During this past year (2011-2012) our laboratory built upon the foundation begun in 2008 to explore the interface between the malaria parasite and the host immune system. We have continued to collaborate with Dr. Rick Fairhurst (LMVR) and Dr. Mahamadou Diakite (MRTC) on a 4-year longitudinal study of 1400 children in 3 villages in Mali. 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 from 2009 to the present, with blood samples being obtained before and after the transmission season as well as during bouts of clinical malaria infection; these samples provide a unique and valuable resource for studies on the development of humoral and cellular responses to blood-stage antigens of malaria parasites. Analysis has shown that children with HbAS are significantly protected against malaria in this population; this was not true for children with the HbAC genotype. Increasing age, a surrogate of acquired immunity, was also protective. Because long-standing data has shown that antibodies can play a significant role in protection against erythrocytic stages of infection, we have pursued a detailed characterization of the antibody populations present in the Malian children. We have already shown that children with HbAS genotype show lower responses by ELISA to a number of merozoite antigens relative to age-matched controls. We believe that the lower titers in the HbAS children may result from their lower incidence of infection and we are analyzing a study of asymptomatic parasitemia in these children. During the past year we have followed these children into a new transmission season and shown that, surprisingly, their IgG levels to specific merozoite proteins do not diminish during the dry season with little malaria transmission. We are continuing to follow the antibody profiles over several transmission seasons using both ELISA and GIA as readouts and are also conducting more in depth studies of the antibodies themselves. In addition, we have developed a flow cytometry assay to profile the antibodies in these children to antigens present on the surface of parasitized erythrocytes. We have investigated changes in these antibody populations with age, with malaria exposure, and with host genotype. Another aspect of our studies of host responses to proteins on the surface of parasitized erythrocytes, we have continued our collaboration with Dr. Kavita Singh (RTB) on a major surface antigen of infected red cells, viz., VAR2CSA. This protein is a member of the large PfEMP1 family and has been implicated in pregnancy associated malaria through binding to chondroitin sulfate A (CSA) in the placenta. Dr. Singh has produced 6 domains of the VAR2CSA protein in recombinant E. coli and we have tested their recognition by antisera from Malian adults. Antibodies from multiparous Malian women but not men recognize some of these domains by ELISA. We have also standardized an opsonization assay using these IgGs and shown that FCR3 parasitized red cells expressing VAR2CSA can be efficiently opsonized by a human monocytic cell line. We have developed a novel procedure testing the ability of various domains to inhibit opsonization by these antibodies. We have thus identified several domains that are important for recognition by anti-VAR2CSA antibodies in this functional assay. Our second major area of investigation relates to the identification of malaria parasite-encoded antigens which could be the targets of new vaccines or drugs. We have hypothesized that there are conserved epitopes present on the infected red cell which could represent such targets. While most blood-stage vaccine candidates are from merozoites, antigens present on the surface of the infected red cell have significant advantages as targets because of their exposure to the serum for long periods. However, the antigenic complexity and diversity of the known surface molecules (e.g., PfEMP1) have proven daunting for vaccine development. To address this problem, we have pursued a new strategy using DNA aptamers. We had previously prepared several DNA aptamer libraries and performed repetitive selections on various targets, and we analyzed the selected aptamer populations using next-generation sequencing technology. We have focused on a small subset which binds to infected but not uninfected red cells; some of these aptamers inhibit parasite growth in vitro. This year we have completed broader studies on the reactivity profiles of this subset and shown that some recognize all the parasite isolates tested. More recently we have developed methods to affinity purify the parasite targets of the aptamers. This is a complex process, involving preparation of extracts from parasitized erythrocytes, binding to immobilized aptamers, and identification of target molecules by mass spectrometry. However, we now have bands on SDS-PAGE gels and are confident we will have identification of target molecules. Using our standardized blood-stage parasite growth inhibition assay (GIA), we have collaborated with others in analysis of several different human trials of various blood-stage vaccine candidates and in testing of preclinical animal sera. An important step forward has been made in collaboration with Dr. Simon Draper and colleagues (Oxford University), who are using recombinant adenoviruses encoding P. falciparum blood stage antigens. A new potential blood-stage vaccine candidate - PfRH5 - has been identified that elicits antibodies with very high levels of GIA activity. Binding of PfRH5 to the red cell protein basigin has been shown to be essential for merozoite invasion. We have tested antibodies to PfRH5 against parasites from different geographic locations and are evaluating antibodies from an immunization-challenge study in Aotus monkeys for functional activity. We are continuing to collaborate with Dr. James Burns (Drexel University) on his novel fusion protein of P. falciparum MSP1 and MSP8. We have shown that it elicits extremely high levels of antibodies with GIA acitivty in rabbits; this construct is being extended to preclinical studies in Aotus monkeys. We have continued to expand our studies on transmission blocking immunity and in collaboration with PATH/MVI, we are performing mosquito membrane feeding assays (MFA). This involves culturing P. falciparum sexual stages in vitro, feeding these parasites to laboratory-reared mosquitoes in the presence or absence of specific antibodies, and later counting oocysts in the mosquito midgut. To accelerate the development of transmission blocking vaccine, this year we have qualified the MFA to test its reproducibility. Using these results we have worked with Dr. Michael Fay of the Biostatistics group to develop a computerized model of the MFA which allows us to ask questions about assay variables and predict outcomes. In addition, it allows us to test sexual stage vaccine candidates for transmission blocking activity and obtain confidence limits. We have constructed and characterized a new set of mouse monoclonal antibodies to the full-length P. falciparum circumsporozoite protein prepared by Dr. Sanjay Singh at Gennova, India. Seven of these monoclonals have been produced in quantity, and we are working with extramural collaborators (Drs. Fidel Zavala and Chris Ockenhouse) to test these for passive protective activity using transgenic P. berghei parasites. Results have been presented at the American Society for Tropical Medicine and Hygiene (10 talks and posters-Philadelphia, PA)(2011). Results have been presented at the European BioMalPar meeting in Germany (2011), the Japanese Society for Parasitology (2012), a World Health Organization workshop in Geneva, Switzerland (2012), and at Gennova, India.