# Immunoinformatic Design and Evaluation of a Multi-Epitope mRNA Vaccine RP14914P Targeting Latent Tuberculosis Infection

## TL;DR

This paper presents a new mRNA vaccine candidate, RP14914P, designed to prevent the reactivation of latent tuberculosis infection using computational methods.

## Contribution

The novel contribution is the in silico design of a multi-epitope mRNA vaccine targeting LTBI with broad population coverage and predicted strong immune activation.

## Key findings

- RP14914P encodes 14 cytotoxic T lymphocyte, 9 helper T lymphocyte, and 14 B-cell epitopes with high antigenicity and immunogenicity scores.
- Molecular dynamics simulations confirm stable binding of the vaccine to TLR2 and TLR4 with docking energies of −1477.8 and −1480.1 kcal/mol, respectively.
- Immune simulations predict strong Th1 polarization, high cytokine secretion, and durable memory responses.

## Abstract

Background: Latent tuberculosis infection (LTBI) is the principal reservoir for active tuberculosis, with >85% of cases attributable to reactivation. Bacillus Calmette-Guérin fails to block this transition, leaving a critical gap in prevention. Methods: An immunoinformatics/reverse-vaccinology pipeline was applied to seven dormancy-related antigens retrieved from Mycobrowser. T-cell epitopes were predicted with NetMHCI/IIpan-4.1 and B-cell epitopes with ABCpred; antigenicity, allergenicity, and toxicity were evaluated with VaxiJen, AllerTOP, and ToxinPred. Secondary/tertiary structures were modeled with PSIPRED and AlphaFold-3; docking to Toll-like receptors (TLR) 2/4 and 100 ns molecular dynamics simulations assessed complex stability. Immune responses were simulated with C-ImmSim, and the mRNA sequence was human-codon-optimized using ExpOptimizer. Results: The resulting construct, RP14914P, encodes 14 cytotoxic T lymphocyte, 9 helper T lymphocyte, and 14 B-cell epitopes within an 866-aa, 90.4 kDa polypeptide. Antigenicity score = 0.7797, immunogenicity score = 8.58629. and no toxicity or allergenicity was predicted. Physicochemical analysis: instability index = 28.65, and solubility = 0.513. Estimated population coverage is 82.35% and 99.67% for Human Leukocyte Antigen (HLA)-I and HLA-II globally. Docking energies: −1477.8 kcal/mol (TLR2) and −1480.1 kcal/mol (TLR4). Molecular dynamics trajectories confirm stable binding. Immune simulation predicts potent activation of Natural Killer cells, macrophages, and dendritic cells, Th1 polarization, high interferon-γ/interleukin-2 secretion, and durable memory. Conclusions: In silico analyses predict that RP14914P exhibits favorable immunogenicity, safety, and broad population coverage, suggesting its potential as a promising mRNA vaccine candidate to prevent LTBI reactivation. However, these computational predictions require thorough experimental validation to confirm the vaccine’s immunogenicity and protective efficacy.