Sensitive and Specific Serodiagnosis of Leishmania infantum Infection in Dogs by Using Peptides Selected from Hypothetical Proteins Identified by an Immunoproteomic Approach

Abstract

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 dogs.

Subtractive Phage Display Selection from Canine Visceral Leishmaniasis Identifies Novel Epitopes That Mimic Leishmania infantum Antigens with Potential Serodiagnosis Applications

“…Regarding the sensitivity and specificity found in this study for selected phage clones, the high performance observed for phage-ELISA using the A1, A8, C7, E7, E8, F12, and G8 clones is compatible with the diagnostic performance observed in recent studies using recombinant proteins (56,57), multiple-epitope chimeric antigens (58,59), and synthetic peptides (60,61). Nevertheless, the results of this study show improvements in sensitivity (100%) and specificity (100%), given that the cutoff values were selected in an attempt to obtain 100% specificity.…”

Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis

“…In addition, the exclusion of the proteins by their cross-reactivity with antibodies from sera of healthy subjects living in areas of endemicity and those from Chagas disease patients represent a refinement of the present proteomic analysis, allowing the discarding of these antigenic proteins in order to be able to diagnose false-positive patients. The specificity of ELISAs using Leishmania antigenic preparations to diagnose leishmaniasis largely depends on the protein formulation employed, although false-positive and false-negative results have still been encountered in some studies (43)(44)(45). In the present study, 20 proteins were identified as possible diagnostic markers for TL.…”

A recombinant chimeric protein composed of human and mice‐specific CD4+ and CD8+ T‐cell epitopes protects against visceral leishmaniasis

“…Studies evaluating antileishmanial vaccine candidates have advanced in recent decades, due to the understanding of T‐cell‐mediated immunological mechanisms associated with the control of infection . Most of the candidates have been evaluated as single recombinant molecules, being administered associated with immune adjuvants, whereas a mixture composed of different immunogens based on the use of multiproteins or polypeptides‐based vaccines has been less studied, although a formulation composed of immunogenic parts of distinct Leishmania proteins could present advantages, such as comprising a higher number of CD4 + and/or CD8 + T‐cells epitopes of several antigens conserved between different Leishmania species, and could be protective against most of them …”

“…Derived from this line of investigation, detection of the major antigenic determinants within the whole proteins and the identification of epitopes able to stimulate the development of a protective immune response against leishmaniasis are also the focus of current research . It is noted that peptides are usually more stable, easier to produce and cheaper than recombinant proteins, although their isolated use as peptide‐based vaccine usually present low protective efficacy due to the immunological restriction to one protein of origin . In the light of our knowledge, the protective characteristics of these vaccine candidates could be improved by increasing the number of doses of the vaccine, the amount of peptide used in each dose and/or even using carrier molecules .…”