Antigenic Multi-Epitope Vaccine Against Ross River Virus Using Capsid and Envelope Proteins via Immunoinformatics and Reverse Vaccinology Approaches.

Abstract

Ross River virus (RRV), a mosquito-borne alphavirus of the Togaviridae family, is the causative agent of Ross River fever a debilitating infectious disease characterized by fever, rash, and persistent polyarthritis. The virus remains endemic across Australia and the Pacific regions, with sporadic outbreaks reported in new geographic areas due to climate change and increased mosquito activity. Despite its growing public health impact, no licensed vaccine or antiviral therapy currently exists, underscoring the urgent need for an effective preventive strategy. In this study, an immunoinformatics-based approach was employed to design a novel multi-epitope vaccine (MEV) against RRV, where conserved and antigenic cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes were predicted from structural proteins (capsid and envelope glycoproteins E1 and E2) as well as selected non-structural protein regions, followed by their rational assembly into a vaccine construct using appropriate linkers and an immune-stimulatory adjuvant. Conserved and antigenic cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell epitopes were predicted from structural and non-structural proteins and linked with appropriate linkers, while β-defensin was incorporated as an adjuvant to enhance immunogenicity. The vaccine construct underwent modeling, refinement, and structural validation using PSIPRED, AlphaFold, and GalaxyRefine. Molecular docking analyses with immune receptors (TLR8 and HLA) and molecular dynamics (MD) simulations evaluated receptor binding affinity and structural stability. The C-ImmSim server was used to simulate immune responses, and codon optimization ensured suitability for Escherichia coli expression. The designed MEV exhibited strong antigenicity (0.79), stability, and solubility, with a molecular weight of ~30 kDa and theoretical pI of 9.98. Structural validation revealed over 95% of residues in favored Ramachandran regions, and docking analysis showed predicted interaction with TLR8 (- 1712.2 kJ/mol). MD simulations confirmed stable complex formation, while immune simulations predicted robust and long-lasting humoral and cellular immune responses with elevated IgG, IFN-γ, and memory B/T-cell populations. The designed MEV demonstrates promising antigenic, structural, and immunological properties, making it a potential vaccine candidate for further in vitro and in vivo evaluation against Ross River virus infection.