Identification of surface-exposed B-cell epitopes on high molecular-weight adhesion proteins of nontypeable Haemophilus influenzae.
Identification of surface-exposed B-cell epitopes on high molecular-weight adhesion proteins of nontypeable Haemophilus influenzae.
复制标题
不可分型流感嗜血杆菌高分子量粘附蛋白表面暴露 B 细胞表位的鉴定。
DOI：
10.1128/iai.64.8.3032-3037.1996
复制
发表时间：
1996
影响因子：
3.1
通讯作者：
StGeme3rd,JW
中科院分区：
文献类型：
--
作者：
Barenkam,SJ;StGeme3rd,JW
我们以前报道，两个表面暴露的高分子量蛋白质，HMW 1和HMW 2，表达的原型菌株的nontypeable流感嗜血杆菌（NTHI）介导附着到人上皮细胞。这些蛋白质是大多数不可分型嗜血杆菌菌株常见的高免疫原性蛋白质家族的成员。我们还报道了用HMW 1-HMW 2混合物免疫改变了中耳炎动物模型的病程，表明这些蛋白质作为NTHI疫苗组分的潜在用途。表面可及B细胞表位的鉴定对于基于这些高分子量蛋白质开发重组或合成肽疫苗的努力可能是重要的。因此，本研究的目的是通过使用单克隆抗体（MAB），以确定表面可及的HMW 1和HMW 2蛋白的表位，并确定这些表位之间的流行率的高分子量蛋白表达的异源nontypeable嗜血杆菌菌株。通过用从原型菌株纯化的高分子量蛋白免疫小鼠产生单克隆抗体，并通过免疫电子显微镜（IEM）筛选识别表面表位的能力。回收了通过IEM识别表面表位的两种MAb，命名为AD 6和10 C5。为了定位这两种单克隆抗体所识别的抗原表位，我们利用pGEMEX载体构建了一组HMW 1和HMW 2重组融合蛋白，并检测了单克隆抗体与这些融合蛋白的反应性。MAb AD 6识别HMW 1和HMW 2中的表位，其定位于两种蛋白质的羧基末端的最后75个氨基酸。当检查与异源菌株的反应性，单克隆抗体AD 6承认高分子量的蛋白质在75%的125个无关的nontypeable嗜血杆菌菌株，此外，与三个这样的菌株进行反应时，IEM检查。单克隆抗体10 C5识别的表位定位于HMW 1羧基末端附近的155个氨基酸片段。该表位与AD 6表位相邻但不同，并且不存在于HMW 2中。10 C5表位由40%的AD 6反应性菌株表达。高分子量粘附蛋白上共享的表面暴露的表位的鉴定表明开发基于重组或合成肽的疫苗对由大多数NTHI菌株引起的疾病具有保护性的可能性。
We previously reported that two surface-exposed high-molecular-weight proteins, HMW1 and HMW2, expressed by a prototypic strain of nontypeable Haemophilus influenzae (NTHI) mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to most nontypeable Haemophilus strains. We also reported that immunization with an HMW1-HMW2 mixture modified the course of disease in an animal model of otitis media, suggesting the potential usefulness of these proteins as NTHI vaccine components. Identification of surface-accessible B-cell epitopes could be important to efforts to develop recombinant or synthetic peptide vaccines based upon these high-molecular-weight proteins. Thus, the purpose of the present study was to identify surface-accessible epitopes on the HMW1 and HMW2 proteins by using monoclonal antibodies (MAbs) and to determine the prevalence of these epitopes among the high-molecular-weight proteins expressed by heterologous nontypeable Haemophilus strains. MAbs were generated by immunizing mice with high-molecular-weight proteins purified from prototype strains and were screened by immunoelectron microscopy (IEM) for the ability to recognize surface epitopes. Two MAbs, designated AD6 and 10C5, that recognized surface epitopes by IEM were recovered. In order to map the epitopes recognized by these two MAbs, we constructed a set of HMW1 and HMW2 recombinant fusion proteins using the pGEMEX vectors and examined the reactivity of the MAbs with these fusion proteins. MAb AD6 recognized an epitope in both HMW1 and HMW2 which mapped to the last 75 amino acids at the carboxy termini of the two proteins. When examined for reactivity with heterologous strains, MAb AD6 recognized high-molecular-weight proteins in 75% of 125 unrelated nontypeable Haemophilus strains and, in addition, reacted with three of three such strains when examined by IEM. MAb 10C5 recognized an epitope that mapped to a 155-amino-acid segment near the carboxy terminus of HMW1. This epitope was adjacent to but distinct from the AD6 epitope and was absent from HMW2. The 10C5 epitope was expressed by 40% of the AD6 reactive strains. Identification of shared surface-exposed epitopes on the high-molecular-weight adhesion proteins suggests the possibility of developing recombinant or synthetic peptide-based vaccines protective against disease caused by the majority of NTHI strains.