Development of a Recombinant Chimeric Antigen Expressing Different Epitopes of <i>Leishmania</i> spp. For Diagnosis of Human Cutaneous Leishmaniasis

Development of highly sensitive and specific diagnostic tests for cutaneous leishmaniasis (CL) remains a significant challenge. Localized immune response in dermal leishmaniasis results in low systemic antibody production. Consequently, serologic diagnostic platform should focus on detecting Leishmania antigens directly from the skin lesions. Designing multiepitope-based proteins as an alternative to crude antigens can enhance sensitivity and specificity, lower production costs, and facilitate development of more field-amenable diagnostic tools. A chimeric multiepitope protein construct (THAG) was designed from four Leishmania spp. genes (txnpx, gp63, hsp70, and amastin). Bioinformatic tools were employed to ensure that the construct was optimized for expression in Escherichia coli and had favorable physicochemical properties. The gene was synthesized, subcloned into the pET28a(+) vector, and expressed in E. coli BL21 (DE3). Protein purification was carried out using Ni-NTA affinity chromatography under hybrid conditions. The purified protein was used to generate polyclonal antibodies in rabbits, and the immunoreactivity of antiserum was assessed against Leishmania antigens from lesions of 54 volunteers with suspected active skin lesions mimic CL through an in-house ELISA. The THAG multiepitope recombinant protein of Leishmania has successfully designed, expressed, purified and confirmed in our study. The results demonstrated sensitivity, specificity and accuracy of the ELISA test in detection of Leishmania antigens in CL lesions to be 90.91% (75.67%-98.08%), 95.24% (76.18%-99.88%), and 92.59% (82.11%-97.94%), respectively. This multiepitope protein has the potential to be further evaluated as a diagnostic tool for CL patients. While we have evaluated this multiepitope protein in an ELISA format, it might be applied to other test formats especially in immunochromatographic assays for the rapid detection of Leishmania antigens in skin lesions. Furthermore, it has the potential to be used as a tool to detect anti-Leishmania antibodies in serum samples of canine and human visceral leishmaniasis (VL) patients.