van der Ley P, van der Biezen J, Hohenstein P, Peeters C, Poolman J T
National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands.
Infect Immun. 1993 Oct;61(10):4217-24. doi: 10.1128/iai.61.10.4217-4224.1993.
The class 1 protein of Neisseria meningitidis is an important component of candidate outer membrane vaccines against meningococcal meningitis. This porin protein contains two variable regions which determine subtype specificity and provide binding sites for bactericidal monoclonal antibodies. To determine the contribution of each of these variable regions in the induction of bactericidal antibodies, a set of isogenic strains differing only in their class 1 epitopes was constructed. This was done by transformation of meningococcal strain H44/76 with cloned class 1 genes and selection of the desired epitope combinations in a colony blot with subtype-specific monoclonal antibodies. When used for the immunization of mice, outer membrane complexes induced bactericidal antibodies only against meningococcal strains sharing at least one of their class 1 epitopes. The results demonstrate that the P1.2 and P1.16 epitopes, normally located in the fourth exposed loop of the protein, efficiently induce bactericidal antibodies independently of the particular sequence in the first variable region. The P1.5 and P1.7 epitopes, normally located in the first exposed loop, were found to induce lower bactericidal titers. Hybrid class 1 outer membrane proteins were constructed by inserting oligonucleotides encoding the P1.7 and P1.16 epitopes into the porA gene. In this way, we obtained a set of strains which carry the P1.5 epitope in loop 1, P1.2 in loop 4, and P1.7 and P1.16 (separately or in combination) in either loop 5 or loop 6. The additional epitopes were found to be exposed at the cell surface. Outer membrane complexes from several of these strains were found to induce a bactericidal response in mice against the inserted epitopes. These results demonstrate that it is feasible to construct meningococcal strains carrying multivalent class 1 proteins in which multiple subtype-specific epitopes are present in different cell surface-exposed loops.
脑膜炎奈瑟菌1类蛋白是抗脑膜炎球菌性脑膜炎候选外膜疫苗的重要组成部分。这种孔蛋白含有两个可变区，它们决定亚型特异性并为杀菌性单克隆抗体提供结合位点。为了确定这些可变区中每一个在诱导杀菌性抗体方面的作用，构建了一组仅在其1类表位上不同的同基因菌株。这是通过用克隆的1类基因转化脑膜炎球菌菌株H44/76，并在亚型特异性单克隆抗体的菌落印迹中选择所需的表位组合来完成的。当用于免疫小鼠时，外膜复合物仅诱导针对至少共享其一个1类表位的脑膜炎球菌菌株的杀菌性抗体。结果表明，通常位于该蛋白第四个暴露环中的P1.2和P1.16表位可有效诱导杀菌性抗体，而与第一个可变区中的特定序列无关。通常位于第一个暴露环中的P1.5和P1.7表位被发现诱导较低的杀菌效价。通过将编码P1.7和P1.16表位的寡核苷酸插入porA基因，构建了杂交1类外膜蛋白。通过这种方式，我们获得了一组菌株，它们在环1中携带P1.5表位，在环4中携带P1.2表位，在环5或环6中分别或组合携带P1.7和P1.16表位。发现额外的表位暴露在细胞表面。发现这些菌株中的几种菌株的外膜复合物在小鼠中诱导针对插入表位的杀菌反应。这些结果表明，构建携带多价1类蛋白的脑膜炎球菌菌株是可行的，其中多个亚型特异性表位存在于不同的细胞表面暴露环中。
