A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be essential to gain a detailed understanding of the impact of malaria on the generation and maintenance of immunological memory, the basis of all vaccines. Our goal is to gain an understanding of the generation, maintenance and activation of immunological memory in response to natural malaria infection, focusing on B cells and the function of Pf-specific antibodies. Although long-lived classical MBCs (CD19+/CD20+/CD21+/CD27+/CD10-) are gradually acquired in response to natural infection, exposure to P. falciparum also results in a large expansion of what we have termed atypical MBCs (CD19+/CD20+/CD21-/CD27-/CD10-). At present, the function of atypical MBCs in malaria is not known nor are the factors that drive their differentiation. We recently carried out studies to establish the relationship between atypical MBC and classical MBCs and provided evidence that atypical MBCs and classical MBCs have undergone the same number of cell divisions and that the V gene repertoires and somatic hypermutation rates are indistinguishable suggesting a common developmental history. We showed that atypical MBCs have lost two key adaptive immune cell functions, namely the ability to signal through the BCR and to differentiate into Ab secreting cells. Atypical MBCs express an array of inhibitory receptors and BCR signaling is blocked at the level of phosphorylation of one of the early kinases in the BCR signaling pathway (Syk) resulting in impaired B cell responses. Moreover, atypical MBCs are unable to respond to a variety of stimuli that triggered the differentiation of classical MBCs to differentiate into Ab secreting cells. One of the defining features of atypical MBCs in HIV-infected individuals is the expression of FcRL4. In collaboration with Dr. Moir we provided evidence that silencing FcRL4 in atypical MBC B cells from HIV-infected individuals resulting in restoration of BCR signaling and that conversely expressing FcRL4 in B cell lines blocked BCR signaling to Syk phosphorylation indicating that FcRL4 expression was central to atypical MBCs dysfunction. In contrast, we showed that aMBCs in malaria do not express FcRL4 but rather upregulate FcRL5. FcRL5 has different signaling potential and ligand specificity as compared to FcRL4 and it is of interest that these phenotypically similar atypical MBCs may differentiate differently in response to P. falciparum versus HIV infection. We have recently expanded our analysis of the antibody repertoire to describe the structure of the entire human antibody repertoire in response to malaria in collaboration with Dr. Ning Jiang, University of Texas. To do so we will use high-throughput-long read sequencing to characterize the expressed antibody repertoire in children and adults living in malaria-endemic Mali and the impact of malaria on this repertoire. We will also clone VH and VL from individual B cells and express those as antibodies to determine which sequences contribute to the Pf-specific immune response. Over the last year we also explored the relationship between Pf infections and autoimmune disease. These studies are based on a long standing observation of a link between autoimmune disease and malaria, namely that in malaria endemic regions of Africa there is very little autoimmune disease. This observation suggests that susceptibility to autoimmune disease may be modulated by malaria and/or that susceptibility to autoimmune disease is protective against sever malaria. In collaboration with Dr. Silvia Bolland, we provided evidence that a genetic susceptibility to systemic lupus erythematosus (SLE) protects against cerebral malaria in a mouse model. Protection appears to be by immune mechanisms that allow SLE-prone mice to better control their overall inflammatory responses to parasite infections. We are exploring the possibility that anti-inflammatory therapy may treat cerebral malaria. In addition, in collaboration with Dr. Inaki Sanz (Emory University) we are characterizing our Malian cohort for autoantibodies associated with human SLE. VH4-34-containing antibodies are auto-antibodies and can be identified using the VH4-34-specific monoclonal antibody 9G4. 9G4+ memory B cells and serum antibodies are prevalent in SLE but not in healthy controls. We have recently determined that in malaria-exposed Malians 9G4+ antibodies rise after clinical malaria but are controlled and rapidly disappear and do not appear in the memory responses. These observations suggest a special but distinct regulation of 9G4+ B cells and antibodies in SLE and in malaria. To better characterize the sequence of events that contribute to cerebral malaria we have established a collaboration with Dr. Dorian McGavern to use two photon, intravital imaging to view the cellular events that occur following infection with a mouse Plasmodium that results in cerebral disease using fluorescently labeled parasites, T cell, mononuclear cells and markers for vascular permeability. These studies have provided strong evidence that the interaction of parasite-specific CD8+ T cells with brain endothelium is necessary for the pathology of cerebral malaria. These results have led us to test two inhibitors of T cell metabolism as adjunctive therapy for cerebral malaria. The first, rapamycin, an mTOR inhibitor, protected against cerebral malaria when administered within the first four days of infection. Treatment with rapamycin increased survival, blocked breakdown of the blood brain barrier and brain hemorrhaging, decreased the influx of both CD4+ and CD8+ T cells into the brain and the accumulation of parasitized red blood cells in the brain. Encouraged by our findings with rapamycin we treated PbA-infected mice with the glutamine analogue, 6-diazo-5-oxo-L-norleucine (DON), in collaboration with Dr. Jonathan Powell at Johns Hopkins who has had experience using DON as a therapy in graft rejection. Remarkably we found that DON rescues mice from CM, when administered late in the infection when mice already show neurological signs of the infection. At the time of treatment mice are suffering blood-brain barrier dysfunction, brain swelling and hemorrhaging accompanied by accumulation of parasite-specific CD8+ effector T cells and infected red blood cells in the brain and perturbation of brain metabolism. Remarkably, DON-treatment restored blood-brain barrier integrity, reduced brain swelling, decreased the function of activated effector CD8+ T cells in the brain and returned brain metabolites to uninfected levels. These results suggest DON as a strong candidate for an effective adjunctive therapy for CM in African children. We are in the process of carrying out experiments to determine the optimal dosing and schedules and pharmacokinetics for effective treatment of ECM and determine the mechanism of DON protection. We are also in the process of organizing and designing a clinical trial to test the ability of DON to prevent deaths from pediatric CM. We have established a collaboration with Terrie Taylor, an expert in CM in children who heads a pediatric clinic in Malawi that treats children with cerebral disease, to explore the potential of DON as an adjunctive therapy of cerebral disease in children.