Sioud M, Dybwad A, Jespersen L, Suleyman S, Natvig J B, Førre O
Institute of Immunology and Rheumatology, University of Oslo, Norway.
Clin Exp Immunol. 1994 Dec;98(3):520-5. doi: 10.1111/j.1365-2249.1994.tb05522.x.
Naturally occurring autoantibodies against cytokines exist in the sera of patients with autoimmune diseases as well as in the sera of normal individuals. We report here that affinity-purified autoantibodies against human TNF-alpha from one rheumatoid arthritis (RA) patient inhibited the cytotoxic effect of TNF-alpha on the mouse fibrosarcoma cell line WEHI 164, by 50%. In an attempt to predict the autoantibodies' recognition site on TNF-alpha protein we screened a random nanopeptide phage library with the affinity-purified TNF-alpha autoantibodies. Among 63 random selected clones, 46 clones carried the sequence ASSLLASSP, NSSPYLNTK or PQSPGSSFP. Frequency analysis of the relative occurrence of the 20 amino acids in the nanopeptides displayed by 50 random bacteriophages picked before selection and 63 after selection to bind to TNF-alpha autoantibodies indicated that proline (P < 0.0003) and serine (P < 0.04) are involved in the binding of the autoantibodies to the phages. Furthermore, we demonstrated that three synthetic peptides (ASSLLASSP, NSSPYLNTK and PPLKPVIDE) displayed by the selected phages reduced the binding of the autoantibodies to TNF-alpha protein by 50%. Interestingly, the sera of mice (BALB/c) immunized with phages displaying ASSLLASSP and NSSPYLNTK peptide showed an anti-TNF-alpha response as detected by ELISA. This response was not found in mice immunized with the wild type phage. Thus, the recombinant phages selected from the phage libraries could be used as carrier for immunization, and therefore as a tool for vaccine development. This work sets the stage for experiments designed to isolate ligands for protective antibodies.
自身免疫性疾病患者血清以及正常个体血清中均存在天然产生的抗细胞因子自身抗体。我们在此报告，从一名类风湿关节炎（RA）患者血清中亲和纯化得到的抗人肿瘤坏死因子-α（TNF-α）自身抗体，可使TNF-α对小鼠纤维肉瘤细胞系WEHI 164的细胞毒性作用降低50%。为了预测自身抗体在TNF-α蛋白上的识别位点，我们用亲和纯化的TNF-α自身抗体筛选了一个随机纳米肽噬菌体文库。在随机挑选的63个克隆中，46个克隆携带序列ASSLLASSP、NSSPYLNTK或PQSPGSSFP。对筛选前随机挑选的50个噬菌体和筛选后与TNF-α自身抗体结合的63个噬菌体所展示的纳米肽中20种氨基酸相对出现频率的分析表明，脯氨酸（P < 0.0003）和丝氨酸（P < 0.04）参与了自身抗体与噬菌体的结合。此外，我们证明，所选噬菌体展示的三种合成肽（ASSLLASSP、NSSPYLNTK和PPLKPVIDE）可使自身抗体与TNF-α蛋白的结合减少50%。有趣的是，用展示ASSLLASSP和NSSPYLNTK肽的噬菌体免疫的小鼠（BALB/c）血清通过ELISA检测显示出抗TNF-α反应。在用野生型噬菌体免疫的小鼠中未发现这种反应。因此，从噬菌体文库中筛选出的重组噬菌体可作为免疫载体，从而作为疫苗开发的工具。这项工作为旨在分离保护性抗体配体的实验奠定了基础。
