Elegant transmission electron microscope (TEM) studies showed that upon entrance of the parasite, and similar to which forms protrusions at the plasma membrane where variant surface virulent determinants are located, induced the formation of Caveola-Vesicle-Complex (CVC) where parasite proteins are located (Aikawa et?al., 1975; Akinyi et?al., 2012). transmembrane and extracellular regions. (C) Pie chart showing peptides coverage of CD71 cytoplasmic and extracellular regions in the CD71+ EVs mass spectrometry analysis. Image_2.tiff (731K) GUID:?E5039E68-00DF-4794-BA55-C0B4CCB60427 Supplementary Figure?3: Humoral immune response Nylidrin Hydrochloride of vivax malaria primo-infected individuals against three abundant antigens detected in CD71+ EVs. Luminex analysis showing antibody levels [represented as mean fluorescence intensity (MFI)] against three antigens (MSP-19, MSP-3, and PHIST). Dotted lines represent cut-off point established for each antigen by calculating the mean +2 standard deviation of the signal obtained in healthy controls (n=8) used as negative controls. Pv, is the most widely distributed human malaria parasite with 7 million annual clinical cases and 2.5 billion people living under risk of infection. There is an urgent need to discover new antigens for vaccination as only two vaccine candidates are currently in clinical trials. Extracellular vesicles (EVs) are small membrane-bound vesicles involved in intercellular communication and initially described in reticulocytes, the host cell of proteins associated to circulating EVs in patients using size exclusion chromatography followed by mass spectrometry (MS). Parasite proteins were detected in only two out of ten patients. To increase the MS signal, we have implemented the direct immuno-affinity capture (DIC) technique to enrich in EVs derived from CD71-expressing cells. Remarkably, we identified parasite proteins in all patients totaling 48 proteins and including several previously identified vaccine candidate antigens (MSP1, MSP3, MSP7, MSP9, Serine-repeat antigen 1, and HSP70) as well as membrane, cytosolic and exported proteins. Notably, a member of the helical interspersed sub-telomeric (PHIST-c) family and a member of the exported proteins, were detected in five out of six analyzed patients. Humoral immune response analysis using sera from vivax patients confirmed the antigenicity of the PHIST-c protein. Collectively, we showed that enrichment of EVs by CD71-DIC from plasma of patients, allows a robust identification of immunogenic proteins. This study represents a significant advance in identifying new antigens for vaccination against this human malaria parasite. Keywords: and is the most Nylidrin Hydrochloride widely distributed representing 53% of malaria burden in the South-East Asia region and the most predominant species in the region of the Americas (WHO, 2020). Recent estimates of its burden indicate that close to 3.3 billion of people are under risk of infection and near 7?million yearly clinical cases. Major biological differences between and will not work against is rising-up (Price et?al., 2020), and this includes sub-Saharan Africa where was considered to be mostly absent (Twohig et?al., 2019). Among control tools, vaccines remain the most cost-effective public health measurement. Remarkably, WHO has recently announced the approval of the RTS,S (Mosquirix) as a vaccine against recommended for young children in Africa under moderate to high risk of transmission. In spite Nylidrin Hydrochloride of this major historical achievement, this vaccine does not cross-protect against (Toda et?al., 2020), now known to represent the largest cryptic parasite biomass during chronic infections (Kho et?al., 2021a; Kho et?al., 2021b). preferentially, if not exclusively, invades reticulocytes, young red cells where exosomes were first described as a cargo-disposal mechanism that selectively remove proteins such as the transferrin receptor CD71 during the maturation of reticulocytes to erythrocytes (Harding et?al., 1983; Pan and Johnstone, 1983). Using a Nylidrin Hydrochloride reticulocyte-prone rodent malaria model, we previously demonstrated the presence of parasite proteins in plasma-derived EVs and showed that reticulocyte-derived exosomes from infected mice protected immunized animals against lethal XL infections (Martin-Jaular et?al., 2011). Moreover, such protection was spleen-dependent and involved CD8 T cell mediated immune response (Martin-Jaular et?al., 2016). Interestingly, reticulocyte-derived exosomes contain HLA-I molecules suggesting a potential capacity for antigen presentation (Raposo Nylidrin Hydrochloride and Stoorvogel, 2013; Diaz-Varela et?al., 2018). More recently, we also demonstrated the presence of proteins associated to plasma-derived EVs from vivax malaria patients isolated by size-exclusion chromatography (SEC) and using mass spectrometry (Toda et?al., 2020). While three proteins were unequivocally identified (PHISTc, MSP3.1, GAPDH), we detected them in only two out of ten patients. Here, we have implemented immune-capture of CD71+-EVs from plasma of patients. This strategy greatly improved the enrichment of reticulocyte-specific EV markers and the coverage of EV-associated parasite proteins detected by mass spectrometry. Mouse monoclonal to CD37.COPO reacts with CD37 (a.k.a. gp52-40 ), a 40-52 kDa molecule, which is strongly expressed on B cells from the pre-B cell sTage, but not on plasma cells. It is also present at low levels on some T cells, monocytes and granulocytes. CD37 is a stable marker for malignancies derived from mature B cells, such as B-CLL, HCL and all types of B-NHL. CD37 is involved in signal transduction Many of the parasite proteins identified are known to be immunogenic during natural infections and some of them represent vaccine candidates. Thus, this study is a step-forward to identify new antigens for vaccination against this human malaria parasite. Material and Methods Malaria Patients and.