Mycobacterial EsxG·EsxH (TB9.8·TB10.4) peptides as a subunit vaccine to booster BCG vaccination in an experimental model of pulmonary Tuberculosis. Data

The attenuated <i>Mycobacterium bovis</i> bacillus Calmette-Guérin (BCG) vaccine is currently the only validated vaccine against tuberculosis (TB). In a previous study, we conducted an <i>in-silico</i> selection of four peptides (G1, G2, H1, and H2) derived from the mycobacterial protein antigens TB10.9·TB10.4 (EsxG·EsxH). Bioinformatic analysis and molecular dynamic simulations predicted these epitopes could be loaded into a MHC-II complex, inducing T and B cell activation. The present study aimed to experimentally validate these peptides as subunit vaccines by determining their cytotoxicity, immunogenicity, and protective efficacy against <i>Mycobacterium tuberculosis</i> (Mtb) in mice when administered as a booster to BCG vaccination. Mice were vaccinated with BCG and, two months later, were subcutaneously immunized with either peptide G1, G2, H1, or H2. One-month post-immunization, mice were challenged with the reference strain H37Rv of moderate virulence or the hypervirulent clinical isolate 09005186. After vaccination and before the challenge, the spleen and lung cells were harvested and stimulated <i>in vitro</i> with the corresponding peptide to measure cytokine expression in CD4⁺, and CD8⁺ T cells, as well as the phenotypes of activated effector T cells, proliferative senescence, central and periphery memory CD4⁺ and CD8⁺ cells. Additionally, specific IgG antibody titers elicited by each peptide were measured using ELISA. Compared with animals vaccinated only with BCG, boosting BCG vaccination with these peptides provided enhanced protection by significantly prolonging the mice survival, reducing the bacillary load, and decreasing tissue damage (pneumonia). These findings contribute to the broader understanding of peptide-based subunit vaccines and highlight the potential for tailored approaches to enhance protective immunity.

减毒牛分枝杆菌（Mycobacterium bovis）卡介苗（bacillus Calmette-Guérin, BCG）是目前唯一经证实有效的结核病（tuberculosis, TB）疫苗。在既往研究中，我们通过计算机模拟实验（in silico）从分枝杆菌蛋白抗原TB10.9·TB10.4（EsxG·EsxH）中筛选得到4个肽段（G1、G2、H1、H2）。生物信息学分析与分子动力学模拟预测，这些表位可装载于主要组织相容性复合体II类（MHC-II）分子中，进而诱导T、B细胞活化。本研究旨在通过实验验证上述肽段作为亚单位疫苗的潜力：将其作为卡介苗免疫后的加强免疫制剂，在小鼠模型中评估其抗结核分枝杆菌（Mycobacterium tuberculosis, Mtb）的细胞毒性、免疫原性与保护效力。实验中，小鼠先接受卡介苗免疫，2个月后分别皮下免疫G1、G2、H1或H2肽段。免疫后1个月，小鼠分别被中等毒力参考毒株H37Rv，或高毒力临床分离株09005186攻毒。在免疫后至攻毒前，采集小鼠脾细胞与肺细胞，使用对应肽段进行体外刺激，以检测CD4+、CD8+ T细胞的细胞因子表达水平，以及活化效应T细胞、增殖性衰老细胞、中枢记忆与外周记忆CD4+、CD8+ T细胞的表型。此外，通过酶联免疫吸附试验（ELISA）检测各肽段诱导的特异性IgG抗体滴度。相较于仅接受卡介苗免疫的小鼠，以上述肽段进行加强免疫的小鼠存活时间显著延长、菌负荷降低、组织损伤（肺炎病变）减轻，保护效果得到增强。本研究结果有助于深化对肽类亚单位疫苗的认知，并凸显定制化策略提升保护性免疫的应用潜力。