Malaria 2012: Drugs, Vaccines, and Pathogenesis

Journal Articles

Johanna Daily

Daily JP, Scanfeld D, Pochet N, et al. Distinct physiological states of Plasmodium falciparum in malaria-infected patients. Nature 2007. 450(7172):1091-5.

LeRoux M, Lakshmanan V, Daily JP. Plasmodium falciparum biology: analysis of in vitro versus in vivo growth conditions. Trends Parasitol. 2009. 25(10):474-81.

Wirth D, Daily J, Winzeler E, et al. In vivo profiles in malaria are consistent with a novel physiological state. Proc. Natl. Acad. Sci. USA 2009. 106(27):E70.

Krupka M, Seydel K, Feintuch CM, et al. Mild Plasmodium falciparum malaria following an episode of severe malaria is associated with induction of the interferon pathway in Malawian children. Infect. Immun. 2012 80(3):1150-5.

Kirk Deitsch

Dzikowski R, Deitsch KW. Active transcription is required for maintenance of epigenetic memory in the malaria parasite Plasmodium falciparum. J. Mol. Biol. 2008. 382(2):288-97.

Kirkman LA, Deitsch KW. Antigenic variation and the generation of diversity in malaria parasites. Curr. Opin. Microbiol. 2012 Apr 12.

Howitt CA, Wilinski D, Llinás M, et al. Clonally variant gene families in Plasmodium falciparum share a common activation factor. Mol. Microbiol. 2009. 73(6):1171-85.

Kishore SP, Perkins SL, Templeton TJ, et al. An unusual recent expansion of the C-terminal domain of RNA polymerase II in primate malaria parasites features a motif otherwise found only in mammalian polymerases. J. Mol. Evol. 2009. 68(6):706-14.

Dzikowski R, Deitsch KW. Genetics of antigenic variation in Plasmodium falciparum. Curr. Genet. 2009. 55(2):103-10.

David Fidock

O'Brien C, Henrich PP, Passi N, et al. Recent clinical and molecular insights into emerging artemisinin resistance in Plasmodium falciparum. Curr. Opin. Infect. Dis. 2011. 24(6):570-7.

Adjalley SH, Johnston GL, Li T, et al. Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue. Proc. Natl. Acad. Sci. USA 2011. 108(47):E1214-23.

Anderson T, Nkhoma S, Ecker A, et al. How can we identify parasite genes that underlie antimalarial drug resistance? Pharmacogenomics 2011. 12(1):59-85.

Adjalley SH, Lee MC, Fidock DA. A method for rapid genetic integration into Plasmodium falciparum utilizing mycobacteriophage Bxb1 integrase. Methods Mol. Biol. 2010. 634:87-100.

Manuel Llinás

Ochoa A, Llinás M, Singh M. Using context to improve protein domain identification. BMC Bioinformatics 2011. 12:90.

Olszewski KL, Llinás M. Central carbon metabolism of Plasmodium parasites. Mol. Biochem. Parasitol. 2011. 175(2):95-103.

Olszewski KL, Morrisey JM, Wilinski D, et al. Host-parasite interactions revealed by Plasmodium falciparum metabolomics. Cell Host Microbe 2009. 19;5(2):191-9.

Otto TD, Wilinski D, Assefa S, et al. New insights into the blood-stage transcriptome of Plasmodium falciparum using RNA-Seq. Mol. Microbiol. 2010. 76(1):12-24.

Victor Nussenzweig

Zhang M, Mishra S, Sakthivel R, et al. PK4, a eukaryotic initiation factor 2α(eIF2α) kinase, is essential for the development of the erythrocytic cycle of Plasmodium. Proc. Natl. Acad. Sci. USA 2012. 109(10):3956-61.

Zhang M, Fennell C, Ranford-Cartwright L, et al. The Plasmodium eukaryotic initiation factor-2α kinase IK2 controls the latency of sporozoites in the mosquito salivary glands. J. Exp. Med. 2010. 207(7):1465-74.

Ménard R, Heussler V, Yuda M, et al. Plasmodium pre-erythrocytic stages: what's new? Trends Parasitol. 2008. 24(12):564-9.

Singh AP, Buscaglia CA, Wang Q, et al. Plasmodium circumsporozoite protein promotes the development of the liver stages of the parasite. Cell 2007. 131(3):492-504.

Christopher Plowe

Thera MA, Doumbo OK, Coulibaly D, et al. A field trial to assess a blood-stage malaria vaccine. N. Engl. J. Med. 2011. 365(11):1004-13.

Thera MA, Doumbo OK, Coulibaly D, et al. Safety and immunogenicity of an AMA1 malaria vaccine in Malian children: results of a phase 1 randomized controlled trial. PLoS One 2010. 5(2):e9041.

Takala SL, Coulibaly D, Thera MA, et al. Extreme polymorphism in a vaccine antigen and risk of clinical malaria: implications for vaccine development. Sci. Transl. Med. 2009 1(2):2ra5.

Thera MA, Plowe CV. Vaccines for malaria: how close are we? Annu. Rev. Med. 2012;63:345-57.

Plowe CV, Alonso P, Hoffman SL. The potential role of vaccines in the elimination of falciparum malaria and the eventual eradication of malaria. J. Infect. Dis. 2009. 200(11):1646-9.

Ana Rodriguez

Orengo JM, Evans JE, Bettiol E, et al. Plasmodium-induced inflammation by uric acid. PLoS Pathog. 2008. 4(3):e1000013.

Orengo JM, Leliwa-Sytek A, Evans JE, et al. Uric acid is a mediator of the Plasmodium falciparum-induced inflammatory response. PLoS One 2009; 4(4):e5194.

Bettiol E, Carapau D, Galan-Rodriguez C, et al. Dual effect of Plasmodium-infected erythrocytes on dendritic cell maturation. Malar. J. 2010 Mar 1;9:64.

Vernon Schramm

Cassera MB, Hazleton KZ, Merino EF, et al. Plasmodium falciparum parasites are killed by a transition state analogue of purine nucleoside phosphorylase in a primate animal model. PLoS One 2011. 6(11):e26916.

Cassera MB, Zhang Y, Hazleton KZ, et al. Purine and pyrimidine pathways as targets in Plasmodium falciparum. Curr. Top Med. Chem. 2011;11(16):2103-15.

Madrid DC, Ting LM, Waller KL, et al. Plasmodium falciparum purine nucleoside phosphorylase is critical for viability of malaria parasites. J. Biol. Chem. 2008. 283(51):35899-907.

Schramm VL. Immucillins as antibiotics for T-cell proliferation and malaria. Nucleosides Nucleotides Nucleic Acids 2004. 23(8-9):1305-11.

Timothy Wells

Burrows JN, Chibale K, Wells TN. The state of the art in anti-malarial drug discovery and development. Curr. Top Med. Chem. 2011. 11(10):1226-54.

Wells TN, Alonso PL, Gutteridge WE. New medicines to improve control and contribute to the eradication of malaria. Nat. Rev. Drug Discov. 2009. 8(11):879-91.

Olliaro P, Wells TN. The global portfolio of new antimalarial medicines under development. Clin. Pharmacol. Ther. 2009. 85(6):584-95.

Wells TN, Poll EM. When is enough enough? The need for a robust pipeline of high-quality antimalarials. Discov. Med. 2010 May;9(48):389-98.

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Malaria in humans is primarily caused by four species of Plasmodium: P. falciparum, P. vivax, P. ovale and P. malariae. A fifth, P. knowlesi, was initially thought to infect only nonhuman primates but is now known to cause infection and illness in humans as well. Researchers have sequenced the genomes of P. falciparum, P. vivax, and P. knowlesi, which have provided invaluable insight into the parasite's basic biology and have accelerated drug discovery and vaccine development efforts.

Although P. falciparum causes the most severe form of malaria and the greatest number of deaths, a small portion of those individuals infected with the species can remain completely asymptomatic. The host and parasite factors as well as the molecular mechanisms that underlie this variation in disease outcomes are the subject of research by Johanna Daily of the Albert Einstein College of Medicine.

The parasite's complicated life cycle, which anchored many of the talks, alternates between the sporogonic stage, which occurs within a female Anopheles mosquito, and the two stages that occur within the human host: the exo-erythrocytic stage that occurs within the liver and the erythrocytic stage within red blood cells.

The life cycle of the malaria parasite. (Image courtesy of Christopher Plowe)

The malaria cycle begins when sporozoites residing within an infected mosquito's salivary glands enter the human host's skin when the mosquito takes a blood meal. From the skin, they make their way within minutes to the liver, where the worm-like sporozoites transform into schizonts. Thousands of merozoites then develop within each schizont over a period of 48 hours, and when the hepatocytes rupture, these merozoites invade red blood cells (RBCs), where they undergo asexual multiplication at a furious pace.

Inside RBCs, the merozoites first grow into a ring-shaped form, then into a larger trophozoite form before transforming into the schizont stage, during which the parasite divides several times to produce new merozoites. These then burst from this cell to invade new RBCs in a periodic pattern with each cycle lasting two to three days. This stage, which occurs two to three weeks after the host has been bitten by an infected mosquito, is when the clinical symptoms manifest and is the target of vaccines intended to prevent or reduce illness without preventing infection—a topic addressed by Christopher Plowe, a physcian-scientist at the University of Maryland School of Medicine's Center for Vaccine Development. In addition to releasing merozoites, the rupture of the cells also results in the release of factors that trigger inflammation, which in excess can exacerbate pathology, as explained by Ana Rodriguez, from New York University School of Medicine. Her group has identified a new inflammatory factor and a potential new treatment to mitigate the effects of inflammation.

During the red blood cell stage, the parasite effectively hijacks the host cell and its machinery, both to evade the immune response and to scavenge for metabolic precursors and nutrients. The expression, on the surface of infected cells, of the highly variant P. falciparum erythrocytic membrane proteins (PfEMP1) encoded by the multigene var family of genes allows the cells to take themselves out of circulation and to enter into other organs such as the lung, heart, brain, and in the case of pregnant women, the placenta, thereby causing disease progression and often death. Kirk Deitsch, Professor at Weill Cornell Medical College described his group's efforts to understand var gene regulation with the goal of finding ways to disrupt it and to prevent disease progression.

Defining the metabolic interactions between the host red blood cell and the parasite could reveal new biochemical pathways, potential drug targets, and novel antimalarial therapies based on the enzymatic reactions the parasite uses to sustain itself. Both Manuel Llinás of Princeton University and Vernon Schramm of Albert Einstein College of Medicine addressed these aspects of malaria research in their talks. Llinás described new ways of identifying parasitic metabolites and of defining the malarial metabolome, and Schramm explained the development of parasite enzyme inhibitors as antimalarial therapies.

Some merozoites differentiate into male or female gametocytes, infective forms that are responsible for spreading the infection to new victims. When taken up by an uninfected mosquito during a blood meal, these can develop into gametes that undergo fertilization to form ookinetes in the mosquito's mid-gut. Further development into sporozoites is a two-week process, after which these forms migrate to the salivary glands, where they can remain latent for weeks while maintaining their viability, as described by Victor Nussezweig, of New York University Langone Medical Center. His work has revealed several details of the mechanism behind this latency that are being used to develop new classes of antimalarial drugs. The injection of these sporozoites into another human bitten by this infected mosquito starts a new infection.

Researchers are investigating almost every aspect of the multiple developmental stages of the parasite across the vector and human host to identify new vaccine and drug targets. Although artemisinin-based combination therapies (ACTs) proved highly effective at reducing malaria transmission around the globe over the past decades, recent studies point to delayed parasite clearance—an increase in the time required for the number of parasites circulating in the bloodstream to be reduced—in response to ACTs in endemic areas in Southeast Asia, which Timothy Wells of Medicines for Malaria Venture (MMV) referred to as an "early warning system" for the emergence of artemisinin-resistance. While Wells described his organization's efforts to partner with academia and with the pharmaceutical industry to develop and market new drugs aimed at overcoming the resistance problem, David Fidock, from Columbia University Medical Center spoke about his group's efforts to identify the genetic determinants of drug resistance.

Speakers:
Johanna Daily, Albert Einstein College of Medicine
Ana Rodriguez, New York University School of Medicine

Highlights

• One distinct parasite gene expression profile identified in vivo closely resembled the parasite's so-called environmental stress response.
• Transcriptome experiments are helping to define host factors that are associated with mild versus severe disease.
• Infected RBCs are known to become sequestered in the brain's microvasculature and are thought to trigger local inflammation that can lead to blood–brain barrier damage and death in cerebral malaria. The release of precipitates of uric acid by ruptured RBCs could play a role in this damage.
• AT1-blocking drugs, such as Losartan, and AT2-activating drugs might both prove beneficial additions to a treatment regimen of parasite-killing anti-malarial drugs.

Host and parasite factors responsible for varied disease outcomes

Infection with Plasmodium falciparum can have widely different clinical manifestations in humans, ranging from no symptoms at all to mild flu-like symptoms to coma and death. Working with the hypothesis that both host and parasite factors are likely to interact dynamically to determine the outcome of infection, Johanna Daily's group at Albert Einstein College of Medicine has taken a transcriptomics approach to parse these interactions, measuring alterations in gene expression in host and parasite by isolating RNA directly from blood samples from people infected with the parasite.

Daily started off the first session of the Symposium by presenting three recent studies from her group. The first revealed unexpected diversity in the biology of the malaria parasite inside the human host during the parasite's asexual stage. Of the three distinct parasite gene expression profiles that the researchers identified in vivo, one that had not been previously observed under lab culture conditions closely resembled the so-called environmental stress response (Daily JP et al. Nature, 2007). Interestingly, this profile in the parasite was associated with patients who had high levels of inflammation and who were hospitalized with severe malaria. Daily is now trying to recapitulate aspects of this stress response in the lab, hoping to understand how the parasite adapts to stress. She is also working to identify the signals that spur its development from the asexual state into the sexual, transmissible state with the goal of finding ways to halt this transition.

The second study addressed the question of why only a small percentage of P. falciparum infections result in cerebral malaria (CM), which occurs when infected RBCs build up within small blood vessels in the brain causing blockages that in turn lead to swelling and damage. Using retinopathy—which manifests as patchy retinal whitening and focal changes of blood vessel color—as a marker for CM, Daily's group transcriptionally profiled more than 60 children who were enrolled in the Blantyre Malaria Project in Malawi and who were hospitalized with coma. The analysis helped to distinguish between genes associated with brain sequestration and CM—including several from the chemokine and tumor necrosis factor (TNF) family—and those that are associated with retinopathy-negative malaria. This latter group includes the gene for basigin, an immunoglobulin family protein that was previously identified as a receptor that the parasite binds in order to invade erythrocytes. The researchers are now conducting additional experiments to further define host factors that are associated with CM.

To gain broader insight into host responses associated with severe versus mild malaria, and to specifically understand why children in malaria-endemic areas develop protective immunity to severe disease, Daily's group turned again to transcriptional analysis. They investigated differences in each child's transcriptional response between an initial episode of severe malaria and the secondary infection that caused mild malaria in the same child. This study, which included five Malawian children, identified six genes associated with severe disease—including thymidine kinase 1 and carbonic anhydrase 1—that are now being further tested as biomarkers. The analysis also revealed that mild malaria is associated with the induction of the Interferon (IFN)-mediated signaling pathway—previously associated only with antiviral responses—and with T cell activation (Krupka M et al. Infect Immun, 2012). Daily's goal is to build from these data pathogenesis models that could facilitate the development of therapeutics for reducing disease severity.

A new trigger for cerebral malaria

Whereas an early inflammatory response characterized by cytokine release can help clear parasites, a prolonged and excessive inflammatory response can exacerbate disease pathology and lead to severe malaria. Strong inflammation correlates with the rupture of infected erythrocytes during the blood-stage in the malaria cycle. Several parasitic molecules that spill out into the blood stream at this time have been identified as inflammatory triggers, including CpG DNA and agonists of the toll-like receptor 9 (TLR9) such as hemozoin—the parasite pigment formed from heme degradation. In addition, AT-rich DNA, abundant in the malarial genome, escapes into the blood. Recently, Ana Rodriguez and her team at New York University School of Medicine added a new set of inflammatory triggers to the list—precipitates of uric acid.

Uric acid precipitates localize to the cytosol of the parasite, not to the RBC. (Image courtesy of Ana Rodriguez)

After it invades red blood cells (RBCs), the parasite heavily imports hypoxanthine from its host cell, which is needed for DNA synthesis and replication, and converts it to soluble uric acid. Increasing concentrations of uric acid as well as other changes within the infected RBCs might be causing soluble uric acid to precipitate, according to Rodriguez. In contrast to uninfected cells, both mouse red blood cells (RBCs) infected with P. yoelii and P. berghei as well as fresh RBCs from malaria patients in Iquitos, Peru readily stained in tests with antibodies specific for the precipitated, not the soluble, form of uric acid. Electron microscopy experiments helped Rodriguez and colleagues precisely locate the uric acid precipitates in the cytosol of the parasite within the infected cell. The team further found that unlike soluble uric acid, the precipitated uric acid activated human dendritic cells (DCs), up-regulating all the classic cell surface activation markers such as CD11c and CD86, but downregulating HLA-DR, a cell surface receptor whose activity is also downregulated in DCs in malaria-infected children.

Infected RBCs are known to become sequestered in the brain's microvasculature and are thought to trigger local inflammation that can lead to blood–brain barrier damage and death. Rodriguez suggested that the release of precipitates of uric acid by ruptured RBCs could play a role in this context, as preliminary in vitro evidence from her team shows that the precipitates can activate and subsequently damage immortalized human brain endothelial cells, cause the loss of adhesion molecules from the cells' surface, and trigger cell death.

As endothelial damage is also caused by the peptide hormone angiotensin II, which causes blood vessel constriction, the team examined the effect of available drugs that inhibit angiotensin II activity in the context of malaria. These drugs, which either block the angiotensin II receptor AT1 or activate the AT2 receptor, protected human brain endothelial cells from damage in vitro, and protected mice from cerebral malaria. The two receptors of angiotensin II, AT1 and AT2, are antagonistic to each other, with AT1 increasing vascular permeability and inflammation—which Rodriguez calls the key events that need to be avoided in cerebral malaria—in contrast to AT2, which reduces permeability. So AT1-blocking drugs, such as Losartan, and AT2-activating drugs might both prove beneficial additions to a treatment regimen of parasite-killing anti-malarial drugs, a hypothesis that Rodriguez hopes will be tested soon.

Speakers:
Kirk Deitsch, Weill Cornell Medical College
Manuel Llinás, Princeton University

Highlights

• Focusing on a polymorphism within the C-terminal domain of RNA polymerase II of P. falciparum helped identify a histone methyltransferase, PfSet2, that regulates the var gene family.
• PfSet2 might be a good drug target, as blocking its interaction with RNA pol II disrupts var gene regulation.
• Characterizing the malaria 'metabolome' by combining highly accurate mass spectrometry with other approaches such as QTL mapping is revealing new metabolic pathways and compounds that could be targeted by new therapies.

A new modifier of malarial epigenetic memory and potential drug target

To escape destruction in the spleen and instead to sequester themselves in deep tissue microvasculature such as in the brain, as described by Daily and Rodriguez, infected RBCs use the knob-like, cytoadhesive malarial protein PfEMP1 that is expressed on their surface to bind host endothelial receptors. The parasite regulates the transcription of the 60 different var genes in its genome that encode different forms of PfEMP1 such that each parasite only expresses one var gene at a time. This mutually exclusive expression ensures the parasite's ability to dodge the host's antibody response: when parasites expressing one antigenic form of PfEMP1 are wiped out, a new wave of parasites expressing a different PfEMP1 antigen can take over and keep the infection going.

Kirk Deitsch of Weill Cornell Medical College has been working to unravel the regulation of var gene expression. He has shown that the parasite's ability to ensure that every round of cell division produces parasitic progeny that switch on the same var gene depends on cellular memory maintained through active transcription, that is, through the expression of that gene (Dzikowski R, Deitsch KW. J Mol Biol, 2008). If any of the var genes is artificially silenced in one generation, the next generation of parasites "fails to remember which gene they were supposed to activate," said Deitsch. This finding led him to hypothesize that RNA polymerase II, the enzyme that produces mRNA during transcription, was somehow "marking the chromatin epigenetically, or leaving behind a 'footprint' as it moved through the gene, to reinforce memory," a process that Deitsch's team has dubbed "the Hansel-and-Gretel" model in which epigenetic marks on the chromatin are like the bread crumbs in the fairy tale.

A clue to how this model might work came from an astute postdoc's observation of a polymorphism within the C-terminal domain (CTD) of RNA pol II, the end of the molecule that interacts with and that recruits activator proteins and various histone modifiers during transcription (Kishore SP et al. J Mol Evol, 2009). Observing that the CTD is shorter in rodent-specific Plasmodium strains than in the primate-infecting strains, the team wondered if this difference meant that there were transcription factors present in primate parasites that were absent in rodent parasites. Without these posited additional transcription factors, the rodent parasites would not require the extra length of CTD present in primate species. Computational analysis of genomic data sets from three rodent and three primate parasite species confirmed this supposition by identifying a histone methyltransferase called PfSet2 and its counterpart PfJmjC1, which removes methyl marks. Deitsch's team has determined via co-immunoprecipitation experiments that the N terminus of the PfSet2 methyltransferase directly binds the CTD of RNA pol II and that phosphorylation of the CTD eliminates this interaction.

So the model that Deitsch proposed is that as RNA pol II moves along a var gene, it recruits Set2, which marks specific histones, alerting the parasite to the gene that needs to be activated through multiple asexual divisions of the parasite. He speculated that two sites on PfSet2 would make good drug targets: the enzymatic domain required for methyltransferase activity as well as the N-terminal domain that it uses to bind to RNA pol II, as blocking either of these activities disrupted var gene regulation. His team is now making transgenic parasites to investigate these possibilities.

Charting the malarial metabolome to find new drug targets

After infecting RBCs, malarial parasites engage with their hosts metabolically to scavenge nutrients, a process that involves "breaking and making small molecules, which is what ultimately allows the parasite to divide and survive," explained Manuel Llinás of Princeton University. Defining these metabolic interactions could reveal new biochemical pathways, enzymatic reactions, and compounds that could be targeted by novel antimalarial therapies. Llinás is attempting to build a comprehensive picture of the malarial metabolome—the total set of all small molecule metabolites, or breakdown products, and their levels—via several approaches that involve the use of high accuracy mass spectrometry to measure metabolites in extracts from infected RBCs.

Llinás focused on two of these approaches during his talk, the first of which used the QTL (Quantitative Trait Loci) method to pinpoint locations in the genome that regulate parasite metabolism. In these experiments, a wild-type parasite strain called HB3 was crossed with a multi-drug-resistant strain called Dd2. Instead of assaying the recombinant progeny—which carry a mixture of genetic markers from both parents—for just one phenotype and asking how a particular marker segregates for that phenotype, Llinás used metabolomics tools to probe thousands of phenotypes simultaneously. "We are essentially performing a multidimensional QTL experiment in which we are assaying thousands of metabolites, with each metabolite representing one phenotype, and asking which locus in the genome is responsible for that metabolite," he elaborated.

Mapping a phenotype with quantitative trait loci (QTL). (Image courtesy of Manuel Llinás)

QTL analysis revealed 561 linkages with LOD (logarithm base 10 of odds) scores greater than three (signifying strong evidence of linkage between the phenotype and a genetic locus). The three most significant linkages involved unknown compounds that mapped to a region containing the Chloroquine Resistance Transporter (CRT) on chromosome seven. When mutated, the CRT protein, which is found in the membrane of the parasite's food vacuole, makes the parasite resistant to the drug chloroquine. After walking his audience through a complicated "detective story that figured out what these compounds are and why they map to this locus," Llinás revealed that the compounds were dipeptides resulting from the breakdown of hemoglobin, which the parasite scavenges from the RBC and imports into its food vacuole. The CRT protein transports the dipeptides back out into the cytosol for further processing into amino acids. According to Llinás, mutations in the CRT that prevent this transport could put pressure on the parasite's survival. One of Llinás's plans is to expand this approach to find out if there are differences in the metabolites that are generated at different stages of the parasite cycle within infected RBCs.

Another of Llinás's major goals is to identify compounds that are produced exclusively by the parasite (and therefore not an artifact present in culture media or produced by host cells). His team is approaching this question through a system of mixing specific ratios of amounts of different extracts—including culture medium, uninfected cells, infected cells, and purified parasite—and analyzing each mixture by mass spectrometry to measure the contribution of each extract. Identifying metabolites that are unique to Plasmodium could provide new opportunities to kill it by targeting metabolic pathways used only by the parasite.

Speakers:
Christopher Plowe, University of Maryland School of Medicine
Victor Nussenzweig, New York University Langone Medical Center
Vernon Schramm, Albert Einstein College of Medicine

Highlights

• Although blood-stage vaccines against MSP1 did not show efficacy against clinical malaria in Kenya, a monovalent recombinant protein vaccine against AMA1 has demonstrated up to 60% efficacy in a phase II trial in Mali.
• The best bet for a highly efficacious vaccine might lie in attenuated whole-organism vaccines, an approach that was first tested in the 1940s.
• The parasite kinase PK4, which is required for the completion of the parasite development cycle within RBCs, is a new target for drug development.
• Purine nucleoside phosphorylases (PNPs), which help the parasite salvage purine for survival, are another drug target against which inhibitors called transition state analogs are in development.
• DADMe-Immucilin G (BCX4945) is a promising TS analog that has been shown to clear parasites in primate models following oral treatment for seven days.

Improving the recipe for a high efficacy, blood-stage malaria vaccine

Disease symptoms occur only during the blood stage of malaria infection, when the rupture of infected liver cells releases thousands of merozoites into circulation that then infect RBCs. So vaccines that target the blood stage parasite are considered to be anti-disease vaccines that are meant to prevent or reduce clinical illness without actually preventing infection. This is in contrast to pre-erythrocytic vaccines that would block infection by preventing malarial parasites from invading blood cells and from causing disease. Despite decades of work and several dozen candidates tested, only one pre-erythrocytic vaccine has reached phase III in clinical development, reported Christopher Plowe of the University of Maryland School of Medicine, who began the second half of the symposium with an extensive overview of the history of vaccine development. Plowe showed that there is still a huge chasm to cross, as even RTS,S—the vaccine that has reached phase III testing—demonstrated only 26% efficacy in preventing all malarial episodes over a 4-year period (Thera MA, Plowe CV. Annu Rev Med, 2012).

Although early blood-stage vaccines that target the merozoite surface protein 1 (MSP1) disappointingly showed no efficacy against clinical malaria in Kenyan children, Plowe expressed hope that two other vaccines that target the blood-stage parasite's apical membrane antigen 1 (AMA1) that are being tested by his team in Mali will be the first of their kind to show clinical efficacy in humans. But sequencing of the AMA1 gene from 500 infected patients in a single Malian village revealed a potential stumbling block, as Plowe's team identified more than 200 unique AMA1 strain variants. This raised the daunting question of whether a >200-valent AMA1 vaccine—a vaccine designed to immunize against 200 different strains of the parasite—would be needed to provide broad efficacy (Takala SL et al. Sci Transl Med, 2009). However, the team's molecular epidemiological analyses showed that a cluster of just eight amino acids that lie next to the presumed RBC-binding site of AMA1 are largely responsible for the parasite's ability to escape the host's immune system. Plowe therefore suggested that a vaccine that contained as few as eight "serotypes" of AMA1 might protect against the majority of the strains.

The blood-stage parasite's apical membrane antigen 1 (AMA1), a target of recent vaccine attempts, showing a highly variable amino acid position at residue 197. (Image courtesy of Christopher Plowe)

Plowe also told the audience that a monovalent recombinant protein AMA1 vaccine FMP2.1, based on the 3D7 strain co-administered with the same adjuvant system being used with RTS,S, is being tested at Bandiagara in Mali. In a phase II trial, FMP2.1 showed strong efficacy of approximately 60% against clinical malaria caused by strains that matched the vaccine at the 8 previously-identified polymorphic amino acid positions (Tera MA et al. N Engl J Med, 2011). By identifying immune correlates of blood-stage vaccine protection, Plowe's team is now trying to understand how the vaccine works.

Plowe also remarked that the vaccine development field seems to be moving "back to the future" with revived interest in attenuated whole-organism vaccines, which were shown to provide protection in birds and monkeys in tests conducted more than 60 years ago. When a radiation-attenuated, metabolically active but nonreplicating sporozoite vaccine that was manufactured in aseptic mosquitoes failed to deliver significant protective efficacy two years ago, researchers identified the mode of delivery (subcutaneous injection) as a potential cause of failure. So efforts are underway to improve delivery to mimic more closely the efficiency of a mosquito bite at injecting the required number of sporozites.

An Achilles heel in the blood stage parasite becomes a new drug target

The malaria parasite undergoes obligatory episodes of "latency" during its lifecycle, such as during the sporozoite stage in mosquito salivary glands. This latency is part of a stress response that is characterized by a temporary halt in protein synthesis. This inhibition is achieved by the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α) at a single amino acid, Serine 59, by three parasite kinases IK1, IK2, and PK4 at different developmental stages. Victor Nussenzweig of New York University Langone Medical Center has been investigating the functions performed by these kinases during parasite development with the aim of developing kinase inhibitors as a new class of antimalarial drugs.

While investigating the role of IK2 in sporozoite latency using IK2-knockout parasites a few years ago, his team showed that IK2 blocks the development of sporozoites in mosquito salivary glands by phosphorylating eIF2α, halting the synthesis of proteins that are required for the parasite's liver stage and causing transcripts of these proteins to accumulate. But when injected into mammalian hosts, this translational repression is undone by the dephosphorylation of eIF2α by an as-yet-unknown phosphatase, which is inactive in the salivary gland but which is activated when the parasite enters the host. This workaround allows sporozoites to transform into liver-stage parasites (Zhang M et al. J Exp Med, 2010).

Life cycle stage-specific transcription of the PbeIF2α kinases. (Image courtesy of Victor Nussenzweig)

More recently, Nussenzweig's group has focused on defining the function of PK4, the kinase that was known to be essential in blood-stage parasites (Zhang M et al. Proc Natl Acad Sci USA, 2012). By constructing a conditional PK4 mutant, his group confirmed that PK4 activity is essential only in the erthyrocytic stage. Site-directed mutagenesis of Serine 59 of eIF2α showed that phosphorylation of this serine by PK4 is a prerequisite for the successful completion of the parasite's developmental cycle in RBCs.

The team tracked PK4 activity within the lifecycle to the schizont stage and to the gametocytes that infect mosquitoes. Hypothesizing that inhibiting PK4 might inhibit transmission, Nussenzweig is collaborating with a group at the National Institutes of Health to develop an assay for high-throughout screening for PK4 inhibitors. He hopes that the minimal similarity between PK4 and its mammalian counterparts, as well as the short lifecycle of the parasite in the blood (necessitating only a short treatment course), will overcome the problems of specificity and the dangers of off-target effects of the drugs on host kinases.

Killing parasites with drugs that block purine salvage

Vernon Schramm of Albert Einstein College of Medicine is also developing a new class of antimalarials: enzyme-inhibiting drugs that are transition state (TS) analogs, compounds that bind to enzymes much more strongly than do the enzymes' typical substrates. His group previously developed TS analogs against purine nucleoside phosphorylases (PNPs)—the enzymes involved in purine metabolism—that are currently in clinical trials for treating T cell leukemia and gout.

These successes led Schramm's group and collaborators to focus their efforts on developing antimalarial TS analogs that block both Plasmodium and human PNPs because the parasite—a purine auxotroph—needs both enzymes for the formation of hypoxanthine and salvages purine for survival. Since these enzymes are present at very high levels, effective PNP inhibition requires TS analogs to have extraordinarily high affinity for the enzymes. The team has formulated and tested one such TS analog, DADMe-Immucilin G (BCX4945), an inhibitor with picomolar affinity, in Aotus primates, which are a good model to test PNP targeting because human parasitic strains cause lethal infections in these animals unless treated.

Oral treatment with BCX4945 for seven days resulted in parasite clearance from blood with no toxicity. These features as well as the drug's chemical stability and mechanism of action, which differs from other antimalarials, support its potential use in combination therapies, according to Schramm. His team is now developing TS analog inhibitors for another enzyme in the purine salvage pathway.

Speakers:
Timothy Wells, Medicines for Malaria Venture
Elizabeth Winzeler, The University of California, San Diego
David Fidock, Columbia University Medical Center

Highlights

• Working with academic and pharmaceutical partners to find new drug targets using both whole cell- and target-based screening, MMV is building a strong therapeutic pipeline that includes several candidates that are being validated in patients.
• The emergence of resistance to artemisinin and other drugs is being monitored by watching for delays in the time required to clear parasites following treatment.
• Experiments to identify single nucleotide polymorphisms and copy number variations associated with drug pressure are shedding light on how parasites evolve to evade drugs.
• The development of a zinc-finger nuclease-based technique to edit the parasite genome rapidly and efficiently is helping to deliver genetic and mechanistic insights into the development of multidrug resistance.

Building a robust pipeline for the discovery, development, and delivery of new antimalarials

Although currently available artemisinin-based combination therapies (ACTs) cure 99% of treated patients, drug resistance is an ever-present and inevitable problem, one that might be stemmed or its likelihood reduced with a large pool of first-line anti-malarial drugs that work via different mechanisms. Furnishing such variety and avoiding drug resistance, in addition to enabling "customer choice" and providing inexpensive, extremely safe medicines to pregnant women and children—two of the populations most affected by malaria—are the major raisons d'être of Medicines for Malaria Venture (MMV), a not-for-profit, public-private partnership established in Switzerland in 1999. With partners from academic and public research institutes as well as from the pharmaceutical industry, MMV has assembled a robust antimalarial portfolio that now includes "4 new approved medicines, 2 pivotal trials, 7 translational projects, and more than 30 projects in the discovery phase," reported Timothy Wells, MMV's Chief Scientific Officer. MMV's goal is to pursue the World Health Organization's 2007 decision to aim for strategies that go beyond curing patients to stopping transmission and preventing reinfection.

He described MMV's efforts to develop pediatric versions of malaria medicines, including the successfully launched Coartem® Dispersible, which is the first high-quality ACT developed especially for children and of which 100 million treatments have been delivered since 2009. He also described the development of Eurartesim™ (dihydroartemisinin/piperaquine), which was launched in late 2011 for adults. To protect more than 25 million women whose pregnancies are threatened by malaria each year and to prevent infant mortality, MMV and two partners have undertaken a pivotal trial that is testing a novel combination therapy of the antibacterial drug azithromycin and chloroquine, which have been shown to work synergistically to boost efficacy beyond 90%. As sexually transmitted bacterial infections are prevalent among populations also most affected by malaria during pregnancy, this combination could also deliver additional public health benefits (Wells TN et al. Nat Rev Drug Discov, 2009).

Wells also touched on the challenge of drug resistance, noting that it was a question of "when, not if" resistance would emerge to artemisinin. He cited a study that documented a two-fold increase in parasite clearance time in infected patients living along the Thailand/Cambodia border in response to the artemisinin derivative artesunate, which is currently working very well in Africa against severe malaria. In Wells's view, delayed clearance times are an early warning sign of imminent treatment failure. Taking this into consideration, MMV integrates drug design that is guided by parasite biology (screening a large library of chemical compounds against the whole parasite to find compounds that are active against more than one molecular target) and drug design guided by structure (to generate second-generation versions of existing drugs to which resistance has emerged). Wells also informed the audience about the success of MMV's "Malaria Box," a freely available "treasure trove" of 400 chemicals with known antimalarial activity that MMV hopes will become an open-source drug discovery platform for researchers.

One of MMV's partners in research is Elizabeth Winzeler of the University of California, San Diego, another speaker at the symposium. Winzeler was involved in the development a synthetic molecule belonging to a class of chemicals called spiroindolones and in the molecule's testing in phase II trials. She is now working on identifying its mechanism of action and its target, with results to be published soon. Her group has also collaborated with industry partners to use a high-content imaging assay of liver-stage activity to identify another class of compounds called imidazolopiperazines as molecules that have activity against both blood stage as well as exo-erythrocytic parasites, a feature necessary for eradication. She reported that her lab is also investigating the imidazolopiperazines' target and mechanism of action. Winzeler also described her group's efforts to model the acquisition of drug resistance in the laboratory. The researchers are growing parasite clones for up to a year in the presence or absence of drug pressure, isolating DNA and performing whole-genome tiling arrays and genome sequencing to compile a list of single nucleotide polymorphisms and copy number variations associated with drug pressure. They are then mining these data for markers that can help predict parasite evolution and for use as a comparative data set for global parasite diversity and drug-resistance mapping studies.

Finding new use for an old drug and editing the parasite genome to understand the basis of drug resistance

Drugs that prevent the transmission of gametocytes—the sexual, blood-stage parasitic forms—from humans to mosquitoes are now part of the malaria eradication agenda. David Fidock of Columbia University Medical Center has developed a robust and sensitive assay for detecting and quantifying the gametocidal activity of known antimalarial drugs. In this system, his team treated a series of transgenic P. falciparum parasite lines that express GFP-luciferase reporters from gene promoters that are active in early-, mid-, or late-stage gametocytes with a panel of first-line antimalarials (Adjalley SH et al. Proc Natl Acad Sci USA, 2011). Whereas the rest acted only on immature gametocytes, the only drug to block these gametocytes as well as the transmission of mature gametocytes to mosquitoes was methylene blue, a dye first shown to have antimalarial activity in 1891. Fidock's team is now investigating this dye's mechanism of antimalarial action and is developing a high-throughout screen to identify other gametocytic compounds.

Methylene Blue potently blocks gametocyte transmission to Anopheles mosquitoes. (Image courtesy of David Fidock)

Fidock is also advancing scientists' ability to rapidly modify the parasite genome through the use of zinc finger nucleases. Zinc finger nucleases (ZFNs) are generated as obligate heterodimers that have to bind to target DNA sequences that are in very close proximity to one another in order to perform the nuclease function and to initiate a double stranded break (DSB). Although most organisms can repair these breaks via two different mechanisms, Fidock's group discovered that the malaria parasite relies on the mechanism of homology-directed recombination, where the homologous sequence inserted to fix the break comes from a donor template. Fidock's team has taken advantage of this mechanism to set up a system to accomplish quick, DSB repair-dependent gene editing in the parasite.

In one experiment, they used zinc finger nucleases to edit the PfCRT gene, which, as Fidock showed many years ago, contains several point mutations that collectively mediate chloroquine resistance. Chloroquine-sensitive parasites that only required one additional point mutation to become chloroquine-resistant were transfected with donor plasmids that carried the resistance-conferring mutation. The plasmids also co-expressed custom-designed ZFNs that could engineer DSBs near the precise region where this mutation needed to be inserted. When these transfected parasites were subjected to selection pressure via treatment with chloroquine, a homologous population of resistant parasites emerged within two to four weeks, a record time for a process that takes up to six months with conventional allele exchange methods. Fidock's team has continued to tweak this system so that they are now able to accomplish gene editing without depending on drug selection. "This means that we can edit genes that produce non-selectable phenotypes, which really opens up the field," said Fidock. Through this system, Fidock hopes to define in great detail the genetic and mechanistic basis of multidrug resistance and to gain insight into how resistance spreads.