In silico design of bivalent multi-epitope mRNA vaccine candidates against Mycoplasma pneumoniae and Chlamydia pneumoniae
Community-acquired pneumonia (CAP) remains a major global health concern. Mycoplasma pneumoniae ( M. pneumoniae ) and Chlamydia pneumoniae ( C. pneumoniae ) are important atypical CAP pathogens, and reported coinfection may complicate diagnosis and management. However, no licensed vaccine simultaneously targeting both pathogens is currently available. In this context, mRNA-based multi-epitope vaccines represent a promising platform for bivalent vaccine design because they allow the integration of multiple epitopes into a single construct and may support both humoral and cellular immune responses. This study aimed to design bivalent multi-epitope mRNA vaccine candidates against M. pneumoniae and C. pneumoniae using an immunoinformatics-based strategy. Conserved cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and linear B lymphocyte (LBL) epitopes were screened from selected antigenic proteins. Two constructs, MCV1 and MCV2, were assembled with distinct N-terminal immunostimulatory or helper-epitope modules and evaluated for antigenicity, safety, physicochemical and structural properties, receptor docking, three independent 100-ns molecular dynamics simulations, replicate-level MM-PBSA, immune simulation, population coverage, human codon optimization, and RNA secondary structure. A total of 11 CTL, 5 HTL, and 14 LBL epitopes with ≥ 90% conservancy among the analyzed strain sequences were selected. Both core constructs were predicted to be antigenic, non-toxic, non-allergenic, and structurally acceptable. HADDOCK refinement yielded cluster-level scores of − 394.3 ± 7.3 for MCV1–TLR2 and − 399.0 ± 5.5 for the exploratory MCV2–TLR4 complex. Across three independent trajectories, MM-PBSA total binding free energies were − 125.19 ± 20.13 and − 126.87 ± 12.73 kcal/mol, respectively. C-ImmSim generated modeled profiles involving humoral and cellular immune components, and the estimated global population coverage was 97.90%. Human codon optimization yielded codon adaptation index values of 0.92 and 0.91 and GC contents of 61.06% and 60.94% for MCV1 and MCV2, respectively. In this in silico study, two bivalent multi-epitope mRNA vaccine candidates against M. pneumoniae and C. pneumoniae were computationally prioritized. These findings provide a theoretical basis for subsequent experimental validation, while in vitro and in vivo studies are still required to verify their actual immunogenicity, protective efficacy, and safety.