Elsevier

Microbial Pathogenesis

Volume 110, September 2017, Pages 14-22
Microbial Pathogenesis

Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis

https://doi.org/10.1016/j.micpath.2017.06.020Get rights and content

Highlights

  • Two phage clones tested as vaccine were evaluated as diagnostic antigens.

  • Their exogenous peptides were synthesized and also evaluated in ELISA experiments.

  • All antigens showed high sensitivity and specificity values in the assays.

  • They can be used isolate or in association for the diagnosis of visceral leishmaniasis.

Abstract

In the Americas, Brazil is responsible by 90% of the cases registered of visceral leishmaniasis (VL), and Leishmania infantum is the most common parasite species responsible by disease in Brazilian dogs and humans. A precise diagnosis may allow to a faster and more effective treatment against the disease, which increases the possibility of cure, as well as to induce less toxic effects, due to a lower time exposition for the chemotherapeutics. In a previous study, two L. infantum mimotopes, B10 and C01 clones, were recognized by antibodies in VL dogs sera by a phage display technology, and were well-successfully evaluated as vaccine candidates against visceral and tegumentary leishmaniasis. In the present work, the diagnostic efficacy of these clones, as well as of their exogenous peptides (B10: LSFPFPG and C01: FTSFSPY), was evaluated to diagnose canine and human VL. ELISA assays were performed with the four antigens, and results showed that both clones, as well as their synthetic peptides; showed high sensitivity and specificity values to identify VL samples, presenting an excellent performance to serologically diagnose VL-developing humans and dogs. On the other hand, a wild-type phage, a random non-specific clone and a L. infantum antigenic preparation were used as controls, and showed worst sensitivity and specificity results. In conclusion, besides their biological action as vaccine, B10 and C01 phages and their synthetic peptides could be considered as new markers for the serodiagnosis of canine and human VL.

Introduction

Leishmania parasites cause important diseases in dogs and humans, which affect over 12 million people worldwide, with more than 2.0 million new cases of leishmaniasis being registered annually [1]. Clinical disease manifestations range from self-healing cutaneous lesions to visceral leishmaniasis, which is fatal if acute and untreated [2]. There is currently no effective vaccine against Leishmania infection, and current treatment is limited by high toxicity, the cost of the drugs and hospitalization [3]. In addition, the increasing incidence of therapeutic failures and the emergence of drug-resistant parasites highlight the need to diagnoses leishmaniasis as soon as possible, aiming to offer a faster and more effective treatment for the patients.

The rK39 protein has been employed as a diagnostic marker to detect visceral leishmaniasis (VL) cases. This recombinant protein identifies infected patients, with sensitivity values ranging from 82% to 100% [4], [5]. However, cross-reactivity with VL-related diseases, as well as with sera from healthy subjects living in endemic areas of disease have led to false-positive results, hampering the specificity of the tests [6], [7]. Thus, it is necessary to search for new candidates that will serve to design serodiagnostic systems with higher degree of sensitivity and specificity than current kits. In parallel to the use of recombinant proteins, other antigens such as synthetic peptides could be also considered, since these antigens are simpler, specific, stable and cheaper to produce [8], [9]. In addition, polyprotein or polypeptide-based chimera [10], [11], [12] and phage clones [13], [14] have been also used as diagnostic markers on leishmaniasis.

Phage display is a molecular biology technology that consists in the search of molecules against an interest target. Genetically modified bacterial virus, namely phages, express exogenous peptides in their viral capsid [15], [16], which are selected based on their affinity to antibodies, T cell receptor, whole cells, among others [17]. Phage display has been employed to search new vaccine candidates [18], [19], immunotherapeutic [20], as well as diagnostic markers for parasitic diseases [13].

In a previous study, two Leishmania infantum mimotopes were identified by antibodies in VL dogs sera by phage display technology [21]. These clones, namely B10 and C01, were successfully evaluated as vaccine candidates against visceral [21] and tegumentary [22] leishmaniasis. Due to the fact that these molecules were serologically identified by antibodies in VL dogs sera, in the present study, the diagnostic efficacy of these antigens was evaluated for the serodiagnosis of canine and human VL. Aiming to evaluate the specificity of target peptides expressed outside in these molecules, their exogenous sequences were synthesized and also evaluate as diagnostic markers in the serological assays by ELISA technique.

Section snippets

Animals study

A sample of canine sera consisting of 100 dogs (Canis familiaris) composed by males and females of different breeds and species was used. Canine VL (n = 51) was diagnosed by two serological tests (IFAT-CVL® and EIE-CVL® Kits, Bio-Manguinhos, Fiocruz, Brazil), besides positive parasitological results for L. infantum (strain MHOM/BR/1970/BH46) kDNA by a PCR technique. Healthy dogs were selected from endemic (n = 15; Belo Horizonte, Minas Gerais, Brazil) or non-endemic (n = 9; Poços de Caldas,

Antigenicity of B10 and C01 phages and their synthetic peptides in the canine VL

In the present study, four antigens, two representing phage clones (B10 and C01) and two representing synthetic peptides (B10 Peptide: LSFPFPG and C01 Peptide: FTSFSPY), were evaluated as diagnostic markers for VL. Properly, these antigens were used in serological tests based on ELISA assays to detect VL-developing dogs and humans. As controls, a wild-type (WT phage) clone, which is not genetically modified and, therefore, does not express exogenous peptides in its viral capsid, as well as a

Discussion

In the present study, the diagnostic properties of four antigens, represented by two phage clones (B10 and C01) and their respective synthetic peptides were evaluated for the serodiagnosis of both canine and human VL. These phage clones have already been previously showed as effective vaccine candidates against visceral [21] and tegumentary [22] leishmaniasis. Since these molecules were recognized by antibodies in VL dogs sera, it could be speculate about the antigenicity of these clones to

Conflicts of interest

The authors hereby declare that they have no conflicts of interest.

Acknowledgements

This work was supported by grants from Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais (Edital 02/2017), Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroproteção (INCT Nano-Biofar), National Institute of Science and Technology in Theranostics and Nanobiotechnology (CNPq/CAPES/FAPEMIG, Process N. 465669/2014-0), FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014) and CNPq (APQ-482976/2012-8, APQ-488237/2013-0, and APQ-467640/2014-9). EAFC and LRG are grant

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