Malaria immunology

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Uncomplicated malaria To better understand the impact of chronic malaria exposure on the human B cell compartment, we took several approaches. 1) Single cell RNAseq analysis of B cells from malaria-exposed and HIV-infected individuals. We established a collaboration with Dr. Jun Zhu (NHLBI), an expert in RNAs as regulators of cellular differentiation, to carry out single cell RNA seq analysis of B cells from adults from malaria-endemic Mali. In a collaboration with Dr. Susan Moir (NIAID) we also compared our single cell RNAseq results to those of B cells from HIV high viremic individuals. From this analysis we made several important findings. We learned that: the atypical MBCs were composed of two different functional subpopulations defined by the isotype of their BCR; atypical and classical MBCs were derived from naive B cells; and that B cell subpopulations in malaria and HIV are remarkably similar. A manuscript describing these studies is under revision for publication in Nat Immunol. 2) The factors that drive atypical MBC differentiation. We completed a study to define the factors that drive the differentiation of human tonsil B cells to acquire the phenotype of atypical MBCs in vitro. We determined that predominantly naive B cells gave rise to T-bet + atypical MBCs, consistent with our results from single cell RNAseq. Key factors that triggered differentiation included: the form in which antigen was presented to B cells with presentation on stiff membranes that the B cell continually engaged and disengaged but could not internalize being most effective; the presence of gamma IFN; and the presence of the TLR9 agonist CpG. Provided with these stimuli naive B cells expressed a variety of genes associated with atypical MBCs including T-bet and FcRL5 and lost their ability to be triggered through the BCR by soluble antigens (Ambegaonkar et al. 2019 Front Immunol). 3) The functionality of atypical MBCs. We showed earlier that atypical MBCs were unable to respond to soluble antigen. Based on our studies on BCR signaling we were aware that the B cell response to soluble versus membrane antigens were qualitatively different although there were no examples of B cell preferences for membrane-associated versus soluble antigens. We asked: are atypical MBCs able to respond to membrane-associated antigens? We discovered that atypical MBCs are able to robustly signal in response to membrane-associated antigens and to capture and process the antigen. We determined that when engaging membrane-associated antigens B cells spread over the membrane and in the spreading process inhibitory receptors, that are highly expressed on atypical MBCs, were segregated away from the BCRs, allowing BCRs to function. These findings are in press (Ambegaonkar et al. 2020 Sci Advances). We are pursuing this observation to determine the generality of the finding in different B cell subsets. We are testing this novel hypothesis using human Tfh cells and atypical MBCs in vitro. Cerebral malaria The impact of parasite biology on the induction of cerebral malaria Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65) that does not cause ECM differs in only 21 single nucleotide polymorphisms (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. We first showed that a SNP resulting in a single amino acid change (S to F) in an ApiAP2 transcription factor in PbNK65 allowed infected mice to mount a T helper cell 1 (TH1)-type immune response that controlled subsequent infections. As compared to PbNK65S, PbNK65F parasites differentially expressed 46 genes, most of which are predicted to play roles in immune evasion. PbNK65F infections resulted in an early interferon-gamma response and a later expansion of germinal centers, resulted in high levels of infected red blood cell-specific TH1-type immunoglobulin G2b (IgG2b) and IgG2c antibodies. Thus, the Pb ApiAP2 transcription factor functions as a critical parasite virulence factor in malaria infections. A paper describing these findings was recently published (Akkaya et al. 2020 Sci Adv). We subsequently investigated the impact of this S to F SNP in ApiAP2 on the development of ECM. Using CRISPR-Cas9 engineered parasites, we showed that despite its immune modulatory role of this SNP the single amino acid change S to F in ApiAP2 is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in the ability to induce ECM. A manuscript describing this finding is under revision for publication in Sci Reports. Using the ECM mouse model to search for therapies to treat CM in African children Our goal over the last year was to better understand the similarities and differences in the pathology associated with CM in African children and ECM in mice to allow us to better evaluate DON as an adjunctive therapy in CM. To this end we established a collaboration with Dr. Terrie Taylor (Michigan State University), an expert in CM in children who heads a pediatric clinic in Malawi that treats children with CM. We described the immune infiltrates into the brains of children who died of CM using multiplex immunohistochemistry analysis of brain sections. We established for the first time that CD8+ T cells sequester along the abluminal face of blood vessels in the brains of children with CM (Riggle et al. 2020 J Clin Invest). This finding opens up a new world of possible adjunctive therapies and validates the use of DON as a CM therapy. Based on these findings, over the last year, we initiated plans to carry out a phase I/IIa clinical trial of DON as an adjunctive therapy for African children with CM. Our efforts are well underway. We have: identified collaborators and a well-equipped site in Malawi to carry out the trial; written a clinical protocol that will soon be submitted for IRB approval; obtained a sufficient quantity of cGMP DON for our studies; submitted a pre-IND to the FDA for approval of the use of DON in our protocol and applied for funding to support the clinical trial.