# Structural Design of T-Cell Epitope-Based mRNA Vaccine Constructs Determines the Quality of T-Cell Immunity and Protective Efficacy Against SARS-CoV-2 in Mice

## TL;DR

This study shows how the design of mRNA vaccines affects T-cell immunity and protection against SARS-CoV-2 in mice.

## Contribution

The study demonstrates how structural design of mRNA vaccines influences T-cell polarization and protective efficacy.

## Key findings

- Constructs with MHC class I epitopes and no signal peptide induced strong Th1 responses and CD8+ T-cell proliferation.
- Vaccines with elevated IL-10 and IL-4 production provided limited protection, suggesting Th2 or regulatory bias.
- Optimized constructs achieved up to 66% survival in mice after lethal SARS-CoV-2 challenge.

## Abstract

Background/Objectives: 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. Methods: 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. Results: 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. Conclusions: 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.