G-ID 7 Plasmodium vivax and Exosome Research (PVREX)
Hernando A del Portillo, ICREA Research Professor, and Carmen Fernandez-Becerra, Assistant Research Professor and the Plasmodium vivax and Exosome Research Group (PVREX) joined the IGTP in November 2015 through a collaborative agreement between the Barcelona Institute for Global health (ISGlobal) and the IGTP.
The group brings their expertise in malaria and exosome research with a very strong motivation to produce translational impact. The group uses a series of molecular and cellular biology technologies combined with immunoepidemiological studies in endemic regions of vivax malaria (i) to study the biology of the parasite, (ii) to unveil mechanistic insights into anaemia and splenomegaly, and (iii) to discover new antigens for vaccinations. In addition, the group is pioneering the use of reticulocyte-derived exosomes as a new vaccine and platform against malaria. To this end, Dr. del Portillo has co-founded INNOVEX THERAPEUTICS SL, the first Spanish Spin-off devoted to the use of exosomes as new therapeutic agents and diagnostic tools. It is important to note that the Spin-off is presently located at IGTP.
Additionally, the group has implemented small-scale soluble protein production in the cell-free wheat germ system for multiplex immunological assay, in vivo imaging of malaria parasites in mouse models and organs-on-a-chip technology to advanced on their studies of the role of the spleen in malaria and other haemolytic anaemias.
Lastly, due to the lack of an in vitro culture system for vivax malaria, which has a unique tropism for reticulocytes, the group is working on immortalization of human CD34+ capable of expanding and differentiation into these young red blood cells. These research areas and technologies are therefore amenable for collaborations with groups working on extracellular vesicles, vaccines and haematological disorders at IGTP.
The research of the group is fully compatible with the possibility of creating a future horizontal Institutional project to develop vaccines against HIV, TB and malaria, together known as the "big three" and responsible for a staggering 5.6 million deaths annually.
1. Reticulocyte-derived exosomes as a new vaccine and platform against Plasmodium vivax malaria
Exosomes are 30-100-nm membrane vesicles of endocytic origin that are released after the fusion of multivesicular bodies (MVBs) with the plasma membrane. While initial studies suggested that the role of exosomes was limited to the removal of proteins during the maturation of reticulocytes to erythrocytes, recent studies indicate that they are produced by different types of cells and are involved in promoting inter-cellular communication and antigen presentation. We have demonstrated that reticulocyte-derived exosomes (rex) from BALB/c mice infected with the reticulocyte-prone non-lethal Plasmodium yoelii 17X strain contain parasite proteins. Moreover, that immunization of mice with rex in combination with CpG elicited IgG2a and IgG2b antibodies and promoted survival, clearance of parasites and subsequent sterile protection of 83% of the animals challenged with P. yoelli 17XL. This is the first report of immune responses elicited by exosomes derived from reticulocytes opening new avenues for the development of rex as a new vaccine and platform against malaria. Presently, we are looking for the mode-of-action of such vaccination strategy in the rodent model and in extrapolating such results to humans. To translate these results and vaccination approach, a protocol for scalability and production of human reticulocyte-derived exosomes and fulfilment of regulatory for a Phase I clinical trial at Can Ruti are priority goals.
2. Antigen discovery
We have identified vaccine-protective antigens associated with reticulocyte-derived exosomes in experimental infections of a reticulocyte-prone rodent malaria model. As the human malaria caused by P. vivax infects reticulocytes, we are presently identifying parasite antigens associated with rex in natural infections of malaria patients. In addition, through global transcriptional analysis, we have identified P. vivax antigens whose expression is dependent on an intact spleen. To determine which antigens are associated with clinical protection, we have modified vectors for soluble expression of malaria proteins in the wheat germ cell-free system and are testing them in multiplex assays with immune sera of children from prospective longitudinal cohort studies in Brazil and PNG.
3. Role of the spleen, cytoadherence and pathology
It is amply accepted that P. vivax infected-reticulocytes do not cytoadhere in the deep capillaries of inner organs having an obligate passage through the spleen. Questions thus remain as how P. vivax escapes spleen-clearance. To gain insight into the function and structure of the spleen in malaria, we have implemented intravital microscopy and magnetic resonance imaging in a rodent malaria model with tropism for reticulocytes. Noticeably, experimental infections in this model induce remodelling of the spleen facilitating cytoadherence and macrophage-clearance escape. Significantly, we have obtained evidence demonstrating that a similar mechanism occurs in P. vivax, as there is cytoadhesion of P. vivax-infected reticulocytes to the ICAM1 endothelial receptor and to cryostat sections of human spleens. This data is in strong contrast to the dogma that the Plasmodium-infected cells avoid passage through the spleen. Most important, our data strongly suggest that parasite host interaction in the spleen is totally different from what we originally thought in that this organ might play an important role in chronic infection. To further advance our knowledge on the human spleen and malaria, in collaboration with IBEC, we have constructed a human spleen on a chip and are now evaluating its usage in studies of malaria and other haematological disorders.
4. Functional gene studies and in vitro culture of P. vivax
We are interested in functionally characterizing P. vivax genes through heterologous transfection in P. falciparum as presently there is no continuous in vitro culture of P. vivax blood stages. To this end, we have constructed different vectors to stably express transgenes in P. falciparum in all the asexual blood stages at different levels. Using one of such vectors, we have over-expressed virulent genes of P. vivax in P. falciparum and have demonstrated that members of particular variant proteins are ligands of the endothelial ICAM1 receptor, involved in severe malaria. Similar approaches are also being pursued with other putative virulent genes to determine their sub-cellular localization and function. To validate these findings, in collaboration with Fundaçao de Medicina Tropical do Amazonas, we are trying to establish a continuous in vitro culture system for P. vivax to try implementing homologous transfection technology and reverse genetics approaches for this parasite species.
5. Biomarker discovery
We are using mass-spectrometry to identify new markers for identifying P. vivax asymptomatic carriers with the ultimate goal of developing POC diagnostic devices easily deployable in the field to contribute to the elimination of vivax malaria.
6. Exosome-vaccines against neglected tropical diseases
Parasitic diseases have affected humans since their emergence on Earth. These include diseases related to poverty such as the malaria caused by Plasmodium vivax, Chagas disease caused by Trypanosoma cruzi and fascioliasis caused by F. hepatica. We hypothesize that exosomes derived from these three human infections act in inter-cellular communication facilitating the establishment of infections. Moreover, the parasite-specific proteins associated with these exosomes will identify new antigens for vaccination and the use of human reticulocyte-derived exosomes will serve as a new vehicle for antigen delivery and presentation to develop vaccines against these neglected tropical diseases. This is a joint-project with Drs. Antonio Osuna (U. de Granada) and Antonio Marcilla (U. de Valencia) funded through the Fundación Ramón Areces