A multi-epitope approach for development of a universal vaccine against leptospirosis

Introduction Leptospira , a zoonotic pathogen, poses a significant public health threat, causing morbidity and mortality in both humans and animals. Although several bacterin and recombinant vaccines targeting specific serovars have been developed, most provide short-term, serovar-restricted protection and do not consistently induce sterilizing immunity or broad cross-serovar coverage. Materials To address this challenge, we employed comparative pan-genomic analysis and reverse vaccinology to identify conserved potential vaccine candidates (PVCs) and designed a multi-epitope vaccine (MEV) incorporating highly immunogenic B- and T-cell epitopes. Unlike previous studies largely limited to computational prediction or a small number of known antigens, our strategy integrates soft-core genome-wide antigen prioritization with structural validation and experimental evaluation of innate and adaptive immune responses. Selected epitopes were linked with appropriate spacers and fused to a TLR4 agonist (APPHALS) at the N-terminus to enhance immune activation. Results and discussion In silico analyses confirmed the stability and immunogenic potential of the MEV construct. The purified recombinant MEV reacted strongly with hyperimmune and clinical sera. Anti-MEV antibodies agglutinated multiple pathogenic Leptospira serovars and inhibited bacterial growth in vitro . MEV stimulation induced macrophage activation, evidenced by increased proinflammatory cytokine production and upregulation of co-stimulatory molecules. Immunization in mice elicited robust humoral and T-cell responses. Collectively, these findings position MEV as a rationally designed next-generation vaccine candidate with potential for broad cross-protective immunity against leptospirosis.