Loeb M R, Woodin K A
Department of Pediatrics, University of Rochester Medical Center, New York 14642.
Infect Immun. 1987 Dec;55(12):2977-83. doi: 10.1128/iai.55.12.2977-2983.1987.
The cross-reactivity of exposed surface epitopes of outer membrane proteins from a spectrum of Haemophilus influenzae type b isolates that varied in their evolutionary distance from each other and in their outer membrane protein composition was analyzed by using an immunoblot assay. The results for outer membrane proteins a, n, and b/c were as follows. (i) A total of 13 of 14 strains possessing a protein a with similar mobilities on gels (i.e., the same apparent molecular weight) as protein a of strain Eag absorbed antibodies to protein a of strain Eag. These strains represented a broad spectrum on a scale of evolutionary distance. (ii) In contrast, only one of seven strains possessing a protein a with different mobilities absorbed these antibodies. (iii) Of five isolates close to strain Eag on the evolutionary scale, the four with a protein n with the same mobility as protein n of strain Eag absorbed antibodies to protein n of strain Eag. (iv) In contrast, of five isolates distant from strain Eag on the evolutionary scale, none absorbed antibodies to protein n, including one strain that had a protein n of the same mobility as that of strain Eag. (v) All strains that absorbed antibodies to protein b/c also absorbed antibodies to lipopolysaccharide, and the reverse of this was also true. Evolutionary distance and mobility of protein b/c on gels were not factors. Control experiments indicated that this result was an artifact due to the strong association of lipopolysaccharide with protein b/c on the gel and subsequent blot. The important conclusions from these experiments, especially pertinent for consideration of these proteins in either whole or peptide vaccines, are that proteins with apparently identical molecular weights can possess different surface-exposed epitopes, that proteins with different molecular weights can possess cross-reactive surface-exposed epitopes, and that some surface-exposed epitopes have been conserved even though the bacterium has undergone evolutionary divergence. In addition, experiments were also performed to determine whether H. influenzae type b strains maintained their integrity during the absorption step, i.e., incubation in antiserum. Strain Eag, which was used as a prototype type b strain, released a small proportion of its membrane (0.13%), but this did not result in exposure of epitopes that were usually buried. In contrast, strain S2, an unencapsulated mutant of strain Eag, was quite unstable, releasing three times as much membrane and a large proportion of its periplasmic proteins.
利用免疫印迹分析法，分析了一系列b型流感嗜血杆菌分离株外膜蛋白暴露表面表位的交叉反应性。这些分离株在进化距离和外膜蛋白组成上存在差异。外膜蛋白a、n和b/c的结果如下：(i) 在14株凝胶迁移率与Eag菌株蛋白a相似（即表观分子量相同）的菌株中，共有13株能吸收针对Eag菌株蛋白a的抗体。这些菌株在进化距离范围内具有广泛代表性。(ii) 相比之下，在7株蛋白a迁移率不同的菌株中，只有1株能吸收这些抗体。(iii) 在进化尺度上与Eag菌株相近的5株分离株中，4株蛋白n迁移率与Eag菌株蛋白n相同的菌株能吸收针对Eag菌株蛋白n的抗体。(iv) 相比之下，在进化尺度上与Eag菌株距离较远的5株分离株中，没有一株能吸收针对蛋白n的抗体，包括一株蛋白n迁移率与Eag菌株相同的菌株。(v) 所有能吸收针对蛋白b/c抗体的菌株也能吸收针对脂多糖的抗体，反之亦然。蛋白b/c在凝胶上的进化距离和迁移率并非影响因素。对照实验表明，这一结果是由于脂多糖在凝胶和后续印迹上与蛋白b/c紧密结合而产生的假象。这些实验得出的重要结论，对于在全疫苗或肽疫苗中考虑这些蛋白尤为重要，即表观分子量相同的蛋白可能具有不同的表面暴露表位，分子量不同的蛋白可能具有交叉反应性的表面暴露表位，并且即使细菌发生了进化分歧，一些表面暴露表位仍得以保留。此外，还进行了实验以确定b型流感嗜血杆菌菌株在吸收步骤（即在抗血清中孵育）过程中是否保持其完整性。用作b型原型菌株的Eag菌株释放了一小部分其膜（0.13%），但这并未导致通常被掩埋的表位暴露。相比之下，Eag菌株的无荚膜突变株S2相当不稳定，释放的膜是其三倍，且释放了很大一部分周质蛋白。
