Bioinformatics-Guided Design of a Multi-Epitope Vaccine Against Vibrio cholerae Using Graphene Oxide Nanoparticles as a Delivery Platform

Abstract

Background: Cholera, caused by Vibrio cholerae, continues to cause significant morbidity and mortality worldwide, especially in endemic regions. Current vaccines exhibit limitations in durability, strain coverage, and logistic feasibility. This study aimed to design a novel multi-epitope vaccine using bioinformatics tools, coupled with graphene oxide nanoparticles as a delivery vehicle to enhance stability, targeting, and immune activation.

Methods: Complete genomes of V. cholerae were retrieved from NCBI, and open reading frames were identified using ORFfinder. Protein subcellular localization was predicted with PSORT, while signal peptides were removed via SignalP. Epitope allergenicity and toxicity were assessed using AlgPred and ToxinPred, respectively. B-cell and T-cell epitopes were predicted using IEDB tools and validated for antigenicity via VaxJen. Transmembrane helices were screened using TMHMM. Selected epitopes were linked using a PAPAP linker and conjugated to an adjuvant, followed by secondary/tertiary structure prediction and molecular docking with MHC class I and II molecules. Graphene oxide nanoparticles were used for targeted delivery, and in vitro assays were performed to evaluate cytotoxicity, plasmid stability, and anti-V. cholerae activity using real-time PCR.

Results: The final vaccine construct comprised non-allergenic, non-toxic epitopes with high predicted antigenicity, capable of binding multiple MHC alleles. Structural modeling confirmed stability and appropriate folding. Graphene oxide conjugation significantly improved delivery efficiency, maintained plasmid integrity, and reduced expression of key virulence genes in V. cholerae cultures.

Conclusions: This study demonstrates the potential of combining immunoinformatics with nanotechnology to develop next-generation cholera vaccines. The proposed graphene oxide-based multi-epitope nanovaccine offers a promising candidate for further preclinical evaluation, with potential applicability against other bacterial pathogens.