Biology subjects

Popovici, J.

Publications and source records attributed to Popovici, J..

11 recordsLinked to original sources

bioRxiv2026

Functional analysis of novel microneme proteins from Plasmodium vivax blood stages identifies vaccine candidates

Host cell invasion by malaria parasites requires specific molecular interactions with host receptors. Plasmodium vivax merozoite invasion of reticulocytes is mediated by P. vivax Duffy binding protein (PvDBP) and its homolog, P. vivax erythrocyte binding protein (PvEBP). Here, we identify and characterize two novel P. vivax merozoite proteins, PvMP45 and PvMP36, which co- localize with PvDBP and PvEBP in the micronemes and bind reticulocyte receptors. PvMP45 and PvMP36 share high sequence identity with their P. knowlesi homologs, PkMP45 and PkMP36, which form a complex with other invasion related proteins. Field studies reveal that naturally acquired antibodies against PvMP36, PvEBP and PvDBP are associated with protection against clinical P. vivax malaria. We demonstrate that naturally acquired antibodies to PvEBP bind Fcy receptors and likely mediate protection by enabling opsonic phagocytosis. In addition, we show that combining antibodies against PvDBP and PvMP36 results in an additive invasion inhibitory effect against P. vivax blood stages. These results suggest that combining PvDBP, PvEBP and PvMP36 in a multivalent blood stage vaccine could elicit diverse immune mechanisms against P. vivax to achieve high efficacy. ImportanceAll the clinical symptoms of malaria are attributed to the blood stage of malaria parasites during which merozoites invade and multiply within red blood cells. A clear understanding of the host- parasite interactions that enable invasion can open paths for development of novel methods to block parasite growth and prevent malaria. Here, we identify and characterize two novel invasion related proteins from P. vivax merozoites that form an invasion complex and bind host RBC receptors. We demonstrate that antibodies targeting these proteins can block RBC invasion by P. vivax and naturally acquired antibodies that develop following P. vivax infection against one of these proteins are associated with protection against P. vivax malaria. These studies not only expand our understanding of the molecular mechanisms that enable host cell invasion by P. vivax but open new avenues for development of vaccines to protect against P. vivax malaria.

microbiology↗

bioRxiv2026

Stage-aware transcriptomics reveals selective haplotype persistence in short-term ex vivo cultured Plasmodium vivax

Plasmodium vivax (Pv) infections are developmentally asynchronous and often polyclonal, complicating interpretation of bulk parasite transcriptomes. Here, we analyzed paired in vivo and short-term ex vivo transcriptomes from Ethiopian clinical isolates using stage deconvolution and PvMSP1 haplotyping. Ex vivo maturation modestly increased inferred schizont representation while largely preserving the proportion of trophozoites and gametocytes. After adjustment for parasite stage composition, in vivo and ex vivo transcriptomes remained globally similar, with no genes significantly differentially expressed, indicating the absence of major culture-induced transcriptional response. In contrast, short-term culture reduced multiplicity of infection, contracted within-host haplotype diversity, and non-randomly depleted specific haplotypes, consistent with a clonal bottleneck. In a subset of low-complexity infections, residual expression patterns were clustered by dominant haplotype, suggesting genotype-associated transcriptional heterogeneity independent of developmental stage. Together, these findings indicate that short-term ex vivo culture enriches late asexual stages and selectively filters clones rather than inducing a common transcriptional program. These results shows that ex vivo cultures are reliable way to study gene expression, especially for late stages. However, these needs explicitly model developmental composition and infection complexity when interpreting Pv transcriptomes from natural infections Author summaryMalaria caused by Plasmodium vivax is difficult to study because this parasite cannot yet be grown continuously in the laboratory and infections in patients often contain parasites at different developmental stages and multiple parasite lineages at the same time. In this study, we wanted to understand how much of the parasite gene-expression signal reflects true biological differences, and how much is explained by parasite development or changes that occur during short-term laboratory maturation. We compared parasites collected directly from patients in Ethiopia with matched parasite matured briefly outside the body. We found that short-term culture mainly increased the proportion of later-stage parasites, but after accounting for developmental stage, the overall gene-expression patterns remained very similar. However, culture reduced the diversity of parasite lineages within infections, suggesting that some parasite lineages survive better than others under laboratory conditions. Our findings highlight that natural Pv infections are complex mixtures of parasite stages and lineages. Accounting for this complexity will improve how researchers interpret parasite gene-expression studies and design future studies of parasite invasion, transmission, and survival.

cell biology↗

bioRxiv2026

MaRNAV-1 infection of Plasmodium vivax is associated with increased parasite transmission and host inflammatory responses

MaRNAV-1 is an RNA virus recently identified in Plasmodium vivax-infected samples, but definitive evidence that it infects the parasite and influences malaria pathogenesis remains unknown. Here, we demonstrate that MaRNAV-1 is an intracellular virus that is present in P. vivax at various stages of its life cycle, including blood, sporozoite, and liver stages. Viral prevalence varied geographically between Cambodian and Ethiopian parasites. MaRNAV-1 presence and load were positively associated with parasite transmission potential, as reflected by increased gametocyte abundance and higher oocyst prevalence and intensity in membrane feeding assays. MaRNAV-1 loads were higher in symptomatic compared to asymptomatic infections, and higher MaRNAV-1 loads were associated with elevated body temperature, independently of parasitemia. MaRNAV-1 infection elicits an antibody response and is associated with dendritic cell activation, a shift from Th2 to a Th1-driven immune response, and an increased frequency of double-negative B cells. Accordingly, MaRNAV-1-infected patients had higher concentrations of circulating cytokines, such as IFN-{gamma}, CXCL10, IL-1RA, and IL-6, independently of parasitemia. Together, these findings demonstrate that MaRNAV-1 is a genuine parasite-infecting virus associated with increased parasite transmission potential and with modulation of clinical outcomes in, and host immune response to, P. vivax infections. Our study broadens the conventional view of host-pathogen interactions in malaria by revealing complex virus-parasite-host relationships.

microbiology↗

bioRxiv2026

Comprehensive characterization of Plasmodium vivax antigens using a high-density peptide array

Plasmodium vivax is the second most prevalent Plasmodium species, with 2.5 billion people at risk of infection worldwide and around 10 million cases of clinical vivax malaria every year. Despite the clinical importance of this pathogen, very little is known about the P. vivax proteins recognized by the host immune system, which hinders our ability to select vaccine candidates or develop efficient serological markers. To comprehensively characterize immunogenic P. vivax proteins, we designed a high-density peptide array containing 4.2 million peptides covering the entire protein sequence of all P. vivax genes and analyzed antibody responses of infected and malaria-naive individuals. We identified a total of 283 proteins that are commonly immunogenic in symptomatic individuals. These proteins included most proteins known to be involved in erythrocyte invasion, a putative new invasion protein, several nucleoporins, and many uncharacterized proteins that should be further investigated for their roles during blood-stage infections. These analyses also revealed a unique pattern of antibody response against PIR proteins in asymptomatic individuals, that could be associated with protection against clinical vivax malaria. Overall, these data provide an agnostic and comprehensive perspective on immunogenic P. vivax proteins and constitute an important resource for the malaria community to develop new tools for better detecting and eliminating this important human pathogen.

microbiology↗

bioRxiv2025

Rapid detection of G6PD deficiency SNPs using a novel amplicon-based MinION Sequencing Assay

Plasmodium vivax malaria remains a significant global health challenge, complicated by the parasites ability to form dormant liver stages (hypnozoites) that cause relapses. Radical cure of P. vivax malaria requires administration of a hypnozoitocidal drug, such as primaquine or tafenoquine. However, these drugs can cause severe haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is caused by more than 230 different variants at the gene level that confer different degrees of deficiency phenotypically. Understanding the distribution of different G6PD variants in affected populations is essential to inform safer antimalarial treatment strategies. This study aimed to develop a cost-effective sequencing assay targeting key regions of the G6PD gene, suitable for field deployment. A novel assay based on Nanopore technology was designed to amplify two amplicons covering exon 3 to exon 13, focusing on known variants associated with enzyme deficiency. A total of 79 samples from individuals in Cambodia, Vietnam, Afghanistan, and China were sequenced, and a bioinformatics pipeline was created for the targeted variant calling of 192 G6PD SNP mutations. The assay demonstrated reliable detection of known variants, with high concordance between runs, within runs, and with Sanger sequencing. The Nanopore MinION long-amplicon sequencing assay offers a robust and portable solution for large-scale G6PD genotyping in low-resource settings, that will improve malaria control and elimination strategies by enabling safer antimalarial treatment.

microbiology↗

bioRxiv2025

PTRAMP, CSS and Ripr form a conserved complex required for merozoite invasion of Plasmodium species into erythrocytes

Invasion of erythrocytes by members of the Plasmodium genus is an essential step of the parasite lifecycle, orchestrated by numerous host-parasite interactions. In P. falciparum Rh5, with PfCyRPA, PfRipr, PfCSS, and PfPTRAMP, forms the essential PCRCR complex which binds basigin on the erythrocyte surface. Rh5 is restricted to P. falciparum and its close relatives; however, PTRAMP, CSS and Ripr orthologs are present across the Plasmodium genus. We investigated PTRAMP, CSS and Ripr orthologs from three species to elucidate common features of the complex. Like P. falciparum, PTRAMP and CSS form a disulfide-linked heterodimer in both P. vivax and P. knowlesi with all three species forming a complex (PCR) with Ripr by binding its C-terminal region. Cross-reactive antibodies targeting the PCR complex differentially inhibit merozoite invasion. Cryo-EM visualization of the P. knowlesi PCR complex confirmed predicted models and revealed a core invasion scaffold in Plasmodium spp. with implications for vaccines targeting multiple species of malaria-causing parasites.

microbiology↗

bioRxiv2024

Copy number variations of Plasmodium vivax DBP1, EBP/DBP2, and RBP2b in Duffy-positive and Duffy-negative Ethiopians

Recent evidence challenges the belief that Duffy-negative individuals are resistant to Plasmodium vivax due to lacking Duffy Antigen Receptor for Chemokines (DARC). Erythrocyte Binding Protein (EBP/DBP2) has shown moderate binding to Duffy-negative erythrocytes in vitro. Reticulocyte Binding Protein 2b (RBP2b) interactions with Transferrin Receptor 1 (TfR1) suggest involvement in Duffy-negative infections. Gene copy number variations in PvDBP1, PvEBP/DBP2, and PvRBP2b were investigated in Duffy-positive and Duffy-negative P. vivax-infected individuals from Ethiopia. Among Duffy-positive samples, 34% displayed PvDBP1 duplications (Cambodian-type). In Duffy-negative infections, 30% showed duplications, mostly Cambodian-type. For PvEBP/DBP2 and PvRBP2b, Duffy-positive samples exhibited higher duplication rates (1-8 copies for PvEBP/DBP2, 1-5 copies for PvRBP2b 46% and 43% respectively) compared to Duffy-negatives (20.8% and 26% respectively). The range of copy number variations was lower in Duffy-negative infections. Demographic and clinical factors associated with gene multiplications in both Duffy types were explored, enhancing understanding of P. vivax evolution in Duffy-negative Africans.

microbiology↗

bioRxiv2024

Potent AMA1-specific human monoclonal antibody against P. vivax Pre-erythrocytic and Blood Stages

New therapeutics are necessary for preventing Plasmodium vivax malaria due to easy transmissibility and dormancy in the liver that increases the clinical burden due to recurrent relapse. We isolated 12 Pv Apical Membrane Antigen 1 (PvAMA1) specific human monoclonal antibodies from Peripheral Blood Mononuclear Cells of a Pv-exposed individual. PvAMA1 is essential for sporozoite and merozoite invasion, making it a unique therapeutic target. HumAb 826827 blocked the invasion of human erythrocytes using Pv clinical isolates and inhibited sporozoite invasion of human hepatocytes in vitro (IC50 of 0.3 - 3.7 {micro}g/mL). It also significantly reduced liver infection of chimeric FRG-humHep mice in vivo. The crystal structure of rPvAMA1 bound to 826827 shows that 826827 partially occupies the highly conserved hydrophobic groove in PvAMA1 that binds its known receptor, RON2. We have isolated a potent humAb that is isolate-transcendent, blocks both pre-erythrocytic and blood stage infection, and could be a new therapy for Pv.

immunology↗

bioRxiv2023

Comparative transcriptomics reveal differential gene expression in Plasmodium vivax geographical isolates and implications on erythrocyte invasion mechanisms

Plasmodium vivax uses Duffy binding protein (PvDBP1) to bind to the Duffy Antigen-Chemokine Receptor (DARC) to invade human erythrocytes. Individuals who lack DARC expression (Duffy-negative) are thought to be resistance to P. vivax. In recent years, P. vivax malaria is becoming more prevalent in Africa with a portion of these cases detected in Duffy-negatives. Apart from DBP1, members of the reticulocyte binding protein (RBP) and tryptophan-rich antigen (TRAg) families may also play a role in erythrocyte invasion. While the transcriptomes of the Southeast Asian and South American P. vivax are well documented, the gene expression profile of P. vivax in Africa and more specifically the expression level of several erythrocyte binding gene candidates as compared to DBP1 are largely unknown. This paper characterized the first P. vivax transcriptome in Africa and compared with those from the Southeast Asian and South American isolates. The expression of 4,404 gene transcripts belong to 12 functional groups including 43 specific erythrocyte binding gene candidates were examined. Overall, there were 10-26% differences in the gene expression profile amongst the geographical isolates, with the Ethiopian and Cambodian P. vivax being most similar. Majority of the gene transcripts involved in protein transportation, housekeeping, and host interaction were highly transcribed in the Ethiopian P. vivax. Erythrocyte binding genes including PvRBP2a and PvRBP3 expressed six-fold higher than PvDBP1and 60-fold higher than PvEBP/DBP2. Other genes including PvRBP1a, PvMSP3.8, PvMSP3.9, PvTRAG2, PvTRAG14, and PvTRAG22 also showed relatively high expression. Differential expression was observed among geographical isolates, e.g., PvDBP1 and PvEBP/DBP2 were highly expressed in the Cambodian but not the Brazilian and Ethiopian isolates, whereas PvRBP2a and PvRBP2b showed higher expression in the Ethiopian and Cambodian than the Brazilian isolates. Compared to Pvs25, the standard biomarker for detecting female gametocytes, PvAP2-G (PVP01_1440800), GAP (PVP01_1403000), and Pvs47 (PVP01_1208000) were highly expressed across geographical samples. These findings provide an important baseline for future comparisons of P. vivax transcriptomes from Duffy-negative infections and highlight potential biomarkers for improved gametocyte detection.

cell biology↗

bioRxiv2018

In vivo gene expression analyses provide unique insights on P. vivax gametocytogenesis and chloroquine response

Studies of gene expression have provided insights on the regulation of Plasmodium parasites. However, few studies have targeted P. vivax, the cause of one third of all human malaria cases outside Africa, due to the lack of in vitro culture system and the difficulties associated with studying clinical samples. Here, we describe robust RNA-seq profiles of P. vivax parasites generated directly from infected patient blood. Gene expression deconvolution analysis reveals that most parasite mRNAs derive from trophozoites and that the asynchronicity of P. vivax infections is unlikely to confound gene expression studies. We also show that gametocyte genes form two clusters of co-regulated genes, possibly indicating the independent regulation of male and female gametocytogeneses. Finally, despite a large effect on parasitemia, we find that chloroquine does not alter trophozoite gene expression. Overall, our study highlights the biological knowledge that can be gathered by directly studying P. vivax patient infections.\n\nImportancePlasmodium vivax is the second most common cause of human malaria worldwide but, since it cannot be cultured in the laboratory, its biology remains poorly understood. In this study, we describe the analysis of the parasite gene expression profiles generated from 26 patient infections. We show that the proportion of male and female parasites varies greatly among infections, suggesting that they are independently regulated. We also compare the gene expression profiles of the same infections before and after treatment with chloroquine, a common antimalarial, and show that the drug efficiently kills most P. vivax parasites but appears to have little effect on one specific parasite stage, the trophozoites, in contrast with the effect of the drug on P. falciparum. Overall, our study exemplifies the biological insights that can be gained from applying modern genomic tools to study this difficult human pathogen.

microbiology↗

bioRxiv2018

Genetic diversity in two Plasmodium vivax protein ligands for reticulocyte invasion

The interaction between Plasmodium vivax Duffy binding protein (PvDBP) and Duffy antigen receptor for chemokines (DARC) has been described as critical for the invasion of human reticulocytes, although increasing reports of P. vivax infections in Duffy-negative individuals questions its unique role. To investigate the genetic diversity of the two main protein ligands for reticulocyte invasion, PvDBP and P. vivax Erythrocyte Binding Protein (PvEBP), we analyzed 458 isolates collected in Cambodia and Madagascar. First, we observed a high proportion of isolates with multiple copies PvEBP from Madagascar (56%) where Duffy negative and positive individuals coexist compared to Cambodia (19%) where Duffy-negative population is virtually absent. Whether the gene amplification observed is responsible for alternate invasion pathways remains to be tested. Second, we found that the PvEBP gene was less diverse than PvDBP gene (12 vs. 33 alleles) but provided evidence for an excess of nonsynonymous mutations with the complete absence of synonymous mutations. This finding reveals that PvEBP is under strong diversifying selection, and confirms the importance of this protein ligand in the invasion process of the human reticulocytes and as a target of acquired immunity. These observations highlight how genomic changes in parasite ligands improve the fitness of P. vivax isolates in the face of immune pressure and receptor polymorphisms.

genetics↗