Elsevier

Cellular Immunology

Volume 334, December 2018, Pages 61-69
Cellular Immunology

Research paper
Diagnostic application of recombinant Leishmania proteins and evaluation of their in vitro immunogenicity after stimulation of immune cells collected from tegumentary leishmaniasis patients and healthy individuals

https://doi.org/10.1016/j.cellimm.2018.09.006Get rights and content

Highlights

  • Proteins showed antigenic in the human TL were evaluated as recombinant molecules.

  • Cytokine production in stimulated PBMC and antibody subclass levels were investigated.

  • Proteins stimulated the development of a Th1 response in the PBMC cultures.

  • High sensitivity and specificity values were found for the serodiagnosis of TL.

  • These antigens can be applied as new biomarkers of human disease.

Abstract

The present study evaluated the cytokine profile in PBMC supernatants and the humoral response in mucosal leishmaniasis (ML) patients and in healthy subjects living in an endemic area. Four proteins, which had previously proven to be antigenic in the human disease, were tested: LiHyM, enolase, eukaryotic initiation factor 5a, and Beta-tubulin. Results showed that all of the proteins stimulated human cells with higher IFN-γ and lower IL-4 and IL-10 levels. The analysis of antibody isotypes correlated with cell response, since the IgG2 production was higher than IgG1 in both groups. By contrast, a Th2 response was found when an antigenic Leishmania extract was used. Serological analyses revealed high sensitivity and specificity values for the serodiagnosis of the disease, when compared to the data obtained using the antigenic preparation. In conclusion, this study presents new candidates to be evaluated as biomarkers in tegumentary leishmaniasis.

Introduction

Leishmaniases are diseases caused by the protozoa of the Leishmania genus. Distinct parasite species can cause a wide range of clinical manifestations of disease, which is also influenced by the genetic background and immune response of the mammalian hosts. The clinical manifestation can vary, from an asymptomatic status to the localized, diffuse, and visceral clinical forms [1], [2]. Approximately 380 million people in 98 countries are at risk of contracting infection, with 12 million people estimated to be clinically affected, with an annual incidence of 1.5 million cases for tegumentary leishmaniasis (TL), and 0.5 million of visceral leishmaniasis (VL) [3]. TL can cause a local cutaneous lesion, known as cutaneous leishmaniasis (CL) or can progress into mucosal lesions leading to mucosal leishmaniasis (ML) [4].

The parasite demonstration is necessary to confirm the diagnosis of disease; however, parasitological methods have shown variable sensitivity [5], [6]. Thus, other assays, such as antileishmanial antibody tests, could also be used. Serological techniques are mainly based on indirect immunofluorescence assays, enzyme-linked immunosorbent assay (ELISA), and Western-blot [7]. Although considered less invasive to obtain samples than parasitological methods, these assays present variable sensitivity and/or specificity, depending on the antigen used, the clinical status of the patients, the co-existence of cross-reactive diseases, among others [8], [9].

As a consequence, it would be desirable to obtain new recombinant antigens to be tested as alternatives to the application of the total, soluble, or crude preparations [10], [11], [12]. Another diagnostic question is related to the clinical form of the disease, since, in the VL serological assays, antigenic molecules are integrated into the laboratory routine, while for the diagnosis of TL there is no consensus on the application of these antigens, since most patients present low antileishmanial serology and are classified as false-negative [13]. In addition, the maintenance of the parasite-specific humoral reactivity after treatment is another problem, making it difficult to differentiate between a past or an active disease [14]. Consequently, new molecules should be tested in an attempt to improve the sensitivity and specificity for the TL diagnosis.

Regarding cell response, in both VL and TL cases, Th1-type cytokines are required for parasitism control and clinical cure [15], [16]. The ability of peripheral blood mononuclear cells (PBMCs) from patients with active disease and healthy individuals to produce Th1 cytokines is a requirement for protection against infection, given that it is based on the production of cytokines, such as IFN-γ, IL-12, GM-CSF, TNF-α, among others; whereas the susceptibility is based on the production of other cytokines, including IL-4, IL-10, IL-13, among others [17], [18]. Studies have shown that patients whose cutaneous lesions have been cured and subjects with no history of disease, but living in endemic areas of leishmaniasis, present high levels of circulating IFN-γ, IL-12, and TNF-α cytokines, while patients developing progressive lesions present higher IL-4 and IL-10 levels in their PBMC culture supernatants [19], [20]. The analysis of antibody production also presents a difference between the patients’ profile, since higher IgG1 and IgG3 levels are found in active disease patients, while higher IgG2 production is found in cured and treated patients [19]. Taking into account these correlates, efforts have been concentrated on identifying molecules able to induce IFN-γ, IL-12, GM-CSF, among other cytokines, in human PBMCs, as well as generate a higher IgG2 production, aimed at reaching a prophylactic vaccination against the disease [21].

In this context, the present study analyzed the cytokine profile (IFN-γ, IL-4, and IL-10 levels) induced after stimulating a PBMC culture derived from ML patients and healthy subjects living in an endemic area of disease, using different Leishmania proteins. The humoral response (IgG1 and IgG2 subclasses) directed against them was also evaluated by using serum samples. In addition, a serological panel was employed to evaluate the diagnostic potential of these antigens for the serodiagnosis of human TL. The selected candidates, LiHyM (XP_001566959.1), enolase (XP_001563419.1), eukaryotic initiation factor 5a (EIF5a) (XP_001565563.1), and beta-tubulin (XP_001567862.1) proteins, were recently identified in an immunoproteomics study, which were recognized by antibodies in TL patients’ sera, when L. braziliensis promastigote and amastigote extracts were used [9], [22].

Section snippets

Parasites and production of recombinant proteins

Leishmania braziliensis (MHOM/BR/1975/M2904) was used in this analysis. Stationary promastigotes were cultured at 24 °C in complete Schneider's medium (Sigma), which was supplemented with 20% inactivated fetal bovine serum (FBS, Sigma-Aldrich), 20 mM l-glutamine, 200 U/mL penicillin, and 100 µg/mL streptomycin, at pH 7.4. The soluble Leishmania antigenic extract (SLA) was prepared as described elsewhere [23]. The LiHyM (XP_001566959.1), enolase (XP_001563419.1), EIF5a (XP_001565563.1), and

Cytokine and anti-Leishmania antibody production

In this study, recombinant proteins, which had previously proven to be antigenic in TL, were used to stimulate PBMCs, which were collected from active ML patients and healthy subjects living in an endemic area of disease, in order to evaluate the protein-specific IFN-γ, IL-4, and IL-10 production in the culture supernatant. In addition, the humoral reactivity by measuring the IgG1 and IgG2 isotype levels was investigated in serum samples of the patients and subjects. Results showed that rLiHyM,

Discussion

Experimental models of Leishmania infection have been used to evaluate the immune response necessary to protect against parasites, whereas a parasite-specific Th1 response has been related to protection against infection, and a Th2 response has been associated with disease susceptibility [26], [27], [28]. Although studies have been developed, they have been performed in murine or canine models [23], [29], [30], with few works being generated by using human cells from leishmaniasis patients or

Conflict of interest

The authors hereby declare that there is no conflict of interest.

Acknowledgments

This work was supported by grants from FAPEMIG (CBB-APQ-00819-12 and CBB-APQ-01778-2014) and CNPq (APQ-467640/2014-9). EAFC, RAMA and ALT are grant recipients of CNPq. MACF is a grant recipient of CAPES/FAPEMIG.

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      Reactions were stopped by adding 25 μL 2 N H2SO4, and the optical density (OD) values were read in an ELISA microplate spectrophotometer (Molecular Devices, Spectra Max Plus, Canada), at 492 nm. Sera samples collected from ML and VL patients (n = 8 in both cases), which donated blood to obtain PBMC cultures, were used to evaluate the protein and parasite-specific antibody production (Lima et al., 2018). For this, ELISA microplates (JetBiofil®, Belo Horizonte) were coated with the rLiHyL, L. braziliensis or L. infantum SLA (1.0, 2.0, and 1.0 μg per well, respectively), diluted in coating buffer pH 9.6 and incubated for 16 h at 4 °C.

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