Mosaic and cocktail capsid-virus-like particle vaccines for induction of antibodies against the EPCR-binding CIDRα1 domain of PfEMP1.

The sequestration of Plasmodium falciparum-infected erythrocytes to the host endothelium is central to the pathogenesis of malaria. The sequestration is mediated by the parasite´s diverse Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variants, which bind select human receptors on the endothelium. Severe malaria is associated with PfEMP1 binding human endothelial protein C receptor (EPCR) via their CIDRα1 domains. Antibodies binding and inhibiting across the sequence diverse CIDRα1 domains are likely important in acquired immunity against severe malaria. In this study, we explored if immunization with AP205 bacteriophage capsid-virus-like particles (cVLPs) presenting a mosaic of diverse CIDRα1 protein variants would stimulate broadly reactive and inhibitory antibody responses in mice. Three different mosaic cVLP vaccines each composed of five CIDRα1 protein variants with varying degrees of sequence conservation of residues at and near the EPCR binding site, were tested. All mosaic cVLP vaccines induced functional antibodies comparable to those induced by matched cocktails of cVLPs decorated with the single CIDRα1 variant. No broadly reactive responses were observed. However, the vaccines did induce some cross-reactivity and inhibition within the CIDRα1 subclasses included in the vaccines, demonstrating potential use of the cVLP vaccine platform for the design of multivalent vaccines.

Differences in phenotype between long-lived memory B cells against Plasmodium falciparum merozoite antigens and variant surface antigens.

Plasmodium falciparum infections elicit strong humoral immune responses to two main groups of antigens expressed by blood-stage parasites: merozoite antigens that are involved in the erythrocyte invasion process and variant surface antigens that mediate endothelial sequestration of infected erythrocytes. Long-lived B cells against both antigen classes can be detected in the circulation for years after exposure, but have not been directly compared. Here, we studied the phenotype of long-lived memory and atypical B cells to merozoite antigens (MSP1 and AMA1) and variant surface antigens (the CIDRα1 domain of PfEMP1) in Ugandan adults before and after local reduction of P. falciparum transmission. After a median of 1.7 years without P. falciparum infections, the percentage of antigen-specific activated B cells declined, but long-lived antigen-specific B cells were still detectable in all individuals. The majority of MSP1/AMA1-specific B cells were CD95+CD11c+ memory B cells, which are primed for rapid differentiation into antibody-secreting cells, and FcRL5-T-bet- atypical B cells. On the other hand, most CIDRα1-specific B cells were CD95-CD11c- memory B cells. CIDRα1-specific B cells were also enriched among a subset of atypical B cells that seem poised for antigen presentation. These results point to differences in how these antigens are recognized or processed by the immune system and how P. falciparum-specific B cells will respond upon re-infection.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

A novel computational pipeline for var gene expression augments the discovery of changes in the Plasmodium falciparum transcriptome during transition from in vivo to short-term in vitro culture.

The pathogenesis of severe Plasmodium falciparum malaria involves cytoadhesive microvascular sequestration of infected erythrocytes, mediated by P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 variants are encoded by the highly polymorphic family of var genes, the sequences of which are largely unknown in clinical samples. Previously, we published new approaches for var gene profiling and classification of predicted binding phenotypes in clinical P. falciparum isolates (Wichers et al., 2021), which represented a major technical advance. Building on this, we report here a novel method for var gene assembly and multidimensional quantification from RNA-sequencing that outperforms the earlier approach of Wichers et al., 2021, on both laboratory and clinical isolates across a combination of metrics. Importantly, the tool can interrogate the var transcriptome in context with the rest of the transcriptome and can be applied to enhance our understanding of the role of var genes in malaria pathogenesis. We applied this new method to investigate changes in var gene expression through early transition of parasite isolates to in vitro culture, using paired sets of ex vivo samples from our previous study, cultured for up to three generations. In parallel, changes in non-polymorphic core gene expression were investigated. Modest but unpredictable var gene switching and convergence towards var2csa were observed in culture, along with differential expression of 19% of the core transcriptome between paired ex vivo and generation 1 samples. Our results cast doubt on the validity of the common practice of using short-term cultured parasites to make inferences about in vivo phenotype and behaviour.

Endothelial protein C receptor binding induces conformational changes to severe malaria-associated group A PfEMP1.

Severe Plasmodium falciparum malaria infections are caused by microvascular sequestration of parasites binding to the human endothelial protein C receptor (EPCR) via the multi-domain P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands. Using cryogenic electron microscopy (Cryo-EM) and PfEMP1 sequence diversity analysis, we found that group A PfEMP1 CIDRα1 domains interact with the adjacent DBLα1 domain through central, conserved residues of the EPCR-binding site to adopt a compact conformation. Upon EPCR binding, the DBLα1 domain is displaced, and the EPCR-binding helix of CIDRα1 is turned, kinked, and twisted to reach a rearranged, stable EPCR-bound conformation. The unbound conformation and the required transition to the EPCR-bound conformation may represent a conformational masking mechanism of immune evasion for the PfEMP1 family.

Cryo-EM reveals the conformational epitope of human monoclonal antibody PAM1.4 broadly reacting with polymorphic malarial protein VAR2CSA.

Malaria during pregnancy is a major global health problem caused by infection with Plasmodium falciparum parasites. Severe effects arise from the accumulation of infected erythrocytes in the placenta. Here, erythrocytes infected by late blood-stage parasites adhere to placental chondroitin sulphate A (CS) via VAR2CSA-type P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. Immunity to placental malaria is acquired through exposure and mediated through antibodies to VAR2CSA. Through evolution, the VAR2CSA proteins have diversified in sequence to escape immune recognition but retained their overall macromolecular structure to maintain CS binding affinity. This structural conservation may also have allowed development of broadly reactive antibodies to VAR2CSA in immune women. Here we show the negative stain and cryo-EM structure of the only known broadly reactive human monoclonal antibody, PAM1.4, in complex with VAR2CSA. The data shows how PAM1.4's broad VAR2CSA reactivity is achieved through interactions with multiple conserved residues of different sub-domains forming conformational epitope distant from the CS binding site on the VAR2CSA core structure. Thus, while PAM1.4 may represent a class of antibodies mediating placental malaria immunity by inducing phagocytosis or NK cell-mediated cytotoxicity, it is likely that broadly CS binding-inhibitory antibodies target other epitopes at the CS binding site. Insights on both types of broadly reactive monoclonal antibodies may aid the development of a vaccine against placental malaria.

Seroprevalence of SARS-CoV-2 antibodies among children and adolescents recruited in a malariometric survey in north-eastern Tanzania July 2021.

BACKGROUND: African countries stand out globally as the region seemingly least affected by the COVID-19 pandemic, caused by the virus SARS-CoV-2. Besides a younger population and potential pre-existing immunity to a SARS-CoV-2-like virus, it has been hypothesized that co-infection or recent history of Plasmodium falciparum malaria may be protective of COVID-19 severity and mortality. The number of COVID-19 cases and deaths, however, may be vastly undercounted. Very little is known about the extent to which the Tanzanian population has been exposed to SARS-CoV-2. Here, we investigated the seroprevalence of IgG to SARS-CoV-2 spike protein in two Tanzanian rural communities 1½ years into the pandemic and the association of coinciding malaria infection and exposure. METHODS: During a malariometric survey in July 2021 in two villages in north-eastern Tanzania, blood samples were taken from 501 participants (0-19 years old). Malaria was detected by mRDT and microscopy. Levels of IgG against the spike protein of SARS-CoV-2 were measured by ELISA as well as IgG against five different antigens of P. falciparum; CIDRα1.1, CIDRα1.4 and CIDRα1.5 of PfEMP1 and GLURP and MSP3. RESULTS: The seroprevalence of SARS-CoV-2 IgG was 39.7% (106/267) in Kwamasimba and 32.5% (76/234) in Mkokola. In both villages the odds of being seropositive increased significantly with age (AOR = 1.12, 95% CI 1.07-1.17, p < 0.001). P. falciparum malaria prevalence by blood smear microscopy was 7.9% in Kwamasimba and 2.1% in Mkokola. 81.3% and 70.5% in Kwamasimba and Mkokola, respectively, showed recognition of minimum one malaria antigen. Residing in Kwamasimba was associated with a broader recognition (AOR = 1.91, 95% CI 1.34-2.71, p < 0.001). The recognition of malaria antigens increased significantly with age in both villages (AOR = 1.12; 95% CI 1.08-1.16, p < 0.001). Being SARS-CoV-2 seropositive did not associate with the breadth of malaria antigen recognition when adjusting for age (AOR = 0.99; 95% CI 0.83-1.18; p = 0.91). CONCLUSION: More than a third of the children and adolescents in two rural communities in Tanzania had antibodies to SARS-CoV-2. In particular, the adolescents were seropositive but being seropositive did not associate with the status of coinciding malaria infections or previous exposure. In Tanzania, natural immunity may have developed fast, potentially protecting a substantial part of the population from later variants.

Analysis of var Gene Transcript Patterns by Quantitative Real-Time PCR.

Quantitative real-time PCR (qPCR) is a simple and sensitive method for determining the amount of a specific target DNA sequence present in a sample. Compared to RNA-seq, reverse transcription qPCR (RT-qPCR) is fast, requires only low input material and is easy to analyze. Therefore, qPCR is widely used to analyze gene expression in P. falciparum, including analyses of the multicopy gene families encoding variant surface antigens (VSAs), whose expression is clonally variant and prone to changes over time. In the recent years, several P. falciparum genomes of culture-adapted strains have been sequenced, providing the knowledge to design variable gene family-specific qPCR primers for each P. falciparum genetic background. Here, we describe the required materials, methods and key factors to perform RT-qPCR experiments to determine VSA transcript abundances in the P. falciparum clones 3D7/NF54, IT4, HB3, and 7G8.

Mapping the Cord Blood Transcriptome of Pregnancies Affected by Early Maternal Anemia to Identify Signatures of Fetal Programming.

CONTEXT: Anemia during early pregnancy (EP) is common in developing countries and is associated with adverse health consequences for both mothers and children. Offspring of women with EP anemia often have low birth weight, which increases risk for cardiometabolic diseases, including type 2 diabetes (T2D), later in life. OBJECTIVE: We aimed to elucidate mechanisms underlying developmental programming of adult cardiometabolic disease, including epigenetic and transcriptional alterations potentially detectable in umbilical cord blood (UCB) at time of birth. METHODS: We leveraged global transcriptome- and accompanying epigenome-wide changes in 48 UCB from newborns of EP anemic Tanzanian mothers and 50 controls to identify differentially expressed genes (DEGs) in UCB exposed to maternal EP anemia. DEGs were assessed for association with neonatal anthropometry and cord insulin levels. These genes were further studied in expression data from human fetal pancreas and adult islets to understand their role in beta-cell development and/or function. RESULTS: The expression of 137 genes was altered in UCB of newborns exposed to maternal EP anemia. These putative signatures of fetal programming, which included the birth weight locus LCORL, were potentially mediated by epigenetic changes in 27 genes and associated with neonatal anthropometry. Among the DEGs were P2RX7, PIK3C2B, and NUMBL, which potentially influence beta-cell development. Insulin levels were lower in EP anemia-exposed UCB, supporting the notion of developmental programming of pancreatic beta-cell dysfunction and subsequently increased risk of T2D in offspring of mothers with EP anemia. CONCLUSIONS: Our data provide proof-of-concept on distinct transcriptional and epigenetic changes detectable in UCB from newborns exposed to maternal EP anemia.

Varia: a tool for prediction, analysis and visualisation of variable genes.

BACKGROUND: Parasites use polymorphic gene families to evade the immune system or interact with the host. Assessing the diversity and expression of such gene families in pathogens can inform on the repertoire or host interaction phenotypes of clinical relevance. However, obtaining the sequences and quantifying their expression is a challenge. In Plasmodium falciparum, the highly polymorphic var genes encode the major virulence protein, PfEMP1, which bind a range of human receptors through varying combinations of DBL and CIDR domains. Here we present a tool, Varia, to predict near full-length gene sequences and domain compositions of query genes from database genes sharing short sequence tags. Varia generates output through two complementary pipelines. Varia_VIP returns all putative gene sequences and domain compositions of the query gene from any partial sequence provided, thereby enabling experimental validation of specific genes of interest and detailed assessment of their putative domain structure. Varia_GEM accommodates rapid profiling of var gene expression in complex patient samples from DBLα expression sequence tags (EST), by computing a sample overall transcript profile stratified by PfEMP1 domain types. RESULTS: Varia_VIP was tested querying sequence tags from all DBL domain types using different search criteria. On average 92% of query tags had one or more 99% identical database hits, resulting in the full-length query gene sequence being identified (> 99% identical DNA > 80% of query gene) among the five most prominent database hits, for ~ 33% of the query genes. Optimized Varia_GEM settings allowed correct prediction of > 90% of domains placed among the four most N-terminal domains, including the DBLα domain, and > 70% of C-terminal domains. With this accuracy, N-terminal domains could be predicted for > 80% of queries, whereas prediction rates of C-terminal domains dropped with the distance from the DBLα from 70 to 40%. CONCLUSION: Prediction of var sequence and domain composition is possible from short sequence tags. Varia can be used to guide experimental validation of PfEMP1 sequences of interest and conduct high-throughput analysis of var type expression in patient samples.

Sickle-trait hemoglobin reduces adhesion to both CD36 and EPCR by Plasmodium falciparum-infected erythrocytes.

Sickle-trait hemoglobin protects against severe Plasmodium falciparum malaria. Severe malaria is governed in part by the expression of the Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) that are encoded by var genes, specifically those variants that bind Endothelial Protein C Receptor (EPCR). In this study, we investigate the effect of sickle-trait on parasite var gene expression and function in vitro and in field-collected parasites. We mapped var gene reads generated from RNA sequencing in parasite cultures in normal and sickle-cell trait blood throughout the asexual lifecycle. We investigated sickle-trait effect on PfEMP1 interactions with host receptors CD36 and EPCR using static adhesion assays and flow cytometry. Var expression in vivo was compared by assembling var domains sequenced from total RNA in parasites infecting Malian children with HbAA and HbAS. Sickle-trait did not alter the abundance or type of var gene transcripts in vitro, nor the abundance of overall transcripts or of var functional domains in vivo. In adhesion assays using recombinant host receptors, sickle-trait reduced adhesion by 73-86% to CD36 and 83% to EPCR. Similarly, sickle-trait reduced the surface expression of EPCR-binding PfEMP1. In conclusion, Sickle-cell trait does not directly affect var gene transcription but does reduce the surface expression and function of PfEMP1. This provides a direct mechanism for protection against severe malaria conferred by sickle-trait hemoglobin. Trial Registration: ClinicalTrials.gov Identifier: NCT02645604.

Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant.

Cryo-EM reveals the architecture of placental malaria VAR2CSA and provides molecular insight into chondroitin sulfate binding.

Placental malaria can have severe consequences for both mother and child and effective vaccines are lacking. Parasite-infected red blood cells sequester in the placenta through interaction between parasite-expressed protein VAR2CSA and the glycosaminoglycan chondroitin sulfate A (CS) abundantly present in the intervillous space. Here, we report cryo-EM structures of the VAR2CSA ectodomain at up to 3.1 Å resolution revealing an overall V-shaped architecture and a complex domain organization. Notably, the surface displays a single significantly electropositive patch, compatible with binding of negatively charged CS. Using molecular docking and molecular dynamics simulations as well as comparative hydroxyl radical protein foot-printing of VAR2CSA in complex with placental CS, we identify the CS-binding groove, intersecting with the positively charged patch of the central VAR2CSA structure. We identify distinctive conserved structural features upholding the macro-molecular domain complex and CS binding capacity of VAR2CSA as well as divergent elements possibly allowing immune escape at or near the CS binding site. These observations will support rational design of second-generation placental malaria vaccines.

Common virulence gene expression in adult first-time infected malaria patients and severe cases.

Sequestration of Plasmodium falciparum(P. falciparum)-infected erythrocytes to host endothelium through the parasite-derived P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion proteins is central to the development of malaria pathogenesis. PfEMP1 proteins have diversified and expanded to encompass many sequence variants, conferring each parasite a similar array of human endothelial receptor-binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum-infected adult travellers returning to Germany. Patients were categorized into either malaria naive (n = 15) or pre-exposed (n = 17), and into severe (n = 8) or non-severe (n = 24) cases. For differential expression analysis, PfEMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var-expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with PfEMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding PfEMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic PfEMP1 variants are more common in patients with a naive immune status, and/or adverse inflammatory host responses to first infections favor the growth of EPCR-binding parasites.

Structure-Guided Design of a Synthetic Mimic of an Endothelial Protein C Receptor-Binding PfEMP1 Protein.

Structure-guided vaccine design provides a route to elicit a focused immune response against the most functionally important regions of a pathogen surface. This can be achieved by identifying epitopes for neutralizing antibodies through structural methods and recapitulating these epitopes by grafting their core structural features onto smaller scaffolds. In this study, we conducted a modified version of this protocol. We focused on the PfEMP1 protein family found on the surfaces of erythrocytes infected with Plasmodium falciparum A subset of PfEMP1 proteins bind to endothelial protein C receptor (EPCR), and their expression correlates with development of the symptoms of severe malaria. Structural studies revealed that PfEMP1 molecules present a helix-kinked-helix motif that forms the core of the EPCR-binding site. Using Rosetta-based design, we successfully grafted this motif onto a three-helical bundle scaffold. We show that this synthetic binder interacts with EPCR with nanomolar affinity and adopts the expected structure. We also assessed its ability to bind to antibodies found in immunized animals and in humans from malaria-endemic regions. Finally, we tested the capacity of the synthetic binder to effectively elicit antibodies that prevent EPCR binding and analyzed the degree of cross-reactivity of these antibodies across a diverse repertoire of EPCR-binding PfEMP1 proteins. Despite our synthetic binder adopting the correct structure, we find that it is not as effective as the CIDRα domain on which it is based for inducing adhesion-inhibitory antibodies. This cautions against the rational design of focused immunogens that contain the core features of a ligand-binding site of a protein family, rather than those of a neutralizing antibody epitope.IMPORTANCE Vaccines train our immune systems to generate antibodies which recognize pathogens. Some of these antibodies are highly protective, preventing infection, while others are ineffective. Structure-guided rational approaches allow design of synthetic molecules which contain only the regions of a pathogen required to induce production of protective antibodies. On the surfaces of red blood cells infected by the malaria parasite Plasmodium falciparum are parasite molecules called PfEMP1 proteins. PfEMP1 proteins, which bind to human receptor EPCR, are linked to development of severe malaria. We have designed a synthetic protein on which we grafted the EPCR-binding surface of a PfEMP1 protein. We use this molecule to show which fraction of protective antibodies recognize the EPCR-binding surface and test its effectiveness as a vaccine immunogen.

Increased circulation time of Plasmodium falciparum underlies persistent asymptomatic infection in the dry season.

The dry season is a major challenge for Plasmodium falciparum parasites in many malaria endemic regions, where water availability limits mosquito vectors to only part of the year. How P. falciparum bridges two transmission seasons months apart, without being cleared by the human host or compromising host survival, is poorly understood. Here we show that low levels of P. falciparum parasites persist in the blood of asymptomatic Malian individuals during the 5- to 6-month dry season, rarely causing symptoms and minimally affecting the host immune response. Parasites isolated during the dry season are transcriptionally distinct from those of individuals with febrile malaria in the transmission season, coinciding with longer circulation within each replicative cycle of parasitized erythrocytes without adhering to the vascular endothelium. Low parasite levels during the dry season are not due to impaired replication but rather to increased splenic clearance of longer-circulating infected erythrocytes, which likely maintain parasitemias below clinical and immunological radar. We propose that P. falciparum virulence in areas of seasonal malaria transmission is regulated so that the parasite decreases its endothelial binding capacity, allowing increased splenic clearance and enabling several months of subclinical parasite persistence.

Longitudinal analysis of naturally acquired PfEMP1 CIDR domain variant antibodies identifies associations with malaria protection.

BACKGROUNDMalaria pathogenicity is determined, in part, by the adherence of Plasmodium falciparum-infected erythrocytes to the microvasculature mediated via specific interactions between P. falciparum erythrocyte membrane protein (PfEMP1) variant domains and host endothelial receptors. Naturally acquired antibodies against specific PfEMP1 variants can play an important role in clinical protection against malaria.METHODSWe evaluated IgG responses against a repertoire of PfEMP1 CIDR domain variants to determine the rate and order of variant-specific antibody acquisition and their association with protection against febrile malaria in a prospective cohort study conducted in an area of intense, seasonal malaria transmission.RESULTSUsing longitudinal data, we found that IgG antibodies against the pathogenic domain variants CIDRα1.7 and CIDRα1.8 were acquired the earliest. Furthermore, IgG antibodies against CIDRγ3 were associated with reduced prospective risk of febrile malaria and recurrent malaria episodes.CONCLUSIONThis study provides evidence that acquisition of IgG antibodies against PfEMP1 variants is ordered and demonstrates that antibodies against CIDRα1 domains are acquired the earliest in children residing in an area of intense, seasonal malaria transmission. Future studies will need to validate these findings in other transmission settings and determine the functional activity of these naturally acquired CIDR variant-specific antibodies.TRIAL REGISTRATIONClinicalTrials.gov NCT01322581.FUNDINGDivision of Intramural Research, National Institute of Allergy and Infectious Diseases, NIH.

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.

Controlled human malaria infection with Plasmodium falciparum demonstrates impact of naturally acquired immunity on virulence gene expression.

The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen PfEMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against PfEMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized PfEMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P. falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding PfEMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P. falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express PfEMP1 variants unrecognized by the host's immune system, thus promoting overall parasite survival in the face of host immunity.