Structural Design of T-Cell Epitope-Based mRNA Vaccine Constructs Determines the Quality of T-Cell Immunity and...

Epitope-based mRNA vaccines represent a promising strategy for eliciting protective T-cell immunity against SARS-CoV-2 and as well as for non-infectious mRNA-based vaccines. However, how the structural architecture of vaccine constructs (including epitope arrangement, linker composition, signal peptide presence, and the combination of MHC class I and II epitopes) shapes the quality of T-cell responses remains poorly understood.

Ten tandem minigene mRNA constructs (Cons1-10) encoding different combinations of MHC class I and class II epitopes from SARS-CoV-2 proteins (S, N, M, ORF3a) were designed, encapsulated in lipid nanoparticles, and administered to C57BL/6 mice. Immunogenicity was assessed by cytokine profiling (IFN-γ, IL-2, IL-4, IL-10) and T-cell proliferation assays. Protective efficacy was evaluated in K18-hACE2 transgenic mice challenged with SARS-CoV-2.

Constructs lacking a signal peptide and enriched in MHC class I-restricted epitopes induced robust Th1 responses and strong CD8⁺ T-cell proliferation, achieving up to 66% survival following lethal challenge. In contrast, constructs associated with elevated IL-10 and IL-4 production conferred limited protection (11-33%), consistent with functional skewing towards regulatory or Th2-associated immune profiles.

These findings establish a direct link between construct design parameters and T-cell polarization quality, and provide a rational framework for next-generation epitope-based mRNA vaccine development.