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Exosome-mediated mechanisms of non-hypnozoite cryptic infections in P. vivax malaria

Image: Haruka Toda

Strong support of non-hypnozoite cryptic infections, mainly in the spleen and bone marrow, has been published 1-3. Noticeably, extracellular vesicles in the size range of exosomes obtained from human patients facilitated binding to the human spleen 4. We thus hypothesize that EVs, specifically reticulocyte-derived exosomes, from P. vivax infections mediate bone marrow defects on erythropoiesis and spleen extramedullary erythropoiesis facilitating cryptic infections likely responsible for asymptomatic transmission. As this species cannot be continuously culture in vitro, proving this hypothesis represents a major technical challenge. Two different approaches implementing new human science technological frontiers will be pursued, organs-on-a-chip and humanized mouse models of these human organs, thus facilitating to stringently test the role of exosomes as intercellular communicators in a space and velocity that will facilitate their uptake by cells in these hemopoietic tissues. 

Functional analysis of subtelomeric variant genes

As infected reticulocytes with mature developmental stages of P. vivax are detected in the peripheral circulation, it has been amply accepted that there is no cytoadherence, hence no sequestration in this species. Against this dogma, our group identified the largest subtelomeric multigene family of human malaria parasites, the vir multigene family with expressed VIR proteins, likely involved in vivax pathology through cytoadherence of (pRetics) 5. Proof-of-principle of vivax pRetics cytoadherence partly mediated by VIR proteins was later demonstrated in vitro using endothelial, placental and brain cells 6, and more recently we demonstrated that a P. falciparum transgenic line expressing a VIR protein was able to cytoadhere to human spleen fibroblasts 3. We will pursue functional binding assay of variant surface proteins expressed in the human bone marrow and spleen during natural infections using CRISPR/cas-9 technologies to produce new transgenic lines of P. falciparum.

Image: Carmen Fernandez-Becerra

Reticulocyte-derived exosomes (Rex) vaccines against P. vivax

Image: Carmen Fernandez-Becerra

T-cell responses were believed not to play a major role in natural immunity against blood stages of malaria as P. falciparum, the most virulent human malaria parasite, predominantly invades mature red blood cells which lack antigen presenting machinery. However, our recent study demonstrated that reticulocyte exosomes contain HLA Class I antigens actively uptaken by dendritic cells 7 and that circulating extracellular vesicles directly obtained from patients contained novel parasite antigens 4. These results support further studies of reticulocyte exosomes from vivax infections to discover new antigens for vaccination and of human reticulocyte exoxomes as a potential vaccine delivery platform for eliciting cytotoxic T-cell responses against vivax malaria. 

Hypnozoite biomarker discovery

We are using human liver-chimeric FRG-KO-(huHep) mice treated with schizonticidal drugs to determine the proteome composition of plasma-derived exosomes from animals exclusively infected with P. vivax hypnozoites. Moreover, our previous proteome analysis 8 showed the presence of unique human proteins that are presently being used to highly enriched exclusively for exosomes coming from human hepatocytes; thus, increasing the signal for biomarker discovery. We will produce 3-5 unique parasite proteins and monoclonal antibodies against them to facilitate the construction of microfuidic devices with the ultimate goal of developing POC diagnostic device easily deployable in the field to contribute to the elimination of vivax malaria. 

Image: Iris Aparici-Herraiz

1. Machado Siqueira A, Lopes Magalhaes BM, Cardoso Melo G, et al. Spleen rupture in a case of untreated Plasmodium vivax infection. PLoS Negl Trop Dis 2012;6:e1934.

2. Kho S, Qotrunnada L, Leonardo L, et al. Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study. PLoS Med 2021;18:e1003632.

3. Fernandez-Becerra C, Bernabeu M, Castellanos A, et al. Plasmodium vivax spleen-dependent genes encode antigens associated with cytoadhesion and clinical protection. Proceedings of the National Academy of Sciences of the United States of America 2020;117:13056-65.

4. Toda H, Diaz-Varela M, Segui-Barber J, et al. Plasma-derived extracellular vesicles from Plasmodium vivax patients signal spleen fibroblasts via NF-kB facilitating parasite cytoadherence. Nat Commun 2020;11:2761.

5. del Portillo HA, Fernandez-Becerra C, Bowman S, et al. A superfamily of variant genes encoded in the subtelomeric region of Plasmodium vivax. Nature 2001;410:839-42.

6. Carvalho BO, Lopes SC, Nogueira PA, et al. On the cytoadhesion of Plasmodium vivax-infected erythrocytes. J Infect Dis 2010;202:638-47.

7. Diaz-Varela M, de Menezes-Neto A, Perez-Zsolt D, et al. Proteomics study of human cord blood reticulocyte-derived exosomes. Sci Rep 2018;8:14046.

8. Gualdron-Lopez M, Flannery EL, Kangwanrangsan N, et al. Characterization of Plasmodium vivax Proteins in Plasma-Derived Exosomes From Malaria-Infected Liver-Chimeric Humanized Mice. Frontiers in microbiology 2018;9:1271.