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

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)