Heinz F X, Berger R, Tuma W, Kunz C
Virology. 1983 Oct 30;130(2):485-501. doi: 10.1016/0042-6822(83)90102-2.
A model showing the topological distribution, functions, and serological specificities of eight distinct, monoclonal antibody-defined epitopes on the tick-borne encephalitis (TBE) virus glycoprotein has been presented in a previous publication (F. X. Heinz, R. Berger, W. Tuma, and Ch. Kunz (1983). Virology 126, 525-537.) In the present report the influence of conformational change, chemical modification, and fragmentation on the antigenic reactivity of each epitope has been analyzed by the use of blocking enzyme immunoassays and "Western blotting." One of the two major antigenic domains (A), composed of three different epitopes, completely lost its antigenicity upon incubation at pH 5.0 or by treatment with guanidine-HCl/urea, SDS, reduction and carboxymethylation, as well as by proteolytic (trypsin, alpha-chymotrypsin, thermolysin) and chemical (CNBr) fragmentation. The second major antigenic domain (B), however, defined by four distinct monoclonal antibodies, three of which are hemagglutination (HA)-inhibiting, neutralizing, and protective, was shown to be resistant to low pH, guanidine-HCl/urea treatment, and proteolytic cleavage of the native protein. Also, polyclonal immune sera from mice and rabbits contained antibody populations reactive with antigenic determinants which are resistant and others which are sensitive to conformational change and fragmentation. Glycoprotein fragments with molecular weights of about 9000, generated by proteolysis of the native protein, were immunoreactive with neutralizing and protective monoclonal antibodies (defining domain B) as well as with a polyclonal mouse immune serum. Thus, these fragments appear to contain antigenic determinants which are immunodominant on the native protein and play an important role in the induction of a protective immune response against TBE virus. In addition, these results show that antibody binding to antigenic domains which are topologically and structurally completely unrelated may result in neutralization and/or HA inhibition. As the presence of two receptor-binding sites is unlikely, different effector mechanisms may account for the effects of these antibodies. The antigenic reactivity of domain A is sensitive to the same treatments which also inactivate HA activity of TBE virus, whereas domain B is resistant. These treatments include a change of domain A induced by incubation at slightly acidic pH which also results in inactivation of virus infectivity. Antibodies to domain A therefore presumably block viral activities by direct binding at or near the putative receptor-binding site whereas antibodies to domain B may cause loss of biological activities by inducing a conformational change of the receptor-binding site.
先前的一篇出版物（F. X. 海因茨、R. 伯杰、W. 图马和Ch. 昆茨（1983年）。《病毒学》126卷，525 - 537页）中展示了一个模型，该模型呈现了蜱传脑炎（TBE）病毒糖蛋白上八个不同的、单克隆抗体定义的表位的拓扑分布、功能和血清学特异性。在本报告中，通过使用封闭酶免疫测定法和“蛋白质印迹法”，分析了构象变化、化学修饰和片段化对每个表位抗原反应性的影响。由三个不同表位组成的两个主要抗原结构域之一（A），在pH 5.0孵育、用盐酸胍/尿素、SDS、还原和羧甲基化处理，以及通过蛋白水解（胰蛋白酶、α - 糜蛋白酶、嗜热菌蛋白酶）和化学（溴化氰）片段化处理后，完全丧失了其抗原性。然而，由四种不同单克隆抗体定义的第二个主要抗原结构域（B），其中三种具有血凝（HA）抑制、中和和保护作用，被证明对低pH、盐酸胍/尿素处理以及天然蛋白的蛋白水解切割具有抗性。此外，来自小鼠和兔子的多克隆免疫血清中含有与抗原决定簇反应的抗体群体，其中一些对构象变化和片段化具有抗性，另一些则敏感。通过天然蛋白的蛋白水解产生的分子量约为9000的糖蛋白片段，与中和和保护性单克隆抗体（定义结构域B）以及多克隆小鼠免疫血清具有免疫反应性。因此，这些片段似乎含有在天然蛋白上占主导地位的抗原决定簇，并且在诱导针对TBE病毒的保护性免疫反应中起重要作用。此外，这些结果表明，抗体与拓扑结构和结构上完全不相关的抗原结构域结合可能导致中和和/或HA抑制。由于不太可能存在两个受体结合位点，不同的效应机制可能解释了这些抗体的作用。结构域A的抗原反应性对同样也使TBE病毒HA活性失活的处理敏感，而结构域B则具有抗性。这些处理包括在略酸性pH下孵育诱导的结构域A的变化，这也导致病毒感染性失活。因此，针对结构域A的抗体大概是通过直接结合在假定的受体结合位点或其附近来阻断病毒活性，而针对结构域B的抗体可能是通过诱导受体结合位点的构象变化导致生物活性丧失。
