Search

Genetic variants of SARS-CoV-2 have been emerging and circulating in many places across the world. Rapid detection of these variants is essential since their dissemination can impact transmission rates, diagnostic procedures, disease severity, response to vaccines or patient management. Sanger sequencing has been used as the preferred approach for variant detection among circulating human immunodeficiency and measles virus genotypes. Using primers to amplify a fragment of the SARS-CoV-2 genome encoding part of the Spike protein, we showed that Sanger sequencing allowed us to rapidly detect the introduction and spread of three distinct SARS-CoV-2 variants in two major Brazilian cities. In both cities, after the predominance of variants closely related to the virus first identified in China, the emergence of the P.2 variant was quickly followed by the identification of the P1 variant, which became dominant in less than one month after it was first detected.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other RBP interactions by transferring methyl groups to arginine residues (R-methylation). Herein, we functionally characterized the five predicted PRMTs in

The nucleocapsid (N) and the receptor binding domain (RBD) of the Spike (S) proteins elicit robust antibody and T cell responses either in vaccinated or COVID-19 convalescent individuals. We generated a chimeric protein that comprises the sequences of RBD from S and N antigens (SpiN). SpiN was highly immunogenic and elicited a strong IFNγ response from T cells and high levels of antibodies to the inactivated virus, but no neutralizing antibodies (nAb). Importantly, hamsters and the human Angiotensin Convertase Enzyme-2-transgenic mice immunized with SpiN were highly resistant to challenge with the wild type SARS-CoV-2, as indicated by viral load, clinical outcome, lung inflammation and lethality. This protective immunity was dependent of CD4+ T and CD8+ T cells, but not by transfer of antibody of vaccinated mice. Thus, our experiment provides an example T cell-mediated immunity and reinforce the concept that T cell target antigens other that the S protein maybe considered to improve SARS-CoV-2 vaccines, and eventually circumvent the immune scape by variants.

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are major targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other macromolecular interactions of RBPs by transferring methyl groups to protein arginine residues. Herein, we present the results of a study that functionally characterized the five predicted PRMTs inLeishmania braziliensisby gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that arginine methylation profiles vary amongLeishmaniaspecies and that target protein methylation changes across differentL. braziliensislife cycle stages, with higher PRMT expression in the promastigote stages than in the axenic amastigote stage. Knockout of some of theL. braziliensisPRMTs led to significant changes in global arginine methylation patterns without affecting promastigote axenic growth. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine (MMA) levels, which is mainly catalyzed by PRMT7. Putative targets and/or PRMT-interacting proteins were identified by coimmunoprecipitation using HA-tagged PRMTs, revealing a network of target RBPs and suggesting functional interactions between them and a relevant participation in epigenetic control of gene expression. Finally, we demonstrate thatL. braziliensisPRMT1 and PRMT5 are required for efficient macrophage infectionin vitro, and that in the absence of PRMT1 and PRMT5, axenic amastigote proliferation is impaired. The results indicate that arginine methylation is modulated across life cycle stages inL. braziliensisand show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout theL. braziliensislife cycle, showing that arginine methylation is important for parasite-host cell interactions.

In Brazil, cutaneous leishmaniasis is caused predominantly by L. (V.) braziliensis. The few therapeutic drugs available exhibit several limitations, mainly related to drug toxicity and reduced efficacy in some regions. Miltefosine (MF), the only oral drug available for leishmaniasis treatment, is not widely available and has not yet been approved for human use in Brazil. Our group previously reported the existence of differential susceptibility among L. (V.) braziliensis clinical isolates. In this work, we further characterized three of these isolates of L. (V.) braziliensis chosen because they exhibited the lowest and the highest MF half maximal inhibitory concentrations and were therefore considered less tolerant or more tolerant, respectively. Uptake of MF, and also of phosphocholine, were found to be significantly different in more tolerant parasites compared to the less sensitive isolate, which raised the hypothesis of differences in the MF transport complex Miltefosine Transporter (MT)-Ros3. Although some polymorphisms in those genes were found, they did not correlate with the drug susceptibility phenotype. Drug efflux and compartmentalization were similar in the isolates tested, and amphotericin B susceptibility was retained in MF tolerant parasites, suggesting that increased fitness was also not the basis of observed differences. Transcriptomic analysis revealed that Ros3 mRNA levels were upregulated in the sensitive strain compared to the tolerant ones. Increased mRNA abundance in more tolerant isolates was validated by quantitative PCR. Our results suggest that differential gene expression of the MT transporter complex is the basis of the differential susceptibility in these unselected, naturally occurring parasites., Graphical abstract Image 1, Highlights • Brazilian L. (V.) braziliensis isolates vary in mitefosine susceptibility. • Diminished drug internalization was observed in more tolerant isolates. • Drug susceptibility did not correlate with SNPs in MT-Ros3 genes. • Drug efflux and compartmentalization were similar in the isolates tested. • Increased drug sensitivity is accompanied by Ros3 mRNA upregulation.

Leishmania major is the main causative agent of cutaneous leishmaniasis in the Old World. In Leishmania parasites, the lack of transcriptional control is mostly compensated by post-transcriptional mechanisms. Methylation of arginine is a conserved post-translational modification executed by Protein Arginine Methyltransferase (PRMTs). The genome from L. major encodes five PRMT homologs, including the cytosolic protein associated with several RNA-binding proteins, LmjPRMT7. It has been previously reported that LmjPRMT7 could impact parasite infectivity. In addition, a more recent work has clearly shown the importance of LmjPRMT7 in RNA-binding capacity and protein stability of methylation targets, demonstrating the role of this enzyme as an important epigenetic regulator of mRNA metabolism. In this study, we unveil the impact of PRMT7-mediated methylation on parasite development and virulence. Our data reveals that higher levels of LmjPRMT7 can impair parasite pathogenicity, and that deletion of this enzyme rescues the pathogenic phenotype of an attenuated strain of L. major. Interestingly, lesion formation caused by LmjPRMT7 knockout parasites is associated with an exacerbated inflammatory reaction in the tissue correlated with an excessive neutrophil recruitment. Moreover, the absence of LmjPRMT7 also impairs parasite development within the sand fly vector Phlebotomus duboscqi. Finally, a transcriptome analysis shed light onto possible genes affected by depletion of this enzyme. Taken together, this study highlights how post-transcriptional regulation can affect different aspects of the parasite biology., Author summary Understanding the genetics of Leishmania, a protozoan parasite causing leishmaniasis, is relevant for understanding fundamental questions on the pathogen’s biology and its interaction with hosts. We explore mechanisms used by Leishmania to promptly adapt to different hosts investigating the control of gene expression occurring at the post-transcriptional level in the parasite. Methylation of arginine performed by Protein Arginine Methyltransferase (PRMTs), among other post-translational modifications, may alter the function and interactions of target proteins, some of them are RNA binding proteins, known regulators of gene expression. In this study, we unveil the impact of PRMT7 on parasite development and pathogenicity. In addition to a negative correlation between the levels of LmjPRMT7 and parasite pathogenicity, we observed an impairment of the parasite development in the sand fly vector. Remarkably, despite a severe lesion development in mice, we observed no differences in parasite burden between infections with the pathogenic LmjPRMT7 knockout parasite or the attenuated parental line. Instead, the severe pathology observed is associated with an exacerbated inflammatory response correlated with excessive neutrophil recruitment.

Trypanosoma cruzi trypomastigotes adhere to extracellular matrix (ECM) to invade mammalian host cells regulating intracellular signaling pathways. Herein, resin-assisted enrichment of thiols combined with mass spectrometry were employed to map site-specific S-nitrosylated (SNO) proteins from T. cruzi trypomastigotes incubated (MTy) or not (Ty) with ECM. We confirmed the reduction of S-nitrosylation upon incubation with ECM, associated with a rewiring of the subcellular distribution and intracellular signaling pathways. Forty, 248 and 85 SNO-peptides were identified only in MTy, Ty or in both conditions, respectively. SNO proteins were enriched in ribosome, transport, carbohydrate and lipid metabolisms. Nitrosylation of histones H2B and H3 on Cys64 and Cys126, respectively, is described. Protein-protein interaction networks revealed ribosomal proteins, proteins involved in carbon and fatty acid metabolism to be among the enriched protein complexes. Kinases, phosphatases and enzymes involved in the metabolism of carbohydrates, lipids and amino acids were identified as nitrosylated and phosphorylated, suggesting a post-translational modifications crosstalk. In silico mapping of nitric oxide synthase (NOS) genes, previously uncharacterized, matched to four putative T. cruzi proteins expressing C-terminal NOS domain. Our results provide the first site-specific characterization of S-nitrosylated proteins in T. cruzi and their modulation upon ECM incubation before infection of the mammalian hosts. SIGNIFICANCE: Protein S-nitrosylation represents a major molecular mechanism for signal transduction by nitric oxide. We present for the first time a proteomic profile of S-nitrosylated proteins from infective forms of T. cruzi, showing a decrease in SNO proteins after incubation of the parasite with the extracellular matrix, a necessary step for the parasite invasion of the host mammalian cells. We also show for the first time nitrosylation of H2B (Cys64) and H3 (Cys126) histones, sites not conserved in higher eukaryotic cells, and suggest that some specific histone isoforms are sensitive to NO signaling. S-nitrosylation in H2B and H3 histones are more abundant in MTy. Moreover, proteins involved in translation, glycolytic pathway and fatty acid metabolism are enriched in the present dataset. Comparison of the SNO proteome and the phosphoproteome, obtained previously under the same experimental conditions, show that most of the proteins sharing both modifications are involved in metabolic pathways, transport and ribosome function. The data suggest that both PTMs are involved in reprogramming the metabolism of T. cruzi in response to environmental changes. Although NO synthesis was detected in T. cruzi, the identification of NOS remains elusive. Analysis in silico showed two genes similar in domains to NADPH-dependent cytochrome-P450 reductase and two putative oxidoreductases, but no oxygenase domain of NOS was mapped in the T. cruzi genome. It is tempting to speculate that NO synthase-like from T. cruzi and its early NO-mediated pathways triggered in response to host interaction constitute potential diagnostic and therapeutic targets.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions., Author summary T. cruzi, the etiological agent of Chagas’ disease, interacts with ECM in order to invade cells and establish the infection in the mammalian host. Studying this interaction, our group has recently described changes in the nitration profile of histones upon incubation of the parasite with ECM. Herein we used a proteomic approach to identify nitrated DNA binding proteins in T. cruzi. We also used ChIP-Seq to investigate whether the contact of the parasite with host ECM is capable of promoting changes in the DNA binding pattern of nitrated proteins. We observed significant alterations in the nitration levels and DNA binding profile of T. cruzi histones upon parasite incubation with ECM.

Leishmaniasis is a worldwide public health problem caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the most important species responsible for tegumentary leishmaniases in Brazil. An understanding of the molecular mechanisms underlying the success of this parasite is urgently needed. An in-depth study on the modulation of gene expression across the life cycle stages of L. braziliensis covering coding and noncoding RNAs (ncRNAs) was missing and is presented herein. Analyses of differentially expressed (DE) genes revealed that most prominent differences were observed between the transcriptomes of insect and mammalian proliferative forms (6,576 genes). Gene ontology (GO) analysis indicated stage-specific enriched biological processes. A computational pipeline and 5 ncRNA predictors allowed the identification of 11,372 putative ncRNAs. Most of the DE ncRNAs were found between the transcriptomes of insect and mammalian proliferative stages (38%). Of the DE ncRNAs, 295 were DE in all three stages and displayed a wide range of lengths, chromosomal distributions and locations; many of them had a distinct expression profile compared to that of their protein-coding neighbors. Thirty-five putative ncRNAs were submitted to northern blotting analysis, and one or more hybridization-positive signals were observed in 22 of these ncRNAs. This work presents an overview of the L. braziliensis transcriptome and its adjustments throughout development. In addition to determining the general features of the transcriptome at each life stage and the profile of protein-coding transcripts, we identified and characterized a variety of noncoding transcripts. The novel putative ncRNAs uncovered in L. braziliensis might be regulatory elements to be further investigated.

Trypanosoma cruzi, the etiological agent of Chagas’ disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host., Author summary Adhesion of Trypanosoma cruzi to distinct elements of ECM involving different surface proteins from the infective stage of the parasite has been described. Despite the relevance of ECM for T. cruzi infection, the signaling pathways triggered in trypomastigotes upon interactions with ECM are less well understood. In previous work we demonstrated the dephosphorylation of proteins, such as α-tubulin, paraflagellar rod proteins and ERK 1/2 in trypomastigotes incubated with either laminin or fibronectin. Further, we described changes in the S-nitrosylation and nitration pattern of proteins from trypomastigote incubated with ECM. To expand our knowledge on ECM triggered parasite signaling we applied quantitative proteomic and phosphoproteomic studies to trypomastigotes incubated with ECM (MTy) compared to controls (Ty). Our results indicate relevant changes in total protein and phosphoprotein profiles in MTy. The kinases implicated in the modifications were suggested by bioinformatic analyses, as well as the number of modifications and the frequency of amino acids per peptide that have been modified. Proteins involved in metabolic processes, including enzymes from the glycolytic pathway, phosphatases and kinases were the most representative groups among the proteins modified by phosphorylation. Quantification of metabolites in MTy and Ty also indicated that glucose metabolism is impaired in trypomastigotes incubated with ECM. The significant inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy associated with phosphorylation levels, strongly suggests that trypomastigotes reprogram their metabolism in response to interaction with the extracellular matrix, an obligatory step prior to host cell invasion.

The serodiagnosis of human tegumentary leishmaniasis (TL) presents some problems, such as the low level of antileishmanial antibodies found in most of the patients, as well as the cross-reactivity in subjects infected by other trypanosomatids. In the present study, an immunoproteomic approach was performed aimed at identification of antigens in total extracts of stationaryphase promastigote and amastigote-like forms of Leishmania (Viannia) braziliensis using sera from TL patients. With the purpose of reducing the cross-reactivity of the identified proteins, spots recognized by sera from TL patients, as well as those recognized by antibodies present in sera from noninfected patients living in areas where TL is endemic and sera from Chagas disease patients, were discarded. Two Leishmania hypothetical proteins and 18 proteins with known functions were identified as antigenic. The study was extended with some of them to validate the results of the immunoscreening. The coding regions of five of the characterized antigens (enolase, tryparedoxin peroxidase, eukaryotic initiation factor 5a, β-tubulin, and one of the hypothetical proteins) were cloned in a prokaryotic expression vector, and the corresponding recombinant proteins were purified and evaluated for the serodiagnosis of TL. The antigens presented sensitivity and specificity values ranging from 95.4 to 100% and 82.5 to 100%, respectively. As a comparative antigen, a preparation of Leishmania extract showed sensitivity and specificity values of 65.1 and 57.5%, respectively. The present study has enabled the identification of proteins able to be employed for the serodiagnosis of TL., Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-Nanobiofar), FAPEMIG (CBB-APQ-0496-11 and CBB-APQ-00819-12), and CNPq (APQ-472090/2011-9, APQ-482976/2012-8, and APQ-488237/2013-0). In addition, this study was partially funded in Madrid by a Spanish grant from Ministerio de Economía y Competitividad-FEDER (FISPI14/00366 from the Instituto de Salud Carlos III)

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior As leishmanioses são doenças que afetam indivíduos em cinco continentes, sendo endêmicas em 98 países, incluindo o Brasil. A cada ano são reportados oficialmente 58.000 novos casos de leishmaniose visceral (LV) e 220.000 novos casos de leishmaniose cutânea. A espécie Leishmania amazonensis é um dos agentes causadores das leishmanioses, podendo levar a uma ampla variedade de manifestações clínicas da doença. O objetivo desse estudo foi analisar a relação entre o cultivo axênico de L. amazonensis por longos períodos de tempo e a variação da capacidade infectiva dos parasitas, no intuito de identificar proteínas que possam estar envolvidas nesse processo. Para isso, parasitas em fase estacionária foram cultivados por 150 dias e amostras foram coletadas a cada 50 dias. As amostras foram utilizadas para avaliar a infecciosidade dos parasitas por meio de experimentos in vitro e in vivo, e para uma caracterização proteômica. Em relação aos resultados obtidos, após a realização da eletroforese bidimensional, 315 spots apresentaram variação de intensidade ao longo do cultivo, sendo que 258 spots foram identificados por espectrometria de massa. Das 56 proteínas selecionadas, 37 e 19 apresentaram diminuição ou aumento significativo de sua expressão, respectivamente, ao longo do cultivo. Dessas proteínas, cinco tiveram sua variação de expressão validada por Immunoblotting. As análises da eletroforese bidimensional associada com a espectrometria de massas e estudos por bioinformática indicaram que a diminuição de infecciosidade em parasitas cultivados por longos períodos sob condições axênicas pode ser ocasionada pelo controle de vias metabólicas específicas, da atuação de proteínas envolvidas no sistema redox do parasita, ou de proteínas do citoesqueleto. As informações apresentadas nesse estudo podem contribuir para um melhor entendimento dos processos biológicos envolvidos com a diminuição da infecciosidade de L. amazonensis quando submetida a culturas axênicas por longos períodos de tempo. Essas descobertas representam uma contribuição para o descobrimento de novos candidatos vacinais, agentes para diagnóstico e/ou alvos imunoterapêuticos contra as leishmanioses, usando as proteínas conhecidas e/ou hipotéticas que apresentaram variação significativa de sua expressão ao longo das culturas axênicas. Leishmaniasis is an endemic disease that affects people in five continents and 98 countries, including Brazil, being officially reported 58,000 new cases of visceral leishmaniasis and 220,000 new cases of cutaneous leishmaniasis each year. Leishmania amazonensis , a causative agent of leishmaniasis, can cause a wide variety of clinical manifestations of the disease. The aim of this study was to analyzethe relationship between the L. amazonensis axenic cultivation for long periods with the infectivity ability of the parasite in order to identify proteins that may be involved in this process. For that purpose, L. amazonensis stationary phase was grown for 150 days and aliquots were collected every 50 days, been used to analysis of of infectivity of the parasite by in vitro and in vivo experiments, and to proceed with proteomic characterization. After 2-DE, from the 315 spots with variation in intensity throughout the cultivation, 258 were identified by mass spectrometry. From the 56 selected proteins, 37 have possible decreased of expression over the passages and 19 different proteins showed possible increased of expression during the parasite cultivation. The expression variation of five of these proteins was validated by Immunoblotting. Twodimensional electrophoresis associated with mass spectrometry and bioinformatics analyzes showed that loss of infectivity observed in cultured parasites for prolonged periods under axenic conditions may be caused by the control of specific metabolic pathways, as well as by the control of the performance of the proteins involved with the parasite redox system or by changes in cytoskeletal proteins. The information presented in this study may help to a better understanding of biological processes involved in the reduction of infectivity of L. amazonensis when subjected to axenic cultures for long periods of time. Moreover, these findings may represent a contribution to the elucidation of novel vaccine candidates, diagnostic agents and / or immunotherapeutic targets against the parasite-induced disease, using known and hypothetical proteins with significant changes in their expression along the axenic cultures.

Background:The present study analyzed whether or not the in vitro cultivation for long periods of time of pre-isolated Leishmania amazonensis from lesions of chronically infected BALB/c mice was able to interfere in the parasites' infectivity using in vivo and in vitro experiments. In addition, the proteins that presented a significant decrease or increase in their protein expression content were identified applying a proteomic approach.Methodology/Principal Findings:Parasites were cultured in vitro for 150 days. Aliquots were collected on the day 0 of culture (R0), as well as after ten (R10; 50 days of culture), twenty (R20; 100 days of culture), and thirty (R30; 150 days of culture) passages, and were used to analyze the parasites' in vitro and in vivo infectivity, as well as to perform the proteomic approach. Approximately 837, 967, 935, and 872 spots were found in 2-DE gels prepared from R0, R10, R20, and R30 samples, respectively. A total of 37 spots presented a significant decrease in their intensity of expression, whereas a significant increase in protein content during cultivation could be observed for 19 proteins (both cases >2.0 folds). Some of these identified proteins can be described, such as diagnosis and/or vaccine candidates, while others are involved in the infectivity of Leishmania. It is interesting to note that six proteins, considered hypothetical in Leishmania, showed a significant decrease in their expression and were also identified.Conclusions/Significance:The present study contributes to the understanding that the cultivation of parasites over long periods of time may well be related to the possible loss of infectivity of L. amazonensis. The identified proteins that presented a significant decrease in their expression during cultivation, including the hypothetical, may also be related to this loss of parasites' infectivity, and applied in future studies, including vaccine candidates and/or immunotherapeutic targets against leishmaniasis. © 2014 Magalhães et al., Instituto Nacional de Ciência e Tecnologia em NanoBiofarmacéutica, FAPEMIG (CBB-APQ-00496-11 and CBB-APQ-00819-12), and CNPq (472090/2011-9 and 482976/2012-8);This study was also, in part, supported in Spain by grants from Ministerio de Ciencia e Innovación FIS/PI1100095

Visceral leishmaniasis (VL) is a neglected disease and is fatal if untreated. Dogs serve as reservoirs for Leishmania infantum (syn. L. chagasi) due to their susceptibility to infection and high skin parasitism. Therefore, VL control in Brazil involves the elimination of seropositive dogs, among other actions. However, the most frequently used serological tests have limitations regarding sensitivity and specificity. In this study, we have selected three Leishmania antigens (C1, C8 and C9) and have produced them as recombinant proteins using pET-28a-TEV vector and Escherichia coli BL-21 as expression system. When tested in ELISA with human samples, the C9 antigen was the one showing the most promising results, with 68% sensitivity and 78% specificity. When testing canine samples, the C1, C8 and C9 antigens showed a sensitivity range from 70% to 80% and specificity range from 60% to 90%. The C1 antigen presented higher sensitivity (80%) and the C8 antigen presented higher specificity (90%). Due to it, we decided to mix and test C1 and C8 antigens together, resulting in the C18 antigen. The mix also yielded high percentages of detected symptomatic and asymptomatic dogs however it did not improve the performance of the diagnostic. Comparison of our tests with the tests recommended by the Brazilian Ministry of Health revealed that our antigens' sensitivities and the percentage of detected asymptomatic dogs were much higher. Our results suggest that the C1, C8, C18 and C9 recombinant proteins are good antigens to diagnose canine visceral leishmaniasis and could potentially be used in screening tests. To diagnose human visceral leishmaniasis, the C9 antigen presented reasonable results, but more optimization must be performed for this antigen to provide better performance.