Xu Yun, Wang Xiaochun, Zhong Qiangsen
Department of Pathogen Biology, School of Medicine, Anhui University of Science and Technology, Huainan, China.
Department of Pathogen Biology, School of Medicine, Anhui University of Science and Technology, Huainan, China.
J Microbiol Methods. 2025 Jul 26;236:107202. doi: 10.1016/j.mimet.2025.107202.
Bacillus Calmette-Guérin (BCG) is the only vaccine currently used in clinical practice to prevent tuberculosis (TB), however, it remains limited in preventing latent infection, TB reactivation, and providing comprehensive protection. In this study, the pcaA gene, an antigen associated with persistent infection, was selected, and the recombinant plasmid pET28a-PcaA was successfully constructed for protein expression and purification. The specificity of the antigen was further verified using chemiluminescence, enzyme-linked immunosorbent assay (ELISA), flow cytometry, and mycobacterial growth inhibition assay (MGIA). Significant differences were observed in the expression levels of IFN-γ, IL-2, IL-8, and IgG in the peripheral blood of patients with Mycobacterium tuberculosis (M. tb) following stimulation with the PcaA antigen in the Active Tuberculosis (ATB) and Latent Tuberculosis Infection (LTBI) groups. An IL-8 combined diagnostic model could effectively distinguish between ATB and LTBI, while anti-PcaA IgG demonstrated strong performance in ruling out M. tb infection. Recombinant PcaA protein (rPcaA) was formulated with liposome dimethyl dioctadecylammonium bromide (DDA) / colloidal manganese salt (MnJ)/DM to immunize mice. Serum-specific antibody levels, cytokines secreted by splenocytes, and the number of multifunctional T cells in splenocytes were assessed. The results indicated that the BCG + rPcaA-DM vaccine group exhibited significantly elevated levels of Th1-type cytokines, antibody titers, and the frequencies of IFN-γ/TNF-α single and double-positive CD4 and CD8 T cells compared to the BCG group. Furthermore, splenocytes and lung cells from immunized mice significantly inhibited mycobacterial growth. These findings suggest that the rPcaA-DM vaccine, as a BCG booster, significantly enhances Th1 polarization and provides robust protective efficacy, with potential to prevent LTBI progression to ATB.
卡介苗（BCG）是目前临床实践中唯一用于预防结核病（TB）的疫苗，然而，它在预防潜伏感染、结核再激活以及提供全面保护方面仍然存在局限性。在本研究中，选择了与持续感染相关的抗原pcaA基因，并成功构建了重组质粒pET28a-PcaA用于蛋白表达和纯化。使用化学发光、酶联免疫吸附测定（ELISA）、流式细胞术和分枝杆菌生长抑制试验（MGIA）进一步验证了该抗原的特异性。在活动性结核病（ATB）和潜伏性结核感染（LTBI）组中，用PcaA抗原刺激后，结核分枝杆菌（M. tb）患者外周血中IFN-γ、IL-2、IL-8和IgG的表达水平存在显著差异。IL-8联合诊断模型可以有效区分ATB和LTBI，而抗PcaA IgG在排除M. tb感染方面表现出强大性能。重组PcaA蛋白（rPcaA）与脂质体二甲基二十八烷基溴化铵（DDA）/胶体锰盐（MnJ）/DM制剂一起用于免疫小鼠。评估了血清特异性抗体水平、脾细胞分泌的细胞因子以及脾细胞中多功能T细胞的数量。结果表明，与卡介苗组相比，卡介苗+rPcaA-DM疫苗组的Th1型细胞因子水平、抗体滴度以及IFN-γ/TNF-α单阳性和双阳性CD4和CD8 T细胞频率显著升高。此外，免疫小鼠的脾细胞和肺细胞显著抑制了分枝杆菌的生长。这些发现表明，rPcaA-DM疫苗作为卡介苗加强剂，显著增强了Th1极化并提供了强大的保护效力，具有预防LTBI进展为ATB的潜力。
