Plasmodium vivax clinical burden accounts for 7 million annual clinical cases. Strikingly, only two vaccine candidates based on the Duffy binding protein are currently in clinical trials, thus indicating the need for discovering new antigens. Extracellular vesicles (EVs) are small endocytic double-membrane vesicles demonstrating an important role in intercellular communication during infectious diseases. EVs were initially described in reticulocytes, the host cell of P. vivax, as a selective cargo disposal mechanism to get rid of the transferrin receptor (CD71) among other proteins during the maturation of reticulocytes to erythrocytes. Our group recently reported the parasite protein content of circulating EVs coming from P. vivax patients by size exclusion chromatography (SEC) followed by mass spectrometry (MS). However, we could only detect parasite proteins in two out of ten patients. In order to enrich the EVs derived from CD71+ cells coming from plasma samples of infected patients we have implemented the direct immuno-affinity capture of circulating CD71+ EVs.
A total of 48 parasite proteins were identified in all patients. Many of the parasite proteins identified are known to be immunogenic during natural infections and some of them represent vaccine candidates (MSP1, MSP3, MSP7, MSP9, Serine-repeat antigen 1, and HSP70) as well as membrane, cytosolic and exported proteins. The most abundant parasite proteins identified corresponded to a hypothetical protein, the merozoite surface protein 3.3 and a member of the PHIST family, PHISTc. Remarkably, antibody responses were detected above negative control cut-off for MSP3 and PHIST recombinant P. vivax proteins in patients confirming their antigenicity. Therefore, CD71+-EVs from plasma of patients isolated by DIC allows a robust identification of P. vivax immunogenic proteins. This study represents a step forward to identify new antigens for vaccination against this human malaria parasite.