Phogat Supriya, Yadav Jyoti, Chaudhary Darshna, Jaiwal Ranjana, Jaiwal Pawan K
Department of Zoology, M. D. University, Rohtak, 124001, India.
Centre for Biotechnology, M. D. University, Rohtak, 124001, India.
Mol Biotechnol. 2025 Apr 2. doi: 10.1007/s12033-025-01431-7.
A novel tuberculosis subunit vaccine specific for Mycobacterium tuberculosis dual antigens, culture filtrate protein-10 (CFP-10) and antigen 85B (Ag85B) conjugated with cholera toxin non-toxic B subunit (CTB), was expressed as a single polypeptide in high amounts and cost-effectively in Escherichia coli. The recovery and purification conditions for the recombinant fusion protein were established. This simple peptide vaccine required no exogenous adjuvant as it contained CTB, a potent immune modulator. The vaccine's physiochemical, structural, and immunological properties were determined using the in-silico tools. It was highly antigenic, non-allergenic, and non-toxic. Its BlastP search with human proteomes excluded the chances of autoimmune reactions. The tertiary structure model (3D) was validated by Ramachandran plot assessment. The 3D structure docking with Toll-like receptors, TLR-1, 2, 4, and 6, showed that the binding affinity between the vaccine peptide and TLRs was high, and their complex was stable, indicating a strong immune response. The in-silico immune simulation revealed the vaccine-induced both innate and adaptive immune responses. In-vivo validation of the immunogenicity of CTB.CFP10.Ag85B in Wistar rats revealed higher activation of IgG immune response compared to either antigen protein. Similar results were also obtained using the C-ImmSim simulation online server. A comparison of immunogenicity of CTB.CFP10.Ag85B with the only available TB vaccine, Bacillus Calmette-Guérin (BCG) or as a booster after vaccination of Wistar rats with BCG, indicated that the IgG levels were the highest in rats vaccinated with BCG, followed by a booster dose of CTB.CFP10.Ag85B fusion protein. The fusion protein would be a safe potential vaccine booster candidate in BCG-primed individuals against TB.
一种新型的结核分枝杆菌双抗原特异性亚单位疫苗，即与霍乱毒素无毒B亚基（CTB）偶联的培养滤液蛋白10（CFP-10）和抗原85B（Ag85B），在大肠杆菌中以单一多肽的形式大量且经济高效地表达。建立了重组融合蛋白的回收和纯化条件。这种简单的肽疫苗无需外源性佐剂，因为它含有强效免疫调节剂CTB。使用计算机模拟工具确定了该疫苗的物理化学、结构和免疫学特性。它具有高度抗原性、无致敏性且无毒。通过与人类蛋白质组进行BlastP搜索排除了自身免疫反应的可能性。通过拉氏图评估验证了三级结构模型（3D）。疫苗肽与Toll样受体TLR-1、2、4和6的3D结构对接表明，疫苗肽与TLR之间的结合亲和力很高，且它们的复合物很稳定，表明会产生强烈的免疫反应。计算机免疫模拟显示该疫苗可诱导先天免疫和适应性免疫反应。对Wistar大鼠体内CTB.CFP10.Ag85B免疫原性的验证表明，与任一抗原蛋白相比，其IgG免疫反应的激活程度更高。使用在线C-ImmSim模拟服务器也获得了类似结果。将CTB.CFP10.Ag85B与唯一可用的结核病疫苗卡介苗（BCG）的免疫原性进行比较，或将其作为Wistar大鼠接种BCG后的加强剂，结果表明，接种BCG的大鼠IgG水平最高，其次是接种CTB.CFP10.Ag85B融合蛋白加强剂量的大鼠。该融合蛋白将是在接种BCG的个体中作为抗结核安全潜在疫苗加强剂的候选物。
