During the past fiscal year the Long laboratory has continued to integrate into the Laboratory of Malaria and Vector Research (LMVR) and to expand our investigations into the interface between the malaria parasite and the host innate and adaptive immune systems. To accomplish this we have employed both clinical research studies in Mali and several different rodent models of malaria infection.? In this context we have continued our ongoing analyses of animal and human sera directed to malaria parasite antigens using a standardized parasite growth inhibition assay (GIA) that we have developed. Results from preclinical and clinical trials using this assay have contributed significantly to decisions about clinical development of various blood-stage antigens. In addition, we have explored the interaction of anti-merozoite antibodies of different specificities. We had previously shown that naive US volunteers vaccinated with AMA1 produce antibodies which inhibit merozoite invasion. However, when the same formulation was taken to Mali for a similar clinical trial in adults, antibodies were elicited by the vaccine but no increase in the GIA was seen. In collaboration with Dr. Kazutoyo Miura of MVDB, we tested two hypotheses: 1) that the antibodies produced by malaria-experienced Africans were qualitatively different so that they were not effective in GIA, or 2) that other antibodies in the sera of malaria experienced Africans interfered with the biological activity of the anti-AMA1 antibodies. We affinity purified AMA1 specific antibodies from US and Malian sera and, while there were small differences, we showed that in general anti-AMA1 antibodies elicited by vaccination or by infection with parasites had comparable biological activity. We then addressed the second hypothesis and showed that non-AMA1 IgGs from Malians could interfere with the GIA activity of anti-AMA1 antibodies of Malians or US volunteers. These results likely explain the failure to see increased GIA in immunized Africans and also have implications for laboratory evaluation of blood stage malaria vaccine trials. We also propose that this interference by other anti-malaria antibodies is a mechanism by which the parasite maintains a balance within the vertebrate host. Finally,we have begun to apply the GIA assay as a research tool to identify novel erythrocytic-stage vaccine candidates. ? On the cellular level we have established techniques for analysis of human and mouse CD4+ T cell responses to malaria antigens including identification of T memory cells, T cells with a regulatory phenotype, and Th17 cells. For the rodent studies, we have collaborated with Drs. James Burns and Robert Seder in identifying and characterizing multifunctional CD4+ T lymphocytes specific for P. yoelii MSP8. We are now determining the fate of these cells, which produce more than one cytokine, after parasite infection. We are also extending these studies to other parasite antigens and to other rodent models of malaria infection. ? In regard to CD4+ T cells in humans, we have continued to refine the analysis of different cell subtypes including T memory cells and multifunctional T cells. We have completed a study of kinetics of CD4+ T cell responses to AMA1 in a clinical trial and we are currently completing analysis of another clinical trial employing Alhydrogel with CPG. To extend these studies to malaria endemic areas, we have collaborated with Dr. Rick Fairhurst of LMVR and Dr.Mahamadou Diakite to initiate a longitudinal study of 1260 children of various ages in 3 villages in Mali. A subset of these children is being followed for in depth T cell responses, as well as development of humoral immune responses. In addition, we have initiated studies on innate immune responses in these children.
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