Research paper
Serodiagnosis of canine leishmaniasis using a novel recombinant chimeric protein constructed with distinct B-cell epitopes from antigenic Leishmania infantum proteins

https://doi.org/10.1016/j.vetpar.2021.109513Get rights and content

Highlights

  • Bioinformatics tools were applied in four antigenic Leishmania proteins.

  • Seven specific-B cell epitopes were identified and used to construct a chimera.

  • The recombinant chimera was tested in ELISA experiments against canine sera.

  • High sensitivity and specificity values were found for this recombinant antigen.

  • Better performance was obtained in comparison to a commercial kit.

Abstract

Visceral leishmaniasis (VL) is an important public health problem in the world, and control measures are insufficient to avoid the spread of this neglected disease. Dogs are important domestic reservoirs of Leishmania parasites in countries where VL is a zoonosis, representing a major source of infection between sand fly vectors and humans. In this context, a precise diagnosis of canine leishmaniasis (CanL) could help to reduce the number of human cases. Distinct approaches for the diagnosis of CanL have used recombinant proteins in serological assays. However, variable results of the antigens have been found, mainly to diagnosis asymptomatic cases. The present study used bioinformatics to select specific B-cell epitopes of four Leishmania infantum proteins, which had previously been proven to be antigenic in VL, aiming to produce a novel chimeric protein and to evaluate it for the diagnosis of CanL. Seven B-cell epitopes were identified and used to construct the chimera, which was analyzed in a recombinant format through an ELISA assay against a canine serological panel. A soluble Leishmania antigenic extract (SLA) was used as an antigen control. Results showed 100 % sensitivity and specificity for chimera, while when using SLA the values were 26.0 % and 96.4 %, respectively. The performance of chimera was compared with a commercial kit using asymptomatic and symptomatic dog sera, and the data showed that no false-negative result was found when the recombinant protein was used. However, when using the commercial kit, 40.0 % and 16.0 % of the false-negative results were found, respectively. In conclusion, the recombinant chimera showed an antigenic potential to be evaluated in new studies against a larger serological panel for the diagnosis of CanL.

Introduction

Visceral leishmaniasis (VL) is a neglected tropical disease that presents a significant impact on global public health, where 0.5 million new cases are registered per year (WHO, 2018). In the Americas, the infection is caused by the parasite Leishmania infantum, with dogs being the main domestic reservoirs and representing the major source of infection for sand fly vectors (Alvar et al., 2004; Giunchetti et al., 2006). Measures to control disease dissemination have not contributed to a reduction in the number of human cases (Dantas-Torres et al., 2019). In addition, canine leishmaniasis (CanL) presents a more relevant epidemiological context due to the higher number of the canine disease cases, when compared to human VL, and, in this context, the improvement in diagnosis of CanL is urgently required to avoid the spread of disease for both mammalian hosts (Costa et al., 2018; Ribeiro et al., 2018).

The diagnosis of CanL is conducted by means of serological, molecular, and parasitological tests, with parasitological methods being the reference standard. However, in endemic areas, serological tests can be better used in epidemiological surveys to facilitate diagnosis (Borja et al., 2018; Pessoa-E-Silva et al., 2019). In this context, recombinant proteins have overcome the limitations for VL diagnosis in the serological assays due to the use of crude antigen extracts, and such antigens have been tested in prototypes to detect humoral reactivity in L. infantum-infected dogs (Souza et al., 2012; Fraga et al., 2014; Oliveira et al., 2015; Coelho et al., 2016; Dias et al., 2018). Otherwise, commercial kits have been produced to detect CanL; however, although they present high sensitivity to detect symptomatic cases, the sensitivity is reduced when asymptomatic animals are evaluated, due to the lower presence of specific antibodies in the sera of these animals (Costa et al., 2013; Souza-Filho et al., 2016), which is an important problem due to the higher prevalence of asymptomatic disease among the infected animals. In addition, these kits present also cross-reactions, mainly when sera samples from dogs infected by other pathogens, such as Trypanosoma cruzi, Ehrlichia canis and Toxoplasma gondii, among others, are evaluated (Troncarelli et al., 2009; Pessoa-E-Silva et al., 2019).

In an immunoproteomic approach performed in L. infantum promastigote and amastigote protein extracts with the sera of asymptomatic and symptomatic VL dogs, animal antibodies identified parasite proteins that could be considered for the improvement of the diagnosis of VL (Coelho et al., 2012). In fact, some of these proteins were individually tested in recombinant formats for this biological purpose. LiHyD (XP_001468360.1), a Leishmania-specific hypothetical protein, was evaluated as a diagnostic marker for CanL, and successful results were obtained (Lage et al., 2016). Another protein, the IgE-dependent histamine-releasing factor (HRF; XP_001465979.1), was also identified in the study and showed high values of sensitivity and specificity for the diagnosis of CanL (Coelho et al., 2016). A hypothetical Leishmania amastigote-specific protein, called LiHyp1 (XP_001468941.1), which presents homology to the super-oxygenase gene family, was also well-evaluated for the diagnosis of CanL (Martins et al., 2013). The β-tubulin (XP_001468164.1) protein also identified in the cited immunoproteomics study was evaluated in a recombinant version for the diagnosis of leishmaniasis (Duarte et al., 2015).

Based on the possibilities to construct novel recombinant proteins for the diagnosis of CanL, chimeric proteins have shown advantages when compared to the use of individual proteins, since they are single molecules consisting of distinctly specific B-cell epitopes of different parasite proteins (Boarino et al., 2005; Santos et al., 2020). In this context, chimera could help to improve the sensitivity and specificity of the tests, mainly by eliminating the presence of other undesired dominant epitopes. Therefore, in the present study, specific B-cell epitopes were selected from amino acid sequences of the four L. infantum proteins (LiHyD, LiHyp1, HRF, and β-tubulin). The gene codifying a novel chimeric protein was designed, and the protein was expressed, purified, and evaluated as a CanL marker. In addition, the performance of this chimeric protein was compared with a commercial kit to identify asymptomatic and symptomatic cases of the disease.

Section snippets

Ethics

The Ethical Handling of Research Animals (CEUA) from the Federal University of Minas Gerais (UFMG; Belo Horizonte, Minas Gerais, Brazil) approved this study with the protocol number 144/2020.

Dog samples

The sample size consisted of 190 dogs (Canis familiaris), consisting of males (n = 111) and females (n = 79) of different breeds and ages, which were collected from an endemic area of VL (Belo Horizonte, Minas Gerais, Brazil). The samples used here were derived from a sera bank located at the Laboratory of

Bioinformatics, construction of chimera, and immunoblottings

The following B-cell epitopes were identified by bioinformatics assays in the four target proteins: YEPGDFIRAHIDNL and LPVEAQRFSLVFRR for LiHyp1, CPFDVEGDIMYVVNGRYIDVGGEDYGISA for HRF, VPRAVLMDLEPGTM and AKNMMQAADPRHGR for β-tubulin, and IGQQPGGGMMDGLS and DVQDGHMGPQQADA for LiHyD. The secondary structure of the chimera was predicted using the JPRED program (Fig. 1). The presence of alpha-helix is indicated in red color, while beta-leaves are represented in blue color. The figure also shows the

Discussion

An immunoproteomics approach performed in L. infantum antigenic extracts against sera samples from asymptomatic and symptomatic CanL identified parasite proteins that could be tested as diagnostic antigens for disease (Coelho et al., 2012). In fact, identified antigens, such as LiHyD, LiHyp1, HRF, and β-tubulin were individually tested as recombinant proteins for the diagnosis of canine and/or human disease, and results were satisfactory (Martins et al., 2013; Duarte et al., 2015; Coelho et

Author contributions

Conceived and designed the experiments: EAFC VTM RAMA DO NCG. Performed the experiments: DLV DPL ASM CSF BBF GPL JSO JAOS FFR TTOS WFS. Analyzed the data: EAFC RTF LLB MACF. Contributed reagents/materials/analysis tools: LAA DFMS JAGS LLB. Wrote the paper: EAFC VTM RAMA RTF.

Financial support

This work was supported by grant MR/R005850/1 from the Medical Research Council (VAccine deveLopment for complex Intracellular neglecteD pAThogEns - VALIDATE), UK, and grant APQ-408675/2018-7 from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The authors also thank the Brazilian agencies Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), CNPq and the Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the student

Declaration of Competing Interest

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

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