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Abstract Background Subtelomeric multigene families of malaria parasites encode virulent determinants. The published genome sequence of Plasmodium vivax revealed the largest subtelomeric multigene family of human malaria parasites, the vir super-family, presently composed of 346 vir genes subdivided into 12 different subfamilies based on sequence homologies detected by BLAST. Results A novel computational approach was used to redefine vir genes. First, a protein-weighted graph was built based on BLAST alignments. This graph was processed to ensure that edge weights are not exclusively based on the BLAST score between the two corresponding proteins, but strongly dependant on their graph neighbours and their associations. Then the Markov Clustering Algorithm was applied to the protein graph. Next, the Homology Block concept was used to further validate this clustering approach. Finally, proteome-wide analysis was carried out to predict new VIR members. Results showed that (i) three previous subfamilies cannot longer be classified as vir genes; (ii) most previously unclustered vir genes were clustered into vir subfamilies; (iii) 39 hypothetical proteins were predicted as VIR proteins; (iv) many of these findings are supported by a number of structural and functional evidences, sub-cellular localization studies, gene expression analysis and chromosome localization (v) this approach can be used to study other multigene families in malaria. Conclusions This methodology, resource and new classification of vir genes will contribute to a new structural framing of this multigene family and other multigene families of malaria parasites, facilitating the design of experiments to understand their role in pathology, which in turn may help furthering vaccine development.

Abstract Background Plasmodium vivax has traditionally been considered virtually absent from Western and Central Africa, due to the absence of the Duffy blood group in most of the population living in these areas. Recent reports, however, suggest the circulation of P. vivax in sub-Saharan Africa. Methods Giemsa/Field-stained smears from febrile patients recruited in five different cities (Goundam, Tombouctou, Gao, Bourem and Kidal) pertaining to three regions from Northern Mali were examined. Nested-PCR and DNA sequence analyses of selected samples were performed to fully confirm the presence of P. vivax infections. Results Results demonstrated the presence of P. vivax infections in close to 30% of the cases as detected by Giemsa/Field-stained smears and nested-PCR and DNA-sequence analyses of selected samples unequivocally confirmed the presence of P. vivax. Conclusions The diagnostics of this human malaria parasite should be taken into account in the context of malaria control and elimination efforts, not only in Mali, but also in sub-Saharan Africa.

Abstract Background In vitro cell-free systems for protein expression with extracts from prokaryotic (Escherichia coli) or eukaryotic (wheat germ) cells coupled to solid matrices have offered a valid approach for antigen discovery in malaria research. However, no comparative analysis of both systems is presently available nor the usage of suspension array technologies, which offer nearly solution phase kinetics. Methods Five Plasmodium vivax antigens representing leading vaccine candidates were expressed in the E. coli and wheat germ cell-free systems at a 50 μl scale. Products were affinity purified in a single-step and coupled to luminex beads to measure antibody reactivity of human immune sera. Results Both systems readily produced detectable proteins; proteins produced in wheat germ, however, were mostly soluble and intact as opposed to proteins produced in E. coli, which remained mostly insoluble and highly degraded. Noticeably, wheat germ proteins were recognized in significantly higher numbers by sera of P. vivax patients than identical proteins produced in E. coli. Conclusions The wheat germ cell-free system offers the possibility of expressing soluble P. vivax proteins in a small-scale for antigen discovery and immuno-epidemiological studies using suspension array technology.

Abstract Background Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. Methods Glutathione S-transferase (GST) and GST-fusion proteins representing the N- terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation. Results The results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay (ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea (PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG. Conclusions This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.

Abstract Background There are increasing reports of severe clinical cases exclusively associated with Plasmodium vivax infections. Notably, this severity has been recently suggested to be associated with chloroquine resistance. Patients Two different patients presented at the Hospital Clinic in Barcelona with P. vivax malaria episodes. One patient had severe symptoms and the other mild symptoms. Both patients traveled through the Brazilian Amazon (Manaus) in 2007. For both patients the current diagnosis of malaria was the first. Two other patients with mild symptoms presented to the "Centro de Pesquisa em Medicina Tropical", also in the Brazilian Amazon (Rondônia) in 2000. Methods To exclude the possibility that the patient's severe symptoms were due to Plasmodium falciparum, a nested PCR was performed. A magnetic method was used to purify P. vivax free of human leukocytes. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters likely to be involved in chloroquine resistance in P. vivax, namely the P. vivax chloroquine resistance transporter, pvcrt-o, and the P. vivax multidrug resistance transporter, pvmdr 1. Results Results demonstrated that the severe clinical symptoms were exclusively due to P. vivax. The patient presented acute respiratory conditions requiring admission to the intensive care unit. The magnetic method showed highly purified infected-reticulocytes with mature stages. In addition, it was found that parasites obtained from the severe patient had up to 2.9-fold increase in pvmdr1 levels and up to 21.9-fold increase in pvcrt-o levels compared to expression levels of parasites from the other patients with mild symptoms. Conclusion This is the first clinical case of severe disease exclusively associated with vivax malaria in Spain. Moreover, these findings suggest that clinical severity could be associated with increased expression levels of parasite genes likely involved in chloroquine resistance. It is necessary to further explore the potential of pvmdr1 and particularly pvcrt-o expression levels as molecular markers of severe disease in P. vivax.

Abstract Background The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil. Methods Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection. Results The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals. Conclusion This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.

Abstract Background Plasmodium vivax is the most widely distributed human malaria, responsible for 70–80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. Methods A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of -30 was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology Results A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. Conclusion These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

Extracellular vesicles (EVs) released by virus-infected cells have the potential to encapsulate viral peptides, a characteristic that could facilitate vaccine development. Furthermore, plasma-derived EVs may elucidate pathological changes occurring in distal tissues during viral infections. We hypothesized that molecular characterization of EVs isolated from COVID-19 patients would reveal peptides suitable for vaccine development. Blood samples were collected from three cohorts: severe COVID-19 patients (G1), mild/asymptomatic cases (G2), and SARS-CoV-2-negative healthcare workers (G3). Samples were obtained at two time points: during the initial phase of the pandemic in early 2020 (m0) and eight months later (m8). Clinical data analysis revealed elevated inflammatory markers in G1. Notably, non-vaccinated individuals in G1 exhibited increased levels of neutralizing antibodies at m8, suggesting prolonged exposure to viral antigens. Proteomic profiling of EVs was performed using three distinct methods: immunocapture (targeting CD9), ganglioside-capture (utilizing Siglec-1) and size-exclusion chromatography (SEC). Contrary to our hypothesis, this analysis failed to identify viral peptides. These findings were subsequently validated through Western blot analysis targeting the RBD of the SARS-CoV-2 Spike protein's and comparative studies using samples from experimentally infected Syrian hamsters. Furthermore, analysis of the EV cargo revealed a diverse molecular profile, including components involved in the regulation of viral replication, systemic inflammation, antigen presentation, and stress responses. These findings underscore the potential significance of EVs in the pathogenesis and progression of COVID-19., Competing Interests: HP, MMo, and LF are shareholders of Innovex Therapeutics. SM-T was a former employee of Innovex Therapeutics. JM-P has received institutional grants and educational/consultancy fees from AbiVax; AstraZeneca; Gilead Sciences; Grifols; Janssen; Merck Sharp & Dohme; and ViiV Healthcare; all outside the submitted work. JC and JB are shareholders of Albajuna Therapeutics SL, NI-U reports institutional grants from Grifols, Dentaid, Hipra and Amassence. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Gualdrón-López, Ayllon-Hermida, Cortes-Serra, Resa-Infante, Bech-Serra, Aparici-Herraiz, Nicolau-Fernandez, Erkizia, Gutierrez-Chamorro, Marfil, Pradenas, Ávila Nieto, Cucurull, Montaner-Tarbés, Muelas, Sotil, Ballana, Urrea, Fraile, Montoya, Vergara, Segales, Carrillo, Izquierdo-Useros, Blanco, Fernandez-Becerra, de La Torre, Pinazo, Martinez-Picado and del Portillo.)

Recent studies indicate that human spleen contains over 95% of the total parasite biomass during chronic asymptomatic infections caused by Plasmodium vivax . Previous studies have demonstrated that extracellular vesicles (EVs) secreted from infected reticulocytes facilitate binding to human spleen fibroblasts (hSFs) and identified parasite genes whose expression was dependent on an intact spleen. Here, we characterize the P. vivax spleen-dependent hypothetical gene (PVX_114580). Using CRISPR/Cas9, PVX_114580 was integrated into P. falciparum 3D7 genome and expressed during asexual stages. Immunofluorescence analysis demonstrated that the protein, which we named P. vivax Spleen-Dependent Protein 1 (PvSDP1), was located at the surface of infected red blood cells in the transgenic line and this localization was later confirmed in natural infections. Plasma-derived EVs from P. vivax -infected individuals (PvEVs) significantly increased cytoadherence of 3D7_PvSDP1 transgenic line to hSFs and this binding was inhibited by anti-PvSDP1 antibodies. Single-cell RNAseq of PvEVs-treated hSFs revealed increased expression of adhesion-related genes. These findings demonstrate the importance of parasite spleen-dependent genes and EVs from natural infections in the formation of intrasplenic niches in P. vivax , a major challenge for malaria elimination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Ayllon-Hermida, Nicolau-Fernandez, Larrinaga, Aparici-Herraiz, Tintó-Font, Llorà-Batlle, Orban, Yasnot, Graupera, Esteller, Popovici, Cortés, del Portillo and Fernandez-Becerra.)

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells., Competing Interests: No potential conflicts of interest were reported by the authors., (© 2023 The Authors. Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.)

Dogs are highly valued companions and work animals that are susceptible to many life-threatening conditions such as canine leishmaniosis (CanL). Plasma-derived extracellular vesicles (EVs), exploited extensively in biomarker discovery, constitute a mostly untapped resource in veterinary sciences. Thus, the definition of proteins associated with plasma EVs recovered from healthy and diseased dogs with a relevant pathogen would be important for biomarker development. For this, we recovered, using size-exclusion chromatography (SEC), EVs from 19 healthy and 20 CanL dogs' plasma and performed proteomic analysis by LC-MS/MS to define their core proteomic composition and search for CanL-associated alterations. EVs-specific markers were identified in all preparations and also non-EVs proteins. Some EVs markers such as CD82 were specific to the healthy animals, while others, such as the Integrin beta 3 were identified in most samples. The EVs-enriched preparations allowed the identification of 529 canine proteins that were identified in both groups, while 465 and 154 were only identified in healthy or CanL samples, respectively. A GO enrichment analysis revealed few CanL-specific terms. Leishmania spp. protein identifications were also found, although with only one unique peptide. Ultimately, CanL-associated proteins of interest were identified and a core proteome was revealed that will be available for intra- and inter-species comparisons.

Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs., Competing Interests: Conflict of interest V. C., S. A. M., and E. L. F. are employed by and/or is a shareholder of Novartis Pharma AG. Other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Plasmodium vivax is the most widely distributed human malaria parasite representing 36.3% of disease burden in the South-East Asia region and the most predominant species in the region of the Americas. Recent estimates indicate that 3.3 billion of people are under risk of infection with circa 7 million clinical cases reported each year. This burden is certainly underestimated as the vast majority of chronic infections are asymptomatic. For centuries, it has been widely accepted that the only source of cryptic parasites is the liver dormant stages known as hypnozoites. However, recent evidence indicates that niches outside the liver, in particular in the spleen and the bone marrow, can represent a major source of cryptic chronic erythrocytic infections. The origin of such chronic infections is highly controversial as many key knowledge gaps remain unanswered. Yet, as parasites in these niches seem to be sheltered from immune response and antimalarial drugs, research on this area should be reinforced if elimination of malaria is to be achieved. Due to ethical and technical considerations, working with the liver, bone marrow and spleen from natural infections is very difficult. Recent advances in the development of humanized mouse models and organs-on-a-chip models, offer novel technological frontiers to study human diseases, vaccine validation and drug discovery. Here, we review current data of these frontier technologies in malaria, highlighting major challenges ahead to study P. vivax cryptic niches, which perpetuate transmission and burden., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aparici Herraiz, Caires, Castillo-Fernández, Sima, Méndez-Mora, Risueño, Sattabongkot, Roobsoong, Hernández-Machado, Fernandez-Becerra, Barrias and del Portillo.)

Plasmodium vivax 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 P. vivax , as a selective disposal mechanism of the transferrin receptor (CD71) in the maturation of reticulocytes to erythrocytes. We have recently reported the proteomics identification of P. vivax proteins associated to circulating EVs in P. vivax 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 P. vivax 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 Plasmodium helical interspersed sub-telomeric (PHIST-c) family and a member of the Plasmodium 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 P. vivax immunogenic proteins. This study represents a significant advance in identifying new antigens for vaccination against this human malaria parasite., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Aparici-Herraiz, Gualdrón-López, Castro-Cavadía, Carmona-Fonseca, Yasnot, Fernandez-Becerra and del Portillo.)

Plasmodium malariae has a wide geographic distribution, but mainly at very low parasitemias and in co-infections, leading to an underestimated prevalence of this species. Studies for the detection of antibodies against Plasmodium recombinant proteins are increasingly used to map geographical distributions, seroprevalence and transmission intensities of malaria infection. However, no seroepidemiological survey using recombinant P. malariae proteins has been conducted in Brazil. This work evaluated the antibody response in serum samples of individuals from endemic regions of Brazil (the Amazon region and Atlantic Forest) against five recombinant proteins of P. malariae merozoite surface protein 1 (MSP1), and the MSP1 C-terminal portions of P. vivax and P. falciparum , in a multiplex assay. The positivity was 69.5% of samples recognizing at least one MSP1 recombinant protein. The mean of the Reactivity Index for the C-terminal portion of the P. falciparum was significantly higher compared to the other recombinant proteins, followed by the C-terminal of P. vivax and the N-terminal of P. malariae . Among the recombinant P. malariae proteins, the N-terminal of P. malariae showed the highest Reactivity Index alone. This study validates the use of the multiplex assay to measure naturally acquired IgG antibodies against Plasmodium MSP1 proteins and demonstrate that these proteins are important tools for seroepidemiological surveys and could be used in malaria surveillance.

Background: A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival., Methods and Findings: We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted., Conclusions: Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen., Competing Interests: The authors have declared that no competing interests exist.

The spleen is a secondary lymphoid organ with multiple functions including the removal of senescent red blood cells and the coordination of immune responses against blood-borne pathogens, such as malaria parasites. Despite the major role of the spleen, the study of its function in humans is limited by ethical implications to access human tissues. Here, we employed multiparameter flow cytometry combined with cell purification techniques to determine human spleen cell populations from transplantation donors. Spleen immuno-phenotyping showed that CD45 + cells included B (30%), CD4 + T (16%), CD8 + T (10%), NK (6%) and NKT (2%) lymphocytes. Myeloid cells comprised neutrophils (16%), monocytes (2%) and DCs (0.3%). Erythrocytes represented 70%, reticulocytes 0.7% and hematopoietic stem cells 0.02%. Extracellular vesicles (EVs) are membrane-bound nanoparticles involved in intercellular communication and secreted by almost all cell types. EVs play several roles in malaria that range from modulation of immune responses to vascular alterations. To investigate interactions of plasma-derived EVs from Plasmodium vivax infected patients (PvEVs) with human spleen cells, we used size-exclusion chromatography (SEC) to separate EVs from the bulk of soluble plasma proteins and stained isolated EVs with fluorescent lipophilic dyes. The integrated cellular analysis of the human spleen and the methodology employed here allowed in vitro interaction studies of human spleen cells and EVs that showed an increased proportion of T cells (CD4+ 3 fold and CD8+ 4 fold), monocytes (1.51 fold), B cells (2.3 fold) and erythrocytes (3 fold) interacting with PvEVs as compared to plasma-derived EVs from healthy volunteers (hEVs). Future functional studies of these interactions can contribute to unveil pathophysiological processes involving the spleen in vivax malaria., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Gualdrón-López, Díaz-Varela, Toda, Aparici-Herraiz, Pedró-Cos, Lauzurica, Lacerda, Fernández-Sanmartín, Fernandez-Becerra and del Portillo.)

Cryptic Plasmodium niches outside the liver possibly represent a major source of hypnozoite-unrelated recrudescences in malaria. Maurizio Ascoli, an Italian physician and scientist, suggested that infection was maintained as a result of the persistence of endoerythrocytic parasites in the circulatory bed of some internal organs, mainly the spleen. This would explain a proportion of the recurrences in patients, regardless of the Plasmodium species. Ascoli proposed a method that included the co-administration of adrenaline, in order to induce splenic contraction, and quinine to clear expelled forms in major vessels. Driven by controversy regarding safety and effectiveness, along with the introduction of new drugs, the Ascoli method was abandoned and mostly forgotten by the malaria research community. To date, however, the existence of cryptic parasites outside the liver is gaining supportive data. This work is a historical retrospective of cryptic malaria infections and the Ascoli method, highlighting key knowledge gaps regarding these possible parasite reservoirs.

Chagas disease is emerging in countries to which it is not endemic. Biomarkers for earlier therapeutic response assessment in patients with chronic Chagas disease are needed. We profiled plasma-derived extracellular vesicles from a heart transplant patient with chronic Chagas disease and showed the potential of this approach for discovering such biomarkers.

Chagas disease is caused by infection with the parasite Trypanosoma cruzi, which might lead to a chronic disease state and drive to irreversible damage to the heart and/or digestive tract tissues. Endemic in 21 countries in the Americas, it is the neglected disease with a highest burden in the region. Current estimates point at ~6 million people infected, of which ~30% will progress onto the symptomatic tissue disruptive stage. There is no vaccine but there are two anti-parasitic drugs available: benznidazole and nifurtimox. However, their efficacy is variable at the chronic symptomatic stage and both have frequent adverse effects. Since there are no prognosis markers, drugs should be administered to all T. cruzi-infected individuals in the indeterminate and early symptomatic stages. Nowadays, there are no tests-of-cure either, which greatly undermines patients follow-up and the search of safer and more efficacious drugs. Therefore, the identification and validation of biomarkers of disease progression and/or treatment response on which to develop tests of prognosis and/or cure is a major research priority. Both parasite- and host-derived markers have been investigated. In the present manuscript we present an updated outlook of the latter., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Non-human primates (NHPs) have been shown to be infected by parasites of the genus Plasmodium , the etiological agent of malaria in humans, creating potential risks of zoonotic transmission. Plasmodium brasilianum , a parasite species similar to P. malariae of humans, have been described in NHPs from Central and South America, including Brazil. The merozoite surface protein 1 (MSP1), besides being a malaria vaccine candidate, is highly immunogenic. Due to such properties, we tested this protein for the diagnosis of parasite infection. We used recombinant proteins of P. malariae MSP1, as well as of P. falciparum and P. vivax , for the detection of antibodies anti-MSP1 of these parasite species, in the sera of NHPs collected in different regions of Brazil. About 40% of the NHP sera were confirmed as reactive to the proteins of one or more parasite species. A relatively higher number of reactive sera was found in animals from the Atlantic Forest than those from the Amazon region, possibly reflecting the former more intense parasite circulation among NHPs due to their proximity to humans at a higher populational density. The presence of Plasmodium positive NHPs in the surveyed areas, being therefore potential parasite reservoirs, needs to be considered in any malaria surveillance program., Competing Interests: The authors declare no conflict of interest.

Plasmodium vivax , the most widely distributed human malaria parasite, causes severe clinical syndromes despite low peripheral blood parasitemia. This conundrum is further complicated as cytoadherence in the microvasculature is still a matter of investigations. Previous reports in Plasmodium knowlesi , another parasite species shown to infect humans, demonstrated that variant genes involved in cytoadherence were dependent on the spleen for their expression. Hence, using a global transcriptional analysis of parasites obtained from spleen-intact and splenectomized monkeys, we identified 67 P. vivax genes whose expression was spleen dependent. To determine their role in cytoadherence, two Plasmodium falciparum transgenic lines expressing two variant proteins pertaining to VIR and Pv-FAM-D multigene families were used. Cytoadherence assays demonstrated specific binding to human spleen but not lung fibroblasts of the transgenic line expressing the VIR14 protein. To gain more insights, we expressed five P. vivax spleen-dependent genes as recombinant proteins, including members of three different multigene families (VIR, Pv-FAM-A, Pv-FAM-D), one membrane transporter (SECY), and one hypothetical protein (HYP1), and determined their immunogenicity and association with clinical protection in a prospective study of 383 children in Papua New Guinea. Results demonstrated that spleen-dependent antigens are immunogenic in natural infections and that antibodies to HYP1 are associated with clinical protection. These results suggest that the spleen plays a major role in expression of parasite proteins involved in cytoadherence and can reveal antigens associated with clinical protection, thus prompting a paradigm shift in P. vivax biology toward deeper studies of the spleen during infections., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Plasmodium vivax is the most widely distributed human malaria parasite. Previous studies have shown that circulating microparticles during P. vivax acute attacks are indirectly associated with severity. Extracellular vesicles (EVs) are therefore major components of circulating plasma holding insights into pathological processes. Here, we demonstrate that plasma-derived EVs from Plasmodium vivax patients (PvEVs) are preferentially uptaken by human spleen fibroblasts (hSFs) as compared to the uptake of EVs from healthy individuals. Moreover, this uptake induces specific upregulation of ICAM-1 associated with the translocation of NF-kB to the nucleus. After this uptake, P. vivax-infected reticulocytes obtained from patients show specific adhesion properties to hSFs, reversed by inhibiting NF-kB translocation to the nucleus. Together, these data provide physiological EV-based insights into the mechanisms of human malaria pathology and support the existence of P. vivax-adherent parasite subpopulations in the microvasculature of the human spleen.

Exosomes are extracellular vesicles of endocytic origin containing molecular signatures implying the cell of origin; thus, they offer a unique opportunity to discover biomarkers of disease. Plasmodium vivax , responsible for more than half of all malaria cases outside Africa, is a major obstacle in the goal of malaria elimination due to the presence of dormant liver stages (hypnozoites), which after the initial infection may reactivate to cause disease. Hypnozoite infection is asymptomatic and there are currently no diagnostic tools to detect their presence. The human liver-chimeric (FRG huHep) mouse is a robust P. vivax infection model for exo-erythrocytic development of liver stages, including hypnozoites. We studied the proteome of plasma-derived exosomes isolated from P. vivax infected FRG huHep mice with the objective of identifying liver-stage expressed parasite proteins indicative of infection. Proteomic analysis of these exosomes showed the presence of 290 and 234 proteins from mouse and human origin, respectively, including canonical exosomal markers. Human proteins include proteins previously detected in liver-derived exosomes, highlighting the potential of this chimeric mouse model to study plasma exosomes derived unequivocally from human hepatocytes. Noticeably, we identified 17 parasite proteins including enzymes, surface proteins, components of the endocytic pathway and translation machinery, as well as uncharacterized proteins. Western blot analysis validated the presence of human arginase-I and an uncharacterized P. vivax protein in plasma-derived exosomes. This study represents a proof-of-principle that plasma-derived exosomes from P. vivax infected FRG-huHep mice contain human hepatocyte and P. vivax proteins with the potential to unveil biological features of liver infection and identify biomarkers of hypnozoite infection.

Plasma-derived vesicles hold a promising potential for use in biomedical applications. Two major challenges, however, hinder their implementation into translational tools: (a) the incomplete characterization of the protein composition of plasma-derived vesicles, in the size range of exosomes, as mass spectrometric analysis of plasma sub-components is recognizably troublesome and (b) the limited reach of vesicle-based studies in settings where the infrastructural demand of ultracentrifugation, the most widely used isolation/purification methodology, is not available. In this study, we have addressed both challenges by carrying-out mass spectrometry (MS) analyses of plasma-derived vesicles, in the size range of exosomes, from healthy donors obtained by 2 alternative methodologies: size-exclusion chromatography (SEC) on sepharose columns and Exo-Spin™. No exosome markers, as opposed to the most abundant plasma proteins, were detected by Exo-Spin™. In contrast, exosomal markers were present in the early fractions of SEC where the most abundant plasma proteins have been largely excluded. Noticeably, after a cross-comparative analysis of all published studies using MS to characterize plasma-derived exosomes from healthy individuals, we also observed a paucity of "classical exosome markers." Independent of the isolation method, however, we consistently identified 2 proteins, CD5 antigen-like (CD5L) and galectin-3-binding protein (LGALS3BP), whose presence was validated by a bead-exosome FACS assay. Altogether, our results support the use of SEC as a stand-alone methodology to obtain preparations of extracellular vesicles, in the size range of exosomes, from plasma and suggest the use of CD5L and LGALS3BP as more suitable markers of plasma-derived vesicles in MS.

Parasitic diseases affect billions of people and are considered a major public health issue. Close to 400 species are estimated to parasitize humans, of which around 90 are responsible for great clinical burden and mortality rates. Unfortunately, they are largely neglected as they are mainly endemic to poor regions. Of relevance to this review, there is accumulating evidence of the release of extracellular vesicles (EVs) in parasitic diseases, acting both in parasite-parasite inter-communication as well as in parasite-host interactions. EVs participate in the dissemination of the pathogen and play a role in the regulation of the host immune systems. Production of EVs from parasites or parasitized cells has been described for a number of parasitic infections. In this review, we provide the most relevant findings of the involvement of EVs in intercellular communication, modulation of immune responses, involvement in pathology, and their potential as new diagnostic tools and therapeutic agents in some of the major human parasitic pathogens.

Molecular markers associated with the increase of chloroquine resistance and disease severity in Plasmodium vivax are needed. The objective of this study was to evaluate the expression levels of pvcrt-o and pvmdr-1 genes in a group of patients presenting CQRPv and patients who developed severe complications triggered exclusively by P. vivax infection. Two different sets of patients were included to this comprehensive study performed in the Brazilian Amazon: 1) patients with clinically characterized chloroquine-resistant P. vivax compared with patients with susceptible parasites from in vivo studies and 2) patients with severe vivax malaria compared with patients without severity. Quantitative real-time PCR was performed to compare the transcript levels of two main transporters genes, P. vivax chloroquine resistance transporter (pvcrt-o) and the P. vivax multidrug resistance transporter (pvmdr-1). Twelve chloroquine resistant cases and other 15 isolates from susceptible cases were included in the first set of patients. For the second set, seven patients with P. vivax-attributed severe and 10 mild manifestations were included. Parasites from patients with chloroquine resistance presented up to 6.1 (95% CI: 3.8-14.3) and 2.4 (95% CI: 0.53-9.1) fold increase in pvcrt-o and pvmdr-1 expression levels, respectively, compared to the susceptible group. Parasites from the severe vivax group had a 2.9 (95% CI: 1.1-8.3) and 4.9 (95% CI: 2.3-18.8) fold increase in pvcrt-o and pvmdr-1 expression levels as compared to the control group with mild disease. These findings suggest that chloroquine resistance and clinical severity in P. vivax infections are strongly associated with increased expression levels of the pvcrt-o and pvmdr-1 genes likely involved in chloroquine resistance.

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34⁺ hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.

Background: In vitro cell-free systems for protein expression with extracts from prokaryotic (Escherichia coli) or eukaryotic (wheat germ) cells coupled to solid matrices have offered a valid approach for antigen discovery in malaria research. However, no comparative analysis of both systems is presently available nor the usage of suspension array technologies, which offer nearly solution phase kinetics., Methods: Five Plasmodium vivax antigens representing leading vaccine candidates were expressed in the E. coli and wheat germ cell-free systems at a 50 μl scale. Products were affinity purified in a single-step and coupled to luminex beads to measure antibody reactivity of human immune sera., Results: Both systems readily produced detectable proteins; proteins produced in wheat germ, however, were mostly soluble and intact as opposed to proteins produced in E. coli, which remained mostly insoluble and highly degraded. Noticeably, wheat germ proteins were recognized in significantly higher numbers by sera of P. vivax patients than identical proteins produced in E. coli., Conclusions: The wheat germ cell-free system offers the possibility of expressing soluble P. vivax proteins in a small-scale for antigen discovery and immuno-epidemiological studies using suspension array technology.

Background: Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic., Methods: Glutathione S-transferase (GST) and GST-fusion proteins representing the N- terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation., Results: The results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay (ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea (PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG., Conclusions: This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.

The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

Background: The naturally-acquired immune response to Plasmodium vivax variant antigens (VIR) was evaluated in individuals exposed to malaria and living in different endemic areas for malaria in the north of Brazil., Methods: Seven recombinant proteins representing four vir subfamilies (A, B, C, and E) obtained from a single patient from the Amazon Region were expressed in Escherichia coli as soluble glutathione S-transferase fusion proteins. The different recombinant proteins were compared by ELISA with regard to the recognition by IgM, IgG, and IgG subclass of antibodies from 200 individuals with patent infection., Results: The frequency of individuals that presented antibodies anti-VIR (IgM plus IgG) during the infection was 49%. The frequencies of individuals that presented IgM or IgG antibodies anti-VIR were 29.6% or 26.0%, respectively. The prevalence of IgG antibodies against recombinant VIR proteins was significantly lower than the prevalence of antibodies against the recombinant proteins representing two surface antigens of merozoites of P. vivax: AMA-1 and MSP119 (57.0% and 90.5%, respectively). The cellular immune response to VIR antigens was evaluated by in vitro proliferative assays in mononuclear cells of the individuals recently exposed to P. vivax. No significant proliferative response to these antigens was observed when comparing malaria-exposed to non-exposed individuals., Conclusion: This study provides evidence that there is a low frequency of individuals responding to each VIR antigens in endemic areas of Brazil. This fact may explain the host susceptibility to new episodes of the disease.

Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. The occurrence of clinical protection in P. vivax malaria in Brazil was first reported among residents of the riverine community of Portuchuelo, in Rondônia, western Amazon. We thus analyzed immune sera from this same human population to determine if naturally acquired humoral immune responses against the merozoite surface protein 1 of P. vivax, PvMSP1, could be associated with reduced risk of infection and/or clinical protection. Our results demonstrated that this association could be established with anti-PvMSP1 antibodies predominantly of the immunoglobulin G3 subclass directed against the N terminus but not against the C terminus, in spite of the latter being more immunogenic and capable of natural boosting. This is the first report of a prospective study of P. vivax malaria demonstrating an association of reduced risk of infection and clinical protection with antibodies against an antigen of this parasite.

Plasmodium vivax is the most widely distributed human malaria parasite and responsible for 70-80 million clinical cases each year and a large socio-economical burden. The sequence of a chromosome end from P. vivax revealed the existence of a multigene superfamily, termed vir (P. vivax variant antigens), that can be subdivided into different subfamilies based on sequence similarity analysis and which represents close to 10-20% of the coding sequences of the parasite. Here we show that there is a vast repertoire of vir genes abundantly expressed in isolates obtained from human patients, that different vir gene subfamilies are transcribed in mature asexual blood stages by individual parasites, that VIR proteins are not clonally expressed and that there is no significant difference in the recognition of VIR-tags by immune sera of first-infected patients compared with sera of multiple-infected patients. These data provide to our knowledge the first comprehensive study of vir genes and their encoding variant proteins in natural infections and thus constitute a baseline for future studies of this multigene superfamily. Moreover, whereas our data are consistent with a major role of vir genes in natural infections, they are inconsistent with a predominant role in the strict sense of antigenic variation.

Background: Plasmodium vivax is the most widely distributed human malaria, responsible for 70-80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected., Methods: A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10(-30) was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology, Results: A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them., Conclusion: These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.

The malarial parasite Plasmodium vivax causes disease in humans, including chronic infections and recurrent relapses, but the course of infection is rarely fatal, unlike that caused by Plasmodium falciparum. To investigate differences in pathogenicity between P. vivax and P. falciparum, we have compared the subtelomeric domains in the DNA of these parasites. In P. falciparum, subtelomeric domains are conserved and contain ordered arrays of members of multigene families, such as var, rif and stevor, encoding virulence determinants of cytoadhesion and antigenic variation. Here we identify, through the analysis of a continuous 155,711-base-pair sequence of a P. vivax chromosome end, a multigene family called vir, which is specific to P. vivax. The vir genes are present at about 600-1,000 copies per haploid genome and encode proteins that are immunovariant in natural infections, indicating that they may have a functional role in establishing chronic infection through antigenic variation.