Transcriptome analysis and functional characterization of Plasmodium vivax spleen and bone marrow dependent genes and the role of the extracellular vesicles in cryptic niche formation

[eng] INTRODUCTION: Plasmodium vivax is the most widespread malaria parasite, with 2.8 billion people at risk across regions from Southeast Asia to the Americas. In 2022 alone, approximately 6.9 million clinical cases were reported worldwide (1). Classically considered less severe than P. falciparum, vivax malaria can lead to serious complications such as severe anaemia, thrombocytopenia, acute respiratory distress, splenic rupture and even death (2). One of the major challenges with controlling P. vivax is its resilience to elimination compared to P. falciparum. This difficulty has driven the need for a deeper understanding of the parasite’s pathobiology and epidemiology (3,4). Among the factors contributing to P. vivax pathophysiology is the subtelomeric multigenic vir family (5). Unlike the clonally expressed var genes in P. falciparum, vir genes from different subfamilies are co-expressed, leading to a broader expression pattern. This broad expression might explain why acquired immunity in vivax malaria develops much faster than in P. falciparum (6,7). Research over the past decade has increasingly shown that P. vivax uses both the bone marrow (BM) and spleen as key organs in its life cycle. The BM serves for sexual differentiation and parasite growth, as evidenced by the presence of infected reticulocytes in BM aspirates (8–10). Similarly, the spleen harbours significantly higher densities of P. vivax-infected red blood cells (RBCs) compared to peripheral blood (11,12). These findings suggest that both the BM and spleen act as cryptic niches for the parasite, contributing to its persistence and the potential for causing anaemia(8,13). Recently, extracellular vesicles (EVs) have emerged as novel intercellular communicators, not only between parasite populations but also between parasites (14) and their human hosts (15). However, the exact role of EVs in 23 vivax malaria, in particular in forming these cryptic niches within hematopoietic organs is not fully understood. HYPOTHESIS AND OBJECTIVES: We hypothesize that during natural P. vivax infections, the spleen and BM, reticulocyte-rich tissues, serve as cryptic niches which favour parasite growth and differentiation. Additionally, we postulate that these processes involve specific P. vivax proteins whose transcription depend on the BM and spleen and that EVs play a crucial role in facilitating cryptic niche formation. The main objective is to identify, validate, and functionally characterize P. vivax genes that are dependent on the spleen and BM for expression. We aim to clarify the role these genes play during acute vivax malaria and to uncover how EVs contribute to cryptic niche formation.