A Mycobacterium tuberculosis multi-epitope DNA vaccine encoding adaptive immune antigens provokes IFNγ/Th1 immunity and confers potential protection

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains a significant global health threat. However, the licensed Bacille Calmette-Guérin (BCG) vaccine provides only limited protection in adults, underscoring the urgent need for more effective preventive strategies. Recent studies have shown that multi-epitope DNA vaccines are superior to traditional vaccines in terms of immunogenicity, safety and stability. In this study, we develop a multi-epitope DNA vaccine that contains CD8+ T-cell epitopes, CD4+ T-cell epitopes, and B-cell epitopes using bioinformatics tools. These epitopes are derived from three genome-encoded proteins, ESAT-6, Rv2660c, and RpfB, which exhibit stage-specific immunodominance in the early, resting, and convalescent stages of MTB infection. Using reverse vaccinology and computational immunomodulation, we demonstrate that the multiepitope vaccine increases antigen-specific antibody titres, activates CD8+ T and CD4+ T cells, and enhances IFN-γ secretion. In vitro validation studies in HEK293T cells confirm high-yield expression of multi-epitope-encoded antigens, whereas in vivo immunization experiments reveal significant expansion of NK cells and Th1-polarized lymphocytes, with concomitant upregulation of pro-inflammatory mediators. Collectively, these results highlight the potent activation of adaptive immunity through Th1-driven mechanisms and IFN-γ-mediated mycobacterial clearance, which are crucial for defending against MTB.

1. Introduction

Tuberculosis (TB) is a major global health issue that affects a quarter of the population and causes 1.5 million deaths annually [1]. While treatable with drugs such as isoniazid and rifampicin, drug resistance leads to low cure rates [2] and high costs [3]. Therefore, TB remains a leading cause of death, with approximately 1.5 million fatalities annually, second only to COVID-19 [4]. Moreover, the Bacille Calmette-Guérin (BCG) vaccine has limited efficacy [5,6], highlighting the urgent need for a better TB vaccine.

TB vaccine development primarily focuses on subunit vaccines utilizing recombinant DNA technology [7]. These vaccines incorporate select antigen components of MTB, which can be divided into protein subunit vaccines and recombinant BCG (rBCG). Although subunit vaccines are safer than whole pathogen vaccines are, they still have limitations, including weaker immunogenicity and the risk for antigen denaturation. Consequently, the development of nucleic acid vaccines has emerged as a promising alternative. These vaccines utilize plasmid vectors encoding Mycobacterium tuberculosis (MTB) antigens to induce host immune responses through genetic transfection [8]. Animal studies have demonstrated that DNA vaccines can induce antigen-specific antibody responses and show great potential in eliciting T-cell-mediated immunity [9]. Although DNA vaccines are generally more cost-effective than are mRNA vaccines and have been shown to be safe in humans, their immunogenicity remains relatively poor [10,11]. Notably, advances in reverse vaccinology and immunoinformatics have lowered epitope screening costs and improved precision, facilitating the design of multi-epitope DNA vaccines with specific immune profiles [12,13]. Despite these advancements, tuberculosis multi-epitope DNA vaccines are still in preclinical stages, with no candidates in clinical trials [14].