The LMVR program in Mali is divided into three main units: malaria pathogenesis and immunology, vector studies and Leishmaniasis. Malaria Immunology and Pathogenesis The goals of the malaria pathogenesis/immunology program are: 1) To understand how Malian children with hemoglobin S, alpha-thalassemia, G6PD deficiency, and ABO blood group polymorphisms are protected from Plasmodium falciparum malaria.2) To develop a profile of the acquisition of malaria cellular and humoral immunity in Malian children and to correlate those results with the studies on blood group polymorphisms. 3) To identify the role of specific PfEMP-1 variants in pathogenesis, including analysis of immune responses to the VAR2CSA variant of PfEMP1 since this variant has been associated with placental malaria.4) To investigate the genetic structure of the malaria parasites in this population and assess how this parasite population changes with time and with selective pressures such as drugs. These goals are being accomplished through a 5-year longitudinal study of 1500 children ranging in age from 6 month to 18 years of age living in 3 villages in Mali (the Kenieroba study) with all children being typed for a series of these red cell and hemoglobin polymorphisms. In the past 3 years, we have diagnosed and treated over 3500 episodes of uncomplicated and severe falciparum malaria. Analysis of our epidemiological data shows that HbS 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. The first 3 years of this study are being prepared for publication.We are now investigating the mechanistic basis for the HbAS protection by testing the effects of HbS and naturally-acquired IgG on the binding of parasitized RBCs to microvascular endothelial cells (MVECs) a critical event in malaria pathogenesis. We are determining whether parasites infecting children with HbAS display particular PfEMP1 variants which might form the basis of a vaccine against severe malaria. We are also profiling serological responses to domains of VAR2CSA, the PfEMP1 variant associated with placental malaria. Finally, we have followed 300 HbA, HbS, and HbC children weekly for parasitemia and malaria symptoms to explore why HbS is so much more protective against malaria. To address the acquisition of immunity, we identified a sub-cohort of children (300 children), selecting those with sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. These children have been followed for development of humoral and cellular responses to blood-stage antigens of malaria parasites. We tested the hypothesis that the HbAS children develop accelerated antibody responses to malaria as a basis for their protection but we have shown has that HbAS humoral responses are lower than their matched controls. Thus, this cannot be responsible for their increased resistance. Other studies of humoral and cellular immune responses to malaria parasites and specific antigens are also being pursued. A major 2010 initiative was the establishment of in vitro and in vivo tests for parasite drug responses to artesunate and amodiaquine the two antimalarial drugs used in Mali. In the 2010 transmission season we monitored parasite clearance rates of artesunate in over 100 children to assess the artemisinin resistance phenotype in Africa. Initial studies found rapid clearance rates and no indication of resistance, and we are continuing these studies in the 2011 transmission season.We have also established a robust in vitro drug susceptibility assay to determine parasite IC50s for these drugs. These responses and parasite genomic data will be followed over time to determine wheether parasite resistance develops in a setting of intense local drug pressure. Another addition in 2011 is a collaboration with Michael Walther of LMIV, who has designed a study to examine the T regulatory responses of children with malaria. This has greatly expanded our efforts since all children with malaria are being bled 21 days after their diagnosis to obtain a convalescent blood sample. Our studies have supported the submission of 8 abstracts to the 2010 ASTMH meeting, with 4 Malian trainees giving posters or oral presentations and additional abstracts have been submitted for the 2011 meeting. All members of the Fairhurst laboratory, many members of Carole Longs laboratory, Dr. Michael Walther (LMIV) and several other trainees in LMVR are presently using data, parasites, or patient blood samples from our Kenieroba protocol. Vectors Malaria vector studies encompass a number of areas of mosquito biology: 1. After we showed that Anopheles gambiae can aestivate (dry season diapause) under natural conditions, we have quantified the importance of aestivation as opposed to long distance migration, demonstrating that aestivation is the most important strategy used by the M form of An. gambiae, but the S form and An. arabiensis rely on long distance migration. These finding have important practical implications for malaria control.2. Studies were conducted on variation in metabolism rate of mosquitoes under natural conditions as part of multi-project investigation of mechanisms to explain the extended survival of aestivating mosquitoes. 3. A survey was undertaken to localize the hidden shelters of mosquitoes during the dry season but despite extensive effort only 3 An. gambiae specimens were found.4. A large scale SNPs study was launched in collaboration with Liverpool School of Tropical Medicine to determine, based on genetic data, if the source of the early wet season population is from a parental generation that existed at the end of the previous wet season. 5. A novel study on the mating biology of An. gambiae was published, extending previous studies on reproductive isolation and speciation.We found that only swarm size predicts mating success. 6. Continued efforts to employ 3D photographic techniques to analyze the structure and dynamics of swarms of male mosquitoes and the consequences of this behavior for reproductive isolation of different An. gambiae molecular forms. Sand fly / Leishmaniasis Program Phlebotomus duboscqi is the principal vector of Leishmania major, the causative agent of cutaneous leishmaniasis (CL), in West Africa and is the suspected vector in Mali. Although found throughout the country the seasonality and infection prevalence of P. duboscqi has not been established in Mali. We conducted a three year study in two neighboring villages, Kemena and Sougoula in Central Mali, an area with a leishmanin skin test positivity of up to 45%. During the first year of 18,595 flies collected, 69% belonged to 12 species of Sergentomyia and 31% to two species of the genus Phlebotomus, P. duboscqi and P. rodhaini. Of those, P. duboscqi represented 99% of the collected Phlebotomus species. Seasonality and infection prevalence of P. duboscqi was monitored over two consecutive years. Using a quasi-Poisson model we observed a significant annual, seasonal and village effect on the number of collected P. duboscqi.The infection status of pooled P. duboscqi females was determined by PCR showing a 2.7% infection prevalence. Based on the PCR product size, L. major was identified as the only species found in flies from the two villages incriminating P. duboscqi as the vector of CL in Mali. A second objective of this study is to investigate the type of cellular immune responses induced by the repeated bites of the sand fly and to determine which of the distinct secreted salivary proteins from this sand fly are responsible for the immune responses observed in a naturally exposed population.PBMC were collected from study subjects and these samples will be tested to characterize human cellular responses to the salivary repertoire.