Identification of Epitope-based vaccine peptides within phatogenic and intermediate Leptospira spp.

Leptospira, a spirochete bacterium causing leptospirosis, is a zoonotic disease transmitted from animals to humans. Its pathophysiological mechanisms remain largely unknown, and symptoms can range from mild flu-like symptoms to severe, even fatal outcomes. The limitations of commercially available Leptospiral vaccines pose significant public health challenges, especially in endemic regions. Epitope- based vaccination offers a promising strategy to identify potential peptides for vaccine development. This research aims to identify potential epitope-based peptides within pathogenic and intermediate Leptospira species using in silico and in vitro methodologies. The study inciated by selecting the outer membrane protein LipL46, conserved in 43 pathogenic and 21 intermediate Leptospira serovars. Multiple sequence alignment of LipL46 revealed nine conserved regions, from which we selected three peptide sequences: vaccine peptide 1: DGESLGSSLLSKTDAFV, vaccine peptide 2: VSREKAISEWA, and vaccine peptide 3: TENNPIVLKFTG. The epitope mapping of these vaccine peptide sequences with the B-cell and T-cell epitopes, considering 90% coverage of major histocompatibility complex (MHC) class 1 and 2 (30 alleles from MHC class I and 38 alleles from MHC class II alleles) for T-cell epitopes. For B-cell epitope mapping, we used three algorithms (Bepipred Liner Epitope Prediction, Chou & Fasman Beta-Turn and Kalplus & Schulz) to obtain consensus results: vaccine peptide 1 was mapped by all three algorithms, while vaccine peptides 2 and 3 were partially mapped. Although vaccine peptides 2 and 3 were partially mapped, they have strong T-cell epitope potential. Hence, it can ensure a more comprehensive immune response that engages B-cells and T-cells. To standardize vaccine peptide length, epitope conservancy extended vaccine peptides 2 and 3 to 15-mers, resulting in vaccine peptide 2 (285 - 300) TQKVSREKAISEWAT and vaccine peptide 3 (313 - 328) WFNLTENNPIVLKFG. These vaccine peptides were stable based on physicohemical analysis, with values less than 40. Further analysis, including immunogenicity, antigenicity, and molecular docking, showed that vaccine peptide 1 had the lowest immunogenicity score (-0.66657), making it the most immunogenic vaccine peptide compared to vaccine peptides 2 (0.05899) and 3 (0.16569). Additionally, vaccine peptide 1 (0.6320) and 3 (0.8643) were antigenic, while vaccine peptide 2 (0.2689) was non-antigenic. Cytokine prediction was performed on interleukins 2, 4, 6, 10, and interferon-gamma. The three peptides induced various cytokines: vaccine peptide 1 triggered IL-6 and IFN-gamma, vaccine peptide 2 induced IL-10 and IFN-gamma, and vaccine peptide 3 induced IL- 2, IL-4 and IFN-gamma. These variations stem from differences in vaccine peptide binding affinity to MHC molecules and T-cell receptors, leading to distinct immune responses and cytokine profiles. Docking simulations were performed to verify the binding affinity between the vaccine peptides and MHC molecules. Therefore, molecular docking was also predicted using three web tools: HDOCK, HPEPDOCK, and DOCKTHOR. The results showed that vaccine peptide 3 had the lowest docking score compared to the other two vaccine peptides. Hence, these score indicates a strong binding affinity with the docked HLA allelesHuman Leukocyte Antigens alleles (HLA-A, HLA-B, HLA-DP, HLA-DQ, and HLA-DR). The docking scores were -200.89 (HLA-A), -186.753 (HLA-B), -225.472 (HLA-DP), -219.22 (HLA-DQ) and -217.239 (HLA-DR). Following the predicted results, we validated the vaccine peptides in vitro using 30 serum samples from the Institute for Medical Research (IMR), comprising 20 positive and ten (10) negative samples. The ELISA assay revealed that vaccine peptide 1 demonstrated the most promising results, with the highest absorbance value (0.279) compared to vaccine peptide 2 (0.159) and vaccine peptide 3 (0.230) at a wavelength of 630 nm. Statistical analysis indicated no significant difference between the vaccine peptides. This might be due to the low number of samples tested. Further in vivo studies are crucial to confirm the safety and efficacy of the vaccine.

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