Design of a novel multi epitope antigen for diagnosis of visceral leishmaniasis using an immunoinformatics approach.

Visceral Leishmaniasis (VL) represents a critical parasitic disease with considerable implications for public health. The predominant etiological agent responsible for most VL cases in the Mediterranean region and Latin America is the Leishmania infantum parasite. Current diagnostic methods, including microscopy and serological assays, have limitations such as low sensitivity and cross-reactivity. The development of multi-epitope antigens presents a promising approach for improving diagnostic accuracy. This study focused on the design of a novel multi-epitope antigen for human VL diagnosis using immunoinformatics tools. Four antigenic proteins, including Prohibitin, Apolipoprotein A-I, Aldehyde Dehydrogenase, and Protein Disulfide-Isomerase, were selected based on an extensive review of the literature and prior immunoproteomic studies of L. infantum conducted by our research team. Conserved B-cell epitopes were identified and linked using EAAAK linkers to ensure structural integrity. The designed antigen was assessed for antigenicity, stability, solubility, and structural integrity. Secondary and tertiary structures were predicted and validated through computational tools. Codon optimization enhanced expression efficiency in Escherichia coli, and the sequence was successfully cloned into the pET-23a(+) vector. Immunoinformatics analysis confirmed the antigen's high immunogenic potential, stability, and suitability for diagnostic applications. These findings suggest that the design of such a multi-epitope antigen may represent a dependable candidate for human VL serodiagnosis, warranting further experimental validations.