Antigenicity of phage clones and their synthetic peptides for the serodiagnosis of canine and human visceral leishmaniasis
Graphical abstract
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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2020, Microbial PathogenesisCitation Excerpt :Other studies using recombinant proteins and synthetic peptides have also showed that depletion studies have efficacy in demonstrate the specificity of the related antigens [65,66]. Synthetic peptides have showed efficacy for the diagnosis of canine and human VL [17,18]. They are simpler to produce, present higher stability and low cost of production, when compared to the recombinant proteins [19].
Evaluation of Leishmania infantum pyridoxal kinase protein for the diagnosis of human and canine visceral leishmaniasis
2020, Immunology LettersCitation Excerpt :In addition, bioinformatics tools identified a specific B-cell epitope in the L. infantum PK sequence, which was synthetized and evaluated with diagnostic purposes. This fact is relevant, since synthetic peptides, when compared to recombinant proteins, are simpler and cheaper to produce, and have been well-evaluated in immunoassays for the diagnosis of such diseases as canine [42,43] and human [44,45] VL. Results presented here showed high sensitivity and specificity values when both the recombinant protein and the synthetic peptide were used, including when asymptomatic VL dog sera were tested, thus demonstrating the feasibility to employ these antigens, in ELISA experiments or incorporated in other diagnostic platforms, for the diagnosis of human and canine VL.
A Leishmania infantum hypothetical protein evaluated as a recombinant protein and specific B-cell epitope for the serodiagnosis and prognosis of visceral leishmaniasis
2020, Acta TropicaCitation Excerpt :One alternative means to search for more sensitive and specific products will be through the use of synthetic peptides (Chávez-Fumagalli et al., 2013). These antigens, as compared to recombinant proteins, are relatively simpler and cheaper to produce (Costa et al., 2017). In this context, synthetic peptides have been evaluated, individually or mixed, in immunoassay experiments for the diagnosis of VL, and satisfactory results have been found (Faria et al., 2011; Costa et al., 2012; Menezes-Souza et al., 2015).
Diagnostic markers selected by immunoproteomics and phage display applied for the serodiagnosis of canine leishmaniosis
2019, Research in Veterinary ScienceCitation Excerpt :Results showed that both clones, as well as their synthetic peptides, presented high sensitivity and specificity values to identify symptomatic and asymptomatic canine disease. On the other hand, using a wild-type phage, a random non-specific clone and Leishmania antigenic extract as controls, lower sensitivity and specificity results were encountered (Costa et al., 2017). A list containing the main phage clones, which were selected by phage display strategy and showed to be antigenic in ELISA assays for the serodiagnosis of CanL is shown (Table 2).