopheles stephensi mosquitoes. After 14â&#x20AC;&#x201C;21 days, five infectious mosquitoes are allowed to feed on malaria-naive human volunteers for 5â&#x20AC;&#x201C;10 minutes. Subsequent development of liver-stage parasites is subclinical and takes approximately 6 days. Parasites can be detected in the blood of unprotected volunteers by microscopy (using a thick blood smear) on average 11 days (range 7â&#x20AC;&#x201C;15 days) after challenge.

A possible solution is the intravenous inoculation of very small numbers of infected erythrocytes, based on the idea that such a low level of sub-microscopic parasitaemia will not result in clinical risks and will allow extended follow-up of parasite replication in erythrocytes. The number of parasites that are inoculated is approximately 1,000 times lower than the estimated number of merozoites released from the liver following a standard experimental sporozoite challenge with bites from five infected mosquitoes. This allows for an extended blood-stage follow-up of approximately three more replication cycles (6 days) before thick blood smear detection thresholds are reached, with obligatory treatment. A master cell bank of infected erythrocytes for human clinical use has been generated by storing infected erythrocytes from two parasitaemic volunteers who were infected by mosquito bites, in compliance with blood bank safety criteria37. Since the 1990s, approximately 50 humans have been infected by direct inoculation of infected erythrocytes from this master cell bank. The length of the prepatent period â&#x20AC;&#x201D; the interval from inoculation until infected erythrocytes are microscopically detectable â&#x20AC;&#x201D; correlates with the number of inoculated parasites7. The sensitivity of the model has been further increased by the administration of very small inoculae of infected erythrocytes, combined with the introduction of the quantitative real-time PCR assay for measuring parasite growth rates during this sub-microscopic period37,38. With inoculae as small as 300 infected erythrocytes, parasite growth curves were generated over a 7â&#x20AC;&#x201C;9-day period before initiation of treatment was required 37.

NATURE REVIEWS | Immunology

The blood-stage challenge model has several potential shortcomings. The viability of the injected parasites can only be determined retrospectively by culture, so it is difficult to standardize the exact number of viable injected parasites. Differences of a factor of ten in terms of the number of viable parasites have been described between inoculae38,39. Furthermore, although the small number of inoculated parasites allows for a long window of observation, it may also boost the immune response, and low-level blood-stage infections are very efficient at inducing complete protection40. Finally, the liver stage of parasite development is circumvented by this model, bypassing potential immune effects induced by the vaccine on liverstage parasites. This may be important, as some asexual erythrocytic stage vaccine candidate antigens can also be expressed during the liver stage36. However, irrespective of these disadvantages, low-dose blood-stage challenges allow sufficient time to monitor several parasite multiplication cycles. With further validation, they might function as a crucial decision point for progress to Phase IIb trials, thereby saving time and money, and decreasing the requirement for Phase IIb trial subjects. So far, only one asexual erythrocytic stage vaccine has been tested by blood-stage challenge24. The results of a second trial with the vaccine AMA1 carried out at the University of Oxford, UK, will soon be reported (ClinicalTrials.gov identifier: NCT00984763). A direct comparison between blood-stage and sporozoite-stage challenge infections will be helpful to determine the most suitable model to test such asexual erythrocytic stage vaccines. VOlUME 11 | jANUARy 2011 | 61

PersPectives

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Plasmodium vivax infection Although the first experimental human malaria infections were carried out with P. vivax41, the standardization of P. vivax challenge for routine use has proven to be much more difficult than for P. falciparum challenge. A major hurdle is the absolute requirement for reticulocytes or young erythrocytes to obtain long-term in vitro growth of P. vivax. Nevertheless, promising results have been obtained through an alternative approach in which experimental infections are initiated using wild-type P. vivax parasites obtained from infected humans in Colombia. Blood from P. vivax-infected patients was assessed using routine blood bank procedures to exclude the presence of other transmissible agents (such as T. pallidum, hepatitis B virus and hepatitis C virus) and was subsequently fed to laboratory-reared Anopheles albimanus mosquitoes. After 14â&#x20AC;&#x201C;15 days, mosquitoes were allowed to feed for 10â&#x20AC;&#x201C;15 minutes on the forearms of healthy human volunteers. A total of 40 non-immune volunteers took part in two different trials, and data from 17 volunteers have been published so far15,42. After microscopic detection of parasites by thick blood smear, all participants were treated with a combination of chloroquine and primaquine. Because a proportion of P. vivax parasites can lay dormant as hypnozoites in the liver or develop slowly in humans, resulting in long prepatent periods23, primaquine is prescribed to

ensure clearance of all liver-stage parasites. This complicated protocol may be further compromised by drug resistance of some P. vivax strains, as is commonly observed in Southeast Asia. The most frequently reported symptoms were myalgia, headache and malaise, without the occurrence of severe or serious adverse events. The prepatent period was 9â&#x20AC;&#x201C;13 days42. The P. vivax challenge model has been further developed in the US Military Malaria Vaccine Program by the transportation of freshly infected Anopheles dirus mosquitoes from the Thaiâ&#x20AC;&#x201C; Burmese border to infect malaria-naive volunteers in the United States (ClinicalTrials. gov identifier: NCT00935623). Currently, the first P. vivax vaccine candidate, based on the P. vivax circumsporozoite protein VMP001, is being tested by such challenge studies in the United States (ClinicalTrials. gov identifier: NCT01157897). Quantitative real-time PCR detection of P. vivax parasite load and genotyping of wild-type parasites will further improve the P. vivax challenge model15. Hopefully, the future development of new laboratory tools, including the use of stem cells as a source for young erythrocytes, will facilitate the long-term in vitro culture of P. vivax. Strengths and limitations Experimental human challenges aim to predict the potential efficacy of vaccine candidates against natural infections in the field. A major strength of the sporozoite

62 | jANUARy 2011 | VOlUME 11

infection model is the use of infectious mosquitoes, mimicking the natural route of infection. Moreover, human experimental challenges are carried out in a controlled environment, allowing detailed evaluation of parasite growth and immunological determinants. In addition to the evaluation of vaccine efficacy, experimental challenges provide the opportunity to study correlates and mechanisms of protection. An example is the induction of immunity using whole parasites, by exposure of malaria-naive volunteers to infectious mosquito bites while using chloroquine prophylaxis43. Chloroquine kills blood-stage parasites but leaves liver-stage parasites unaffected, thereby exposing the liver-stage and early blood-stage antigens to the immune system. Subsequent challenge showed that volunteers were completely protected from infection, and this was associated with multi-functional memory T cell responses. However, such immunization protocols are not practical for use in the field because parasite inoculation cannot be controlled and chloroquine resistance is widespread. Several differences between experimental and naturally acquired infections might limit the interpretation of results from experimental challenge models. First, experimental infections are carried out using one parasite strain only, whereas it is well accepted that P. falciparum field strains are genetically diverse within and between regions44. Genetic diversity of the parasite strains is a major challenge for protein-based vaccines that target strain-specific antigens, and puts limitations on the direct translation of results from Phase IIa trials into the field situation. The availability of a small portfolio of genetically well-characterized P. falciparum strains for experimental infections would be a major asset. Trials to test such strains in humans are currently being carried out. Another potential difference is that in an experimental infection the parasite load is delivered almost instantly by five infected mosquitoes. Such a high parasite burden has been considered unnatural and might be an overly stringent test for the protective capacity of the vaccine-induced immune response45. However, although the frequency of infectious mosquito bites is generally less than this in malaria-endemic areas, intense transmission can occur. A person may be subjected to 35â&#x20AC;&#x201C;96 mosquito bites per night, and in certain areas approximately 10% of mosquitoes are infected with P. falciparum46. www.nature.com/reviews/immunol

PersPectives A final potential limitation of current malaria challenge models involving sporozoite infection relates to the uncontrolled number of sporozoites inoculated by biting mosquitoes. This number is generally thought to vary up to a maximum of several thousand sporozoites47–51. Use of a well-defined number of inoculated sporozoites will strengthen the power of the model, as the dose probably influences the prepatent period7,27,52. In principle, the most accurate way of dosing sporozoites is to inject them directly by needle and syringe, as the number of sporozoites counted in mosquito salivary glands or the number of mosquito bites is a poor predictor of the number of sporozoites injected49. Early in the practice of malaria therapy, sporozoites were extracted from mosquito salivary glands (in 1927)53 and the number of injected sporozoites was determined (in 1937)54. However, standardized sporozoite viability assays are not yet available. Recent progress has been made by Sanaria Inc.55, which has developed technology for the purification and cryopreservation of aseptic sporozoites for use in humans according to the current safety standards. The first results of a human challenge study with aseptic P. falciparum sporozoite-infected mosquitoes indicate that these mosquitoes might be very efficient at conveying infection56. Experimental human infections are underway to test the infectiousness of these cryopreserved sporozoites by needle injection. However, one must bear in mind that needle and syringe administration of a bolus of sporozoites is clearly different from mosquito bite delivery, which may be an important factor to consider particularly for sporozoite vaccines that aim to induce antibodies to immobilize sporozoites. conclusions and perspectives Experimental human infections provide a crude model to predict malaria vaccine efficacy in future field trials in a well-controlled setting. The experimental malaria challenge model in humans using P. falciparuminfected mosquito bites is now well established in several international sites and increasingly used as a crucial check point for the clinical development of pre-erythrocytic stage malaria vaccines. Taking into account the potential limitations, such efficacy data from Phase IIa trials will support the decisionmaking process by ethical boards and communities in malaria-endemic countries regarding whether to further test a candidate vaccine in Phase IIb trials in susceptible populations. In addition to vaccine safety data, the availability of information on potential efficacy is an important asset for ethical

justification to conduct experimental malaria infections in human volunteers. In vaccine research, most risk is borne by study subjects and the benefits accrue mainly to the community in finding safe and protective vaccines57. The only candidate malaria vaccine showing protective efficacy in Phase IIb field trials so far is RTS,S. This candidate vaccine would almost certainly never have been developed without optimization in a series of Phase IIa trials. As is true for any type of clinical research, risks must be minimized and scientific benefits maximized. We believe that the benefits of Phase IIa trials outweigh the potential risks in well-designed studies and will be essential to the development of an effective malaria vaccine, provided that all safeguards are in place for the safety of volunteers58. The more recent introduction of a sensitive PCR assay for parasite detection has enhanced the reproducibility and statistical power of human challenge infections. Statistical models will be applied to further improve the discriminative power between control and test groups as well as to provide biological information about the parasite life cycle (including the duration of liver-stage maturation, number of infected hepatocytes, duration of blood-stage trophozoite maturation and multiplication rates). Initiatives are underway to further strengthen and harmonize the human challenge model, with possible applications for testing asexual erythrocytic stage vaccines and for P. vivax vaccine research15. As there is substantial variation in the numbers of sporozoites that are injected by mosquitoes and this cannot be controlled in the sporozoite challenge model, administration of a known number of sporozoites by needle injection may be a further improvement to the model. In addition, the human challenge model will benefit from the availability of a small portfolio of genetically well-characterized strains to explore immune responses to different strains and heterologous protection. Such advances will accelerate malaria vaccine development, with the aim of meeting the ambitious goals of the Malaria Vaccine Technology Roadmap by 2015–2025. Robert W. Sauerwein and Meta Roestenberg are at the Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, P.O. BOX 9101, 6500 HB Nijmegen, The Netherlands. Vasee S. Moorthy is at the Initiative for Vaccine Research, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland. Correspondence to R.W.S. e‑mail: R.Sauerwein@mmb.umcn.nl doi:10.1038/nri2902

NATURE REVIEWS | Immunology

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Acknowledgements

The views expressed in this manuscript are those of the authors and should not be taken to represent the views or stated policy of the World Health Organization.

Competing interests statement

The authors declare no competing financial interests.

FURtHeR inFoRMation clinicaltrials.gov: http://clinicaltrials.gov icH: http://www.ich.org Malaria vaccine technology roadmap Process: http://www.malariavaccine.org/files/Malaria_vaccine_TrM_ exec_Summary_Final_000.pdf the Malaria Product Pipeline: Planning for the Future: www.policycures.org/downloads/The_malaria_product_ pipeline_planning_for_the_future.pdf WHO Guidelines for the treatment of Malaria: http://www. who.int/malaria/publications/atoz/9789,241,547,925/en/ index.html WHO Malaria vaccine rainbow tables: www.who.int/vaccine_research/links/rainbow/en/index. html WHO World Malaria report 2009: http://www.who.int/malaria All lInkS ARe ACTIVe In The onlIne pdf

IN FOCUS NEWS DIS E ASE CO NTRO L

Mosquitoes score in chemical war Growing resistance is threatening global malaria-control efforts. BY DECLAN BUTLER

Lines says that the malaria-control community felt too many lives were at stake to let the threat of resistance stand in the way of massively scaling up the bed-net and spraying campaigns. Teasing out the impact of resistance on the success of malaria-control interventions is difficult because so many other factors influence their outcome. More systematic and more sophisticated monitoring of resistance is also vital, says Lines. The best surveillance data

SOURCE: WHO; GO.NATURE.COM/EOD8DC

ey weapons in the fight against malaria, pyrethroid insecticides, are losing their edge. Over the past decade, billions of dollars have been spent on distributing longlasting pyrethroid-treated bed nets and on indoor spraying. Focused in Africa, where most malaria deaths occur, these efforts have greatly reduced the disease’s toll. But they have also created intense selection pressure for mosquitoes to develop resistance. “Data are coming in thick and fast indicating increasing levels of resistance, and also of resistance in new places,” says Jo Lines, an entomoData compiled by the WHO last year show that logical epidemiologist and head of pyrethroid resistance in mosquitoes became widespread between 2000 and 2010, and the vector control at the Global Malaria situation is likely to have worsened since. Programme of the World Health Organization (WHO) in Geneva, Switzerland. The WHO now intends to launch a global strategy to tackle the problem by the end of the year. Pyrethroids are the mainstay of Resistant malaria control because they are safe, Resistance suspected cheap, effective and long-lasting. Susceptible Alternatives such as organophosphates and carbamates are available Mosquito strain for indoor spraying, although they Anopheles pharoensis cost more and are less effective. But An. labranchiae pyrethroids are the only insecticides An. funestus approved by the WHO for use in An. arabiensis bed nets. “We have lots of our eggs An. gambiae sensu stricto in the pyrethroid basket,” says RobAn. gambiae sensu lato ert Newman, director of the Global Malaria Programme. The international community has been slow to respond to the threat despite (see ‘Resistance on the rise’), although useful, warnings, says Janet Hemingway, director of do not give a complete picture of where resistthe Liverpool School of Tropical Medicine, UK, ance is emerging and how prevalent it is, he says. and chief executive of the non-profit Innovative Malaria-control programmes often lack insectVector Control Consortium, a public–private resistance monitoring, and detection of all venture set up in 2005 to develop new insec- forms of resistance is not easy. Quick, cheap tests ticides and monitoring tools. “A number of us can pick out gene mutations that help the moshad been banging the drums, saying: ‘As soon as quitoes’ nerve cells withstand pyrethroid attack. you scale up you are going to get resistance.’” But But other forms of resistance, which depend on

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increased levels of mosquito enzymes that can destroy pyrethroids before they reach their target, require more complex tests to detect (H. Ranson et al. Trends Parasitol. 27, 91–98; 2011). But uncertainties about the extent of resistance or its impact are “no excuse for inaction”, says Newman, arguing that the proposed WHO strategy needs to be urgently implemented, and also rolled out preemptively in places where resistance has yet to be detected. The WHO’s plan will recommend, for example, that control programmes rotate insecticides sprayed indoors, using pyrethroids one year and a different class the next. This would be more costly and less effective than relying only on pyrethroids, however, so control programmes may be reluctant to adopt this measure. Lines says that new combinations of insecticides also need to be developed, so that mosquitoes resistant to one would be killed by the other. In areas where pyrethroid bed nets are used, a different class of insecticides should be used for wall spraying, he adds. Ultimately, entirely new classes of insecticides — particularly those that can be applied to bed nets — are needed to alleviate the dependence of malaria-control efforts on pyrethroids. For indoor spraying, some longer-lasting and more costeffective non-pyrethroid insecticides should be available by next year, Hemingway says, although developing wholly new classes will take five to seven years. Repurposed agricultural insecticides might also act as a stopgap were resistance to pyrethroids to develop rapidly. Research targeting mosquito control is “grossly underfunded” compared with that on malaria drugs and vaccines, she adds, which is why control efforts have had so few options to call on. ■

OT H E R NE W S Survey reveals treasure trove of rare-earth elements on sea floor

go.nature.com/ tm23gy

● Technique provides clues to the

shading of fossil animals go.nature.com/

n4dhmh

● Oil-spill aftermath hampers rig

research go.nature.com/tyzhoy ● Better biosurveillance could halt disease spread go.nature.com/5sqj33

ON T H E B LOG The top five efforts in fundamental neutron physics

go.nature.com/ zouq58

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IN FOCUS NEWS

The RTS,S/AS01 candidate vaccine offers poor protection against severe malaria.

G LOBAL HEALTH

Malaria vaccine results face scrutiny Experts question early release of incomplete trial data. BY DECLAN BUTLER

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alaria vaccine could save millions of children’s lives”; “World’s first malaria vaccine works in major trial”; “Malaria vaccine almost here”. To judge from last week’s headlines, scientists had made a big breakthrough in the long campaign to create a malaria vaccine, proving its effectiveness with interim results from a huge phase III clinical trial in Africa1. Yet several leading vaccine researchers, who are critical of the unusual decision to publish partial trial data, argue that the results raise questions about whether the RTS,S/AS01 candidate vaccine can actually win approval. RTS,S has been in development for some 25 years, initially by the US military, and since 2001 by a public–private venture between the PATH Malaria Vaccine Initiative (MVI) and the drug-maker GlaxoSmithKline (GSK), supported by US$200 million in funding from the Bill & Melinda Gates Foundation. Bill Gates himself announced the interim results at the Gates Malaria Forum in Seattle, Washington. Gates’ speech and the MVI’s public-relations material were suitably circumspect about the results, but they were “immediately translated into headlines about [reductions] in death and mortality”, says Andrew Farlow, an economist at the University of Oxford, UK, who has previously assessed the RTS,S programme2. “But the data are not telling you that at all.” Some researchers question whether the results should have been published before all

the data were available; full results are expected in 2014. Interim trial data are usually reported only to regulatory authorities, and clinical trials published only once all the data are in, noted Nicholas White, a malaria expert at Mahidol University in Bangkok, in an editorial3 accompanying the interim results. “There does not seem to be a clear scientific reason why this trial has been reported with less than half the efficacy results available,” he wrote. The publication presents vaccine-efficacy data for infants aged 5–17 months, but not for those aged 6–12 weeks, who are the stated target of the trial: it is this group that would receive the malaria vaccine alongside routine immunizations. The aim of the trial is to provide the World Health Organization (WHO) with the information it needs to consider licensing the vaccine, and recommend it for use in that age group. “What is the point of publishing the interim data on the 5–17-month-olds?” asks Stephen Hoffman, a veteran malaria researcher and chief executive of a rival vaccine effort, Sanaria, based in Rockville, Maryland. The MVI’s director, Christian Loucq, argues that the results were “robust enough to be published. We decided this before we knew the results; we felt it was our scientific and ethical duty to make the results public when they become available.” One of the biggest claims made in the paper is that RTS,S reduced the NATURE.COM total number of episodes Vaccines special: of clinical malaria in the nature.com/vaccines older group by 55.1%,

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NEWS IN FOCUS compared to controls. This measure of efficacy is recommended for assessing a partially effective vaccine4. But the public expects vaccine efficacy to describe protection over a period of time, argues Judith Epstein, a captain and paediatrician at the US Military Malaria Vaccine Program in Silver Spring, Maryland. Recalculating the trial data shows that RTS,S protected just 35–36% after 12 months, she says, adding that the paper should have presented both numbers. The study also showed no detectable impact on mortality, and it is too early to tell whether RTS,S actually protects against malaria, or merely delays infection. The paper did report that RTS,S reduced severe malaria by 47% in the older group. But combining that result with available data from the younger age group cut that number to 34.8% — meaning that for the youngest children, the benefit must be even smaller. “The real question mark is the 34.8% efficacy in severe disease,” says Blaise Genton of the Swiss Tropical and Public Health Institute in Basel, and a member of the WHO technical advisory group for RTS,S. The results suggest that the vaccine might fall short of expectations, laid out in 2006 by a WHO-led consortium5, that it should have a “protective efficacy of more than 50% against severe disease and death and lasts longer than one year”. “If it doesn’t reduce deaths, and has only a modest effect on severe malaria, these are going to be big questions for decision-makers at WHO, GSK and the Gates Foundation,” says Hoffman. Another worrying finding is that the frequency of serious adverse events, such as convulsions and meningitis, was significantly higher in the vaccinated group, although the data are too preliminary to draw firm conclusions. “The severe disease findings are a concern,” says Genton. But Hoffman, like many researchers contacted by Nature, says that RTS,S still marks a significant achievement. It is the first vaccine against a parasite, Plasmodium falciparum, to consistently show a significant protective effect in large-scale trials. The phase III trial of RTS,S resulted in groundbreaking cooperation with African scientists, who led the 11 trials in 7 countries, says Hoffman. “I think that those teams deserve an incredible amount of recognition and congratulation.” ■ 1. The RTS,S Clinical Trials Partnership New Engl. J. Med. http://dx.doi.org/10.1056/ NEJMoa1102287 (2011). 2. Farlow, A. In The Science, Economics, and Politics of Malaria Vaccine Policy (2006). Chapter available at http://go.nature.com/8oxle3 3. White, N. J. New Engl. J. Med. http://dx.doi. org/10.1056/NEJMe1111777 (2011). 4. Moorthy, V. S., Reed, Z. & Smith, P. G. Vaccine 27, 624–628 (2009). 5. World Health Organization et al. Malaria Vaccine Technology Roadmap (2006). Available at http://go.nature.com/i1jdf7

GEN ETI C S

Fetal gene screening comes to market Non-invasive procedure could make prenatal testing easier, but it comes with ethical problems. B Y E R I K A C H E C K H AY D E N

ntil last week, scrutinizing a fetus’s DNA for indications of genetic abnormalities meant tapping into the mother’s womb with a needle. Now there’s a test that can do it using a small sample of the mother’s blood. MaterniT21, a Down’s syndrome test that Sequenom of San Diego, California, launched in major centres across the United States on 17 October, is the first of several such tests expected on the market in the next year. It signals the arrival of a long-anticipated era of non-invasive prenatal genetic screening, with its attendant benefits and ethical complications (see Nature 469, 289–291; 2011). With the technology in place to sequence the fetal DNA carried in a pregnant woman’s bloodstream, geneticists predict the list of conditions that can be detected by non-invasive means will grow rapidly. Another company, Gene Security Network of Redwood City, California, says its forthcoming test will also check for other genetic abnormalities, and Sequenom is studying the feasibility of expanding its test. “There’s every reason to think that in the future you’ll be able to extract an enormous amount of information from that sequencing data,” says Peter Benn, director of the Diagnostic Human Genetics Laboratories at the University of Connecticut Health Center in Farmington. Sequenom’s test sequences 36-base-pair fragments of DNA to identify sections from chromosome 21. Normally, the chromosome contributes 1.35% of the total maternal and fetal DNA in the mother’s blood. An overabundance of this material indicates the genetic abnormality that marks Down’s syndrome. Sequenom is marketing its test as an add-on to current screening methods, which estimate the chance that a woman is carrying a fetus with Down’s syndrome from ultrasound results and protein markers in the blood. Such nongenetic screening can detect 90–95% of Down’s syndrome cases, but falsely indicates that up to 5% of women are carrying a baby affected by the condition. Sequenom’s test could be taken after a positive screening result to help a woman decide whether to undergo amniocentesis, a test that extracts amniotic fluid with a needle and carries a small risk of miscarriage. A study published this month, and paid for by

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Sequenom, found that the company’s test has a false positive rate of 0.2% (G. E. Palomaki et al. Genet. Med. http://dx.doi.org/10.1097/ GIM.0b013e3182368a0e; 2011). It could spare some women from having amniocentesis after a false-positive screening result. But Benn says that the test will also pose difficulties. For instance, because it would take 8–10 days to get the results of Sequenom’s test, if a woman did still opt for amniocentesis, and the result confirms that the baby has Down’s syndrome, there would be little time left to decide whether to terminate the pregnancy. And some women who test positive on MaterniT21 will probably choose to terminate pregnancies immediately rather than have amniocentesis. “Inserting this new test in the way that Sequenom is proposing is very difficult, from the patient perspective, and difficult for physicians and counsellors to manage,” Benn says. Ethicists also caution that using such easy screening methods ever “In the future earlier in pregnancy you’ll be able might worsen the gender to extract an imbalance seen in counenormous tries such as China and amount of India. And if it becomes routine to check for many information different kinds of genetic from that abnormalities, ethicists sequencing predict that more couples data.” may face the quandary of whether to carry an ‘unhealthy’ fetus to term. “The idea that couples have choices about whether to continue their pregnancies may become strained because parents may be seen as irresponsible for allowing ‘defective’ pregnancies to go to term,” says Mildred Cho, an ethicist at Stanford University in Palo Alto, California. Other ethicists worry that fears of eugenics will be raised if testing can be done for less-serious conditions. Sequenom is solely focused on developing tests for conditions that are already part of prenatal screening programmes, says Mathias Ehrich, the company’s senior director for research and development diagnostics. “We do not want to invent new applications. Our focus is on making existing clinical applications safer,” he says. “I don’t think that we are in a position to say that we should determine what hair colour the baby has.” ■