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Summary The PfEMP1 family of surface proteins is central for Plasmodium falciparum virulence and must retain the ability to bind to host receptors while also diversifying to aid immune evasion. The interaction between CIDRα1 domains of PfEMP1 and endothelial protein C receptor (EPCR) is associated with severe childhood malaria. We combine crystal structures of CIDRα1:EPCR complexes with analysis of 885 CIDRα1 sequences, showing that the EPCR-binding surfaces of CIDRα1 domains are conserved in shape and bonding potential, despite dramatic sequence diversity. Additionally, these domains mimic features of the natural EPCR ligand and can block this ligand interaction. Using peptides corresponding to the EPCR-binding region, antibodies can be purified from individuals in malaria-endemic regions that block EPCR binding of diverse CIDRα1 variants. This highlights the extent to which such a surface protein family can diversify while maintaining ligand-binding capacity and identifies features that should be mimicked in immunogens to prevent EPCR binding., Graphical Abstract, Highlights • EPCR binding is retained by PfEMP1 CIDRα1 domains despite huge sequence variation • Diverse CIDRα1 domains retain structural and chemical features to bind to EPCR • CIDRα1 domains mimic features of a natural ligand of EPCR and block its binding • Patient sera contain neutralizing antibodies that prevent parasite binding to EPCR, PfEMP1 proteins of Plasmodium must retain binding to host receptor EPCR while diversifying for immune evasion. Using structural studies, Lau et al. show that EPCR-binding surfaces of PfEMP1 are conserved in shape and bonding potential, despite sequence diversity, and identify features that should be mimicked in immunogens preventing EPCR binding.

Previous microsatellite analyses of sympatric populations of Plasmodium vivax and Plasmodium falciparum in Brazil revealed higher diversity in the former species. However, it remains unclear whether regional species-specific differences in prevalence and transmission levels might account for these findings. Here, we examine sympatric populations of P. vivax ( n = 87) and P. falciparum ( n = 164) parasites from Pursat province, Western Cambodia, where both species are similarly prevalent. Using 10 genome-wide microsatellites for P. falciparum and 13 for P. vivax , we found that the P. vivax population was more diverse than the sympatric P. falciparum population (average virtual heterozygosity [ H E ], 0.87 vs. 0.66, P = 0.003), with more multiple-clone infections (89.6% vs. 47.6%) and larger mean number of alleles per marker (16.2 vs. 11.1, P = 0.07). Both populations showed significant multi-locus linkage disequilibrium suggestive of a predominantly clonal mode of parasite reproduction. The higher microsatellite diversity found in P. vivax isolates, compared to sympatric P. falciparum isolates, does not necessarily result from local differences in transmission level and may reflect differences in population history between species or increased mutation rates in P. vivax .

Immunity to sand fly saliva in rodents induces a T(H)1 delayed-type hypersensitivity (DTH) response conferring protection against leishmaniasis. The relevance of DTH to sand fly bites in humans living in a leishmaniasis-endemic area remains unknown. Here, we describe the duration and nature of DTH to sand fly saliva in humans from an endemic area of Mali. DTH was assessed at 24, 48, 72, and 96 hours post bite in volunteers exposed to colony-bred sand flies. Dermal biopsies were obtained 48 hours post bite; cytokines were quantified from peripheral blood mononuclear cells (PBMCs) stimulated with sand fly saliva in vitro. A DTH response to bites was observed in 75% of individuals aged 1-15 years, decreasing gradually to 48% by age 45, and dropping to 21% thereafter. Dermal biopsies were dominated by T lymphocytes and macrophages. Abundant expression of IFN-γ and absence of T(H)2 cytokines establishes the T(H)1 nature of this DTH response. PBMCs from 98% of individuals responded to sand fly saliva. Of these, 23% were polarized to a T(H)1 and 25% to a T(H)2 response. We demonstrate the durability and T(H)1 nature of DTH to sand fly bites in humans living in a cutaneous leishmaniasis-endemic area. A systemic T(H)2 response may explain why some individuals remain susceptible to disease.

Hemoglobin (Hb) variants are associated with reduced risk of life-threatening Plasmodium falciparum malaria syndromes, including cerebral malaria and severe malarial anemia. Despite decades of research, the mechanisms by which common Hb variants – sickle HbS, HbC, α-thalassemia, fetal HbF – protect African children against severe and fatal malaria have not been fully elucidated. In vitro experimental and epidemiological data have long suggested that Hb variants do not confer malaria protection by restricting the growth of parasites in red blood cells (RBCs). Recently, four Hb variants were found to impair cytoadherence, the binding of P. falciparum-infected RBCs (PfRBCs) to microvascular endothelial cells (MVECs), a centrally important event in both parasite survival and malaria pathogenesis in humans. Impaired cytoadherence is associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite’s major cytoadherence ligand and virulence factor, on the surface of host RBCs. We propose a model in which Hb variants allow parasites to display relatively low levels of PfEMP1, sufficient for sequestering PfRBCs in microvessels and avoiding their clearance from the bloodstream by the spleen. By preventing the display of high levels of PfEMP1, Hb variants may weaken the binding of PfRBCs to MVECs, compromising their ability to activate endothelium and initiate the downstream microvascular events that drive the pathogenesis of malaria.

With the paradigm shift from the reduction of morbidity and mortality to the interruption of transmission, the focus of malaria control broadens from symptomatic infections in children ≤ 5 years of age to include asymptomatic infections in older children and adults. In addition, as control efforts intensify and the number of interventions increases, there will be decreases in prevalence, incidence and transmission with additional decreases in morbidity and mortality. Expected secondary consequences of these changes include upward shifts in the peak ages for infection (parasitemia) and disease, increases in the ages for acquisition of antiparasite humoral and cellular immune responses and increases in false-negative blood smears and rapid diagnostic tests. Strategies to monitor these changes must include: 1] studies of the entire population (that are not restricted to children ≤ 5 or ≤ 10 years of age), 2] study sites in both cities and rural areas (because of increasing urbanization across sub-Saharan Africa) and 3] innovative strategies for surveillance as the prevalence of infection decreases and the frequency of false-negative smears and rapid diagnostic tests increases.

There is a well-established clinical association between hemoglobin genotype and innate protection against Plasmodium falciparum malaria. In contrast to normal hemoglobin A, mutant hemoglobin C is associated with substantial reductions in the risk of severe malaria in both heterozygous AC and homozygous CC individuals. Irrespective of hemoglobin genotype, parasites may induce knob-like projections on the erythrocyte surface. The knobs play a major role in the pathogenesis of severe malaria by serving as points of adherence for P. falciparum-infected erythrocytes to microvascular endothelia. To evaluate the influence of hemoglobin genotype on knob formation, we used a combination of atomic force and light microscopy for concomitant topographic and wide-field fluorescence imaging. Parasitized AA, AC, and CC erythrocytes showed a population of knobs with a mean width of approximately 70 nm. Parasitized AC and CC erythrocytes showed a second population of large knobs with a mean width of approximately 120 nm. Furthermore, spatial knob distribution analyses demonstrated that knobs on AC and CC erythrocytes were more aggregated than on AA erythrocytes. These data support a model in which large knobs and their aggregates are promoted by hemoglobin C, reducing the adherence of parasitized erythrocytes in the microvasculature and ameliorating the severity of a malaria infection.

Non-tuberculous mycobacteria are becoming increasingly important pathogens among transplant recipients. We report a case of disseminated Mycobacterium haemophilum infection in a heart transplant recipient, manifesting as cellulitis, subcutaneous nodules, septic arthritis, and pneumonitis. Our case illustrates diverse challenges in the identification and treatment of this pathogen, such as its unique culture requirements and variable antimicrobial susceptibilities. Heightened clinical suspicion is necessary to establish a timely diagnosis so that optimal treatment can be administered.

Non-tuberculous mycobacteria (NTM) have emerged as important pathogens in organ transplant recipients. Because NTM pulmonary infections vary in their clinical and radiographic presentations, heightened clinical suspicion is necessary for accurate diagnosis. We report a case of Mycobacterium abscessus empyema in a lung transplant recipient. Repeated attempts at identifying the organism from a variety of clinical specimens led to the correct diagnosis and treatment.

Merozoite invasion of erythrocytes is a crucial step for the asexual cycle of Plasmodium falciparum. Multiple invasion pathways, which involve different ligand-receptor interactions, have been identified in P. falciparum by examining the entry of purified parasite into erythrocytes with different surface receptors, either mutant or under different enzyme treatments. The most critical step for a successful invasion assay is the isolation of erythrocytes infected with viable schizonts. Here, we applied a magnetic column to purify the schizonts for the erythrocyte invasion assay. Comparing to Percoll-sorbitol purification method, this modified approach showed great improvement on reproducibility and reliability of invasion assay, particularly for short-term, culture-adapted parasite isolates. The magnetic purification method is an excellent alternative for parasite isolates that do not tolerate or with unknown sensitivity to Percoll-sorbitol exposure.

In 2012, an estimated 207 million malaria cases and 627,000 malaria deaths were reported worldwide (World Health Organization, 2013). For millennia, Plasmodium falciparum malaria has exerted significant evolutionary pressure on the human genome, selecting for balanced polymorphisms that confer disease protection. This possibility was first proposed in 1949 by J. B. S. Haldane, who suggested that reduced fitness in β-thalassemia homozygotes due to severe anemia was offset by increased fitness in heterozygotes due to P. falciparum malaria protection (Haldane, 1949). In 1954, A. S. Allison demonstrated in Kenya that hemoglobin S (HbS) heterozygosity (HbAS, sickle-cell trait) conferred protection from malaria, and proposed that this protection balanced the severe anemia morbidity and premature fatality associated with HbS homozygosity (HbSS, sickle-cell disease) (Allison, 1954). Clinical studies have since associated HbC and α+thalassemia with malaria protection (Williams, 2006, Taylor et al., 2012), and shown that α+thalassemia antagonizes HbAS; that is, HbAS children who co-inherit α+thalassemia are not protected from malaria (Williams et al., 2005). While the mechanisms by which hemoglobinopathies confer malaria protection are debated (Taylor et al., 2013), with the notable exception of HbAS, they do not seem to protect by suppressing parasite densities. Epidemiological observations, together with in-vitro data, support alternative mechanisms of protection that enable individuals to “tolerate” parasitemia without developing symptoms. In one mechanism, HbS and α+thalassemia each impairs binding of parasitized red blood cells (pRBCs) to microvascular endothelial cells (MVECs, cytoadherence) and uninfected RBCs (rosetting), two virulence traits mediated by interactions between P. falciparum erythrocyte membrane protein 1 (PfEMP1) ligands on pRBCs and various receptors (CD36, ICAM1, endothelial protein C receptor [EPCR]) on host cells. We have always believed that future experimental data consistent with this mechanism are more likely to be biologically and clinically relevant if they reconcile with robust epidemiological findings. In this issue of EBioMedicine, Opi et al. (2014) meet exactly this type of criterion in proposing a mechanism to explain their original 2005 report of negative epistasis between α+thalassemia and HbAS in Kenya (Williams et al., 2005). Specifically, they hypothesized that if α+thalassemia antagonizes malaria-protective effects of HbAS in the field, then it should also antagonize cytoadherence-weakening effects of HbAS in the laboratory. Using RBCs from individuals with HbAS, α+thalassemia, or both, they performed experiments that measure cytoadherence, rosetting, and PfEMP1 expression of four P. falciparum lines. While their reductionist approach of using few parasite strains is a minor limitation of this study, they made up for this by using a large panel of RBCs from hemoglobinopathic donors (10–20 of each genotype) (Opi et al., 2014). Their findings largely confirm earlier reports that HbAS and α+thalassemia are each associated with impaired cytoadherence, rosetting, and PfEMP1 expression (Cholera et al., 2008, Krause et al., 2012), with two exceptions. For one parasite strain, α+thalassemia increased PfEMP1 expression without affecting rosetting; for another, α+thalassemia increased PfEMP1 expression but paradoxically decreased cytoadherence. These findings contrast with those of Krause et al. (2012), who generally found that α+thalassemia reduced both PfEMP1 expression and cytoadherence (α+thalassemia increased the cytoadherence of some parasite isolates) (see Fig. 1A–B of reference Krause et al., 2012). These apparent discrepancies may result from marked differences in the authors' use of (i) antibodies specific for PfEMP1 variants expressed by parasite lines ITvar9 and TM284var1 (vs. FVO, A4tres, and FCR3CSA); (ii) four laboratory-adapted parasite lines (vs. multiple naturally-circulating parasite isolates from Malian children with malaria); and (iii) purified CD36 and ICAM1 proteins as binding substrates (vs. CD36-expressing MVECs and monocytes). As Opi et al. (2014) emphasize, future studies should test large numbers of both RBC donors and P. falciparum strains to help resolve these differences, which will hopefully provide more consistent experimental data that adequately explain well-established field observations in Africa (reviewed in Taylor et al., 2012). These differences notwithstanding, the major new finding of this study is that α+thalassemia consistently reverses HbAS-mediated reductions in cytoadherence, rosetting, and PfEMP1 expression in vitro. This solid result now provides impetus for exploring how α+thalassemia antagonizes the effects of HbAS on PfEMP1-mediated phenomena. One possibility is that α+thalassemia selectively reduces the amount of HbS in HbAS RBCs, for example, by reducing the number of α-globins that bind βs globins to form stable hemoglobin α2β2 tetramers. Opi et al. (2014) provide preliminary evidence to support this, showing that the proportion of intracellular HbS significantly decreases as the degree of α+thalassemia increases. How well these HbS proportions correlate with cytoadherence, rosetting, PfEMP1 expression, and “knob” densities (Cholera et al., 2008) remains to be determined. Nevertheless, this study further highlights in-vitro binding assays as valuable tools in defining how hemoglobinopathies confer malaria protection. Additional experiments using naturally-circulating isolates and hemoglobinopathic RBCs from areas where α+thalassemia is common are needed to confirm this study's findings, to investigate the lack of epistasis between α+thalassemia and HbC in clinical studies (reviewed in Taylor et al., 2012), and the effects of α+thalassemia and HbAS on pRBC binding to EPCR — a phenotype strongly associated with cerebral malaria syndromes that are rarely seen in HbAS children.

Clinical immunity to Plasmodium falciparum malaria takes years to develop and is never complete. One explanation for these observations is that antigenic variation enables malaria parasites to evade humoral immunity; another is that P. falciparum induces immune dysregulation, which inhibits the development of protective cellular immunity. Research described by D'Ombrain et al. in this Cell Host & Microbe issue probes how the parasite's main virulence factor Pf EMP-1 might significantly alter human innate immune responses.

Introduction Despite the marked reduction in reported cases of P. falciparum malaria, this disease remains a major global health threat claiming the lives of 655,000 people and putting an estimated 3.3 billion of the world’s population at risk of illness. Although people living in malaria-endemic areas mount a broad range of immune responses to a great variety of parasite antigens, protective immunity to clinical malaria develops only slowly, and some argue the duration of such responses is comparatively short-lived. The concept of original antigenic sin, whereby an initial infection imprints a certain response pattern that prevents the immune system from mounting more efficient responses upon subsequent infections, may explain these observations. Methods In a longitudinal cohort study of Malian children aged 0.5 to 10 years, we investigate whether a child experiencing repeated malaria episodes mounts similar immunological responses each time. We also investigated whether these responses can be grouped into distinct patterns, and whether any distinct patterns associate with disease outcome. Results We are currently characterizing the quality and magnitude of parasite antigen-specific T cell responses after in vitro stimulation of peripheral blood mononuclear cells collected during an acute malaria episode using multi-parameter flow cytometry. Based on cell surface phenotype we classify memory CD4 + and CD8 + T cells into effector memory (CD45RO + CCR7 − ) and central memory (CD45RO + CCR7 + ) T cells and characterize their function by measuring the production of IFN ϒ , TNF α , IL-2 and IL-10. We also quantify cytokine levels in the corresponding supernatants and plasma samples using multi-parameter Bioplex assays. Conclusion The results of this study will enable us to determine whether original antigenic sin influences malaria incidence, and may reveal an association between a specific immunological response pattern and enhanced protection from a subsequent malaria episode, which can be further explored in identifying candidate vaccine antigens.