Abstract
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.