Despite the high burden exerted, Plasmodium vivax has been a ‘neglected’ human malaria parasite. Most of such neglect is due to the fact that infection with P. vivax has been associated with a benign course. In the last decade, however, there have been numerous reports of severe manifestations associated with exclusive P. vivax infection (Mueller et al., 2009 Lancet Inf. Dis.). Central to this pathogenesis are the phenomena of antigenic variation and cytoadherence. Noticeably, our group first identified a subtelomeric variant multigene superfamily in P. vivax that we termed vir (vivax variant genes) (del Portillo HA et al., 2001 Nature; Carlton et al., 2008 Nature). Since, we have hypothesized that it is involved in a novel spleen evasion mechanism by mediating cytoadherence to barrier cells of fibroblastic origin; thus, avoiding complete macrophage destruction and establishing chronic and cryptic infections (Fernandez-Becerra C et al., & del Portillo HA 2008 Trends Parasitol.). To prove this hypothesis, we first used a rodent malaria model and implemented in vivo imaging of the spleen to study the dynamic passage of the parasite through this organ. Our results indeed fully support the hypothesis and a new model of spleen immune evasion in malaria (Martin-Jaular L et al., 2011 Cell Microbiol.). To extrapolate these results to humans, we performed a global transcriptional analysis of parasites obtained from experimental infections on monkeys that had an intact spleen or had been splenectomised. Remarkably, several VIR proteins were shown to be fully dependent on an intact spleen for expression (unpublished). In the absence of a continuous in vitro culture system for blood stages of P. vivax, one of such proteins was expressed in P. falciparum by constructing a transgenic line strain constitutively expressing such VIR protein at the surface of infected RBC. Functional cytoadherence assays under flow physiological conditions demonstrated binding to the ICAM-1 receptor, known to be involved in severe malaria (Bernabeu et al., 20 Cell Microbiol). Next, we demonstrated that it binds to cryostat sections of human spleen and that this binding is partially inhibited by antibodies against VIR proteins (unpublished). Last, to pursue functional studies we have constructed a spleen-on-a-chip (Rigat-Brugarolas et al., 2014 Lab Chip) and are constructing humanized mouse models. The impact of this research area is several fold: (i) demonstration of spleen cytoadherence is against the dogma that the spleen plays only a removal role of parasitized red blood cells in malaria; (ii) demonstration of cryptic stages of P. vivax in the human spleen will discover a new parasite life stage in malaria; (iii) active adherence of P. vivax-infected reticulocytes mediated by spleen-dependent variant proteins will validate their use as novel antigens for vaccination.
Cytoadhesion assay of P. falciparum transgenic line 3D7_vir14-3HA to CHO cells expressing human endothelial receptors (Source: Bernabeu and Fernandez-Becerra, unpublished).
Of all the clinical syndromes associated with severe vivax disease, anaemia is one of the most prevalent worldwide. However, unlike the anaemia caused by P. falciparum, the most virulent human malaria parasite, very little is known about anaemia in P. vivax. Of interest, P. vivax has a predominant, if not unique, tropism for reticulocytes and our own and other studies have demonstrated that reticulocyte-prone malarial parasites preferentially invade reticulocytes highly expressing the CD71 receptor (CD71hi) in haematopoietic tissues (Fernandez-Becerra C et al., 2013 Mem Inst Oswaldo Cruz). However, even if the presence of P. vivax in the bone marrow has been suggested in studies since the 19th Century, all studies of this species in this tissue were limited by the lack of molecular tools to unequivocally demonstrate the presence of P. vivax and different stages, particularly gametocytes often morphologically confounded as schizonts. Remarkably, post-mortem studies in different tissues of P. falciparum, the most virulent species largely present in sub-Saharan Africa, showed that early stage gametocytes are predominantly found in the bone marrow (Aguilar et al., 2014 Blood; Joyce et al., 2014 Sci Transl Med). These observations have led the suggestion that early stage gametocytes and maturation takes place in an immunopriviledge side before releasing mature stages into peripheral blood circulation for transmission to mosquitoes.
Recently, for the first time, we carried-out a collaborative study with Dr. Marcus V Lacerda (Fundaçao de Medicina Tropical Dr Heitor Vieira Dourado, Manaus, Brazil) on the bone marrow from a P. vivax-patient which unequivocally demonstrated enrichment of ring-stages and gametocytes in this tissue as well as morphological changes of erythroid precursor cells indicating an altered erythropoiesis during an active infection. Moreover, transcriptional analysis of bone marrow aspirates during infection and at convalescence of this same patient demonstrated transcriptional changes of miRNAs related to erythropoiesis (Baro B et al., & del Portillo HA 2017 PLoS Neg Trop. Dis. Accepted for publication). Together with the morphological studies of P. falciparum gametocytes in the bone marrow, these data indeed support the view that transmission stages of malaria parasites reside in an immunopriviledge niche for maturation and open new possibilities for a better understanding of survival mechanisms of gametocytes thus contributing to the goal of malaria eradication.
Presently, there are only two P. vivax subunit vaccine candidates undergoing clinical trials and a modest number of other candidates are being tested in preclinical trials. Novel approaches are therefore needed to advance vaccines against this species. Reticulocyte-derived exosomes (rex) are 30-100-nm membrane vesicles of endocytic origin released during the maturation of reticulocytes to erythrocytes upon fusion of multivesicular bodies with the plasma membrane. We previously found that immunization of mice with rex from BALB/c mice infected with the reticulocyte-prone non-lethal P.yoelii 17X strain (rexPy), thus resembling P. vivax, in combination with CpG-ODN promoted survival and long lasting protection of mice subsequently challenged with a lethal strain (Martin-Jaular et al., 2011 PLoS One). More recently, we have unveiled the mode-of-action of such vaccination approach. Thus, we show that rexPy-mediated protection is completely lost in splenectomised animals and such protection is achieved after passive transfer of splenocytes obtained from animals immunized with rexPy+CpG. Notably, rexPy immunization of mice induced non-exhausted memory T cell expansion with effector phenotype. Proteomics analysis of rexPy confirmed their reticulocyte origin and demonstrated the presence of parasite antigens. Our studies thus prove, for what we believe is the first time, that rex from reticulocyte-prone malarial infections are able to induce non-exhausted splenic long-lasting memory responses (Martin-Jaular et al., 2016 Front Cell Dev Biol.). The impact of this research area is also several fold: (i) we propose that early presence of non-exhausted effector memory T-cells in the spleen could facilitate the building of a protective and long-lasting immune response against infection; (ii) determining the protein composition of rex from infections will discover new antigens for vaccinations; (iii) these data reinforce the value of exosomes as inter-cellular communicators and modulators of immune responses; (iv) the data provide a rational basis for the development of a new vaccine and delivery platform against vivax malaria.
Transmission Electron microscopy of P. vivax infected reticulocyte showing the release of membrane vesicles (Source: Fernandez-Becerra and del Portillo, unpublished).
4. Development of a continuous in vitro culture system for blood stages of P. vivax
The development of a continuous in vitro culture system for P. vivax that could generate unrestricted numbers of P. vivax blood-stage parasites is among the most important enabling technologies to be developed to advance research on P. vivax. However, this goal faces major inherent biological hurdles, especially due to the requirement for reticulocyte host cells. We thus concentrated efforts in using CD34+ human hematopoietic stem cells (hHSCs) from bone marrow and peripheral blood (not from cord blood as most other groups) for expansion and differentiation to reticulocytes using a novel stromal cell (Fernandez-Becerra et al., 2013 MIOC). We demonstrated that such reticulocytes express surface markers for entrance of malaria parasites, contain adult haemoglobin and are also permissive to invasion by P. vivax. Unfortunately, like all other previous reports using reticulocytes from cord blood, we never achieve exponential growth of the parasite. An alternative approach is being undertaken by several groups trying to immortalize CD34+ hHSCs to obtain lines capable of expanding and differentiating to reticulocytes; thus, providing an unlimited source of these young red cells for invasion assays. We are also trying this approach using different methodologies and in collaboration with different international groups. The development of a continuous in vitro culture system for blood stages of P. vivax will open avenues for reverse genetic approaches, for developing high-throughput assays for drug and vaccine screenings, and for having an unlimited supply of parasites, including gametocytes, to advance different control strategies.
Giemsa stained smears of RBCs derived from PB CD34+ hHSCs infected with Plasmodium falciparum (1) and Plasmodium vivax (2) after 24 h post infection (Source:Fernandez-Becerra et al., MIOC 2013)
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.
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