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Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

ABSTRACT: We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repeat between L. chagasi and Leishmania donovani. Serological evaluation revealed that 98% (56 of 57) of Brazilian and 100% (52 of 52) of Sudanese visceral leishmaniasis patients have high antibody levels to the rK39 repeat. Detectable anti-K39 antibody was virtually absent in cutaneous and mucosal leishmaniasis patients and in individuals infected with Trypanosoma cruzi. The data show that rK39 may replace crude parasite antigens as a basis for serological diagnosis of visceral leishmaniasis.

SUBMITTER: Burns JM

PROVIDER: S-EPMC45748 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

Burns J M JM Shreffler W G WG Benson D R DR Ghalib H W HW Badaro R R Reed S G SG

Proceedings of the National Academy of Sciences of the United States of America 19930101 2

We report the cloning of a Leishmania chagasi antigen gene and an evaluation of leishmaniasis patient antibody responses to the recombinant protein, rK39. rK39 contains a 39-amino acid repeat that is part of a 230-kDa protein predominant in L. chagasi tissue amastigotes. Sequence analyses showed this protein, LcKin, to be related to the kinesin superfamily of motor proteins. Southern blot analyses demonstrated LcKin-related sequences in seven species of Leishmania, with conservation of the repea ...[more]

PMID: 8421715

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Project description:Visceral leishmaniasis (VL) or kala-azar, the most severe form of leishmaniasis, can lead to death if not properly diagnosed and treated. Correct identification of infected patients and reservoirs is vital for controlling the spread of leishmaniasis. Current diagnostic kits for leishmaniasis show high sensitivity and specificity, but can also result in false negatives and cross reactions with related parasitic infections. New diagnostic methods with greater accuracy are urgently needed for diagnosis of leishmaniasis. In this study, we aimed to uncover a new highly effective antigen for the diagnosis of visceral leishmaniasis in dogs and humans, aiming to improve the accuracy compared with those of current methods of diagnosis. Initially, in-silico epitope prediction analyses identified several potential B-cell epitopes in the repetitive region of Leishmania infantum kinesin, which co-localized with predicted structural disordered regions, suggesting high potential for antigenicity. Based on this analysis, 8.5 genomic motifs, which encode the repetitive sequence of 39 degenerate amino acids, were selected for recombinant expression. BLASTn analysis of this repetitive region indicated that it is absent in the T. cruzi parasite, which is closely related to Leishmania, indicating the specificity of this region. This potentially antigenic protein, named recombinant kinesin degenerated derived repeat (rKDDR), was recombinantly expressed in Escherichia coli BL21-Star using the pET28a-TEV expression vector. We then evaluated the performance of rKDDR in correctly diagnosing Leishmania infection and compared this new assay with currently used diagnostic tests for leishmaniasis. rKDDR showed greater sensitivity and specificity in correctly diagnosing leishmaniasis both in human (sensitivity 92.86% and specificity 100%) and canine (sensitivity 88.54% and specificity 97.30%) sera compared with those of rK39 (human: sensitivity 90.48% and specificity 97.92%; canine: sensitivity 78.13% and specificity 90.09%). In addition, the rKDDR-ELISA outperformed the EIE-LVC kit, which is the serologic kit recommended by the Brazilian Ministry of Health for the diagnosis of canine visceral leishmaniasis. These results indicate that rKDDR is a highly promising candidate for diagnosis of visceral leishmaniasis, and is more accurate than the currently used gold-standard antigens.

Project description:BackgroundA recombinant cysteine proteinase from Leishmania (Leishmania) infantum chagasi (rLdccys1) was previously shown to induce protective immune responses against murine and canine visceral leishmaniasis. These findings encouraged us to use rLdccys1 in the immunotherapy of naturally infected dogs from Teresina, Piauí, a region of high incidence of visceral leishmaniasis in Brazil.Methodology/principal findingsThirty naturally infected mongrel dogs displaying clinical signs of visceral leishmaniasis were randomly divided in three groups: one group received three doses of rLdccys1 in combination with the adjuvant Propionibacterium acnes at one month interval between each dose; a second group received three doses of P. acnes alone; a third group received saline. The main findings were: 1) dogs that received rLdccys1 with P. acnes did not display increase of the following clinical signs: weight loss, alopecia, onychogryphosis, cachexia, anorexia, apathy, skin lesions, hyperkeratosis, ocular secretion, and enlarged lymph nodes; they also exhibited a significant reduction in the spleen parasite load in comparison to the control dogs; 2) rLdccys1-treated dogs exhibited a significant delayed type cutaneous hypersensitivity elicited by the recombinant antigen, as well as high IgG2 serum titers and low IgG1 serum titers; sera from rLdccys1-treated dogs also contained high IFN-? and low IL-10 concentrations; 3) control dogs exhibited all of the clinical signs of visceral leishmaniasis and had low serum IgG2 and IFN-? levels and high concentrations of IgG1 and IL-10; 4) all of the dogs treated with rLdccys1 were alive 12 months after treatment, whereas dogs which received either saline or P. acnes alone died within 3 to 7 months.Conclusions/significanceThese findings illustrate the potential use of rLdccys1 as an additional tool for the immunotherapy of canine visceral leishmaniasis and support further studies designed to improve the efficacy of this recombinant antigen for the treatment of this neglected disease.

Project description:The unreliability of most of the existing antibody-based diagnostic kits to discriminate between active and treated VL cases, relapse situation and reinfection are a major hurdle in controlling the cases of Kala-azar in an endemic area. An antigen targeted diagnostic approaches can be an attractive strategy to overcome these problems. Hence, this study was focused on identifying the Leishmania antigens, lies in circulating immune complex (CICs), can be used for diagnostic as well as prognostic purposes. The present study was conducted on peripheral blood samples of 115 human subjects, based on isolation of CICs. The SDS-PAGE patterns showed an up-regulated expression of 55 kDa and 23 kDa fractions in an antigens obtained from CICs of all clinical and parasitologically proven untreated visceral leishmaniasis patients before treatment (VL-BT), which ensured absolute sensitivity. However, light expressions of these bands were observed in some VL treated cases. To ascertain the prognostic value, 2D expression profiles of circulating antigens were carried out, which revealed 3 upregulated and 12 induced immunoreactive spots. Out of these, ten prominent spots were excised and subjected for enzymatic digestion to generate peptides. Mass spectrometry (MS) analysis successfully explored 20 peptides derived from kinase, kinesin, acetyl Co-A carboxylase, dynein heavy chains (cytoplasmic and axonemal/flagellar), 60S ribosomal protein, nucleoporin protein, RNA polymeraseII, protease gp63, tubulin, DNA polymerase epsilon subunit, GTP-binding protein and tyrosyl-methionyl t-RNA synthetase-like protein and 19 hypothetical protein of unknown function. Presence of L. donovani proteins in circulating antigens were further validated using anti-Ld actin and anti-? tubulin antibody. Besides, MS derived peptides confirmed its reactivity with patients' sera. Therefore, these shortlisted potential antigens can be explored as antigen-based diagnostic as well as prognostic kit.

Project description:Wild canids and domestic dogs are the main reservoir of zoonotic visceral leishmaniasis (VL) caused by Leishmania infantum (syn.: Leishmania chagasi). Serological diagnosis of VL is therefore important in both human and dog leishmaniasis from a clinical and epidemiological point of view. Routine diagnosis of VL is traditionally carried out by immunofluorescent antibody test (IFAT), which is laborious and difficult to standardize and to interpret. In the last decade, however, several specific antigens of Leishmania infantum have been characterized, allowing the development of a recombinant-based immunoassay. Among them, the whole open reading frame encoding K9 antigen, the gene fragment encoding the repetitive sequence of K26, and the 3'-terminal gene fragment of the kinesin-related protein (K39sub) were previously evaluated as diagnostic markers for canine leishmaniasis and proved to be independent in their antibody reactivity. Since sensitivity of serological test is usually higher in multiple-epitope format, in this study the relevant epitopes of K9, K26, and K39 antigens were joined by PCR strategy to produce the chimeric recombinant protein. The resulting mosaic antigen was found highly expressed in Escherichia coli and efficiently purified by affinity chromatography. Antigenic properties of this recombinant antigen were evaluated by indirect enzyme-linked immunosorbent assay (ELISA) using a panel of human and dog sera previously characterized by parasitological and/or serological techniques. Chimeric ELISA showed 99% specificity in both human (n = 180) and canine (n = 343) control groups, while sensitivity was higher in canine VL (96%, n = 213) than in human VL (82%, n = 185). Accordingly, concordance between IFAT and canine chimeric ELISA (k = 0.95, 95% confidence interval = 0.93 to 0.98) was higher than between IFAT and human chimeric ELISA (k = 0.81, 95% confidence interval = 0.76 to 0.87). Results suggest the potential use of this new antigen for routine serodiagnosis of VL in both human and canine hosts.

Dataset Information

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

Publications

Molecular characterization of a kinesin-related antigen of Leishmania chagasi that detects specific antibody in African and American visceral leishmaniasis.

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