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

Strong support of non-hypnozoite cryptic infections, mainly in the spleen and bone marrow, has been published. Noticeably, extracellular vesicles in the size range of exosomes obtained from human patients facilitated binding to the human spleen. The group thus hypothesizes 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 are being 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. 

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

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, the group's recent study demonstrated that reticulocyte exosomes contain HLA Class I antigens actively up taken by dendritic cells 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 exosomes as a potential vaccine delivery platform for eliciting cytotoxic T-cell responses against vivax malaria.

Hypnozoite biomarker discovery

The team is 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, their previous proteome analysis 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. They will produce 3-5 unique parasite proteins and monoclonal antibodies against them to facilitate the construction of microfluidic devices with the ultimate goal of developing POC diagnostic device easily deployable in the field to contribute to the elimination of vivax malaria.

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, this research 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). Proof-of-principle of vivax pRetics cytoadherence partly mediated by VIR proteins was later demonstrated in vitro using endothelial, placental and brain cells, and more recently the group demonstrated that a P. falciparum transgenic line expressing a VIR protein was able to cytoadhere to human spleen fibroblasts. Its researchers are performing 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.

Extracellular vesicles (EVs) as potential new biomarkers in parasitic infections

The fact that EVs have been found in most biological fluids makes them an important target for the identification of new biomarkers in parasitic infections. In Chagas disease, the group is exploring EVs for identifying novel biomarkers specifically in the context of therapeutic response and disease progression during the chronic phase of the disease. In the field of Leishmaniasis, PvREX has recently started a collaborative project, planned to characterize EVs recovered from plasma in different clinical groups of human leishmaniasis (symptomatic, asymptomatic, treated). In parallel, the group also intends to evaluate the potential of extracellular vesicles as biomarkers for canine leishmaniasis. More recently, it has started a new collaborative project to identifying EV-Biomarkers in the context of severe P. falciparum malaria in children.

Plasmodium vivax and Extracellular Vesicles Research Group (PVREX) recognised as a Consolidated Research Group (GRC)

PI: Carmen Fernández Becerra, Hernando A. Del Portillo
Funding agency: Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR)
Agency code: 2021 SGR 01554 G65341695
Start date: 2023
End date: 2025

Novel organ-on-a-chip technology to study extracellular vesicles-mediated cryptic infections in Plasmodium vivax malaria

PI: Hernando A. Del Portillo
Funding agency: Fundación "laCaixa"
Agency code: LCF/PR/HR21/52410021
Start date: 2021
End date: 2024

CIBER en Enfermedades Infecciosas (CIBERINFEC)

PI: Joaquim Gascon
Funding agency: Instituto de Salud Carlos III (ISCIII)
Agency code: INFECG21/2022
Start date: 2021
End date: 2024

Uso de Vesículas extracelulares (EVs) como herramienta para la identificación de nuevos biomarcadores en la enfermedad de Chagas

PI: Carmen Fernández Becerra, Joaquim Gascon
Funding agency: Mundo Sano
Date: 2027

Extracellular Vesicles as Intercellular Communicators and Biomarkers of Cryptic Erythrocytic Infections in Plasmodium vivax malaria (VivaxEVTalk)

PIs: Carmen Fernández Becerra, Hernando A. Del Portillo
Funding agency: MICINN
Agency code: PID2022-142908OB-I00
Start date: 2023
End date: 2026

Plasmodium vivax: a cryptic parasite challenging malaria elimination

PI: Carmen Fernández Becerra, Hernando A. Del Portillo
Funding agency: Ministerio de Ciencia e Innovación Español (MICINN)
Agency code: PID2019-111795RB-I00
Start date: 2020
End date: 2023

Extracellular vesicles as novel prognostic biomarkers for Chagas disease, an emerging chronic infection in Catalonia

PI: Carmen Fernández Becerra
Funding agency: La Marató de TV3
Agency code: 566/U/2018
Start date: 2019
End date: 2023

Title: Evaluation of Extracellular Vesicles Potential as Biomarkers for Canine Leishmaniasis
Author: Sofia Sampaio Esteves
Supervisors: Carmen Fernández Becerra (co-supervisor)
University: Universidade de Porto
Date of defence: 19/04/2023

Title: Extracellular vesicles as tool for the identification of new biomarkers in Chagas disease
Author: Núria Cortés Serra
Supervisors: Carmen Fernández Becerra
University: Universitat de Barcelona
Date of defence: 24/11/2022

Title: Extracellular vesicles as intercellular communicators in cryptic erythrocytic infections in Plasmodium vivax malaria
Author: Iris Aparici Herraiz
Supervisors: Hernando A. del Portillo, Carmen Fernández Becerra 
University: Universitat de Barcelona
Date of defence: 15/09/2022