9th International Conference on Plasmodium vivax Research (ICPvR) – 2025: Day 2

MESA Correspondents bring you cutting-edge coverage from the ICPvR 2025 “Plasmodium vivax: Science and tools for elimination”.

Special Invited Session

Andrea Bosman (Global Malaria Programme, World Health Organization – WHO, Switzerland) discussed the new WHO recommendations regarding near-patient G6PD tests and radical treatment for Plasmodium vivax and P. ovale. In G6PD testing studies, severe adverse events, mainly severe hemolysis, occurred in 11.2% of deficient individuals but were nearly absent in those with normal levels. Bosman emphasized the WHO recommendations of using semi-quantitative near-patient tests with fixed standards for deficient, intermediate, and normal G6PD activity to help inform the administration of specific treatment regimens. Bosman highlighted the practical guidance of near-patient G6PD testing in managing the anti-relapse treatment of P. vivax and P. ovale with primaquine (PQ) and tafenoquine (TQ). The dosing recommendations are as follows: 1 mg/kg/day of PQ for 7 days or a single dose of TQ should only be administered to individuals whose G6PD activity is above the threshold corresponding to greater than 70% of normal levels. For those with G6PD activity above the threshold of 30% of normal levels, 0.5 mg/kg/day of PQ for 14 days, or 0.5 mg/kg/day for 7 days, can be given to prevent relapses of P. vivax and P. ovale. Since WHO recommendations are a living document, dosing guidelines for radical cure, such as for malaria caused by P. vivax and P. ovale, can change based on new evidence and ongoing research.

Session 5 – Targeting vivax: Decoding Host-Parasite Interactions

Manoj Duraisingh (Harvard T.H. Chan School of Public Health, United States) emphasized that a blood-stage vaccine is a potential tool for malaria elimination, as vaccination with Plasmodium vivax Duffy-binding protein (DBP) has been shown to inhibit parasite growth during controlled human malaria infection. However, challenges persist, such as tissue sequestration and reduced accessibility to drugs in younger reticulocytes. The study presented explored alternative invasion pathways, essential ligands, and critical receptors using P. knowlesi models. The hypothesis posits that targeting ligand-receptor interactions could lead to effective therapeutic strategies like vaccines or monoclonal antibodies. Parasite biomass, associated with disease severity, indicates sequestration in tissues like bone marrow and spleen, where parasites preferentially invade younger reticulocytes. Screening revealed that reticulocyte binding-like (RBL) and erythrocyte binding-like (EBL) superfamilies mediate host cell selection, with P. vivax relying on DARC and transferrin receptor (TFRC) for invasion. Antibody inhibition assays in P. knowlesi and P. cynomolgi identified potential antigen targets. Duraisingh concluded that targeting multiple pathways could improve vaccine efficacy and support the development of resistance-proof therapeutics.

Vineeta Singh (Indian Council of Medical Research – National Institute of Malaria Research – ICMR-NIMR, India) talked about the molecular markers and genetic diversity of Plasmodium vivax infections in India, highlighting challenges in malaria control. India contributes nearly half of the global P. vivax burden, with high genetic variability, multiple malaria vectors, and varying relapse phenotypes that complicate elimination efforts. Singh analyzed drug resistance mutations in Pvmdr1, Pvdhfr, Pvdhps, Pvk12, and Pvcrt-o using PCR and sequencing. A phylogenetic analysis mapped genetic diversity among Indian isolates, offering insights into regional variations. At Safdarjung Hospital, a large tertiary care hospital in Delhi, Singh’s study found no correlation between thrombocytopenia or cytokine levels and parasitemia, while severe P. vivax cases rose from 32.8% in 2020 to 65.7% in 2024. The study highlighted the increasing severity of P. vivax infections and the urgent need for intervention. Singh emphasized the need for molecular surveillance, improved diagnostics, and targeted interventions to combat P. vivax malaria effectively and prevent its resurgence.

Carmen Fernández-Becerra (Barcelona Institute for Global Health  – ISGlobal and Germans Trias i Pujol Research Institute – IGTP, Spain) discussed insights into the mechanisms of human malaria pathology through the study of extracellular vesicles (EVs). She supported the idea that there are subpopulations of P. vivax-adherent parasites present in the microvasculature of the human spleen. The cytoadherence of P. vivax-infected reticulocytes to human spleen fibroblasts (hSFs) is facilitated by plasma-derived EVs. Immunofluorescence analysis showed that the P. vivax spleen-dependent protein 1 (PvSDP1) is located on the surface of infected red blood cells in a transgenic line, a finding that was later confirmed in natural infections. Plasma-derived EVs from individuals infected with P. vivax (PvEVs) significantly enhanced the cytoadherence of the 3D7_PvSDP1 transgenic line to hSFs, and this binding was inhibited by the presence of anti-PvSDP1 antibodies. Single-cell RNA sequencing of hSFs treated with PvEVs revealed an increased expression of genes related to adhesion. This highlights the significance of parasite spleen-dependent genes and EVs from natural infections in establishing intrasplenic niches in P. vivax, posing a major challenge for malaria elimination.

Chris Drakeley (Armauer Hansen Research Institute – AHRI, Ethiopia and London School of Hygiene & Tropical Medicine – LSHTM, UK) presented a multisite study in Ethiopia examining factors influencing Plasmodium vivax transmission to mosquitoes. The study found that transmission is driven by parasite and gametocyte densities, with both symptomatic and asymptomatic infections contributing to the infectious reservoir. Recurrent infections remain highly infectious, further sustaining transmission. Additionally, mixed and heterologous infections complicate disease dynamics, while drug efficacy and immune responses play a role in transmission. Drakeley concluded that targeting both infection types with genetic and immunological insights is essential for malaria control and elimination.

Ashis Das (Birla Institute of Technology and Science – BITS-Pilani, India) presented the difference in the transcriptome profile of Plasmodium interspersed repeat (pir) genes in Plasmodium vivax cerebral malaria and hepatic dysfunction cases, namely hepatic dysfunction and cerebral malaria focusing on the vir genes, some of which have been postulated to be involved in cytoadherence. The study involved 23 patients, with 17 categorized as complicated cases (12 with hepatic dysfunction and 5 with cerebral malaria) and 6 as uncomplicated. Real-time qPCR confirmed the upregulation of two genes and the downregulation of two others. The upregulation of these vir/pvpir genes may be important in disease pathogenesis, particularly in their role in cytoadherence to host cell receptors.

Session 6 –  Immunology Unmasked: Natural Infections, Cryptic Reservoirs

Luzia H. Carvalho (Molecular Biology and Immunology of Malaria – BMIM, Brazil) presented a study on Plasmodium vivax vaccine strategies, focusing on Duffy Binding Protein II (DBPII), a key protein for red blood cell invasion via the Duffy Antigen Receptor for Chemokines (DARC). While people in malaria-endemic areas develop antibodies against DBPII, only some produce broadly protective antibodies, limiting immunity. To address this, DEKnull variants were designed to target conserved DBPII regions, inducing stronger and broader immunity. Long-term studies show that some individuals maintain immune memory, while others lose it or fail to develop a response. Notably, IgM antibodies play a crucial role in malaria immunity, challenging the belief that IgG is the main protective antibody. While DBPII remains a promising vaccine target, its strain-specific nature reduces effectiveness. Carvalho concluded by stating that future vaccine strategies should focus on optimizing DEKnull-2 to enhance cross-strain protection, ensuring long-lasting immunity against diverse P. vivax variants in endemic populations.

Hernando del Portillo (Barcelona Institute for Global Health – ISGlobal and Germans Trias i Pujol Research Institute – IGTP, Spain) presented on the role of reticulocyte-derived extracellular vesicles (EVs) in establishing cryptic Plasmodium vivax infections within human bone marrow and spleen. Del Portillo’s study employed organ-on-a-chip technology to replicate the bone marrow and spleen, demonstrating that EVs from malaria patients, but not from healthy individuals or P. falciparum patients, induced erythropoiesis and facilitated parasite migration to the bone marrow. High EVs concentrations were found in malaria patient plasma, suggesting a mechanism for parasite survival and persistence. Del Portillo’s team successfully generated the erythroid lineage on the chip, with cell viability exceeding 95% after five days. Ongoing single-cell RNA sequencing and spatial transcriptomics aim to uncover the signaling pathways involved in these interactions. Additionally, a humanized mouse model using CD34+ cells and fetal tissues, providing insights into parasite dynamics and tissue-specific growth, was also developed. The findings highlighted the importance of bone marrow and spleen as reservoirs for malaria persistence and pointed to EV-mediated signaling as a potential target for therapeutic interventions.

Flora Kano (Molecular Biology and Immunology of Malaria – BMIM, Brazil) stated that the DEKnull-2 vaccine, designed by altering polymorphic residues, targets conserved B-cell epitopes, generating high-titer, stable antibodies. The study hypothesized that T follicular helper (Tfh) cells and B-cell interactions in persistent responders (PR) enhance protective antibody responses. A long-term follow-up in the Brazilian Amazon categorized individuals into persistent, temporary, and non-responders based on DBPII-erythrocyte binding inhibition. PR had more activated and Th2-like Tfh cells, providing stronger B-cell help. PR also had more Tbet+ atypical memory B cells, secreting IgG with superior binding and inhibitory activity than classical memory B cells. Kano underscored the role of these immune subsets in a stable, strain-transcending antibody response and their relevance for next-generation DBP vaccine development as part of a multi-stage, multivalent malaria vaccine strategy.

Steven Kho (Menzies School of Health Research and Charles Darwin University, Australia) presented the first prospective study quantifying splenic biomass in acute Plasmodium vivax malaria. Due to limited spleen histopathology in untreated P. vivax cases, Kho compared blood markers in splenectomized and spleen-intact patients in Timika, Indonesia, using ELISA for plasma lactate dehydrogenase (LDH) and microscopy for circulating P. vivax biomass. Kho found that circulating parasites were higher in patients without a spleen, while total parasite biomass was greater in those with an intact spleen, indicating a large hidden reservoir. A case study further confirmed these findings, highlighting the spleen’s role in P. vivax infections.

Upninder Kaur (Post Graduate Institute of Medical Education & Research – PGIMER, India) investigated the role of polymorphisms in TLR1, TLR6, and TLR9, with TLR9 T-(1486)-C in the promoter region being the most frequent, particularly in complicated cases. Cytokine levels (IFN-γ, TNF-α, IL-6, IL-10) measured via ELISA showed a significant link between TLR9 polymorphisms and cytokine production, with lower IL-10 and IFN-γ levels in TLR9 (1486T) carriers, indicating an altered immune response. These findings highlighted TLR9‘s role in immune modulation, influencing disease severity and offering potential targets for vaccine development and treatment strategies.

Session 7 – Bridging the Gap: Advanced Model Systems for Plasmodium vivax

Anthony Ruberto (University of Georgia, United States) discussed the challenges and opportunities in studying Plasmodium vivax liver stages, particularly hypnozoites, to aid drug discovery. The challenges include the lack of continuous blood-stage parasite cultures, limited genetic tools, and difficulties in identifying the mechanism of action (MoA) of compounds targeting hypnozoites. Additionally, the host hepatocyte signal may mask the hypnozoite protein signal, making it harder to detect. Chemoproteomic profiling revealed disruptions in proteins related to biological processes in infected hepatocytes. The development of an in vitro P. vivax liver stage model is crucial for testing compounds that target hypnozoites and understanding both parasite and host responses. Ruberto highlighted the success of the first non-8-AQ hit project for P. vivax hypnozoites, which established the foundation for further research and potential drug development strategies for anti-hypnozoite therapies.

Melanie J. Shears (University of Washington, United States) discussed the development of humanized mouse and nonhuman primate models for studying Plasmodium vivax, focusing on vaccine and monoclonal antibody (mAb) development. The FRG humanized liver mice model, engrafted with human hepatocytes, supports liver stages and hypnozoite formation. Using PvCSP mAbs, researchers observed reduced schizont formation and relapse rate. In FRG-NOD mice, depleting macrophages and neutrophils enabled complete blood-stage cycles and gametocyte observation, with PvDBP mAbs reducing parasitemia by over 95%. HIS-HEry mice, with human hematopoietic stem cells, offer insights into bone marrow and transmission-blocking strategies. Shears also highlighted the macaque model with P. cynomolgi, a close relative of P. vivax, which mimics relapse and reticulocyte preferences. The novel Pc-PvCSP challenge line facilitates testing of PvCSP-based vaccines, while the PbViVac transgenic parasite, capable of infecting human liver cells but not RBCs, shows promise for vaccine development. Shears concluded that these models are essential for advancing therapeutic interventions against P. vivax malaria.

Robert W. Moon (London School of Hygiene and Tropical Medicine – LSHTM, United Kingdom) presented a study using transgenic Plasmodium knowlesi to explore the role of P. vivax duffy-binding protein (DBP) and reticulocyte binding protein 2b (RBP2b) in erythrocyte invasion. Both unmodified P. knowlesi and modified lines with the P. vivax DBP orthologue showed preferential binding to one allele, with a stronger effect in P. knowlesi. This research offers insights into DBP-based vaccine design for both species. The study also enabled the characterization of parasite-derived full-length RBP2b, aiding in the validation of human monoclonal antibodies. Using orthologous replacement techniques for non-Laveranian parasites like P. vivax continues to be a valuable tool for studying invasion mechanisms, identifying vaccine antigens, and validating inhibitory antibodies. The inclusion of PvRBP2b is crucial for understanding P. vivax invasion, especially its restriction of reticulocytes.

Jenna Oberstaller (University of South Florida, United States) highlighted the importance of understanding Plasmodium vivax biology for malaria control, emphasizing the parasites’ genetic diversity. The study utilized P. knowlesi as a model for genome-wide functional characterization through piggyBac transposon mutagenesis, saturating 98% of genes to assess their essentiality for blood-stage growth. Results revealed significant metabolic differences despite conserved genes, indicating species-specific adaptations for survival in different hosts. For instance, genes essential in P. falciparum were dispensable in P. knowlesi, demonstrating metabolic rewiring. The study also identified parasite-specific pathways involved in intracellular organization, receptor signaling, and nutrient acquisition. Validation experiments showed that genes like APkicm1 and APkrp6 differ in their importance across species. Oberstaller concluded that such genetic rewiring affects drug target efficacy, underscoring the necessity of testing antimalarial drugs across multiple malaria species to ensure broad-spectrum effectiveness. Ongoing work aims to further dissect these metabolic pathways for potential therapeutic interventions.

Jeremy Salvador (Institut Pasteur du Cambodge, Cambodia) highlighted that P. vivax primarily resides in the bone marrow (BM), with low parasitemia in peripheral blood but high density in organs such as the spleen and BM. The BM provides a unique environment with hypoxia, mesenchymal stem cells (MSC), and soluble factors like hematopoietic growth factors and metabolites. The study hypothesized that specific BM conditions support P. vivax survival and growth. Short-term high-oxygen exposure did not significantly impact parasite survival, development, or oxidative stress markers in vitro. Further, experiments with MSCs and a conditioned medium showed no notable effect on parasite survival. However, a high concentration of BM serum tended to reduce P. vivax viability. Preliminary results suggest that while hypoxic conditions might reduce oxidative stress, the BM microenvironment is not essential for P. vivax development in vitro. The study continues with additional biological samples to confirm these findings.

Clara Champagne (Swiss Tropical and Public Health Institute – Swiss TPH, Switzerland) evaluated Plasmodium vivax elimination in Honduras using mathematical modeling to assess intervention strategies in Gracias a Dios, the most endemic region. The study considered improving case management through increased healthcare access, reducing treatment delays, and enhancing primaquine adherence, along with minimizing imported cases from Nicaragua. The presentation underscored that increasing access to care and better adherence to primaquine significantly contribute to reducing malaria cases. Champagne stressed the importance of reducing case importation for successful malaria elimination and emphasized the need to strengthen health systems and enhance cross-border collaboration to achieve this goal in Honduras.

Session 8 – Duffy Enigma: Epidemiology & Invasion Mechanism

Isaac Quaye (Regent University College of Science and Technology, Ghana) presented research demonstrating Plasmodium vivax infection in Duffy-negative individuals, challenging the notion of their inherent resistance. The study demonstrated that P. vivax bypasses Duffy receptor dependency by selectively invading CD71+ reticulocytes, which are highly enriched in the spleen and bone marrow. Furthermore, the absence of atypical chemokine receptor 1 (ACKR1) in Duffy-negative individuals disrupts neutrophil homeostasis, leading to reduced immune cell counts and potentially heightened susceptibility to infections. P. vivax exploits immunosuppressed niches in the bone marrow and spleen, establishing reservoirs that support persistence and transmission. These findings redefine malaria epidemiology, demonstrating that Duffy negativity does not confer immunity against P. vivax. Quaye concluded that understanding ACKR1’s role in immune regulation and parasite invasion is crucial for refining malaria control strategies, particularly in high-prevalence regions such as Africa.

Amy Ibrahim (London School of Hygiene and Tropical Medicine – LSHTM, UK) presented a study on Duffy-negative erythrocyte invasion in Plasmodium knowlesi. The P. knowlesi A1-H.1 parasite line was exposed to a mix of Duffy-negative and Duffy-positive erythrocytes and after six months, a stable in vitro line (Z1H1) was established with Duffy-negative erythrocytes. Genome sequencing revealed that the invasion pathway is independent of the Duffy antigen receptor for chemokines (DARC) binding and is driven by genomic recombination between the DBPα and DBPγ genes. This study demonstrates P. knowlesi’s ability to invade Duffy-negative erythrocytes, with implications for parasite control. It also offers a new tool for studying Duffy-negative invasion in P. vivax, which lacks a stable in vitro culture system.

Johnsy Mary Louis (Kangwon National University, South Korea) presented a study on identifying and characterizing how Duffy binding ligands and receptors facilitate invasion in non-Laverania Plasmodium species. The study confirmed that Duffy Binding Protein (DBP)-DARC interactions are critical for invasion in P. vivax, P. knowlesi, and P. cynomolgi. Co-immunoprecipitation (Co-IP) results showed species-specific binding: PvDBP-RII interacts with human DARC, PcyDBP1-RII with human DARC, and PkDBPα-RII with both human and monkey DARC. Structural analysis revealed mutations in PkDBPα-RII that affect affinity and key residues like D24 in DARC influence binding. These findings suggest that specific DBP residues affect binding affinity, host selection, and interaction preferences, which could inform vaccine design for P. vivax and related species.

Ababio Grace K (Regent University College of Science and Technology, Ghana) presented a study on Duffy gene polymorphisms in Ghana, Namibia, and Zambia. The research identified -67T>C and 125 G>A mutations, with uninfected individuals showing a fixed Duffy-negative genotype, while infected individuals displayed variations. This suggests P. vivax may use co-infections and alternative invasion pathways. Grace K also highlighted that Plasmodium infections may trigger genetic changes near codon 125, while ACKR1 may also impact hematopoiesis and immune responses, underscoring the need for further research on parasite-host interactions.