A novel micronemal protein MP38 is involved in the invasion of merozoites into erythrocytes

The absence of an <i>in vitro</i> cultivation system for <i>Plasmodium vivax</i> hinders the exploration of molecular targets for vaccine development. To address this, functional studies often rely on alternative models, such as <i>P. knowlesi</i>, due to its genetic similarity to <i>P. vivax</i>. This study investigated the role of a novel micronemal protein, PvMP38 (PVX_110945), in both <i>P. vivax</i> and <i>P. knowlesi</i> merozoite invasion of erythrocytes. The full-length ectodomain of PvMP38 was expressed, and polyclonal antibodies were generated to assess its function. PvMP38 was confirmed to localize on micronemal organelle in both <i>P. vivax</i> and <i>P. knowlesi</i> merozoites. <i>In vitro</i> protein-protein interaction assays revealed that PvMP38 binds to Pv12 with high-affinity interaction. A conserved novel complex of Pv12-Pv41-PvMP38 was identified by immunoprecipitation of <i>P. vivax</i> antibodies on <i>P. knowlesi</i> schizont lysates. Linear epitopes of PvMP38 with high and moderate antigenicity were identified in clinical isolates of both species. Invasion inhibition assays demonstrated that a triple antibody combination targeting the PvMP38, Pv12, and Pv41 significantly reduced <i>P. knowlesi</i> merozoite invasion of erythrocytes compared to a single antibody. In addition, CRISPR/Cas9-mediated knockout of <i>P. knowlesi mp38</i> markedly impaired parasite growth, underscoring its essential role during the asexual stage. These findings identify PvMP38 and its associated complex as promising targets for malaria interventions and highlight the utility of <i>P. knowlesi</i> as a model for investigating <i>P. vivax</i> erythrocyte invasion mechanisms.IMPORTANCEThis manuscript reported an effort in malaria eradication by identifying and functionally characterizing a novel <i>Plasmodium vivax</i> micronemal protein, PvMP38, involved in erythrocyte invasion. A narrow repertoire of an efficacious vaccine targeting <i>P. vivax</i> candidates is being developed due to the lack of continuous <i>in vitro</i> culture. This study addresses a gap in <i>P. vivax</i> research using <i>P. knowlesi</i> as a model for both genome editing and antibody functionality validation. By enhancing the protein-protein interaction screening framework, this study demonstrated that PvMP38 forms a complex with Pv12 and Pv41, opening the approaches to multi-antigen vaccines. The successful application of CRISPR/Cas9 gene editing techniques to disrupt its homolog, the <i>pkmp38</i> gene, further assesses the protein's significance in the growth and invasion of the parasite. These findings provided valuable insights into the biology of <i>P. vivax</i> and proposed PvMP38 as a promising candidate for malaria intervention strategies.