A Sequence in Subdomain 2 of DBL1a of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies

Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1a previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1a-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1a antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surfac

A Molecular Link between Malaria and Epstein–Barr Virus Reactivation

Although malaria and Epstein–Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1α (CIDR1α) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1α and EBV in the context of B cells. We show that CIDR1α binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1α-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1α stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1α can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication

Clinical development of placental malaria vaccines and immunoassays harmonization:a workshop report

International audiencePlacental malaria caused by Plasmodium falciparum infection constitutes a major health problem manifesting as severe disease and anaemia in the mother, impaired fetal development, low birth weight or spontaneous abortion. Prevention of placental malaria currently relies on two key strategies that are losing efficacy due to spread of resistance: long-lasting insecticide-treated nets and intermittent preventive treatment during pregnancy. A placental malaria vaccine would be an attractive, cost-effective complement to the existing control tools. Two placental malaria vaccine candidates are currently in Phase Ia/b clinical trials. During two workshops hosted by the European Vaccine Initiative, one in Paris in April 2014 and the other in Brussels in November 2014, the main actors in placental malaria vaccine research discussed the harmonization of clinical development plans and of the immunoassays with a goal to define standards that will allow comparative assessment of different placental malaria vaccine candidates. The recommendations of these workshops should guide researchers and clinicians in the further development of placental malaria vaccines

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation

Effect of Acute Plasmodium falciparum Malaria on Reactivation and Shedding of the Eight Human Herpes Viruses

Human herpes viruses (HHVs) are widely distributed pathogens. In immuno-competent individuals their clinical outcomes are generally benign but in immuno-compromised hosts, primary infection or extensive viral reactivation can lead to critical diseases. Plasmodium falciparum malaria profoundly affects the host immune system. In this retrospective study, we evaluated the direct effect of acute P. falciparum infection on reactivation and shedding of all known human herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-7, HHV-8). We monitored their presence by real time PCR in plasma and saliva of Ugandan children with malaria at the day of admission to the hospital (day-0) and 14 days later (after treatment), or in children with mild infections unrelated to malaria. For each child screened in this study, at least one type of HHV was detected in the saliva. HHV-7 and HHV-6 were detected in more than 70% of the samples and CMV in approximately half. HSV-1, HSV-2, VZV and HHV-8 were detected at lower frequency. During salivary shedding the highest mean viral load was observed for HSV-1 followed by EBV, HHV-7, HHV-6, CMV and HHV-8. After anti-malarial treatment the salivary HSV-1 levels were profoundly diminished or totally cleared. Similarly, four children with malaria had high levels of circulating EBV at day-0, levels that were cleared after anti-malarial treatment confirming the association between P. falciparum infection and EBV reactivation. This study shows that acute P. falciparum infection can contribute to EBV reactivation in the blood and HSV-1 reactivation in the oral cavity. Taken together our results call for further studies investigating the potential clinical implications of HHVs reactivation in children suffering from malaria

A Novel DBL-Domain of the P. falciparum 332 Molecule Possibly Involved in Erythrocyte Adhesion

Plasmodium falciparum malaria is brought about by the asexual stages of the parasite residing in human red blood cells (RBC). Contact between the erythrocyte surface and the merozoite is the first step for successful invasion and proliferation of the parasite. A number of different pathways utilised by the parasite to adhere and invade the host RBC have been characterized, but the complete biology of this process remains elusive. We here report the identification of an open reading frame (ORF) representing a hitherto unknown second exon of the Pf332 gene that encodes a cysteine-rich polypeptide with a high degree of similarity to the Duffy-binding-like (DBL) domain of the erythrocyte-binding-ligand (EBL) family. The sequence of this DBL-domain is conserved and expressed in all parasite clones/strains investigated. In addition, the expression level of Pf332 correlates with proliferation efficiency of the parasites in vitro. Antibodies raised against the DBL-domain are able to reduce the invasion efficiency of different parasite clones/strains. Analysis of the DBL-domain revealed its ability to bind to uninfected human RBC, and moreover demonstrated association with the iRBC surface. Thus, Pf332 is a molecule with a potential role to support merozoite invasion. Due to the high level of conservation in sequence, the novel DBL-domain of Pf332 is of possible importance for development of novel anti-malaria drugs and vaccines

Impact of malaria on B-cell homeostasis and Epstein-Barr virus reactivation. Endemic Burkitt's lymphoma pathogenesis

Over recent years, the concept that many diseases can be aetiologically linked to infection by more than one pathogen has gained increased attention and awareness. Plasmodium falciparum (P.f) and Epstein-Barr virus (EBV) are recognized co-factors in the genesis of endemic Burkitt s lymphoma (eBL), a high grade B cell malignancy, accounting for up to 74% of childhood cancers in equatorial Africa. In this thesis, we have investigated the interactions existing between P.f. and EBV that could lead to the emergence of eBL. A special emphasis was given to the effect of malarial antigens on B cell homeostasis and EBV reactivation. During intra-erythrocytic proliferation of P.f, parasite-derived proteins are successively expressed, exported and presented at the surface of the human red blood cell membrane. The cysteine-rich interdomain region 1 alpha (CIDR1alpha) of the P.f erythrocyte membrane protein 1 (PfEMP1) harbors a multi-adhesive phenotype able to bind to CD36, CD31 and immunoglobulins (Ig). This led us to investigate the effect of P.f infected erythrocyte (IE) and CIDR1alpha, on purified human B cell preparations. Both IE and CIDR1alpha bound to non-immune B cells and induced polyclonal activation accompanied by the production of cytokines and antibodies. This effect was partially mediated by B cell surface immunoglobulins (a constitutive part of the B cell receptor). Nevertheless, the different gene expression profiles obtained comparing the activation signature of CIDR1alpha and anti-Ig stimuli suggested that other molecules/signaling pathways were implicated in this activation process. CIDR1alpha preferentially activated the memory B cell compartment and was capable of rescuing germinal center B cells from spontaneous apoptosis, promoting cell cycle entry. Polyclonal B cell activation is a prominent feature of malaria. We have identified the CIDR1alpha domain of PfEMP1 as a T cell-independent antigen that induces polyclonal B cell activation, shedding light on a possible molecular mechanism leading to hyper-gammaglobulinemia during malaria infection. After primary infection, EBV establishes a life long persistence in the host, residing in a latent state in memory B cells. P.f infection is associated with elevated EBV loads. Different but not exclusive causative effects have been proposed: i) the impairment of the EBV-specific T cell response resulting from malaria-induced immunosuppression and ii) the expansion of the pool of EBV-carrying B cells resulting from malaria-induced polyclonal B cell activation. We and others have recently demonstrated that children living in malaria endemic areas have elevated EBV levels in the plasma indicating that the high EBV loads observed during malaria infection could also result from active viral replication. This led us to assess the impact of CIDR1alpha on EBV reactivation using the EBV positive BL cell line Akata as a model. Upon stimulation with CIDR1alpha, quantitative determination by real time PCR revealed an increased EBV load in the Akata cell cultures. The increased viral load resulted from lytic cycle reactivation as confirmed by experiments performed using an Akata line-based system in which the induction of lytic cycle was reflected by an enhanced expression of green fluorescent protein (GFP). CIDR1alpha stimulation led to an augmentation of GFP positive cells. Moreover, the virus production in CIDR1alpha-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to increased expression of the EBV lytic promoter BZLF1. CIDR1alpha also induced the production of EBV in peripheral blood mononuclear cells derived from healthy donors and from children with eBL. Our results demonstrate that P.f antigens, such as CIDR1alpha, can directly drive an EBV latently infected B cell into lytic cycle. We subsequently monitored the activity of all known herpes viruses (HHVs) infecting humans in saliva and plasma samples from children having acute malaria (day-0) and 14 days after they received anti-malaria treatment (day-14). Children with acute P.f malaria infection had elevated levels of circulating EBV, these levels being cleared after recovery. Acute malaria infection was not associated to an increased plasma load of HSV-1, CMV, HHV.6 or HHV-7, as compared to the control groups (malaria day-14 and malaria negative). However, we observed a profound reduction of HSV-1 levels in the saliva after anti-malarial treatment whereas the salivary loads of other HHVs, including EBV, were unchanged. Due to the low detection rates of HSV-2, VZV and HHV-8 in our study, we couldn t draw any significant conclusions on their activity during P.f infection. Taken together our results suggest the existence of an intimate link between malaria and EBV. The elevated EBV loads observed during malaria infection seems to result not only from an impairment of the EBV-specific T cell response and polyclonal B cell activation but also from viral reactivation directly driven by malarial antigens. In conclusion, this thesis provides unique insights on the molecular mechanisms underlying polyclonal B cell activation and EBV reactivation during Plasmodium falciparum malaria infection and on how two pathogens can co-operate in lymphoma pathogenesis

Conception de protéines bimodulaires réorientant une réponse anticorps préexistante vers des cellules pathogéniques

International audienceNo abstract availabl

VAR2CSA-Mediated Host Defense Evasion of Plasmodium falciparum Infected Erythrocytes in Placental Malaria

International audienceOver 30 million women living in P. falciparum endemic areas are at risk of developing malaria during pregnancy every year. Placental malaria is characterized by massive accumulation of infected erythrocytes in the intervillous space of the placenta, accompanied by infiltration of immune cells, particularly monocytes. The consequent local inflammation and the obstruction of the maternofetal exchanges can lead to severe clinical outcomes for both mother and child. Even if protection against the disease can gradually be acquired following successive pregnancies, the malaria parasite has developed a large panel of evasion mechanisms to escape from host defense mechanisms and manipulate the immune system to its advantage. Infected erythrocytes isolated from placentas of women suffering from placental malaria present a unique phenotype and express the pregnancy-specific variant VAR2CSA of the Plasmodium falciparum Erythrocyte Membrane Protein (PfEMP1) family at their surface. The polymorphic VAR2CSA protein is able to mediate the interaction of infected erythrocytes with a variety of host cells including placental syncytiotrophoblasts and leukocytes but also with components of the immune system such as non-specific IgM. This review summarizes the described VAR2CSA-mediated host defense evasion mechanisms employed by the parasite during placental malaria to ensure its survival and persistence