Fernanda de Moraes Maia¹, Angela da Silva Barbosa², Isabela Ferreira Soares³, Cassia Moreira Santos², Daniella dos Santos Courrol², Josué da Costa Lima Júnior³, Rodrigo Nunes Rodrigues da Silva¹

Leptospirosis is the most widespread zoonoses in the world, with more than 1 million reported cases and 60,000 deaths annually. In Brazil, more than 3,500 cases are reported annually, of which approximately 75% require hospitalization, and 11% result in death. Currently, there is no effective vaccine licensed for humans. In the search for vaccine antigens, proteases associated with disease severity and the bacterial evasion of the innate immune response have been proposed as promising vaccine candidates. Considering the spectrum of proteases exclusive to pathogenic leptospires, and their overlapping functions, this study proposed the development and validation of a chimeric vaccine (CV), composed of epitopes from four proteases, conserved among pathogenic leptospires, and related to the bacterium immune evasion. Using immunoinformatics algorithms, we selected 8 antibody target epitopes and 5 TCD4 cell response targets, recognized by HLAs with broad population coverage and compatible with the murine immune system. The epitopes were connected by flexible linkers, constituting a chimeric protein fused to the universal epitope PADRE and the structural adjuvant HbHA (heparin-binding haemagglutinin). In silico analysis of the critical quality attributes of the generated protein showed a thermostable, soluble, antigenic and safe structure (non-toxic and non-allergenic), capable of interacting properly with TLR-2 and TLR-4. The recombinant protein was expressed in E. coli and formulated with Addavax adjuvant and used to immunize BALB/c mice in three doses (days 0, 21 and 42), with monitoring until day 133. The immune response was compared with control groups, immunized with: synthetic peptide pools (B and TCD4 epitopes) + Addavax, PBS + Addavax and PBS. ELISA analysis demonstrated the high immunogenicity of the CV, with detectable IgG titers reaching 1:300,000 following the third immunization, while the isolated peptides induced titers of less than 1:100. In addition, the humoral immune response elicited by immunization against each individual epitope incorporated into the vaccine formulation was evaluated, revealing antibody titers ranging from 1:25 to 1:800, demonstrating variable but measurable immunoreactivity across all epitopes tested. The cellular response evaluated by ELISpot showed that CV induces a specific T response, although the peptide group showed a greater number of IFN-γ, IL-2 and IL-4 secreting cells when stimulated with TCD4 epitopes. Finally, the serum of the animals immunized with CV recognized the native secreted proteins of L. interrogans Copenhagi (Western blotting), demonstrating its ability to recognize the native protein. The results show that the proposed chimeric vaccine was able to induce a robust humoral response and the recognition of native antigens of the bacteria. These findings reinforce its potential as a promising candidate for the prevention of human leptospirosis and justify preclinical tests followed by challenge experiments with Leptospira spp. to evaluate its protective efficacy. In addition, our data validate the efficacy of the immunoinformatics-based approach for the development of new vaccine antigens.

Agradecimentos: Development agencies – CAPES, FAPERJ, INOVA FIOCRUZ, BIO-MANGUINHOS