Kwon Kee Woong, Choi Eunsol, Kim Hagyu, Kim Hyeong Woo, Choi Sangwon, Lee Seunghyun, Ha Sang-Jun, Shin Sung Jae
Department of Microbiology and Convergence of Medical Science, College of Medicine, Gyeongsang National University, Jinju, 52727, Republic of Korea.
Department of Microbiology, Graduate School of Medical Science, Brain Korea 21 Project, Yonsei University College of Medicine, Seoul, 03722, South Korea.
J Biomed Sci. 2025 May 26;32(1):52. doi: 10.1186/s12929-025-01144-8.
Effective subunit vaccine development requires selecting appropriate adjuvant formulations to trigger desired adaptive immune responses. This study explores the immunogenicity and tuberculosis (TB) vaccine potential of antigens (Ags) combined with Toll-like receptor 4 (TLR4) adjuvants and a stimulator of interferon genes (STING) agonist.
In this work, we investigated the combination of Ags with TLR4 adjuvants (monophosphoryl lipid A / dimethyldioctadecylammonium bromide; MPL/DDA or glucopyranosyl lipid adjuvant-stable emulsion; GLA-SE) and a STING agonist, c-di-GMP (CDG). Mice were immunized three times by intramuscular injections at 3-week intervals. The effects of integrating Ags in these adjuvant formulations on the immune response were evaluated, focusing on the generation of Th1-biased, polyfunctional Ag-specific CD4 T cells and their localization in the lung and spleen. To assess protection, immunized mice were aerogenically challenged with either conventional or ultra-low doses of Mycobacterium tuberculosis (Mtb) 4 weeks after the last immunization. Subsequently, bacterial load and pulmonary inflammation were assessed.
Integrating ESAT6 Ag in TLR4 and CDG adjuvant formulations remarkably boosted Th1-biased, polyfunctional ESAT6-specific CD4 T cells in the lungs and spleen, providing durable protection against Mtb infection. The inclusion of CDG promoted mucosal localization of ESAT6-specific CD4 T cells resembling resident memory phenotypes in the lung parenchyma and increased Ag-specific CD4 T cells in lung vasculature. Immunization with another vaccine Ag candidate, Ag85B, in GLA-SE plus CDG similarly increased Ag85B-specific CD4 T cells in the spleen and both lung compartments. Following ultra-low dose Mtb challenge, ESAT6 or Ag85B/GLA-SE/CDG immunizations significantly reduced bacterial loads compared to non-, Bacillus Calmette-Guérin (BCG)-, and ESAT6 or Ag85B/GLA-SE-immunized groups. Importantly, the inclusion of CDG decreased killer cell lectin-like receptor subfamily G member 1 (KLRG1) expression among Ag-specific CD4 T cells in the lung, correlating with enhanced lung-homing evidenced by expanded lung parenchyma Ag-specific CD4 T cells, including less-differentiated Th1 cells.
This study highlights that CDG, when used in combination with TLR4 adjuvants, enhances long-term protective immunity, offering a promising strategy for subunit TB vaccine development.
有效的亚单位疫苗研发需要选择合适的佐剂配方来触发期望的适应性免疫反应。本研究探讨了抗原（Ags）与Toll样受体4（TLR4）佐剂及干扰素基因刺激剂（STING）激动剂联合使用时的免疫原性及结核病（TB）疫苗潜力。
在本研究中，我们研究了抗原与TLR4佐剂（单磷酰脂质A/二甲基二十八烷基溴化铵；MPL/DDA或吡喃葡萄糖基脂质佐剂稳定乳剂；GLA-SE）以及STING激动剂环二鸟苷酸（c-di-GMP，CDG）的联合使用情况。小鼠每隔3周进行3次肌肉注射免疫。评估了在这些佐剂配方中加入抗原对免疫反应的影响，重点关注Th1偏向性、多功能抗原特异性CD4 T细胞的产生及其在肺和脾中的定位。为评估保护效果，在最后一次免疫后4周，用常规剂量或超低剂量的结核分枝杆菌（Mtb）对免疫小鼠进行气溶胶攻击。随后，评估细菌载量和肺部炎症。
将早期分泌性抗原靶6（ESAT6）抗原整合到TLR4和CDG佐剂配方中，可显著增强肺和脾中Th1偏向性、多功能ESAT6特异性CD4 T细胞，为抵抗Mtb感染提供持久保护。加入CDG可促进ESAT6特异性CD4 T细胞在肺实质中的黏膜定位，类似于驻留记忆表型，并增加肺血管中的抗原特异性CD4 T细胞。用另一种候选疫苗抗原Ag85B在GLA-SE加CDG中进行免疫，同样增加了脾和两个肺区室中Ag85B特异性CD4 T细胞。在超低剂量Mtb攻击后，与未免疫、卡介苗（BCG）免疫、ESAT6或Ag85B/GLA-SE免疫组相比，ESAT6或Ag85B/GLA-SE/CDG免疫显著降低了细菌载量。重要的是，加入CDG可降低肺中抗原特异性CD4 T细胞中杀伤细胞凝集素样受体亚家族G成员1（KLRG1）的表达，这与肺归巢增强相关，表现为肺实质中抗原特异性CD4 T细胞（包括分化程度较低的Th1细胞）增多。
本研究强调，CDG与TLR4佐剂联合使用时可增强长期保护性免疫，为亚单位TB疫苗研发提供了一种有前景的策略。
