Structural basis of LAIR1 targeting by polymorphic Plasmodium RIFINs

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

Do Antibodies to Malaria Surface Antigens Play a Role in Protecting Mothers From Maternal Anemia?

Pregnancy-associated malaria (PAM) caused by Plasmodium falciparum can result in detrimental outcomes for both mother and infant, including low infant birth weight, preterm birth, maternal anemia, spontaneous abortion, and maternal and/or infant mortality. Maternal anemia is a particularly complex outcome, as the body must both maintain erythropoiesis and tolerance of the growing fetus, while directing a Th1 response against the parasite. Underlying the pathogenesis of PAM is the expression of variant surface antigens (VSAPAM) on the surface of infected red blood cells (iRBC) that mediate sequestration of the iRBC in the placenta. Naturally acquired antibodies to VSAPAM can block sequestration and activate opsonic phagocytosis, both associated with improved pregnancy outcomes. In this review, we ask whether VSAPAM antibodies can also protect mothers against malarial anemia. Studies were identified where VSAPAM antibody titres and/or function were associated with higher maternal hemoglobin levels, thus supporting additional protective mechanisms for these antibodies against PAM. Yet these associations were not widely observed, and many studies reported no association between protection from maternal anemia and VSAPAM antibodies. We discuss the epidemiological, biological and technical factors that may explain some of the variability among these studies. We appraise the current evidence of these complex interactions between PAM-specific immunity and maternal anemia, propose potential mechanisms, and discuss knowledge gaps.

Selective Expression of Variant Surface Antigens Enables Plasmodium falciparum to Evade Immune Clearance in vivo

Plasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA and the RIFIN PF3D7_1254800 provides the parasite with protection from macrophage phagocytosis and natural killer cell mediated killing. Taken together, these findings reveal new insights on the molecular and cellular mechanisms that coordinate the immune escape process the parasite utilizes in vivo. As immune evasion may be particularly important during the establishment of the blood stage infection when parasite numbers are still relatively small, identification of specific parasite variant surface antigens provides targets for developing more effective vaccines by targeting parasite immune evasion.

Plasmodium falciparum population genetic complexity influences transcriptional profile and immune recognition of highly related genotypic clusters

As transmission intensity has declined in Senegal, so has the genetic complexity of circulating Plasmodium falciparum parasites, resulting in specific genotypes emerging and persisting over years. We address whether changes in parasite genetic signatures can alter the immune repertoire to variant surface antigens, and whether such responses can influence the expansion or contraction of specific parasite genotypes in the population. We characterize parasites within genotypic clusters, defined as identical by a 24-SNP molecular barcode and a haplotype identifier for other highly polymorphic loci; we measure expression of variant surface antigens (VSA) such as PfEMP-1 by transcript expression typing and expressed var DBL1α sequencing in ex vivo and short-term adapted RNA samples; and we measure IgG responses against VSAs from short-term adapted parasites. We find that parasites within genotypic clusters are genetically identical at other highly polymorphic loci. These parasites express similar Ups var classes and largely the same dominant var DBL1α sequences ex vivo. These parasites are recognized similarly by anti-VSA antibodies after short-term adaptation to culture; however, antibody responses do not correlate with genotype frequencies over time. Both genotype-specific and multiple genotype-reactive surface IgG responses are observed in this population. Parasites with identical genomes are extremely similar in their expression and host antibody recognition of VSAs. Monitoring changes in population-level parasite genomics and transmission dynamics is critical, as fluctuations will influence the breadth of resulting host immune responses to circulating parasite genotypes. These findings suggest shared immune recognition of genetically similar parasites, which has implications for both our understanding of immunity and vaccine development strategies in malaria elimination settings.

Dissecting the Gene Expression, Localization, Membrane Topology, and Function of the Plasmodium falciparum STEVOR Protein Family

ABSTRACT During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparum stevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface. IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum, the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.

The Association of High Prevalence of Trophozoites in Peripheral Blood with Lower Antibody Response toP. falciparumInfected Erythrocytes among Asymptomatic Children in Sudan

Background. The most prominent variant surface antigens (VSAs) ofPlasmodium falciparumare the var gene-encodedPlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) family, which serves as a parasite-sequestering ligand to endothelial cells. In this study we have examined the antibody reactivity of autologous plasma from symptomatic and asymptomatic malaria infected children against the infected erythrocytes’ surface antigens using flow cytometry.Methods. Ethidium-bromide-labelled erythrocytic mature forms ofP. falciparumparasites obtained from symptomatic and asymptomatic children were sequentially incubated with autologous plasma and fluorescein isothiocyanate-conjugated (FITC) antihuman IgG. Plasma antibody reactivity was detected by flow cytometry.Results. Asymptomatic children had more prevalence of trophozoites in peripheral blood (66%) compared to symptomatic children (16%),p=0.002. The mean percentage of infected RBCs reacting with autologous sera was 89.78 among symptomatic children compared to 79.62 among asymptomatic children (p=0.09). Moreover, the mean fluorescence intensity (MFI) in the asymptomatic was significantly higher compared to symptomatic children (pvalue = 0.040).Conclusion. Variant surface antigens onPlasmodium falciparuminfected RBCs from symptomatic malaria children tend to be better recognized by IgG antibodies. This may suggest a role of some IgG antibodies in severity of malaria.

Immune responses during gestational malaria: a review of the current knowledge and future trend of research

Women pregnant with their first child are susceptible to severe P. falciparum disease from placental malaria because they lack immunity to placenta-specific cytoadherence proteins. In subsequent pregnancies, as immunity against placental parasites is acquired, there is a reduced risk of adverse effects of malaria on the mother and fetus and asymptomatic parasitaemia is common. In the case of vivax malaria, with increasing reports of severe cases in Asia and South America, the effects of infection by this species during pregnancy remain to be elucidated. This review summarized the main aspects involved in the acquisition of specific antimalarial immune responses during pregnancy with emphasis in research carried out in America and Asia, in order to offer a framework of interpretation for studies on pregnant women with malaria which are recently being produced in these regions. The authors conclude that (1) Effective humoral responses during gestational malaria are mainly directed against variant surface antigens codified by genes of the var2Csa family of P. falciparum; (2) Acquisition of immunity against these variant antigens depends on the degree and intensity of transmission, and the chance increases with age and successive pregnancies; (3) Antibody development is guided by specific cellular immune responses in cases of placental and maternal infection, and (4) The study of the significance of acquisition of specific immunity against both P. falciparum and P. vivax in America, should be performed.