Mapping B-Cell Epitopes for the Peroxidoxin of Leishmania (Viannia) braziliensis and Its Potential for the Clinical Diagnosis of Tegumentary and Visceral Leishmaniasis

Abstract

The search toward the establishment of novel serological tests for the diagnosis of leishmaniasis and proper differential diagnosis may represent one alternative to the invasive parasitological methods currently used to identify infected individuals. In the present work, we investigated the potential use of recombinant peroxidoxin (rPeroxidoxin) of Leishmania (Viannia) braziliensis as a potential antigen for the immunodiagnosis of human tegumentary (TL) and visceral leishmaniasis (VL) and canine visceral leishmaniasis (CVL). Linear B-cell epitope mapping was performed to identify polymorphic epitopes when comparing orthologous sequences present in Trypanosoma cruzi, the agent for Chagas disease (CD), and the Homo sapiens and Canis familiaris hosts. The serological assay (ELISA) demonstrated that TL, VL and CVL individuals showed high levels of antibodies against rPeroxidoxin, allowing identification of infected ones with considerable sensitivity and great ability to discriminate (specificity) between non-infected and CD individuals (98.46% and 100%; 98.18% and 95.71%; 95.79% and 100%, respectively). An rPeroxidoxin ELISA also showed a greater ability to discriminate between vaccinated and infected animals, which is an important requirement for the public campaign control of CVL. A depletion ELISA assay using soluble peptides of this B-cell epitope confirmed the recognition of these sites only by Leishmania-infected individuals. Moreover, this work identifies two antigenic polymorphic linear B-cell epitopes of L. braziliensis. Specific recognition of TL and VL patients was confirmed by significantly decreased IgG reactivity against rPeroxidoxin after depletion of peptide-1- and peptide-2-specific antibodies (peptide 1: reduced by 32%, 42% and 5% for CL, ML and VL, respectively; peptide-2: reduced by 24%, 22% and 13% for CL, ML and VL, respectively) and only peptide-2 for CVL (reduced 9%). Overall, rPeroxidoxin may be a potential antigen for the immunodiagnosis of TL, VL or CVL, as it has a higher agreement with parasitological assays and is better than other reference tests that use soluble Leishmania antigens for diagnosing CVL in Brazil (EIE-LVC, Bio-manguinhos, FIOCRUZ).

Proteins Selected in Leishmania (Viannia) braziliensis by an Immunoproteomic Approach with Potential Serodiagnosis Applications for Tegumentary Leishmaniasis

“…Tryparedoxin peroxidase belongs to a family of proteins called peroxiredoxins, which are associated with parasite virulence and have a high antigenicity during the active disease. Previous studies have shown the antigenicity of peroxiredoxins in dogs and humans infected by Leishmania infantum or L. braziliensis (46,47). Khosravi et al showed that a realtime PCR using the tryparedoxin peroxidase gene was specific for CL diagnosis (48).…”

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

“…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.…”

Leishmania infantum recombinant kinesin degenerated derived repeat (rKDDR): A novel potential antigen for serodiagnosis of visceral leishmaniasis

“…The Bio-Manguinhos EIE-LVC kit is recommended by the Brazilian Ministry of Health for diagnosis of VL in dogs [44]. However, comparative serological studies have shown that the EIE-LVC kit needs to be used in conjunction with another diagnostic method to reduce false-positive results and to compensate for high potential for cross-reactivity [45,46].…”