The sequence of the Plasmodium falciparum genome was published in 2002 and is being mined for new drug and vaccine targets. Our research is aimed at accelerating the translation of genomics into health interventions. We hope to exploit genomic advances to improve malaria drugs and vaccines that are already well along in clinical development but whose efficacy is threatened by the parasite's genetic diversity.
The sequencing of the P. falciparum genome also presents the opportunity to understand at a molecular level how malaria parasites evolve in response to forces that can be measured and controlled in clinical trials, namely drugs and vaccines. New sequencing technologies permit genome-wide characterization of malaria parasites on an epidemiological scale. Our interdisciplinary, collaborative research program relies on clinical trials of malaria drugs and vaccines at field sites in Africa and Asia to help translate genomic science into tools to reduce malaria disease and death and to explore the molecular evolution of malaria parasites.
Genetic Diversity and Efficacy of Malaria Drugs
P. falciparum has evolved resistance to nearly every class of antimalarial drugs within 10–15 years of their wide deployment. Resistance to drugs for which molecular mechanisms of parasite resistance are known has arisen as rare mutational events in limited, distinct geographic foci and spread across continents in ancestral lineages that can be identified through genotyping. In combinations of two or more, new drugs that simultaneously target different parasite mechanisms are being used to try to overcome this rapid evolution of resistance.
Clinical trial of chloroquine combinations. Chloroquine is an extraordinarily safe, effective, and cheap antimalarial drug, and chloroquine-resistant malaria results in increased disease and death. Resistance to chloroquine is caused by a mutation in the P. falciparum chloroquine resistance transporter gene. We developed methods to use mutation as a molecular marker to detect and track resistance in the field. Malawi, a Central African country, stopped using chloroquine in 1993 because of high rates of resistance. Unexpectedly, we found that the molecular marker for chloroquine-resistant malaria declined and disappeared there over a period of eight years following cessation of chloroquine use (see Figure 1). In 2005, in a controlled clinical trial, we confirmed that chloroquine efficacy had returned.
We then undertook a longitudinal clinical trial (supported by the National Institutes of Health) of chloroquine alone and in combination with drugs intended to block reemergence of chloroquine resistance in Malawi. This trial tested strategies for deterring the emergence/reemergence and spread of drug-resistant malaria and we are applying these strategies to develop and deploy the next generation of combination therapies. By combining chloroquine with drugs with increasingly long durations of action, and measuring the ability of these drugs to block the emergence of parasites carrying the molecular marker for chloroquine resistance, we hope to define the "selection window," or time during which chloroquine needs to be protected by a partner drug to deter reemergence of resistance. These studies will provide the evidence needed to design combination therapies that are refractory to drug resistance, and may open up the possibility of reintroducing chloroquine for malaria treatment or prevention in Africa.
Using single-nucleotide polymorphisms (SNPs), microsatellites, and other genetic markers, we previously described the origin and dissemination of antifolate-resistant malaria in South America. The present situation in Malawi offers an opportunity to understand the population genetics of drug-resistant malaria. We subjected archived filter paper and frozen blood samples from our clinical trials at the Blantyre Malaria Project in Malawi to SNP and microsatellite typing. This showed that the return of chloroquine-sensitive malaria occurred not as a single genetic sweep nor as reversion of resistant forms to sensitive genetic forms, but rather as selection of preexisting, susceptible parasites that had persisted in the population. Measuring effective population sizes and understanding gene flow over time as drug pressure is applied and withdrawn will increase our understanding of malaria population genetics and molecular evolution, and may lead to new strategies for deterring or containing drug resistance.
Molecular basis of resistance to combination therapies. We and others have used molecular markers of drug resistance as tools to monitor resistance and to control a malaria epidemic. Chloroquine and the antifolates have now been replaced in most countries by more effective artemisinin-based combination therapies (ACTs). Resistance now seems to be emerging to the artemisinins in Southeast Asia, and efforts to contain this resistance are hindered by the lack of tools for surveillance. It has been hard to identify simple molecular markers of resistance to many of these newer malaria drugs, suggesting possible involvement of multiple genes.
With support from the World Health Organization through a grant from the Bill and Melinda Gates Foundation and in collaboration with a network of research partners in Southeast Asia, we are conducting genome-wide association studies and other genomic analyses to identify the molecular mechanisms by which malaria parasites from cases of clinical resistance to artemisinins evolve the ability to resist drugs. We hope to develop and validate new molecular markers that can be used to track and contain emerging resistance to current first-line antimalarial drugs.
Genetic Diversity and Malaria Vaccine Efficacy
As with vaccines against HIV, Streptococcus pneumoniae, and influenza virus, malaria vaccine development is complicated by genetic diversity in vaccine antigens. For example, we measured polymorphism within a surface protein vaccine antigen (merozoite surface protein 1, or MSP1) in more than 1,300 malaria infections occurring over three years among children living at our vaccine testing site in Mali, West Africa. We found a high degree of diversity (see Figure 2), with the leading vaccine strain accounting for only a small minority of parasites. This suggests that the subsequent failure of this vaccine might have been predicted had such studies been used to select the vaccine strain early in vaccine development.
Another leading vaccine candidate, the apical membrane antigen 1 (AMA1) is highly polymorphic, with more than 70 SNP sites. If immunity elicited by an AMA1–based vaccine is allele-specific, then vaccination will likely result in directional selection favoring alleles that are different from those targeted by the vaccine, resulting in reduced efficacy.
With support from the National Institutes of Health and the U.S. Agency for International Development, our team established the Bandiagara Malaria Project as a clinical trials site in Mali. There, we have conducted clinical trials in adults of blood-stage malaria vaccines developed by the U.S. Army and GlaxoSmithKline Biologicals. These vaccines are being developed with the goal of building a multistage, multiantigen malaria vaccine that will prevent malaria caused by the genetically diverse malaria parasites found in natural populations.
We have evaluated the safety and immunogenicity of one AMA1-based vaccine in small clinical trials in adults and children, and we recently completed a larger efficacy trial in children. To measure polymorphism and dynamics of AMA1 in preparation for efficacy trials, we have sequenced P. falciparum AMA1 from filter paper blood samples collected during a three-year prospective cohort study in Mali. We found an extraordinary degree of diversity in this vaccine antigen, with more than 200 variants in infected children living in a single village. We were able to identify the key residues for allele-specific protection by mapping these residues on the three-dimensional structure of the protein and performing tests of association of changes in amino acid at specific SNP sites with risk of clinical symptoms (see Figures 3 and 4). Longitudinal analyses of the within-host dynamics of AMA1 polymorphisms indicate that changes in amino acids at specific SNP sites are associated with clinical symptoms. This suggests that these particular amino acid residues may be the most important ones to consider in designing next-generation multivalent or chimeric vaccines that elicit cross-protection against different allelic forms of AMA1.
The translational clinical research program has been supported by the National Institutes of Health, the Bill and Melinda Gates Foundation, the U.S. Department of Defense, and the U.S. Agency for International Development. The genomic and molecular evolution studies have been supported by grants from the National Academies of Science, the W.M. Keck Foundation, and the University of Maryland, and a Distinguished Clinical Scientist Award from the Doris Duke Charitable Foundation.
As of March 10, 2011