Malaria Pathogenesis in young children and vaccine discovery

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In FY20, we reported human and animal studies to investigate malaria immunology and pathogenesis. Highlighted in this years summary are results from our publications. 1. Rodrigues DSA, Prestes EB, Silva LDS, Pinheiro AAS, Francischetti I, Saraiva E, Neto HAP, Bozza MT. 'CXCR4 and MIF are required for neutrophil extracellular trap release triggered by Plasmodium-infected erythrocytes in press with PLoS Pathogens. In press. Neutrophil extracellular trap (NET) evolved as an unique effector mechanism contributing to resistance against infection, that can also promote tissue damage in inflammatory conditions. Malaria infection can trigger NET release, but the mechanisms and consequences of NET formation in this context remain poorly characterized. Here we show that Plasmodium-infected erythrocytes cause the formation of NET by neutrophils from healthy donors in a mechanism dependent on the C-X-C chemokine receptor type 4 (CXCR4), activated by macrophage migration inhibitory Factor (MIF) release by infected erythrocytes. Patients suffering from cerebral malaria had increased amounts of circulating DNA, paralleled by increased NE activity in plasma. Importantly, mice infected P. berghei ANKA, a model of cerebral malaria, also had high amounts of circulating DNA, while treatment with DNAse increased parasitemia and accelerated mortality, indicating a role for NET in resistance against Plasmodium infection. 2. Raj DK, Mohapatra AD, Jnawali A, Zuromski J, Jha A, Cham-Kpu G, Sherman B, Rudlaff RM, Nixon CE, Hilton N, Oleinikov AV, Chesnokov O, Merritt J, Pond-Tor S, Burns L, Jolly G, Mamoun CB, Kabyemala E, Muehlenbachs A, Lambert L, Orr-Gonzalez S, Gndig NF, Fidock DA, Park S, Dvorin JD, Pardi N, Weissman D, Mui BL, Tam YK, Friedman JF, Fried M, Duffy PE, Kurtis JD. Anti-PfGARP activates programmed cell death of parasites and reduces severe malaria. Nature. 2020 Jun;582(7810):104-108. doi: 10.1038/s41586-020-2220-1. Epub 2020 Apr 22. Malaria caused by Plasmodium falciparum remains the leading single-agent cause of mortality in children, yet the promise of an effective vaccine has not been fulfilled. Here, using our previously described differential screening method to analyse the proteome of blood-stage P. falciparum parasites, we identify P. falciparum glutamic-acid-rich protein (PfGARP) as a parasite antigen that is recognized by antibodies in the plasma of children who are relatively resistant-but not those who are susceptible-to malaria caused by P. falciparum. PfGARP is a parasite antigen of 80 kDa that is expressed on the exofacial surface of erythrocytes infected by early-to-late-trophozoite-stage parasites. We demonstrate that antibodies against PfGARP kill trophozoite-infected erythrocytes in culture by inducing programmed cell death in the parasites, and that vaccinating non-human primates with PfGARP partially protects against a challenge with P. falciparum. Furthermore, our longitudinal cohort studies showed that, compared to individuals who had naturally occurring anti-PfGARP antibodies, Tanzanian children without anti-PfGARP antibodies had a 2.5-fold-higher risk of severe malaria and Kenyan adolescents and adults without these antibodies had a twofold-higher parasite density. By killing trophozoite-infected erythrocytes, PfGARP could synergize with other vaccines that target parasite invasion of hepatocytes or the invasion of and egress from erythrocytes. 3. Raja AI, Brickley EB, Taaffe J, Ton T, Zhao Z, Bock KW, Orr-Gonzalez S, Thomas ML, Lambert LE, Moore IN, Duffy PE. A primate model of severe malarial anemia: a comparative pathogenesis study. Scientific Reports. 2019 Dec 12;9(1):18965. doi: 10.1038/s41598-019-55377-3. Severe malarial anaemia (SMA) is the most common life-threatening complication of Plasmodium falciparum infection in African children. SMA is characterised by haemolysis and inadequate erythropoiesis, and is associated with dysregulated inflammatory responses and reduced complement regulatory protein levels (including CD35). However, a deeper mechanistic understanding of the pathogenesis requires improved animal models. In this comparative study of two closely related macaque species, we interrogated potential causal factors for their differential and temporal relationships to onset of SMA. We found that rhesus macaques inoculated with blood-stage Plasmodium coatneyi developed SMA within 2 weeks, with no other severe outcomes, whereas infected cynomolgus macaques experienced only mild/ moderate anaemia. The abrupt drop in haematocrit in rhesus was accompanied by consumption of haptoglobin (haemolysis) and poor reticulocyte production. Rhesus developed a greater inflammatory response than cynomolgus macaques, and had lower baseline levels of CD35 on red blood cells (RBCs) leading to a significant reduction in the proportion of CD35+ RBCs during infection. Overall, severe anaemia in rhesus macaques infected with P. coatneyi has similar features to SMA in children. Our comparisons are consistent with an association of low baseline CD35 levels on RBCs and of early inflammatory responses with the pathogenesis of SMA. 4. Duffy PE. Immunity to Severe Malaria: PfEMP1 tags tell a tale. Cell Host & Microbe. 2019 Nov 13;26(5):571-573. doi: 10.1016/j.chom.2019.10.021. PfEMP1 is the major surface antigen of P. falciparum-infected erythrocytes, mediates endothelial adhesion, and displays extreme sequence diversity that underpins antigenic variation. In this issue of Cell Host & Microbe, Tessema et al. (2019) find that antibodies in sera from a pediatric cohort that bind a discrete PfEMP1 subset associate with protection from severe malaria and predict future risk. 5. Duffy PE. Structure solves the problem with malaria merozoite vaccines. Trends in Parasitology. 2019 Nov;35(11):855-857. doi: 10.1016/j.pt.2019.09.004. Malaria vaccines targeting merozoite invasion of erythrocytes have long held appeal but failed in clinical trials. Three structural studies of antibody-antigen complexes by Alanine et al., Urusova et al., and Rawlinson et al. define neutralizing and non-neutralizing epitopes in essential invasion proteins, leading to rational design of improved merozoite vaccines.