Immunoinformatics-based design of a multi-epitope vaccine against Fusobacterium nucleatum-associated colorectal cancer

A multi-epitope vaccine candidate, A-20EFγ, was rationally designed against Fusobacterium nucleatum using proteomic and genomic information. The construct comprises a B-cell epitope (epitope 20) derived from the ABC transporter permease, along with cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes (epitopes E, F, and γ) selected from the nickel ABC transporter permease. All epitopes demonstrated favorable immunological properties, including high antigenicity, surface accessibility, and the absence of allergenicity and toxicity. These epitopes were assembled using suitable linkers, and outer membrane protein A was incorporated as an adjuvant to enhance immunogenicity. Additionally, the PADRE sequence was included to improve immune stimulation and population coverage, resulting in a remarkable increase in predicted MHC class II and combined population coverage to 99.98%. The three-dimensional structure of the finalized vaccine was modeled using multiple computational approaches and validated through structural quality assessments and molecular dynamics simulations, with the RoseTTAFold model showing superior stability. Molecular docking, network normal mode, and binding free energy analyses revealed stable interactions between the vaccine and Toll-like receptors 2 and 4. Immune simulations further suggested that A-20EFγ could elicit a robust and long-lasting humoral immune response, characterized by high IgG and IgM levels and enhanced memory B-cell activity. Lastly, codon optimization and in silico cloning were conducted to facilitate in vitro synthesis and predict efficient expression in E. coli using the pET-28a (+) vector. Overall, these preliminary findings suggest A-20EFγ as a potential prophylactic vaccine candidate for the prevention of F. nucleatum-associated colorectal cancer.