Parasitology
Leishmania infantum mimotopes and a phage–ELISA assay as tools for a sensitive and specific serodiagnosis of human visceral leishmaniasis

https://doi.org/10.1016/j.diagmicrobio.2016.11.012Get rights and content

Highlights

  • A subtractive phage display selection was performed.

  • Eight Leishmania infantum specific mimotopes were selected.

  • Phage clones expressing these mimotopes were amplified.

  • Phages were evaluated for the serodiagnosis of human visceral leishmaniasis.

  • They showed high sensitivity and specificity values in a phage-ELISA assay.

Abstract

Serological methods used to diagnose visceral leishmaniasis (VL) are considered minimally invasive, but they present problems related with their sensitivity and/or specificity. In this study, a subtractive selection using the phage display technology against antibodies from healthy subjects living in endemic and non-endemic areas of disease, as well as from Chagas disease patients and those developing active VL, was developed. The aim of this study was to select bacteriophage-fused epitopes to be used in the serodiagnosis of human VL. Eight phage clones were selected after the bio-panning rounds, and their reactivity was evaluated in a phage-ELISA assay against a human serological panel. A wild-type clone and the recombinant K39-based immunochromatographic test were used as controls. In the results, it was shown that all clones showed an excellent performance to serologically identify VL patients, demonstrating the feasibility of the isolated phages for developing a specific and sensitive serodiagnosis of human VL.

Introduction

Leishmaniasis is a parasitic disease complex occurring in tropical and subtropical countries worldwide (World Health Organization, 2010). Among the 2 million of cases of the human leishmaniasis, between 200,000 and 400,000 correspond to cases from visceral leishmaniasis (VL), culminating in approximately 20,000 to 30,000 deaths annually (Alvar et al., 2012, Desjeux, 2004). Leishmania parasites are distributed in the world, and in some geographic areas, more than one species can be found causing distinct clinical manifestations of the disease. In the Americas, Brazil is responsible by 90% of the cases registered of VL, and Leishmania infantum species is the most common parasite responsible by this disease in Brazilian dogs and humans (Alvar et al., 2012).

The human infection outcome varies from an asymptomatic and/or subclinical disease to the acute symptomatic disease. While infected subjects developing asymptomatic VL present apparently no impact on their healthy state, symptomatic patients usually develop diverse clinical manifestations, such as lymphadenopathy, fever, diarrhea, malaise, hepatomegaly, and splenomegaly (Michel et al., 2011, Sundar and Chakravarty, 2010). Chemotherapy treatment for VL has been based on parenteral administration of pentavalent antimonials, but several side effects such as anorexia, myalgia, fever, urticaria, and arthralgia, besides toxicity in the liver, spleen, and kidneys, have been registered in the patients (Chávez-Fumagalli et al., 2015, Moore and Lockwoodm, 2010).

A precise diagnosis of VL 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. However, the parasitological diagnosis based on direct demonstration of Leishmania amastigotes presents low sensitivity and requires invasive samples collection procedures, which limit their use (Srividya et al., 2012, Tavares et al., 2003). Although the detection of Leishmania DNA by polymerase chain reaction (PCR) technique possesses high specificity, its sensitivity remains variable due to the random dissemination of the parasites in the collected aspirates (Chatzis et al., 2014). Therefore, antileishmanial serology becomes an ideal diagnostic tool for detection of anti-parasite antibodies in sera of patients, due to its simplicity and low cost, besides being considered minimally invasive (Tavares et al., 2003).

In this context, recombinant antigens have been evaluated for the serodiagnosis of VL, showing satisfactory results when compared to crude, soluble and/or semi-purified Leishmania antigenic preparations. Among the most promising molecules, recombinant K39 (rK39) protein has shown good results for the serodiagnosis of disease (Singh et al., 2010). A commercial kit, namely Kalazar Detect™ Test (InBios International, Inc., Seattle, WA, USA), was developed and is considered a non-invasive immunochromatographic strip assay for the qualitative detection of rK39-specific antibodies for the L. donovani complex in patients sera. However, problems related to cross-reactivity with serum samples from patients with related-VL diseases, such as Chagas disease, as well as false-positive results found in healthy subjects living in endemic areas of the disease; have been registered (Chappuis et al., 2007, Singh and Sundar, 2015, Srivastava et al., 2011, Sundar et al., 2002). When comparing rK39 strip test and the direct agglutination test (DAT), it was verified that the last one presents the ability to detect patients with low levels of antileishmanial antibodies, due to the mosaic of antigens present in the antigenic preparations; although its specificity is hampered due to the fact that antibodies can also interact with cross-reactive antigens (Diro et al., 2007). Strip tests present have limitations, since patients can develop high levels of anti-Leishmania antibodies for months after the treatments, as well as false-negative results are possible to be detected (Maia et al., 2012, Sundar and Rai, 2002). Thus, there is an urgent need to identify new antigenic markers to be tested in a more sensitive and specific serodiagnosis of VL.

Efforts to identify novel diagnostic antigens have recently relied on the phage display, which is a subtractive proteomic approach employed to select random peptides expressed in fusion with the outer surface of phage clones (Smith, 1985). In this technology, bacteriophage-fused peptide libraries are submitted to successive bio-selection rounds, when specific phage clones are selected and their exogenous peptides are further identified by DNA sequencing (Barbas et al., 2001, Smith and Petrenko, 1997, Wang and Yu, 2004). Phage display generates short peptides that mimic epitopes (namely mimotopes), which present antigenic and immunogenic potential to be applied as diagnostic markers and/or vaccine candidates against diseases, such as malaria (Demangel et al., 1996, Greenwood et al., 1991, Monette et al., 2001), toxoplasmosis (Beghetto et al., 2003, Cunha-Junior et al., 2010), canine VL (Costa et al., 2014a, Costa et al., 2014b, Costa et al., 2015), Chagas disease (Pitcovsky et al., 2001), strongyloidiasis (Feliciano et al., 2014) and others.

In this context, in the present study, a stringent subtractive phage display selection was employed to identify new mimotopes to be applied as antigens for the serodiagnosis of human VL. Besides the non-described subtractive selection procedure, the selected mimotopes proved to be highly relevant for the serological analysis, when evaluated by a phage–ELISA technique. This study presents novel diagnostic markers using a simple phage–ELISA test showing high accuracy, and which could well be considered as an alternative tool for the sensitive and specific serodiagnosis of VL.

Section snippets

Ethics statement

This study was approved by Ethics Committee from Federal University of Minas Gerais (COEP/UFMG, protocol CAAE–32343114.9.0000.5149), Belo Horizonte, Minas Gerais, Brazil, and was conducted according to the Declaration of Helsinki principles. A written informed consent was obtained from all patients, who received an individual copy of the study policy, which was reviewed by an independent person.

Parasite and antigen preparation

L. infantum (MHOM/BR/1970/BH46) strain was used. Stationary-phase promastigotes of the parasites were

Selection and identification of the specific peptides

Initially, a subtractive phage display selection was performed. For this, a negative selection was developed and consisted in the exclusion of the phages presenting cross-reactivity with antibodies isolated from sera of healthy individuals or Chagas disease patients. After, a positive selection consisting of keeping the clones specific to interact with antibodies in sera of VL patients was performed. After three positive bio-selection rounds, 96 phage clones were isolated and evaluated by a

Discussion

Immunological methods have been employed as an important strategy for VL diagnosis, due to their advantages in comparison to parasitological methods, presenting an improved sensitivity and specificity, and being considered less invasive than aspirates necessary to be parasitological exams (Mohebali et al., 2010), although important cross-reactivity problems still remain. In this context, in the present study, we have used the phage display technology to develop a highly stringent selection

Competing interests

The authors declare to have no competing interests.

Acknowledgments

This work was supported by grants from Instituto Nacional de Ciência e Tecnologia em Nano-Biofarmacêutica, Rede Nanobiotec/Brasil-UFU (CAPES), PRONEX-FAPEMIG (APQ-01019-09), 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 recipients of CNPq. MACF is a grant recipient of FAPEMIG/CAPES.

References (62)

  • J. Greenwood et al.

    Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens

    J Mol Biol

    (1991)
  • G. Michel et al.

    Importance of worldwide asymptomatic carriers of Leishmania infantum (L. chagasi) in human

    Acta Trop

    (2011)
  • D.J. Rodi et al.

    Phage-display technology-finding a needle in a vast molecular haystack

    Curr Opin Biotechnol

    (1999)
  • R. Sivakumar et al.

    Cloning, expression, and purification of a novel recombinant antigen from Leishmania donovani

    Protein Expr Purif

    (2006)
  • P. Srivastava et al.

    Diagnosis of visceral leishmaniasis

    Trans R Soc Trop Med Hyg

    (2011)
  • E. Abass et al.

    rKLO8, a novel Leishmania donovani-derived recombinant immunodominant protein for sensitive detection of visceral leishmaniasis in Sudan

    PLoS Negl Trop Dis

    (2013)
  • S.M. Alban et al.

    Phage display and synthetic peptides as promising biotechnological tools for the serological diagnosis of leprosy

    PLoS One

    (2014)
  • J. Alvar et al.

    WHO Leishmaniasis Control Team. Leishmaniasis worldwide and global estimates of its incidence

    PLoS One

    (2012)
  • T.S.M. Assis et al.

    Multi-centric prospective evaluation of rK39 rapid test and direct agglutination test for the diagnosis of visceral leishmaniasis in Brazil

    Trans R Soc Trop Med Hyg

    (2011)
  • R. Badaro et al.

    rK39, a cloned antigen of Leishmania chagasi that predicts active visceral leishmaniasis

    J Infect Dis

    (1996)
  • C.F. Barbas et al.

    Phage display: a laboratory manual

    (2001)
  • M. Boelaert et al.

    Rapid tests for the diagnosis of visceral leishmaniasis in patients with suspected disease

    Cochrane Database Syst Rev

    (2014)
  • S.F.G. Carvalho et al.

    Performance of recombinant K39 antigen in the diagnosis of Brazilian visceral leishmaniasis

    Am J Trop Med Hyg

    (2003)
  • B.J. Celeste et al.

    Recombinant Leishmania infantum heat shock protein 83 for the serodiagnosis of cutaneous, mucosal, and visceral leishmaniasis

    Am J Trop Med Hyg

    (2014)
  • F. Chappuis et al.

    Visceral leishmaniasis: what are the needs for diagnosis, treatment and control?

    Nat Rev Microbiol

    (2007)
  • M.A. Chávez-Fumagalli et al.

    New delivery systems for amphotericin B applied to the improvement of leishmaniasis treatment

    Rev Soc Bras Med Trop

    (2015)
  • E.A.F. Coelho et al.

    Immune responses induced by the Leishmania (Leishmania) donovani A2 antigen, but not by the LACK antigen, are protective against experimental Leishmania (Leishmania) amazonensis infection

    Infect Immun

    (2003)
  • L.E. Costa et al.

    Subtractive phage display selection from canine visceral leishmaniasis identifies novel epitopes that mimic Leishmania infantum antigens with potential serodiagnosis applications

    Clin Vaccine Immunol

    (2014)
  • L.E. Costa et al.

    Mimotope-based vaccines of Leishmania infantum antigens and their protective efficacy against visceral leishmaniasis

    PLoS One

    (2014)
  • L.E. Costa et al.

    Phage-fused epitopes from Leishmania infantum used as immunogenic vaccines confer partial protection against Leishmania amazonensis infection

    Parasitology

    (2015)
  • J. Cunningham et al.

    A global comparative evaluation of commercial immunochromatographic rapid diagnostic tests for visceral leishmaniasis

    Clin Infect Dis

    (2012)
  • Cited by (0)

    1

    These authors contributed equally to this work.

    2

    Co-senior authors.

    View full text