Department of Biology, Shahed University, Tehran, Iran.
Molecular Microbiology Research Center, Shahed University, Tehran, Iran.
Front Immunol. 2020 Feb 7;11:158. doi: 10.3389/fimmu.2020.00158. eCollection 2020.
is an important human pathogen causing substantial mortality in hospitalized patients for which treatment with antibiotics has become problematic due to growing antibiotic resistance. In an attempt to develop alternative strategies for dealing with these serious infections surface antigens are being considered as targets for vaccines or immunotherapy. The surface receptor proteins required for zinc acquisition in Gram-negative bacterial pathogens have been proposed as vaccine targets due to their crucial role for growth in the human host. In this study we selected the putative ZnuD outer membrane receptor from as a target for vaccine development. Due to challenges in production of an integral outer membrane protein for vaccine production, we adopted a recently described hybrid antigen approach in which surface epitopes from the TbpA receptor protein were displayed on a derivative of the C-lobe of the surface lipoprotein TbpB, named the loopless C-lobe (LCL). A structural model for ZnuD was generated and four surface loops were selected for hybrid antigen production by computational approaches. Hybrid antigens were designed displaying the four selected loops (2, 5, 7, and 11) individually or together in a single hybrid antigen. The hybrid antigens along with ZnuD and the LCL scaffold were produced in the cytoplasm either as soluble antigens or as inclusion bodies, that were used to generate soluble antigens upon refolding. Mice were immunized with the hybrid antigens, ZnuD or LCL and then used in an sepsis model to evaluate their ability to protect against infection. As expected, the LCL scaffold did not induce a protective immune response, enabling us to attribute observed protection to the displayed loops. Immunization with the refolded ZnuD protein protected 63% of the mice while immunization with hybrid antigens displaying individual loops achieved between 25 and 50% protection. Notably, the mice immunized with the hybrid antigen displaying the four loops were completely protected from infection.
是一种重要的人类病原体，在住院患者中引起大量死亡，由于抗生素耐药性的增加，抗生素治疗已成为问题。为了寻找应对这些严重感染的替代策略，表面抗原被认为是疫苗或免疫疗法的靶点。革兰氏阴性细菌病原体获取锌所需的表面受体蛋白由于其在人体宿主中生长的关键作用，已被提议作为疫苗靶标。在这项研究中，我们选择了来自的假定 ZnuD 外膜受体作为疫苗开发的靶标。由于生产完整的外膜蛋白用于疫苗生产存在挑战，我们采用了最近描述的混合抗原方法，其中来自 TbpA 受体蛋白的表面表位在表面脂蛋白 TbpB 的 C 结构域衍生物上展示，命名为无环 C 结构域 (LCL)。生成了 ZnuD 的结构模型，并通过计算方法选择了四个表面环用于混合抗原生产。设计了混合抗原，单独或一起在单个混合抗原中显示四个选定的环（2、5、7 和 11）。混合抗原与 ZnuD 和 LCL 支架一起在细胞质中产生，要么作为可溶性抗原，要么作为包涵体，在复性后用于产生可溶性抗原。用混合抗原、ZnuD 或 LCL 免疫小鼠，然后在败血症模型中评估它们抵抗感染的能力。正如预期的那样，LCL 支架不会引起保护性免疫反应，使我们能够将观察到的保护归因于显示的环。复性 ZnuD 蛋白免疫保护了 63%的小鼠，而单独显示环的混合抗原免疫则实现了 25%至 50%的保护。值得注意的是，用显示四个环的混合抗原免疫的小鼠完全免受感染。
