Malaria 2014: Advances in Pathophysiology, Biology and Drug Development

Abstracts

Keynote Presentation:

The Pathogenesis of Fatal Cerebral Malaria: A Few More Pieces of the Puzzle
Terrie E. Taylor, Michigan State University

Malaria control and prevention efforts have not yet had a sustained impact in the epicenter of the disease, sub-Saharan Africa. Even now, more than half a million lives are lost each year. Cerebral malaria (CM) is a severe complication clinically characterized by coma and seizures. The case fatality rate is 15-25% and the annual incidence ranges from 1-12 cases per 1,000 children in malaria-endemic region. Most (85%) of the morbidity and mortality occur in young African children. In an autopsy-based investigation of clinicopathological correlates, we described the pathologies of fatal CM, but could not identify a pathologic cause of death. We learned that the standard clinical case definition misclassifies ~25% of patients, and we characterized a constellation of ophthalmologic findings, collectively termed “malarial retinopathy”. Including these eye findings greatly improves the specificity of the clinical diagnosis of CM. In 2009, we added magnetic resonance imaging (MRI) to our characterization of CM patients and learned that marked acute increases in BV are the single most important prognostic feature. Importantly, the brain volumes decrease over 24-48 hours in survivors, suggesting that any interventions targeting this phenomenon would only need to diminish, and not entirely normalize, the volume - - an important consideration in terms of the timing of a trial targeting children with CM. The next step will be to identify the mechanisms responsible for the markedly increased brain volumes seen in fatal CM. These findings will inform the design of a pivotal clinical trial of potentially life-saving interventions.
 

Neuropathology of Experimental Cerebral Malaria
Mahalia S. Desruisseaux, Albert Einstein College of Medicine

Infection with P. falciparum can result in significant morbidity, including adverse long-term neurological sequelae despite successful anti-parasitic treatment. Using a mouse model of experimental cerebral malaria, we previously demonstrated that cerebral vasculopathy was associated with neuronal damage and subsequent neuro-cognitive deficits. The vasoactive peptide, endothelin-1 (ET-1), has been shown to regulate blood-brain barrier (BBB) permeability, inflammation, and vascular tone, and we have demonstrated, in our experimental model, that this peptide may be important in the pathogenesis of cerebral malaria. We also previously reported that experimental CM is associated with abnormal regulation of the microtubule- associated protein, tau, and that dysregulated tau may be involved in the mediation of long-term neuro-cognitive deficits. Here we present data demonstrating that not only is ET-1 important in the regulation of cerebral blood flow, blood-brain barrier integrity and inflammation, but that it is also intimately involved in the development of disease severity and mortality; likely via its regulations of several cellular signaling pathways, including mitogen-activated protein kinase and protein kinase B (or Akt) signaling, pathways which have been implicated in the abnormal regulation of tau. We propose that both long-term morbidity in CM survivors and mortality during acute disease result, in part, from ET-1-mediated disruption of the BBB, its role in the cerebral inflammatory process, and its regulation of cellular signaling pathways, particularly in neural cells, leading to gliosis, neuronal degeneration, and subsequent adverse neuro-cognitive sequelae. The peptide and its downstream substrates may present potential therapeutic targets for adjunctive therapy in cases of cerebral malaria.
 

Keynote Presentation:

The Biology of P. vivax Explored through Genomics
Jane M. Carlton, New York University

Vivax malaria is a serious global health issue, and yet studies on the parasite are fewer and more limited in scope compared to Plasmodium falciparum. The first P. vivax genome, published in 2008, revealed a wealth of information that significantly propelled our understanding of the unique biology and pathology of the species. What has been achieved in the six years since? Here I present a comparative analysis of P. vivax genomes from Brazil, Mauritania, India and North Korea with the previously published El Salvador strain. Approximately twice as much genetic diversity is observed among these isolates as among a comparable collection of isolates of P. falciparum. This diversity, as well as gene family variability, suggests the capacity for greater functional variation within the global population of P. vivax, and serves as a warning that P. vivax could present a qualitatively different eradication task. I will also present preliminary analysis of ~150 global P. vivax isolates collected as part of the International Centers of Excellence in Malaria Research. Finally, we have sequenced several strains of Plasmodium cynomolgi, a simian malaria parasite and the close taxon to P. vivax. A map of genetic variation provides a resource for identifying P. cynomolgi traits and studying parasite populations. We show that genomes of the monkey malaria clade can be characterized almost exclusively by CNVs in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. Together these new datasets drive our knowledge of P. vivax biology significantly forward.
 

Translational Regulation during Stage Transition in the Malaria Parasites
Liwang Cui, PhD, The Pennsylvania State University

Plasmodium falciparum is the causative agent of the deadliest malaria, which causes nearly one million deaths each year. The malaria parasite completes its life cycle in two hosts, a mosquito and a human. Gametocyte, a specialized form of the malaria parasite, mediates transmission from human to mosquito. In malaria parasites, translational regulation is critical during the development of specialized transition stages between the vertebrate host and mosquito vector. Many genes including two transmission-blocking vaccine candidates pfs25 and pfs28 are translationally regulated during gametocyte formation and transmission. Our study of the Pumilio/FBF (Puf) family RNA-binding protein family in malaria parasites revealed they serves as master translation regulators of a large number of genes during the transition stage between human host and mosquito vector. The two Puf members display different yet related functions during gametocyte development. The molecular mechanism is conserved, supporting the paradigm of Puf-mediated translation regulation through 3' untranslated regions (UTRs) of target mRNAs. Furthermore, our study provides first evidence for a functional Puf-binding element in the 5' UTR of a target gene. This study provides a renewed view of Pufs as versatile translation regulators and sheds lights on their functions in the development of lower branches of eukaryotes.
 

The Plasmodium Sporozoite’s Journey and Beyond
Photini Sinnis, MD, Johns Hopkins University

Plasmodium sporozoites are inoculated by infected mosquitoes into the dermis of the mammalian host. Using the rodent malaria model, we have elucidated how the major surface protein of the sporozoite functions in the parasite’s journey from mosquito to mammalian host. More recently we have found that exit from the skin is a bottleneck for the parasite and possibly one of our best opportunities to intervene. I will discuss our data as to how two abundant surface proteins function in sporozoite exit from the dermis. These studies utilize the rodent malaria model and combine cell biology studies with intravital imaging of mutant parasites in a variety of transgenic mice.
 

Sporozoite Stages in the Liver: Development of Novel Therapies against Malaria
Purnima Bhanot, UMDNJ - New Jersey Medical School

The first obligatory developmental step in Plasmodium’s human cycle is the infection of the liver by sporozoites. Within the hepatocyte, sporozoites differentiate and divide to form liver stages. Liver stages eventually enter the bloodstream and infect erythrocytes causing disease. Therefore, inhibiting sporozoite infection and liver stage development (together termed pre-erythrocytic stages) would block malaria at an early step. However, most current drugs do not target pre-erythrocytic stages. We seek to fill this gap by providing mechanistic insights into sporozoite infection of hepatocytes and intrahepatic development. This will help identify novel drug targets and drugs for malaria prevention. We have demonstrated that a tri-substituted pyrolle (Tsp) molecule is a potent inhibitor of both sporozoite infection of hepatocytes and liver stage development. Tsp blocks sporozoite invasion, including that of P. falciparum, and has a partial effect on sporozoite motility. Treatment of sporozoites with Tsp significantly reduces the number of liver stages that develop both in tissue culture and in mice. In addition, Tsp arrests liver stage development prior to the parasite’s exit from the hepatocyte. Importantly, the Tsp-mediated block in liver stage infection leads to a significant decrease in blood stage parasitemia. A major target of Tsp is the parasite’s cGMP dependent protein kinase (PKG). Transgenic sporozoites expressing a Tsp-resistant allele of PKG have reduced sensitivity to Tsp and develop into liver stages even in the presence of Tsp. PKG conditional ‘knockout’ sporozoites become developmentally arrested in the liver. Our work provides proof-of-principle that inhibition of the Plasmodium PKG in pre-erythrocytic stages significantly reduces liver and blood-stage infection.
 

Keynote Presentation:

Artemisinin-Resistant Malaria in Southeast Asia: A Tale of Patients, Parasites and a "Propeller"
Rick M. Fairhurst, MD, PhD, National Institute of Allergy and Infectious Disease, NIH

Plasmodium falciparum resistance to frontline antimalarial drugs has emerged repeatedly in Southeast Asia and spread to Africa, prompting the World Health Organization to recommend the worldwide use of artemisinin (ART)-based combination therapies (ACTs) for falciparum malaria in 2005. ART and its derivatives are highly potent, short-acting antimalarial drugs that must be used in combination with less potent, long-acting partner drugs (e.g., lumefantrine, piperaquine) to completely eliminate parasites from patients. ART resistance in P. falciparum was first reported in 2009 from Pailin Province, Western Cambodia, where it manifested as delayed parasite clearance in patients treated with an ART derivative or ACT. ART-resistant parasites have since become entrenched throughout Western Cambodia, have emerged elsewhere in Southeast Asia, and are now threatening the efficacy of all ACTs worldwide. In a highly collaborative international effort, a series of in-vitro, genomic, epidemiological and clinical studies have rapidly increased our knowledge of ART resistance. These collaborations have (i) defined clinical ART resistance as a long parasite clearance half-life in patients, (ii) mapped the emergence and spread of ART resistance across Southeast Asia, (iii) developed an in-vitro assay for the study of ART resistance in the laboratory, and (iv) associated mutations in a parasite kelch protein "propeller" with ART resistance in Cambodia. Efforts are now underway to elucidate the molecular mechanism of ART resistance, to map the geographic distribution of "propeller" mutations, and to eliminate ART-resistant parasites from Southeast Asia and prevent their global spread.
 

Targeting New Pathways in Plasmodium to Eliminate Malaria
Marcus Lee, PhD, Columbia University Medical Center

To eliminate malaria, medicines must be developed that are not only curative against the pathogenic asexual blood stage but that also prevent the preceding liver stage infection from causing relapses and block transmission to the mosquito host, which occurs via sexual forms known as gametocytes. There are currently no known universal, drug-able and chemically validated targets for these multiple malarial life-stages. We have identified a parasite phosphatidylinositol-4 kinase, a conserved eukaryotic enzyme that modifies cellular lipids to regulate intracellular signaling and trafficking, as a key Plasmodium vulnerability that is the target of imidazopyrazines, a novel class of compound that act across all lifecycle stages. Evolved resistance, full genome-scanning, and genome editing experiments in intra-erythrocytic stages, as well as biochemical data, show that imidazopyrazines exert their potent antimalarial activity through interaction with the ATP-binding pocket of this lipid kinase. In asexual blood stages, imidazopyrazines block a late step in parasite development by disrupting the formation of new membranes around developing daughter parasites, a likely result of perturbed phosphatidylinositol 4-phosphate (PI4P) pools and disrupted vesicular trafficking. Our findings validate PfPI4K as the first drug target that appears to be required across all Plasmodium lifecycle stages.
 

The Molecular Basis of Antifolate Resistance
Laura Kirkman, MD, Weill Cornell Medical College

Drugs targeting the folate biosynthesis pathway were a crucial part of malaria control until widespread resistance undermined the use of these agents. The correlation between point mutations in target enzymes and anti-folate resistance has been well described. We investigated the additional contribution of the first and rate-limiting enzyme of the folate biosynthesis pathway, GTP-cyclohydrolase (GCH1). Plasmodium is able to both synthesize folate de novo as well as scavenge from its environment and we propose that the parasite strives to maintain a balanced flux through the folate pathway. I will present our work investigating the role of gene amplification of gch1, first identified in field isolates that are highly resistant to anti-folates, as well as the contribution of regulation of the protein itself by the extended N terminal region to anti-folate resistance. We propose that modifications in GCH1 copy number and activity potentially facilitate fixation of downstream resistant alleles in the parasite population.
 

Antimalarial Drug Resistance in Africa
Miriam K. Laufer, University of Maryland School of Medicine

Although resistance to antimalarial drugs has historically emerged in Southeast Asia and then spread to Africa, the burden of antimalarial drug resistance is the greatest in high transmission settings in Africa where it leads to increased disease severity and death. There are unique human, vector and parasite factors that contribute to the emergence, spread and disappearance of antimalarial drug resistance in Africa. I will describe our studies of the molecular epidemiology, genetics and clinical implications of drug resistance in Malawi, one of the high transmission regions in Africa, where we have found surprising differences between chloroquine and sulfadoxine-pyrimethamine resistance. I will discuss our results in light of current concerns about artemisinin-resistant malaria in Southeast Asia. Our findings may help to predict the behavior of resistance to new anti-malarial medications and inform strategies to prevent the spread of drug-resistant malaria in Africa the future.
 

Malaria Equilibrative Nucleoside Transporters: A Potential Path to Novel Anti-Malarial Drugs
Myles Akabas, MD, Phd, Albert Einstein College of Medicine

Malaria remains a major public health illness. The development of resistance to current antimalarial medications makes it essential to identify new antimalarial drug targets and therapies. Plasmodium parasites are purine auxotrophs. Thus, purine import is essential for growth. Genetic, biochemical, and physiologic evidence suggests that the Plasmodium falciparum Equilibrative Nucleoside Transporter 1 (PfENT1) is the primary purine transporter. PfENT1 inhibitors might serve as novel antimalarial drugs but no PfENT1 inhibitors have been identified. We developed a novel yeast-based high throughput screen that relies on the 5-fluoruridine sensitivity of PfENT1-expressing fui1Δ yeast to provide a powerful positive selection for inhibitors of PfENT1. We screened ~64,000 compounds in 384 well plates and identified 171 small molecule inhibitors of PfENT1 (0.26% hit rate). We have further characterized nine of the highest activity compounds that constitute five distinct chemotypes in a series of secondary assays. All nine compounds were positive in an orthogonal assay based on the adenosine dependence of growth of PfENT1-expressing purine auxotrophic yeast. All nine also blocked [³H]adenosine uptake into PfENT1-expressing yeast and into RBC-free trophozoite-stage parasites. None of the nine compounds kill yeast but all kill P. falciparum in culture. Compound efficacy is similar for chloroquine sensitive and resistant strains. Twenty-four hour treatment with a 10xIC₅₀ concentration of compound is sufficient to kill parasites. All nine compounds inhibit the homologous P. vivax transporter with similar efficacy. Our data support the hypothesis that blocking purine import through PfENT1 is a compelling approach for the development of novel antimalarial drugs.
 

Generation of Humanized Mouse Models for Malaria
Alexander Ploss, Princeton University

Malaria is a life-threatening parasitic disease for which approximately half the world's population is at risk. Although progress has been made in preventing and treating malaria, more effective, tolerable, and affordable therapies and vaccines are needed. A small animal model would dramatically speed the development of anti-malarial drugs and vaccines. Although plasmodial parasites species exist which efficiently infect rodent they are genetically distinct from parasites that cause malaria in human, which limits their predictive value. Plasmodium falciparum and P. vivax, which contribute most substantially to disease in humans exhibit a mechanistically undefined human tropism and animal models that recapitulate the parasitic life cycle are scarce. To address this challenge we humanize the relevant tissue compartments, i.e. liver and blood in mice. Specifically, transplantation of human hepatocytes into specially conditioned xeno-recipients leads to a robust human hepatic chimerism. Resulting human liver chimeric mice are susceptible to infection with P. falciparum sporozoite and support faithful development of parasitic liver stages. Efforts are ongoing to extent this work to P. vivax. Introduction of human erythrocytes into human liver chimeric mice allows for transition for liver to blood stages. Humanized mice will be useful for studies of malaria pathogenesis and transmission to arthropod hosts. These animals will be immediately applicable for safety and efficacy assessments of genetically attenuated parasites, anti-malarial drugs, and passive immune protection strategies.