Bivalent mRNA Vaccine Candidate Targeting Mycoplasma and Chlamydia Pneumonia Designed via Immunoinformatics
Background
Community-Acquired Pneumonia (CAP) is a major acute respiratory disease threatening global public health. In clinical settings, infections by atypical pathogens are frequently confirmed, making the identification of causative agents difficult. Representative causative agents, Mycoplasma pneumoniae and Chlamydia pneumoniae, typically begin like a common cold but can escalate into severe pneumonia. Cases of co-infection due to simultaneous exposure to both bacteria are not uncommon, and the resulting complex symptoms have frequently led to difficulties in antibiotic prescription and establishing treatment directions.
Currently, there are no approved vaccines globally that simultaneously prevent both pathogens. The previous approach of manufacturing separate single vaccines for each strain has imposed burdens such as causing immune interference between antigens or increasing production costs. As an alternative, the multi-epitope-based mRNA platform is gaining attention. This is because it can evenly activate both humoral and cellular immunity by precisely placing the core antigenic epitopes within a single polypeptide chain. Computer algorithm-based immunoinformatics computation has emerged as a powerful tool for rapidly identifying safe vaccine candidates by screening extensive strain sequences.
Key Findings
The researchers identified epitopes with over 90% conservation across the analyzed strain sequences. A total of 11 Cytotoxic T-Lymphocyte (CTL) epitopes, 5 Helper T-Lymphocyte (HTL) epitopes, and 14 Linear B Lymphocyte (LBL) epitopes were derived.
The selected epitopes were connected with immunostimulatory sequences and linkers to complete two vaccine candidate sequences, MCV1 and MCV2. The two sequences were designed to compare differences in immune activation pathways by arranging different immune-stimulating peptide modules at the N-terminus. Toxicity and allergy evaluations showed that both substances are safe for human cells, and they also exhibited physicochemical stability in terms of the protein's tertiary structure.
In molecular docking experiments to verify binding stability with human immune receptors, favorable binding profiles were observed. When applying the precision docking tool HADDOCK, the cluster binding score for the MCV1 and Toll-Like Receptor 2 (TLR2) complex was calculated as -394.3 ± 7.3. The score for the MCV2-TLR4 complex of the exploratory model was also -399.0 ± 5.5, indicating tight binding to the receptor.
In Molecular Dynamics (MD) simulations repeated three times for 100 nanoseconds (ns), the conformation remained stable without fluctuation. The binding free energies (MM-PBSA) of the MCV1-TLR2 and MCV2-TLR4 complexes were calculated to be -125.19 ± 20.13 kcal/mol and -126.87 ± 12.73 kcal/mol, respectively. The results of the virtual immune simulation (C-ImmSim) showed active B-cell antibody production and memory T-cell responses occurring simultaneously. The predicted inclusivity index of the global population reached 97.90%. Codon optimization to enhance human cell expression efficiency and the design of a stable mRNA secondary structure were also completed.
Significance and Outlook
The vaccine design technique applying immunoinformatics has achieved the result of shortening the initial antigen discovery period, which used to take years of manual experimentation, to within a few weeks. Since sequences with over 90% conservation were used as a common reference, it is expected to form a barrier that prevents immune evasion even against pathogen mutations. Arranging the antigen information of Mycoplasma and Chlamydia, which belong to different genera, harmoniously into a single mRNA molecule is regarded as an advanced engineering achievement in the field of multivalent vaccine development.
This achievement is inherently limited by its nature as an in silico study based on advanced software prediction and physical simulation. Whether the binding affinity and antibody formation prediction profiles derived from computer screens will function identically in actual living tissues is a separate issue. To ensure efficacy as an actual vaccine, in vivo efficacy evaluations using infected animal models must follow. Securing preclinical data to demonstrate the formulation and in vivo delivery stability of lipid nanoparticles (LNPs) is expected to be a critical milestone for commercialization.
BACKGROUND: 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. METHODS: 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. RESULTS: 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 opti
The bivalent mRNA design proposed in this study provides a concrete solution for clinical and industrial applications in the management of complex respiratory infectious diseases. Because Mycoplasma and Chlamydia have initial symptoms similar to a cold, rapid diagnosis in the field is difficult, and in children or the elderly, co-infection can easily lead to severe complications such as acute respiratory distress syndrome. Pharmaceutical and biotech companies can immediately commence synthetic biology production processes based on the optimized codon sequences and epitope arrangement information derived from the paper. If a vaccine that simultaneously protects against two bacterial pathogens with a single dose is commercialized, it is expected to lower ICU admission rates and significantly reduce the financial burden on the healthcare system.