Epitope Mapping of the HSP83.1 Protein of Leishmania braziliensis Discloses Novel Targets for Immunodiagnosis of Tegumentary and Visceral Clinical Forms of Leishmaniasis

Abstract

Gold standard serological diagnostic methods focus on antigens that elicit a strong humoral immune response that is specific to a certain pathogen. In this study, we used bioinformatics approaches to identify linear B-cell epitopes that are conserved among Leishmania species but are divergent from the host species Homo sapiens and Canis familiaris and from Trypanosoma cruzi, the parasite that causes Chagas disease, to select potential targets for the immunodiagnosis of leishmaniasis. Using these criteria, we selected heat shock protein 83.1 of Leishmania braziliensis for this study. We predicted three linear B-cell epitopes in its sequence. These peptides and the recombinant heat shock protein 83.1 (rHSP83.1) were tested in enzyme-linked immunosorbent assays (ELISAs) against serum samples from patients with tegumentary leishmaniasis (TL) and visceral leishmaniasis (VL) and from dogs infected with Leishmania infantum (canine VL [CVL]). Our data show that rHSP83.1 is a promising target in the diagnosis of TL. We also identified specific epitopes derived from HSP83.1 that can be used in the diagnosis of human TL (peptide 3), both human and canine VL (peptides 1 and 3), and all TL, VL, and CVL clinical manifestations (peptide 3). Receiver operating characteristic (ROC) curves confirmed the superior performance of rHSP83.1 and peptides 1 and 3 compared to that of the soluble L. braziliensis antigen and the reference test kit for the diagnosis of CVL in Brazil (EIE-LVC kit; Bio-Manguinhos, Fiocruz). Our study thus provides proof-of-principle evidence of the feasibility of using bioinformatics to identify novel targets for the immunodiagnosis of parasitic diseases using proteins that are highly conserved throughout evolution.

Improving Serodiagnosis of Human and Canine Leishmaniasis with Recombinant Leishmania braziliensis Cathepsin L-like Protein and a Synthetic Peptide Containing Its Linear B-cell Epitope

“…Depletion ELISA was performed to confirm that the synthetic peptide represents a human and canine B-cell linear epitope in CatL. This technique is based on the reduction of serum reactivity via the depletion of peptide-specific antibodies; in this case, the sample is incubated with the synthetic peptide prior to ELISA with a known antigen [23] , [24] . The depleted sample was tested for reactivity against the recombinant protein, and the reduction was proportional to antibody levels that bind to the similar peptide within the protein sequence.…”

“…The reaction was stopped after 30 min with 4 N H 2 SO 4 , and the absorbance was measured at 450 nm. For the depletion ELISA, the sera was incubated in peptide-coated and blocked plates at a 1∶100 dilution overnight at 2–8°C [23] , [24] . Depleted and undepleted samples were transferred to plates coated overnight with r CatL (50 ng/well) and blocked.…”

“…Recombinant Leishmania proteins have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization [17] . In this context, various recombinant proteins, among them k39, KMPII, Peroxidoxins, LACK, nucleosomal histones (H2A, H2B, H3 and H4) and heat shock proteins (families 60, 70 and 83) have been tested in the diagnosis of visceral and tegumentary leishmaniasis and obtained promising results for development of diagnosis kits using these antigens [17] – [24] . Moreover, in silico and experimental methods for epitope mapping and peptide synthesis have great potential for the discovery of new potential pathogen antigens [25] , [26] .…”

Laboratory Diagnosis of Cutaneous and Visceral Leishmaniasis: Current and Future Methods

“…Recombinant proteins of L. ( L. ) infantum , such as HSP70, histone proteins, and a protein of the kinetoplast (KMP11), were also tested using sera from patients with CL, with HSP70 showing the best performance [ 170 ]. Another study using the recombinant version of the HSP83.1 protein from L. ( V. ) braziliensis showed that it is a promising antigen for the immunodiagnosis of both clinical forms of leishmaniasis [ 192 ].…”

Estudo dos efeitos da proteína LaLRR17 de >i<Leishmania (L.) amazonensis >/i<na infecção do macrófago e identificação de seus possíveis ligantes.>i<

“…Para identificar ligantes da LaLRR17 optamos por utilizar a proteína recombinante. De fato, a maior parte dos estudos focados em diagnóstico, cristalografia e análise funcional de uma proteína dependem de sua produção de forma recombinante (MENEZES-SOUZA et al,2014;BATISTA et al,2015;SERAPHIM et al,2015). O sistema bacteriano é o método mais eficaz para produção de proteína recombinante, pois a bactéria Escherichia coli é bem caracterizada em termos genéticos e bioquímicos e pode ser manipulada de forma segura e com baixo custo quando comparada a outros sistemas de expressão (CHAO et al,2002).…”