The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility.

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80-85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.

Reduced Birth Weight Caused by Sextuple Drug-Resistant Plasmodium falciparum Infection in Early Second Trimester.

Mutations in the Plasmodium falciparum genes Pfdhfr and Pfdhps, particularly the sextuple mutant haplotype threatens the antimalarial effectiveness of sulfadoxine-pyrimethamine (SP) as intermittent preventive treatment during pregnancy (IPTp). To explore the impact of sextuple mutant haplotype infections on outcome measures after provision of IPTp with SP, we monitored birth outcomes in women followed up from before conception or from the first trimester until delivery. Women infected with sextuple haplotypes, in the early second trimester specifically, delivered newborns with a lower birth weight compared with women who did not have malaria during pregnancy (difference, -267 g; 95% confidence interval, -454 to -59; P = .01) and women infected with less SP-resistant haplotypes (-461 g; -877 to -44; P = .03). Thus, sextuple haplotype infections seem to affect the effectiveness of SP for IPTp and directly affect birth outcome by lowering birth weight. Close monitoring and targeted malaria control during early pregnancy is therefore crucial to improving birth outcomes.

Immunization with virus-like particles conjugated to CIDRα1 domain of Plasmodium falciparum erythrocyte membrane protein 1 induces inhibitory antibodies.

BACKGROUND: During the erythrocytic cycle, Plasmodium falciparum malaria parasites express P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that anchor the infected erythrocytes (IE) to the vascular lining of the host. The CIDRα1 domain of PfEMP1 is responsible for binding host endothelial protein C receptor (EPCR), and increasing evidence support that this interaction triggers severe malaria, accounting for the majority of malaria-related deaths. In high transmission regions, children develop immunity to severe malaria after the first few infections. This immunity is believed to be mediated by antibodies targeting and inhibiting PfEMP1, causing infected erythrocytes to circulate and be cleared in the spleen. The development of immunity to malaria coincides with acquisition of broad antibody reactivity across the CIDRα1 protein family. Altogether, this identifies CIDRα1 as an important vaccine target. However, the antigenic diversity of the CIDRα1 domain family is a challenge for vaccine development. METHODS: Immune responses in mice vaccinated with Virus-Like Particles (VLP) presenting CIDRα1 antigens were investigated. Antibody reactivity was tested to a panel of recombinant CIDRα1 domains, and the antibodies ability to inhibit EPCR binding by the recombinant CIDRα1 domains was tested in Luminex-based multiplex assays. RESULTS: VLP-presented CIDRα1.4 antigens induced a rapid and strong IgG response capable of inhibiting EPCR-binding of multiple CIDRα1 domains mainly within the group A CIDRα1.4-7 subgroups. CONCLUSIONS: The study observations mirror those from previous CIDRα1 vaccine studies using other vaccine constructs and platforms. This suggests that broad CIDRα1 antibody reactivity may be achieved through vaccination with a limited number of CIDRα1 variants. In addition, this study suggest that this may be achieved through vaccination with a human compatible VLP vaccine platform.

Optimization of rVAR2-Based Isolation of Cancer Cells in Blood for Building a Robust Assay for Clinical Detection of Circulating Tumor Cells.

Early detection and monitoring of cancer progression is key to successful treatment. Therefore, much research is invested in developing technologies, enabling effective and valuable use of non-invasive liquid biopsies. This includes the detection and analysis of circulating tumor cells (CTCs) from blood samples. Recombinant malaria protein VAR2CSA (rVAR2) binds a unique chondroitin sulfate modification present on the vast majority of cancers and thereby holds promise as a near-universal tumor cell-targeting reagent to isolate CTCs from complex blood samples. This study describes a technical approach for optimizing the coupling of rVAR2 to magnetic beads and the development of a CTC isolation platform targeting a range of different cancer cell lines. We investigate both direct and indirect approaches for rVAR2-mediated bead retrieval of cancer cells and conclude that an indirect capture approach is most effective for rVAR2-based cancer cell retrieval.

Acquisition of Antibodies Against Endothelial Protein C Receptor-Binding Domains of Plasmodium falciparum Erythrocyte Membrane Protein 1 in Children with Severe Malaria.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates parasite sequestration in postcapillary venules in P. falciparum malaria. PfEMP1 types can be classified based on their cysteine-rich interdomain region (CIDR) domains. Antibodies to different PfEMP1 types develop gradually after repeated infections as children age, and antibodies to specific CIDR types may confer protection. METHODS: Levels of immunoglobulin G to 35 recombinant CIDR domains were measured by means of Luminex assay in acute-stage (baseline) and convalescent-stage plasma samples from Papua New Guinean children with severe or uncomplicated malaria and in healthy age-matched community controls. RESULTS: At baseline, antibody levels were similar across the 3 groups. After infection, children with severe malaria had higher antibody levels than those with uncomplicated malaria against the endothelial protein C receptor (EPCR) binding CIDRα1 domains, and this difference was largely confined to older children. Antibodies to EPCR-binding domains increased from presentation to follow-up in severe malaria, but not in uncomplicated malaria. CONCLUSIONS: The acquisition of antibodies against EPCR-binding CIDRα1 domains of PfEMP1 after a severe malaria episode suggest that EPCR-binding PfEMP1 may have a role in the pathogenesis of severe malaria in Papua New Guinea.

Malaria in Early Pregnancy and the Development of the Placental Vasculature.

BACKGROUND: Pregnancy malaria has a negative impact on fetal outcome. It is uncertain whether infections in early pregnancy have a clinical impact by impeding the development of the placental vasculature. METHODS: Tanzanian women (n = 138) were closely monitored during pregnancy. Placentas collected at birth were investigated using stereology to establish the characteristics of placental villi and vessels. Placental vasculature measures were compared between women infected with malaria and controls. RESULTS: Compared with controls, placentas from women infected with malaria before a gestational age (GA) of 15 weeks had a decreased volume of transport villi (mean decrease [standard deviation], 12.45 [5.39] cm3; P = .02), an increased diffusion distance in diffusion vessels (mean increase, 3.33 [1.27] µm; P = .01), and a compensatory increase in diffusion vessel surface area (mean increase, 1.81 [0.74 m2]; P = .02). In women who had malaria before a GA of 15 weeks diffusion vessel surface area and transport vessel length distance were positive predictors for birth weight (multilinear regression: P = .007 and P = .055 for diffusion surface area and transport length, respectively) and GA at delivery (P = .005 and P = .04). CONCLUSIONS: Malaria infection in early pregnancy impedes placental vascular development. The resulting phenotypic changes, which can be detected at delivery, are associated with birth weight and gestational length. CLINICAL TRIALS REGISTRATION: NCT02191683.

First-in-human, Randomized, Double-blind Clinical Trial of Differentially Adjuvanted PAMVAC, A Vaccine Candidate to Prevent Pregnancy-associated Malaria.

BACKGROUND: Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual "blood-stage" parasites in the placenta, the major virulence mechanism. METHODS: The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 µg (n = 9) or 50 µg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization. RESULTS: All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing P. falciparum-infected erythrocytes to CSA in a standardized functional assay. CONCLUSIONS: PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area. CLINICAL TRIALS REGISTRATION: EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489.

Severe malaria is associated with parasite binding to endothelial protein C receptor.

Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions.

PfSETvs methylation of histone H3K36 represses virulence genes in Plasmodium falciparum.

The variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), which is expressed on the surface of P. falciparum-infected red blood cells, is a critical virulence factor for malaria. Each parasite has 60 antigenically distinct var genes that each code for a different PfEMP1 protein. During infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune-evasion mechanism to avoid the host antibody response. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. Here we show that knocking out the P. falciparum variant-silencing SET gene (here termed PfSETvs), which encodes an orthologue of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual infected red blood cells. PfSETvs-dependent H3K36me3 is present along the entire gene body, including the transcription start site, to silence var genes. With low occupancy of PfSETvs at both the transcription start site of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long noncoding RNA. These results uncover a previously unknown role of PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologues of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1 proteins may also be applied to the development of a malaria vaccine.

Immunization with Recombinant Plasmodium falciparum Erythrocyte Membrane Protein 1 CIDRα1 Domains Induces Domain Subtype Inhibitory Antibodies.

Plasmodium falciparum malaria pathogenesis is tied to the sequestration of parasites in the microvasculature. Parasite sequestration leading to severe malaria is mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1) binding to endothelial protein C receptor (EPCR) via its CIDRα1 domains. CIDRα1 domains are targets of naturally acquired immunity, and a vaccine eliciting antibodies inhibiting the EPCR binding of CIDRα1 could potentially prevent disease and death from malaria. CIDRα1 domains have diversified in sequence to escape immune recognition but preserved structure to maintain EPCR binding. The EPCR-binding CIDRα1 domains separate into six major sequence types predicted to form a conserved structure in which only the amino acids essential for EPCR binding are highly conserved. Here, we investigated whether antibodies elicited by vaccination with single or multiple recombinant CIDRα1 domains are able to bind and inhibit diverse CIDRα1 domains. We found that EPCR binding-inhibitory antibodies to CIDRα1 variants closely related to those used for vaccination are readily elicited, whereas antibodies binding distant CIDRα1 variants are sporadically generated and are rarely inhibitory. Despite this, sequence similarity correlated poorly with the ability of induced antibodies to inhibit across diverse variants, and no continuous sequence regions of importance for cross-inhibitory antibodies could be identified. This suggested that epitopes of cross-variant inhibitory antibodies were predominantly conformational. Vaccination with immunogens engineered to focus immune responses to specific epitopes or an optimal choice of multiple CIDRα1 variants may improve elicitation of broadly reactive and inhibitory antibody responses.

Plasmodium falciparum Infection Early in Pregnancy has Profound Consequences for Fetal Growth.

Malaria during pregnancy constitutes a large health problem in areas of endemicity. The World Health Organization recommends that interventions are initiated at the first antenatal visit, and these improve pregnancy outcomes. This study evaluated fetal growth by ultrasonography and birth outcomes in women who were infected prior to the first antenatal visit (gestational age, <120 days) and not later in pregnancy. Compared with uninfected controls, women with early Plasmodium falciparum exposure had retarded intrauterine growth between gestational ages of 212 and 253 days (difference between means, 107 g [95% confidence interval {CI}, 26-188]; P = .0099) and a shorter pregnancy duration (difference between means, 6.6 days [95% CI, 1.0-112.5]; P = .0087). The birth weight (difference between means, 221 g [95% CI, 6-436]; P = .044) and the placental weight (difference between means, 84 g [95% CI, 18-150]; P = .013) at term were also reduced. The study suggests that early exposure to P. falciparum, which is not targeted for prevention by current control strategies, has a profound impact on fetal growth, pregnancy duration, and placental weight at term.

Parasites Causing Cerebral Falciparum Malaria Bind Multiple Endothelial Receptors and Express EPCR and ICAM-1-Binding PfEMP1.

Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1 (ICAM-1), and endothelial protein C receptor (EPCR); however, cytoadhesion patterns typical for pediatric malaria syndromes and the associated PfEMP1 members are still undefined. Methods: In a cohort of 94 hospitalized children with malaria, we characterized the binding properties of IE collected on admission, and var gene transcription using quantitative polymerase chain reaction. Results: IE from patients with cerebral malaria were more likely to bind EPCR and ICAM-1 than IE from children with uncomplicated malaria (P = .007). The level of transcripts encoding CIDRα1.4 and CIDRα1.5 domain subclasses was higher in patients with severe disease (P < .05). IE populations exhibiting binding to all 3 receptors had higher levels of transcripts encoding PfEMP1 with CIDRα1.4 and Duffy binding-like (DBL)-ß3 domains than parasites, which only bound CD36. Conclusions: These results underpin the significance of EPCR binding in pediatric malaria patients that require hospital admission, and support the notion that complementary receptor interactions of EPCR binding PfEMP1with ICAM-1 amplifies development of severe malaria symptoms.

The Severity of Plasmodium falciparum Infection Is Associated with Transcript Levels of var Genes Encoding Endothelial Protein C Receptor-Binding P. falciparum Erythrocyte Membrane Protein 1.

By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct P. falciparum erythrocyte membrane protein 1 (PfEMP1) subset mediating binding to endothelial receptors. Previous studies indicate that PfEMP1 adhesins with so-called CIDRα1 domains capable of binding endothelial protein C receptor (EPCR) constitute the PfEMP1 subset associated with severe pediatric malaria. To analyze the relative importance of different subtypes of CIDRα1 domains, we compared Pfemp1 transcript levels in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalized with uncomplicated malaria (n = 42), children with mild malaria not requiring hospitalization (n = 10), and children with parasitemia and no ongoing fever (n = 12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections, and the abundance of these transcripts increased with disease severity. The compositions of CIDRα1 subtype transcripts varied markedly between patients, and none of the subtypes were dominant. Transcript-level analyses targeting other domain types indicated that subtypes of DBLß or DBLζ domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. These observations strengthen the rationale for targeting the PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target EPCR binding of all CIDRα1 subtypes.

Burkitt lymphoma expresses oncofetal chondroitin sulfate without being a reservoir for placental malaria sequestration.

Burkitt lymphoma (BL) is a malignant disease, which is frequently found in areas with holoendemic Plasmodium falciparum malaria. We have previously found that the VAR2CSA protein is present on malaria-infected erythrocytes and facilitates a highly specific binding to the placenta. ofCS is absent in other non-malignant tissues and thus VAR2CSA generally facilitates parasite sequestration and accumulation in pregnant women. In this study, we show that the specific receptor for VAR2CSA, the oncofetal chondroitin sulfate (ofCS), is likewise present in BL tissue and cell lines. We therefore explored whether ofCS in BL could act as anchor site for VAR2CSA-expressing infected erythrocytes. In contrast to the placenta, we found no evidence of in vivo sequestering of infected erythrocytes in the BL tissue. Furthermore, we found VAR2CSA-specific antibody titers in children with endemic BL to be lower than in control children from the same malaria endemic region. The abundant presence of ofCS in BL tissue and the absence of ofCS in non-malignant tissue encouraged us to examine whether recombinant VAR2CSA could be used to target BL. We confirmed the binding of VAR2CSA to BL-derived cells and showed that a VAR2CSA drug conjugate efficiently killed the BL-derived cell lines in vitro. These results identify ofCS as a novel therapeutic BL target and highlight how VAR2CSA could be used as a tool for the discovery of novel approaches for directing BL therapy.

Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites.

BACKGROUND: Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. METHODS: In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. RESULTS: The cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission. CONCLUSIONS: Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised.

Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses.

BACKGROUND: Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation system (SpyTag/SpyCatcher) and the immunogenicity is tested in mice. METHODS: Full-length 3d7 CSP protein was genetically fused at the C-terminus to SpyCatcher. The CSP-SpyCatcher antigen was then covalently attached (via the SpyTag/SpyCatcher interaction) to Acinetobacter phage AP205 VLPs which were modified to display one SpyTag per VLP subunit. To evaluate the VLP-display effect, the immunogenicity of the VLP vaccine was tested in mice and compared to a control vaccine containing AP205 VLPs plus unconjugated CSP. RESULTS: Full-length CSP was conjugated at high density (an average of 112 CSP molecules per VLP) to AP205 SpyTag-VLPs. Vaccination of mice with the CSP Spy-VLP vaccine resulted in significantly increased antibody titres over a course of 7 months as compared to the control group (2.6-fold higher at 7 months after immunization). Furthermore, the CSP Spy-VLP vaccine appears to stimulate production of IgG2a antibodies, which has been linked with a more efficient clearing of intracellular parasite infection. CONCLUSION: This study demonstrates that the high-density display of CSP on SpyTag-VLPs, significantly increases the level and quality of the vaccine-induced humoral response, compared to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria.

Adhesion of Plasmodium falciparum infected erythrocytes in ex vivo perfused placental tissue: a novel model of placental malaria.

BACKGROUND: Placental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact placental tissue. RESULTS: The ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes. CONCLUSION: The ex vivo model provides a novel way of studying receptor-ligand interactions and antibody mediated inhibition of binding in placental malaria.

Differences in PfEMP1s recognized by antibodies from patients with uncomplicated or severe malaria.

BACKGROUND: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants are encoded by var genes and mediate pathogenic cytoadhesion and antigenic variation in malaria. PfEMP1s can be broadly divided into three principal groups (A, B and C) and they contain conserved arrangements of functional domains called domain cassettes. Despite their tremendous diversity there is compelling evidence that a restricted subset of PfEMP1s is expressed in severe disease. In this study antibodies from patients with severe and uncomplicated malaria were compared for differences in reactivity with a range of PfEMP1s to determine whether antibodies to particular PfEMP1 domains were associated with severe or uncomplicated malaria. METHODS: Parts of expressed var genes in a severe malaria patient were identified by RNAseq and several of these partial PfEMP1 domains were expressed together with others from laboratory isolates. Antibodies from Papuan patients to these parts of multiple PfEMP1 proteins were measured. RESULTS: Patients with uncomplicated malaria were more likely to have antibodies that recognized PfEMP1 of Group C type and recognized a broader repertoire of group A and B PfEMP1s than patients with severe malaria. CONCLUSION: These data suggest that exposure to a broad range of group A and B PfEMP1s is associated with protection from severe disease in Papua, Indonesia.

Bacterial superglue enables easy development of efficient virus-like particle based vaccines.

BACKGROUND: Virus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches for VLP-based antigen display require labor-intensive trial-and-error optimization, and often fail to generate dense antigen display. Here we utilize the split-intein (SpyTag/SpyCatcher) conjugation system to generate stable isopeptide bound antigen-VLP complexes by simply mixing of the antigen and VLP components. RESULTS: Genetic fusion of SpyTag or SpyCatcher to the N-terminus and/or C-terminus of the Acinetobacter phage AP205 capsid protein resulted in formation of stable, nonaggregated VLPs expressing one SpyCatcher, one SpyTag or two SpyTags per capsid protein. Mixing of spy-VLPs with eleven different vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22-88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity, longevity and functional efficacy compared to corresponding vaccines employing monomeric proteins. The spy-VLP vaccines also effectively broke B cell self-tolerance and induced potent and durable antibody responses upon vaccination with cancer or allergy-associated self-antigens (PD-L1, CTLA-4 and IL-5). CONCLUSIONS: The spy-VLP system constitutes a versatile and rapid method to develop highly immunogenic VLP-based vaccines. Our data provide proof-of-concept for the technology's ability to present complex vaccine antigens to the immune system and elicit robust functional antibody responses as well as to efficiently break B cell self-tolerance. The spy-VLP-system may serve as a generic tool for the cost-effective development of effective VLP-vaccines against both infectious- and non-communicable diseases and could facilitate rapid and unbiased screening of vaccine candidate antigens.

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families.

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite's sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens.