Review

That Was Then But This Is Now: Malaria Research in the Time of an Eradication Agenda

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Science  14 May 2010:
Vol. 328, Issue 5980, pp. 862-866
DOI: 10.1126/science.1184785

Abstract

The global research community must take up the challenge to work toward the eradication of malaria. In the past, malaria research has focused on drugs and vaccines that target the blood stage of infection, and mainly on the most deadly species, Plasmodium falciparum, all of which is justified by the need to prevent and treat the disease. This work remains critically important today. However, an increased research focus is now being placed on potential interventions that aim to kill the parasite stages transmitted to and by the mosquito vector because they may represent more vulnerable targets to stop the spread of malaria. Here, we highlight some of the research into malaria parasite biology that has the potential to provide new intervention targets for antimalarial drugs and vaccines.

More than 40% of the world’s population lives with some risk of contracting malaria, with most recent estimates suggesting several hundred million clinical cases and 800,000 deaths each year. Malaria is caused mainly by four species of Plasmodium parasites, of which Plasmodium falciparum causes most disease and death across sub-Saharan Africa, and Plasmodium vivax is the most prevalent parasite in most other malaria-endemic parts of the world. Vivax malaria has been historically categorized as benign, but recent data indicate that severe disease caused by P. vivax infection is common (1). Current observations showing the increased transmission of nonhuman primate malaria parasites such as Plasmodium knowlesi to humans (2), the possibility that P. falciparum can propagate in great apes, and observations that apes harbour a diversity of parasite species closely allied with P. falciparum all raise concerns that animal reservoirs for human malaria will make control of human infection more difficult (3). Nevertheless, malaria appears to be retreating in some areas of the developing world, including Asia, South America, and even sub-Saharan Africa, where the human toll of malaria infection is highest and malaria transmission by Anopheles gambiae mosquitoes is efficient. This decrease in malaria is mainly attributable to expanded control programs using drug treatment of infected individuals, preventive drug treatment of populations at high risk of infection, and mosquito control with insecticide-treated bed nets and indoor-insecticide spraying.

Recently, a plan was initiated with the ultimate goal of malaria eradication. The objective appears simple: reduce the parasites’ basic reproduction rate, bringing the average number of new malaria cases occurring for a single existing case to less than one. An ideal scenario is interrupting malaria at its life-cycle choke points: when the parasite is transmitted by mosquitoes as a sporozoite, which infects the liver of an individual (the pre-erythrocytic stages), and when sexual-stage gametocytes are taken up by the mosquito from the blood of an infected individual, leading to colonization of the mosquito midgut (Fig. 1). The parasite numbers passing from mosquito to human and back to mosquito are exceedingly low (a few dozen to a few hundred) when compared with parasite populations in an infected individual during asexual blood-stage replication (billions) and thus may be easier to eliminate (Fig. 1). However, research on asexual blood stages of the parasite remains essential for the development of new therapeutics because it is this phase of the life cycle that causes disease and death. Here, we highlight recent progress in understanding the intricate biology of malaria parasites and how some of these findings may lead to new strategies for intervention.

Fig. 1

The Plasmodium life cycle provides numerous potential points of intervention. Upon mosquito injection of motile salivary gland sporozoites into the human skin, parasites migrate in the tissue and invade a blood vessel. Sporozoites are transported to the liver via the blood stream, leave the liver sinusoid, and infect hepatocytes to initiate liver-stage infection. The parasite undergoes massive growth and forms tens of thousands of first-generation merozoites. Merozoites are released into the blood stream. Each merozoite infects an erythrocyte and initiates the intraerythrocytic cycle. The parasites replicate by means of cyclic infection, replication, and release of next-generation merozoites. Some merozoites form sexual-stage gametocytes after invasion, and these can be taken up by a mosquito during a blood meal. Gametes mate, form a zygote, and develop into an ookinete that infects the mosquito midgut. The resulting oocyst produces sporozoites, which invade the salivary glands, ready for transmission to the next host. Initial infection of the human host liver and the transmission of gametocytes to mosquitoes are considered choke points of the parasite life cycle because parasite numbers are lowest at these points. The numbers shown indicate parasite population sizes during life cycle progression.

New Drugs and Novel Targets

The erythrocytic cycle of P. falciparum involves a massive amplification of the parasite population through periodic cycles of invasion, growth, division, and egress from erythrocytes (Fig. 1). To develop new compounds that target the parasite blood stage, an increased understanding of the mode of action of current antimalarials is necessary (4). Plasmodium parasites catabolize erythrocyte hemoglobin in the food vacuole, releasing toxic heme, which is sequestered in the food vacuole as a crystalline structure called hemozoin (Fig. 2). Antimalarials such as chloroquine appear to interfere with heme sequestration, and despite the rise of widespread resistance to chloroquine, heme sequestration remains a viable target for the development of new antimalarials (5). The important artemisinin group of antimalarials are thought to have their effect by activation of the endoperoxide group through the presence of free Fe3+, which is released during digestion of hemoglobin, and new generations of compounds are being developed that target heme metabolism (5). Proteases are responsible for the degradation of hemoglobin, providing a source of amino acids for parasite protein synthesis as well as a supply of metabolic energy (Fig. 2 and Table 1). Plasmepsin protease inhibitors can potently block P. falciparum growth, but many proteases have redundant functions, suggesting that drugs directed to the food vacuole will need to inhibit multiple proteases (6).

Fig. 2

Potential and realized drug targets of asexual P. falciparum blood stages. Shown are parasite organelles including the nucleus, apicoplast, mitochondrion, ER, Golgi (G), and food vacuole. The intraerythrocytic parasite is surrounded by the PV and PVM, through which proteins traffic to reach the erythrocyte. Most proteins for export are recognized by virtue of a PEXEL motif that is cleaved by Plasmepsin V in the ER to reveal the export signal. These proteins are carried to the PV, where they are recognized for export to the erythrocyte by a putative translocon (PTEX) in the PVM. The translocon consists of at least five subunits, including EXP2, HSP101, PTEX150, PTEX88, and TRX2 (inset). Egress of mature merozoites occurs by the breakdown of the PVM and erythrocyte membrane. This is mediated by proteases in the PV, including subtilisin 1, DPAP3, and SERA5 (solid partial circles of various colors). The food vacuole contains hemozoin, consisting of a crystalline form of heme, which results from the degradation of hemoglobin. Hemoglobin from the erythrocyte cytoplasm is endocytosed through the cytosome, and the endocytic vesicles fuse with the food vacuole membrane, releasing their contents into this structure for degradation by proteases such as plasmepsin and falcipains (solid partial circles).

Table 1

Drugs and possible drug targets for antimalarials against blood-stage and liver-stage parasites. ND, life cycle stage not determined; BS, blood stage; LS, liver stage; DHFR, dihydrofolate reductase; DHPS, dihydropteroate synthase; SHMT, serine hydroxymethyltransferase.

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Antifolate compounds, including pyrimethamine and sulfadoxine (Table 1), have long been an important antimalarial combination, although the rapid rise of drug resistance has greatly reduced their effectiveness (7). They inhibit enzymes involved in folate synthesis, which is essential for parasite viability (8), and the combination of pyrimethamine and sulfadoxine show synergism, increasing their effectiveness against P. falciparum. The crystal structure for P. falciparum dihydrofolate reductase/tetrahydrofolate synthetase (9), the target for pyrimethamine, has identified regions that are important for dimerization of this enzyme, and a charged groove is located on the surface of the dimer that appears to be important in channeling substrate between the two active sites of the enzyme’s bifunctional domains. These features provide new strategies to develop novel antimalarials against this target. Additionally, analysis of the folate enzymatic pathway has identified serine hydroxymethyltransferase (SHMT) as a target for development of novel antimalarials that target the folate biosynthetic pathway (10).

Plasmodia actively invade and egress from erythrocytes, and these processes require specialized parasite proteins (Fig. 3). Inhibition of the action of these proteins provides a novel avenue for antimalarial development. The invasion process involves a cascade of ligand-receptor interactions that are required to recognize the appropriate host cell and establish a tight parasite-host contact that ultimately connects with the actomyosin motor inside the parasite to provide the force required for entry (11). Although the ability to invade host cells is an essential step, the parasite also has to escape, and this process of egress represents a potential target for drug development (12). The intraerythrocytic parasite is surrounded by the parasitophorous vacuole (PV) and the erythrocyte membrane. Both of these are ruptured during egress, allowing access to new erythrocytes for invasion. Egress occurs in a cascade of events in which the membranes rupture, resulting in the explosive release of merozoites (13, 14).

Fig. 3

P. falciparum merozoite invasion. The merozoite interacts with the erythrocyte at the apical end via parasite ligands (EBA-175, EBA-140, EBA-181, PfRh1, PfRh2a/b, PfRh4, and PfRh5) that bind to host receptors and activate the invasion process and formation of the tight junction. The tight junction binds the erythrocyte surface to the merozoite and moves across the surface of the invading parasite. The ligand-receptor interactions are released during invasion by cleavage of parasite ligands with rhomboid 4 (ROM4). Subtilisin 2 (SUB-2) is also involved in processing and shedding proteins on the merozoite surface during invasion. PfAMA-1 participates in the tight junction with the parasite RON complex that spans the erythrocyte membrane, tight junction, and merozoite membrane.

After invasion, the parasite begins a remarkable process of remodeling that converts the terminally differentiated erythrocyte, which lacks a nucleus and machinery for functions such as protein trafficking, into a niche in which the parasite obtains nutrients and hides from host protective mechanisms (15). This requires the export of hundreds of parasite proteins through the PV membrane (PVM) to the erythrocyte cytoplasm and, in some cases, to the host-cell membrane. As a result, large parasite-derived structures appear in the infected erythrocyte—for example, Maurer’s clefts, which are important for protein trafficking and sorting, as well as protrusions on the erythrocyte membrane called “knobs” (16). A pentameric motif is located at the N terminus of most exported proteins and is required for targeting beyond the PVM (15). This motif is a protease recognition sequence that is cleaved in the parasite’s endoplasmic reticulum (ER) by an aspartic acid protease, plasmepsin V, revealing the protein’s export signal (17, 18) for subsequent transport most likely through a translocon complex (19), making protein export a novel antimalarial target.

Can We Develop a Blood-Stage Vaccine?

Development of effective blood-stage vaccines is difficult because of antigenic diversity, parasite mechanisms that evade host responses, and the sheer biomass of parasites present within the host during an infection. Despite this, considerable efforts have been made to develop and test potential vaccine candidates from P. falciparum blood stages because evidence shows that repeated infection of humans results in control of blood-stage parasitaemia and effective immunity that reduces clinical disease (20). Antigens on the invasive merozoite (Fig. 3) have been considered prime vaccine candidates, but blood-stage vaccines have yet to show success (21). The stakes for blood-stage vaccines are even higher when malaria eradication is the aim because they must not only reduce disease but also parasite burden to a degree that reduces transmission.

Repeated low-dose infection of human volunteers with P. falciparum blood stages followed by drug treatment induces strong immunity against homologous challenge (22), and recently rodent malaria parasite blood stages were genetically attenuated through gene deletion of purine nucleoside phosphorylase (PNP) or nucleoside transporter 1 (NT1) (23, 24). These knockout strains protect against challenge with virulent parasites, and in the case of the NT1 knockout strain, complete protection was achieved against challenges with different parasite strains. Therefore, the approach of using genetically attenuated P. falciparum blood stages as potential vaccines warrants further research, and such vaccination also presents opportunities to identify correlates of protection.

Targeting the Apicoplast Organelle?

The apicoplast is unique to apicomplexan parasites, including Plasmodium spp. The bacteria-like origin as well as the essential functions contained within the apicoplast—including lipid, heme, and isoprenoid synthesis—represent a slew of potentially attractive drug targets (Fig. 2 and Table 1) (25). The apicoplast contains replication, transcription, and translation machineries, all of which represent targets for currently known inhibitors of these processes (Table 1). The apicoplast is maintained throughout the parasite life cycle; hence, the development of drugs that target its essential functions can potentially act against blood stages and liver stages, but this requires experimental target validation. For example, the utility of fatty-acid biosynthesis as a drug target for asexual blood stages has been recently brought into question because the enzymes in this pathway are not essential for this part of the life cycle (2628). However, fatty-acid biosynthesis is critical for liver-stage development, suggesting that this pathway might be a drug target for pre-erythrocytic infection (26, 27).

Possible Targets for New Pre-Erythrocytic Antimalarials

Human malaria parasite transmission and liver infection is exceedingly dificult to study, and rodent malaria parasite models have proved invaluable for the study of pre-erythrocytic–stage infection. Infectious sporozoites leave the salivary glands of the mosquito during a bite and enter the host, make their way to the liver, invade hepatocytes, and commence development as liver stages (Fig. 1) (29). Sporozoites are injected into the avascular tissue of the skin then invade blood vessels (30), and their extended stay in the skin leaves the sporozoite vulnerable to immune attack. Sporozoites traverse through cells by means of plasma membrane disruption, and they use this process to migrate through tissues and reach the liver (31). Once a sporozoite encounters a suitable hepatocyte in the liver, it invades, forms a PV, and initiates liver-stage development. Tissue migration and hepatocyte invasion is mediated by interactions of sporozoite proteins with host cell receptors, which might provide new avenues to block infection (29). In addition, rapid growth of liver stages depends on host nutrients that are actively taken from the infected hepatocyte. Hence, methods to starve the liver stage might be good avenues to prevent growth. Liver stages ultimately differentiate into tens of thousands of first-generation merozoites, which are released from the infected hepatocyte into the blood stream. The release is controlled by the parasite and occurs in packages that are still surrounded by the hepatocyte membrane (32). Egress from the liver is probably dependent on proteases, and indeed some of the same proteases necessary for blood-stage egress are expressed during late liver-stage development (33). Thus, protease inhibitors that target blood stages might also inhibit parasite release from the liver.

In essence, understanding host-parasite interactions during liver infection has the potential to inform new interventions that block this vulnurable phase of the parasite life cycle, and thus pre-erythrocytic–stage research should be given high priority.

Vaccines Against Pre-Erythrocytic Parasite Stages

A vaccine for the pre-erythrocytic stages is seen as the ideal tool for disease eradication. Furthermore, pre-erythrocytic stages appear not to exhibit substantial antigenic variation, and a vaccine based on a single parasite strain might provide protection from infection with heterologous strains. However, there is no solid evidence for naturally occurring protective immunity to pre-erythrocytic stages in malaria-endemic areas. Perhaps the repeated exposure to small numbers of sporozoites transmitted by each mosquito bite leads to a state of immune tolerance in the liver. Conversely, immunization with substantial numbers of irradiated sporozoites that are able to infect the liver but cannot complete liver-stage development elicits protective immunity that prevents subsequent infection, which has led to recent efforts in the manufacture of an irradiated P. falciparum sporozoite vaccine (34). Furthermore, genetically engineered sporozoites that are attenuated through the knockout of genes that are essential for liver-stage development provide a powerful new avenue for live attenuated parasite vaccine development. Such attenuated sporozoites confer complete protection in mouse models of malaria; recently, a genetically attenuated P. falciparum parasite with deletions in the genes P52 and P36 was developed and will undergo clinical testing in the near future (35).

In parallel to whole-cell irradiated and genetically attenuated parasites, the design of effective subunit vaccines that target the sporozoite and liver stage are a high priority. The major immunodominant antigen of the sporozoite is the circumsporozoite protein (CSP), and a subunit vaccine based on CSP has proved partially successful in clinical trials (36). The major question now is whether one can identify additional protective pre-erythrocytic antigens and combine them with CSP so as to create a multi-subunit vaccine formulation that results in complete protection. The identification of novel targets for humoral protection is important, but even more critical is the identification of parasite targets that allow the immune system to eliminate infected hepatocytes through T cells (37). Novel pre-erythrocytic vaccine candidates can be tested for efficacy early in clinical development by using experimental malaria challenges of volunteers. Importantly, the experimental immunization with live-attenuated sporozoites provides a powerful platform to search for novel protective antigens by use of high-throughput immunological screens.

Transmission Blocking with a Vaccine?

Targeting the sexual stages of the malaria parasite life cycle with subunit vaccines could prevent transmission to the mosquito vector (38), and a greater focus on this life-cycle choke point is essential in an eradication agenda. Target antigens fall into two broad groups: those that are expressed before gamete fertilization and those that are expressed after fertilization on the surface of the ookinete—the invasive form that infects the mosquito midgut (Fig. 1). For example, immunization of nonhuman primates with the prefertilization antigen Pfs48/45 resulted in high antibody levels that block parasite transmission to mosquitoes in an ex vivo experiment that mixes the antisera with P. falciparum–infected blood and feeds the mixture to mosquitoes (39).

Unlike pre-erythrocytic– and erythrocytic-stage vaccine approaches, transmission-blocking vaccines do not protect the vaccinated individual, and some of the antigens are never seen naturally by the human immune system. Thus, the vaccine may not be boosted by malaria infection. As a result, vaccination must be potent and induce high antibody levels that are sustained for at least one transmission season. Moreover, initial testing of transmission-blocking vaccines for efficacy in clinical studies is not as straightforward as the testing of pre-erythrocytic vaccines because protection of the vaccinated individual is not the readout and population-based studies are needed.

Conclusions and Perspectives

Fundamental secrets of malaria parasite biology are now being revealed because of the development of genetic tools and the application of sophisticated laboratory technologies. The recapitulation of complete parasite life cycles in the laboratory through use of rodent malaria models, the routine in vitro culture of P. falciparum blood stages, laboratory infection of mosquitoes with cultured gametocytes, and improved in vitro growth systems for liver stages has enabled research that was not previously possible. This, in combination with parasite whole-genome sequences, functional genomic tools, systems biology, and the ability to genetically manipulate the parasite, has led to an unprecedented wealth of data. Despite these advances, approximately 60% of P. falciparum genes encode proteins for which no functional assignments exist. Research on P. vivax, the major cause of malaria outside Africa, has lagged behind because the parasite is difficult to grow in the laboratory. P. vivax liver stages can lie dormant as “hypnozoites” and reactivate to cause a blood-stage infection months after the initial infection (Fig. 1 ), making the hypnozoite a high-priority target for research and drug development if malaria eradication is the goal. Currently, we do not have even the most basic understanding of the nature of hypnozoite dormancy. The search for new blood-stage malaria therapeutics continues, and knowledge of the pathophysiology and cell biology of this stage has vastly increased during the past decade, providing new intervention points to improve treatment of malaria.

However, the time has come to broaden our knowledge base of pre-erythrocytic and sexual stages in order to develop novel tools for blocking disease transmission. Promising research findings need to quickly move into human proof-of-concept studies for intervention. If proven, implementation must be accelerated, and this requires large investments. The intervention strategies discussed here need to be combined with other efforts that target the mosquito vector. Only then is it possible to envision a world without malaria within our lifetime.

References and Notes

  1. We apologize to many in the field whose work we could not cite because of space constraints. S.H.I.K. is supported by a grant from the Foundation for the National Institutes of Health through the Grand Challenges in Global Health initiaitve and NIH grant R01 AI053709. A.F.C. is supported by the National Health and Medical Research Council (NHMRC) of Australia, NIH grant RO1 A144008, and an Australia fellowship and is an International Scholar of the Howard Hughes Medical Institute. J.A.B. is supported by a Peter Doherty Postdoctoral Fellowship from NHMRC.
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