In 2009-2010 the Long laboratory built upon the foundation established in the previous 2 years to explore the interface between the malaria parasite and the hosts immune system. To accomplish this we have employed both clinical research studies in Mali and different rodent models of malaria infection. We have collaborated with Dr. Rick Fairhurst and Dr. Mahamadou Diakite of the MRTC in Mali to continue a 5-year longitudinal study of 1400 children of various ages in 3 villages. In 2009 we identified a sub-cohort of these children, selecting those with the sickle cell trait (HbAS) and pairing them with age-matched HbAA controls. These children have been followed through 2009 into 2010 for development of humoral and cellular responses to blood-stage antigens of malaria parasites. From the first year of our study it is already apparent that children with HbAS are significantly protected against malaria in this population. We have tested the hypothesis that the HbAS children develop accelerated antibody responses to blood-stage malaria antigens as a basis for their protection; however, we have shown that in fact their humoral responses are lower than their matched controls. Thus, this cannot be responsible for their increased resistance to infection. In addition to the quantitation of humoral responses by ELISA, we have purified IgG from the same children before and after the rainy season and are testing the ability of these preparations to inhibit parasite growth in vitro. Moreover, we have determined the plasma concentration of a series of cytokines before and after the rainy season. We are currently integrating all these humoral analyses and comparing the responses of children with the sickle cell trait to the control children. These results in children are also being compared with those of adults from three different ethnic groups in Mali. In addition, we are initiating a series of studies on cellular responses of the same children. We have also made significant progress in 2 other areas begun in 2009. The first is the immunologic analysis of antibody responses to the DBL3X domain of the VAR2CSA protein. This PfEMP1 protein has been implicated in pregnancy associated malaria through binding to chondroitin sulfate A (CSA) in the placenta. In collaboration with Dr. Kavita Singh, who determined the crystal structure of the DBL3X domain, we have produced monoclonal and polyclonal antibodies to this domain and shown that some of these antibodies can inhibit binding of parasitized erythrocytes expressing VAR2CSA to CSA. Thus the antibodies have functional activity. More recently, we have shown that this DBL3X domain is recognized by sera from Malian women and we mapped the CSA binding site to the S3 sub-domain of DBL3x (Singh et al, 2010). The second new area is the use of DNA aptamers to identify novel conserved antigenic determinants on the surface of malaria infected erythrocytes. While most blood-stae vaccine candidates have been directed to antigens of merozoites, we believe that antigens present on the surface of the infected red cell have significant advantages as potential vaccine candidates because of their exposure to the serum for long periods. However, the antigenic complexity and diversity of the surface molecules (e.g., PfEMP1) identified to date have proven daunting in terms of vaccine development. Consequently we have taken a novel approach to the identification of strain-conserved targets on the infected red cell membrane. We have prepared several complex DNA aptamer libraries and conducted a series of selections on various targets. To analyze the selected aptamer populations we have used next-generation sequencing technologies primarily Illumina Solexa sequencing. Using this technology we have identified over 100 aptamers with specificity for infected red cells and we have confirmed this by FACS analysis comparing uninfected and infected red cells; some of these aptamers inhibit parasite growth. Once the characterization is completed, we will proceeding to identify the molecular targets of the selected aptamers. An area which we have expanded this year is transmission blocking immunity. In collaboration with PATH/MVI, we are performing mosquito membrane feeding assays and working to standardize this assay. In addition, using a large amount of historical data, we have established the amount of anti-Pfs25 antibody (Pfs25 is an antigen of mosquito stage P. falciparum parasites) required to neutralize 50% of oocysts (IC50) in various species including humans (Cheru et al). This provides a marker for vaccine development with this antigen. Using our standardized blood-stage parasite growth inhibition assay (GIA), we have also collaborated with LMIV and others in analysis of 4 different human trials of various blood-stage vaccine candidates (Sagara et al.; Pierce et al.; Ellis et al.; El Sahly et al). With collaborators (Bruder et al.), we have also shown that recombinant adenoviruses expressing different P. falciparum blood stage antigens can elicit significant levels of antibodies with functional activity in animal studies. Finally we have continued our studies of rodent models of malaria. We are replicating our previous studies of multi-functional CD4+ T cells specific for P. yoelii MSP-8. We had identified such cells and had described their appearance in different tissues after different numbers of injections and after challenge with viable malaria parasites. We have also collaborated on a study of the importance of early interferon-gamma in a mouse model of cerebral malaria performed with P. berghei. These results have been presented at the American Society for Tropical Medicine and Hygiene (9 talks and posters)(Washington, DC, November, 2009) the Multilateral Initiative on Malaria Meeting, Nairobi, Kenya, November 2009); the American Association of Immunologists (AAI, Baltimore MD, May 2010); the Malaria Vaccines for the World, Washington, DC, Sept. 2010). In addition, 9 abstracts/talks are being presented at the 2010 meeting of the American Society for Tropical Medicine and Hygiene in Atlanta, GA.