Malaria Immunology and Pathogenesis Goals are:1) To understand how Malian children with hemoglobin S, alpha-thalassemia, G6PD deficiency, and ABO polymorphisms are protected from Plasmodium falciparum malaria;2) To develop a profile of the acquisition of malaria immunity in Malian children;3) To examine various aspects of pathogenesis due to malaria in Malian children and adults;4) To characterize the pharmacokinetics of artemisinin (ART) and the mechanisms of parasite clearance after treatment. These goals are being accomplished through a 4-year longitudinal study of 1500 children ranging in age from 6 months to 18 years living in 3 villages in Mali (the Kenieroba study). One major effort is to determine the relative protection against malaria conferred by different red blood cell (RBC) polymorphisms, and all the enrolled children have been typed for a series of these polymorphisms. In 4 years, we have diagnosed and treated 4207 episodes of uncomplicated and severe falciparum malaria. Analysis of our data shows that sickle cell trait (HbAS) confers significantly greater protection against malaria than other RBC polymorphisms, and that age, a surrogate of naturally acquired immunity, is also associated with reduced malaria incidence. We are now investigating the mechanistic basis for this protection in a number of ways: 1) We identified a sub-cohort of children, selecting those with sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. We have found that antibodies to a variety of merozoite proteins are lower in HbAS children so that this is not the basis for their resistance;2) We have developed a plasma reactivity assay using flow cytometry to measure antibody binding to infected red cells and shown that HbAS children do not display increased reactivity to the red cell surface;3) We are testing the effects of HbAS and naturally-acquired IgG on the binding of parasitized RBCs to microvascular endothelial cells (MVECs), a critical event in malaria pathogenesis;4) We are determining whether sera from HbAS children display reactivity to selected PfEMP1 molecules or domains. Some children with malaria display severe pathology, but it has been unclear as to whether certain P. falciparum strains elicit more pathology or whether aspects of the host genetics or responses contribute to susceptibility. We are conducting a series of studies related to pathology during malaria including:1) Reactivity of sera from adult males and females from Mali to various domains of a particular PfEMP1 protein, VAR2CSA, which has been implicated in pregnancy associated malaria. We have identified several domains within the VAR2CSA protein which are particular targets of opsonizing antibodies in multigravid women;2) Exploring the role of uric acid levels in disease severity and endothelial cell pathology;3) Demonstrating that the concentration of microparticles in the plasma is elevated in children with malaria;4) Collaborating with Dr. Michael Walther(LMIV) in examining the role of Tregulatory cells in severe malaria using data and peripheral blood samples from our cohort. We have continued to assess the in vitro and in vivo responses of Malian children to artesunate (ART), an effort prompted by reports from Asia of slow clearance of P. falciparum infections. We have determined the clearance times of parasites in several hundred Malian children with uncomplicated malaria and at present the ART resistant phenotype has not been found in our study population. Importantly, we have also observed that older children exhibit more rapid clearance times than younger children. We are testing the hypothesis that plasma IgG accelerates the parasite clearance rate in response to ART. Malaria Transmission Because transmission of malaria is a critical aspect of the parasite life cycle but is poorly understood, we are increasing our efforts to investigate parasite sexual stages in the field. This year we have enrolled 500 people of all ages, reflecting the age demographics of Kenieroba, in a new protocol. Volunteers are being finger-pricked twice per month for a year and sera collected by venipuncture three times. Samples collected will allow analysis of both DNA and RNA. Previous studies have generally concluded that microscopy is not sufficiently sensitive to identify gametocyte carriers so we will only use molecular techniques for this analysis. Clinical samples will be screened for P. falciparum asexual and sexual stages of infection and parasite profiles will be compared throughout the year, particularly during the dry season. In addition, sera will be tested for the capacity to inhibit transmission in membrane feeding assays. Strategies to impact malaria transmission will be accelerated by a better understanding of the entire process of sexual stage development and transmission to mosquitoes. We have also initiated a new project to investigate the P. falciparum sexual stage antigen Pfs47 and its relationship to the immune system of the infected mosquito. Preliminary data has led to the hypothesis that Pfs47 interacts with a mosquito gene to disrupt the immune response, resulting in more effective transmission, and that different alleles of Pfs47 may work better to evade the immune system of certain mosquitoes. This hypothesis is being tested by collection of infected mosquitoes in Mali and characterizing the allelic forms of Pfs47 in these vectors. This research may provide evidence that Pfs47 could be a novel transmission blocking vaccine candidate. Mosquito Vectors The main projects for this year focused on (1) searching for hidden shelters of the malaria mosquito vectors during the Sahelian dry season and (2) evaluation of long distance migration as a means of re-colonization of Sahelian habitats after population extinction. For the first project, we are evaluating a novel approach to find aestivation shelters by using a trained sniffing dog to locate mosquitoes tagged with a foreign scent. The second project consists of two complementary studies on long-distance migration of msoquitoes, viz., (a) on-the-ground monitoring of vector density and composition along two transects over 100 km long and (b) aerial sampling of mosquitoes. Aerial sampling is aimed at detecting seasonal migrations of mosquitoes at higher altitudes. To date we have captured 9 Anopheles gambiae females at high-altitude (>100 m) above ground. We will determine the species and type (M or S form) of mosquitoes that are found at these heights and whether this correlates with re-colonization of the area after the onset of rains. Sand fly / Leishmaniasis Program The program on leishmaniasis and its sandfly vectors is designed to describe the unexamined epidemiology of cutaneous leishmaniasis (CL) in Mali. Previously we showed that Leishmania major, the causative agent of cutaneous leishmaniasis, was the only parasite species found in sandflies, and that Phlebotomus duboscqi is the principal vector of this disease. Importantly, there is no previous data characterizing cellular immune responses to sand fly salivary proteins in leishmania endemic areas. As in animal models, delayed type hypersensitivity (DTH) to sand fly bites was also observed in Malian volunteers;75% of individuals aged 1-15 years showed this, decreasing gradually to 48% by age 45. This response was determined to be a Th1-type by the presence of interferon-gamma in skin biopsies and persisted for long periods. Tick-borne relapsing fever (TBRF) In collaboration with RML, we have continued a study of TBRF in Mali. TBRF is caused by spirochetes maintained in enzootic foci involving small mammals and argasid ticks. Having sampled small rodents and ticks (Ornithodoros sonrai) in 20 villages, we found 11.3% of rodents and 18% of ticks were seropositive for TBRF.
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