Design and evaluation of potent multiepitope broad spectrum DNA and protein vaccine candidates against leptospirosis

Leptospirosis is a widespread zoonotic disease that causes severe health complications with no approved vaccine which provide broad range protection. In this study, we have focused on LruC protein from the outer membrane of Leptospira spp. LruC protein has been considered as promising target for vaccine due to its immunogenicity and conservancy. We have identified total 13 conserved B-cell, CTL, and HTL epitopes from 22 different pathogenic Leptospira species and serovars, which were linked with 4 linkers and 3 adjuvants (HBHA, CTB, TLR4) to design 36 multiepitope vaccine constructs to study the effect of different components on vaccine effectiveness. The antigenicity, immunogenicity, and non-allergenicity of the constructs were confirmed through computational analyses. Physico-chemical properties, secondary structure, and tertiary models of the vaccine constructs were predicted and validated. Molecular docking studies were conducted with Toll-like receptors (TLR2, TLR4) to assess binding affinity, identifying three top vaccine candidates (HBHA-construct 6, CTB-construct 9, and TLR4-construct 12) for further investigation. Further, these candidates were successfully cloned into pVAX1 and pET30a vectors to prepare DNA and protein vaccines, respectively. Moreover, these multiepitope vaccines were tested in mice models to assess its immunogenicity. ELISA performed with antisera against vaccine antigen, as well as crude extract of pathogenic Leptospira species showed significant IgG responses, particularly in protein vaccines. Flow cytometry revealed increased IFN-γ producing CD4⁺ and CD8⁺ T cells, especially in the TLR4-adjuvanted vaccine groups. The microscopic agglutination test further confirmed the specificity of the antibody response to Leptospira serovars. Overall, this study demonstrates the potential of these multiepitope vaccine constructs in eliciting a robust immune response, laying the foundation for future challenge study and preclinical evaluation.