Optimizing DNA and mRNA vaccine against Human metapneumovirus structural proteins based on rationally designed multi-epitope construct

Nucleic acid-based vaccines have emerged as powerful tools in combating both emerging and re-emerging viral pathogens. Among these, Human metapneumovirus (HMPV) is a respiratory pathogen that predominantly affects children, immunocompromised individuals, and the elderly. A major outbreak in China in 2025 renewed global concern, particularly due to the lack of licensed vaccines or antiviral therapies for HMPV, despite its widespread circulation and clinical significance. In this study, we aimed to design multi-epitope, DNA and mRNA vaccine candidates targeting HMPV structural proteins using a reverse vaccinology approach. Reference sequences of six structural proteins were retrieved from the NCBI Virus database. B-cell, MHC-I, and MHC-II epitopes were predicted using IEDB tools, followed by evaluation of antigenicity, allergenicity, toxicity, and structural stability. Twenty-eight epitopes were selected to construct chimeric proteins, incorporating adjuvants such as PADRE and β-defensin, individually for each protein and in a global construct combining epitopes from all proteins. The constructs showed high predicted antigenicity, no toxicity or allergenicity, and strong binding affinity to innate immune receptors, particularly TLR-2. Immune simulations predicted robust humoral and cellular responses after three doses. In silico cloning into pET-28a(+) enabled heterologous protein expression. Codon optimization for Homo sapiens and in silico cloning of the DNA construct into the pVAX1 vector were realized. Finally, the secondary structure of the mRNA transcript was predicted using RNAfold. These findings support the potential of these in silico-designed vaccines against HMPV, particularly for high-risk populations. Selected epitopes may also contribute to the development of diagnostic tools and enhanced surveillance strategies.