Sensitive and specific serodiagnosis of Leishmania infantum infection in dogs by using peptides selected from hypothetical proteins identified by an immunoproteomic approach

In Brazil, the percentage of infected dogs living in areas where canine visceral leishmaniasis (CVL) is endemic ranges from 10 to 62%; however, the prevalence of infection in dogs is probably higher than figures reported from serological studies. In addition, problems with the occurrence of false-positive or false-negative results in the serodiagnosis of CVL have been reported. The present work analyzed the potential of synthetic peptides mapped from hypothetical proteins for improvement of the serodiagnosis of Leishmania infantum infection in dogs. From 26 identified leishmanial proteins, eight were selected, considering that no homologies between these proteins and others from trypanosomatide sequence databases were encountered. The sequences of these proteins were mapped to identify linear B-cell epitopes, and 17 peptides were synthesized and tested in enzyme-linked immunosorbent assays (ELISAs) for the serodiagnosis of L. infantum infection in dogs. Of these, three exhibited sensitivity and specificity values higher than 75% and 90%, respectively, to differentiate L. infantum-infected animals from Trypanosoma cruziinfected animals and healthy animals. Soluble Leishmania antigen (SLA) showed poor sensitivity (4%) and specificity (36%) to differentiate L. infantum-infected dogs from healthy and T. cruzi-infected dogs. Lastly, the three selected peptides were combined in different mixtures and higher sensitivity and specificity values were obtained, even when sera from T. cruzi-infected dogs were used. The study’s findings suggest that these three peptides can constitute a potential tool for more sensitive and specific serodiagnosis of L. infantum infection in dogsThis work was supported by grants from the Pró-Reitoria de Pesquisa from UFMG (Edital 07/2012), Instituto Nacional de Ciência e Tecnologia em Nano-biofarmacêutica (INCT-NANOBIOFAR, Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (CBB-APQ-02364-08, CBB-APQ-00356-10, CBB-APQ-00496-11, and CBB-APQ-00819-12), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (APQ-472090/2011-9), and the Instituto Nacional de Ciência e Tecnologia em Vacinas (INCT-V). E.A.F.C. and A.P.F. are CNPq grant recipients. M.A.C.-F. is a FAPEMIG/CAPES grant recipient. This study was supported in Spain, in part, by grants from the Ministerio de Ciencia e Innovación (FIS/PI1100095)

Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic leishmania infantum antigens with potential serodiagnosis applications

Visceral leishmaniasis (VL) is a zoonotic disease that is endemic to Brazil, where dogs are the main domestic parasite reservoirs, and the percentages of infected dogs living in regions where canine VL (CVL) is endemic have ranged from 10% to 62%. Despite technological advances, some problems have been reported with CVL serodiagnosis. The present study describes a sequential subtractive selection through phage display technology from polyclonal antibodies of negative and positive sera that resulted in the identification of potential bacteriophage-fused peptides that were highly sensitive and specific to antibodies of CVL. A negative selection was performed in which phage clones were adhered to purified IgGs from healthy and Trypanosoma cruzi-infected dogs to eliminate cross-reactive phages. The remaining supernatant nonadhered phages were submitted to positive selection against IgG from the blood serum of dogs that were infected with Leishmania infantum. Phage clones that adhered to purified IgGs from the CVL-infected serum samples were selected. Eighteen clones were identified and their reactivities tested by a phage enzyme-linked immunosorbent assay (phage-ELISA) against the serum samples from infected dogs (n 31) compared to those from vaccinated dogs (n 21), experimentally infected dogs with cross-reactive parasites (n 23), and healthy controls (n 17). Eight clones presented sensitivity, specificity, and positive and negative predictive values of 100%, and they showed no crossreactivity with T. cruzi- or Ehrlichia canis-infected dogs or with dogs vaccinated with two different commercial CVL vaccines in Brazil. Our study identified eight mimotopes of L. infantum antigens with 100% accuracy for CVL serodiagnosis. The use of these mimotopes by phage-ELISA proved to be an excellent assay that was reproducible, simple, fast, and inexpensive, and it can be applied in CVL-monitoring programsThis work was supported by grants from the Pró-Reitoria de Pesquisa of UFMG (supported 03/2013), the Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica (INCT Nano-Biofar), Rede Nanobiotec/Brasil-UFU (CAPES), PRONEX-FAPEMIG (APQ-01019- 09), FAPEMIG (APQ-00496-11 and APQ-00819-12), and CNPq (APQ- 472090/2011-9 and APQ-482976/2012-8). E.A.F.C. and L.R.G. are recipients of grants from CNPq. M.A.C.-F. is the recipient of a grant from FAPEMIG/CAPE

Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis.

Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-? (IFN-?), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-? (TNF-?), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-? and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-?, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-? production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease

Biotecnologia de phage display aplicada para o desenvolvimento de uma vacina contra as leishmanioses e novas plataformas de diagnóstico sorológico

Submitted by Lourena Costa ([email protected]) on 2020-01-20T15:15:49Z No. of bitstreams: 1 Tese Lourena Costa 2016.pdf: 1689404 bytes, checksum: 311aca3e0681f14a49ab119078e9269a (MD5)Rejected by Eliane Araujo ([email protected]), reason: Prezada Lourena, Conforme as “Diretrizes para Normalização de Trabalhos Acadêmicos da UFMG” detectamos que no Arquivo de sua Tese de DOUTORADO não consta a Ata de Defesa devidamente assinada. Considerando a visibilidade nacional e internacional do RI/UFMG, os trabalhos disponibilizados devem seguir padrões e protocolos de integração qualificados e normalizados. Diante disso, gentileza executar as inserções devidas acessando o link abaixo. Qualquer dúvida, estamos à disposição. Atenciosamente, Eliane Araujo Comunidade - TRABALHOS ACADÊMICOS 3409.4625 - 4620 - 5513 on 2020-01-20T15:45:29Z (GMT)Submitted by Lourena Costa ([email protected]) on 2020-01-20T18:46:54Z No. of bitstreams: 1 tese lourena 2016.pdf: 8662080 bytes, checksum: 353640f037809ba668bc42c5be494a88 (MD5)Approved for entry into archive by Eliane Araujo ([email protected]) on 2020-01-20T19:17:12Z (GMT) No. of bitstreams: 1 tese lourena 2016.pdf: 8662080 bytes, checksum: 353640f037809ba668bc42c5be494a88 (MD5)Made available in DSpace on 2020-01-21T10:28:46Z (GMT). No. of bitstreams: 1 tese lourena 2016.pdf: 8662080 bytes, checksum: 353640f037809ba668bc42c5be494a88 (MD5) Previous issue date: 2016-02-19As leishmanioses são doenças que apresentam uma elevada incidência no Brasil. Em modelos murinos, após a cura da doença causada por Leishmania major, os animais adquirem imunidade duradoura contra a reinfecção. Tal fato tem estimulado a realização de trabalhos objetivando o desenvolvimento de vacinas que possam atuar como uma medida efetiva de controle da doença. Com o avanço das ferramentas biotecnológicas, a pesquisa por novos antígenos, mais refinados, vem se mostrando como uma boa perspectiva para o desenvolvimento de novos produtos. A tecnologia de phage display baseia-se na expressão de peptídeos exógenos na superfície externa de clones de bacteriófagos (fagos) e permite a seleção daqueles que apresentam elevada especificidade para uma molécula alvo. Neste projeto, a técnica de phage display foi utilizada com a finalidade de se selecionar fagos que expressam peptídeos com elevada reatividade frente aos anticorpos IgG purificados de soros de cães com leishmaniose visceral (LV) sintomática e assintomática a fim de desenvolver uma vacina. Vinte clones de fagos foram selecionados após três ciclos de bio-pannings e foram avaliados por meio de ensaios in vitro de imunoestimulação de esplenócitos obtidos de camundongos BALB/c sadios ou cronicamente infectados com Leishmania infantum. Os clones que foram capazes de induzir a uma resposta imune Th1 polarizada, representada por níveis elevados de IFN- e baixos níveis de IL-4; foram selecionados. Por critérios de seletividade e especificidade, dois clones (B10 e C01) foram utilizados nos protocolos de vacinação. Camundongos BALB/c vacinados com os clones mais saponina apresentaram uma produção elevada de IFN-, IL-12, e GM-CSF após a estimulação específica in vitro. Após a infecção, esses animais apresentaram reduções significativas na carga parasitária no fígado, baço, medula óssea e linfonodos drenantes, em comparação aos grupos controles (PBS, saponina e fago selvagem mais saponina). Estes dois mimotopos de Leishmania infantum também foram testados contra a infecção por L.amazonensis a fim de verificar a proteção cruzada-heteróloga entre espécies. Os imunógenos B10 e C01 juntos e separados; com e sem adjuvantes típicos de resposta imune Th1 (saponina), foram usados no experimento. Ambos os clones induziram uma produção de IFN-, IL-12 e GMC-SF. Reduziram a carga parasitária das patas, fígado, baço, linfonodos drenantes e medula óssea. Em ambos os trabalhos a proteção foi associada com a produção de IFN-, dependente de IL-12, mediada principalmente por células T CD8+. Estes animais apresentaram também a predominância de anticorpos IgG2a específicos dos parasitas. Dessa forma, o presente trabalho descreve a seleção e identificação de dois clones de bacteriófagos expressando peptídeos de interesse e sua aplicação como candidatos à vacina contra as leishmanioses

A rapid diagnostic test for human Visceral Leishmaniasis using novel Leishmania antigens in a laser direct-write lateral flow device

Visceral Leishmaniasis (VL) causes high morbidity and mortality in low-to-middle-income countries worldwide. In this study, we used Laser Direct-Write (LDW) technology to develop a new Lateral Flow Device (LFD) with double-channel geometry on a low-cost paper platform as a rapid and accurate serodiagnostic assay for human VL. This Duplex VL-LFD was based on a laser-patterned microfluidic device using two recombinant Leishmania proteins, β-tubulin and LiHyp1, as novel diagnostic antigens. The VL-LFD assay was tested with blood/serum samples from patients diagnosed with VL, Tegumentary Leishmaniasis, Leishmaniasis of unknown identity, other parasitic diseases with similar clinical symptoms, i.e. Leprosy Disease and Chagas Disease, and blood from healthy donors, and compared in parallel with commercial rK39 IT-LEISH® Kit. Clinical diagnosis and real-time Polymerase Chain Reaction assay were used as reference standards. VL-LFD Sensitivity (S ± 95% Confidence Intervals (CI)) of 90.9 (78.9-100) and Specificity (Sp ± 95% CI) of 98.7 (96.1-100) outperformed the IT-LEISH® Kit [S = 77.3 (59.8-94.8), Sp = 94.7 (89.6-99.8)]. This is the first study reporting successful development of an LFD assay using the LDW technology and the VL-LFD warrants comparative testing in larger patient cohorts and in areas with endemic VL in order to improve diagnosis and disease management

Canine visceral leishmaniasis : detection of Leishmania spp. genome in peripheral blood of seropositive dogs by real-time polymerase chain reaction (rt-PCR).

Visceral leishmaniasis (VL) is a zoonosis caused by the protozoa of the genus Leishmania. Among the species, L. infantum and/or L. infantum (chagasi) are the most important species affecting the Americas. Domestic dogs are the main reservoir of the parasite and participate effectively in the parasite' transmission cycle. The Canine Visceral Leishmaniasis Control Program (PCLV) adopted in Brazil present as strategies the vector control, health education and serological diagnosis of CVL in dogs followed by culling of the seropositive ones. The resolution to eliminate seropositive dogs by euthanasia, when necessary, are the most controversial and least accepted by society. The diagnostic methods for canine visceral leishmaniasis, currently indicated and approved in Brazil by the Ministry of Health from Brazil are the Dual Path Platform (DPP)? as a screening test and the Enzyme immunoassay test (ELISA?). This study aimed to verify the presence of Leishmania spp. DNA in peripheral blood samples of dogs presenting positive serological results byDPP? and ELISA? tests,throughreal-time polymerase chain reaction (rt-PCR), using the pair of primers 150?152 already described. For this purpose, were collected blood samples from 185 seropositive dogs among them, 41 (22%) exhibited some clinical signal of disease, whereas 144 (78%) was asymptomatic. The animals were also analyzed according to gender, race and hair size. According to the results of rt-PCR, it was observed that among the185 seropositive dogs analyzed, only 132 (71%) presented positive results for CVL and 53 (29%) presented negative results. From this, 41/41 symptomatic dogs were positive (100%), while among the asymptomatic dogs, 91/144 were positive (63, 2%) and 53/144 were negative (36, 8%). Concerning the hair size of seropositive dogs, we found that 41 (22%) had long hair, while 144 (78%) had short hair. No statistical significance occurred between the results of rt-PCR, ELISA and DPP tests and the profile of the animals (gender, size of the dogs and hair size), probably due to the small number of samples and the sampling differences of each profile. But statistical significance occurred between the results of rt-PCR and the clinical evaluation, since the rt-PCR was positive in all symptomatic dogs. Thus, through these results, we reached at the following question, which may contribute to an important current debate: the dogs presenting CVL seropositive diagnosis confirmed by tests distributed by the Ministry of Health were in reality ill or were they seropositive by living in an endemic area of the disease? Would these asymptomatic seropositive dogs spread the disease to the inhabitants even presenting a low parasite charge circulating in the blood

Strychnos pseudoquina

The development of new and cost-effective alternative therapeutic strategies to treat leishmaniasis has become a high priority. In the present study, the antileishmanial activity of Strychnos pseudoquina St. Hil. was investigated and pure compounds that presented this biological effect were isolated. An ethyl acetate extract was prepared, and it proved to be effective against Leishmania amazonensis. A bioactivity-guided fractionation was performed, and two flavonoids were identified, quercetin 3-O-methyl ether and strychnobiflavone, which presented an effective antileishmanial activity against L. amazonensis, and studies were extended to establish their minimum inhibitory concentrations (IC50), their leishmanicidal effects on the intra-macrophage Leishmania stage, as well as their cytotoxic effects on murine macrophages (CC50), and in O+ human red blood cells. The data presented in this study showed the potential of an ethyl acetate extract of S. pseudoquina, as well as two flavonoids purified from it, which can be used as a therapeutic alternative on its own, or in association with other drugs, to treat disease evoked by L. amazonensis