McMichael J C, Fiske M J, Fredenburg R A, Chakravarti D N, VanDerMeid K R, Barniak V, Caplan J, Bortell E, Baker S, Arumugham R, Chen D
Wyeth-Lederle Vaccines and Pediatrics, West Henrietta, New York 14586-9728, USA.
Infect Immun. 1998 Sep;66(9):4374-81. doi: 10.1128/IAI.66.9.4374-4381.1998.
The UspA1 and UspA2 proteins of Moraxella catarrhalis are potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350, 000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100 degreesC. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.
卡他莫拉菌的UspA1和UspA2蛋白是预防该菌所致疾病的潜在疫苗候选物。我们已对这两种蛋白进行了特性鉴定，并通过体外和体内试验评估了它们的疫苗潜力。两种蛋白均通过Triton X - 100抽提从O35E分离株中纯化出来，随后进行离子交换和羟基磷灰石层析。对通过酶切和化学裂解蛋白制备的内部肽段序列进行分析，结果显示UspA1和UspA2呈现出明显的结构差异，但共有一个包含单克隆抗体17C7识别的表位的共同序列。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE），纯化的UspA1在未加热时分子量约为350,000，在100℃加热10分钟后分子量为100,000。相比之下，纯化的UspA2经SDS - PAGE显示表观分子量为240,000，且不随加热时间长短而变化。通过凝胶过滤测定，UspA1和UspA2的大小分别为1,150,000和830,000。初步结果表明这两种蛋白在细菌致病过程中具有不同功能。发现纯化的UspA1能结合HEp - 2细胞，且抗UspA1血清而非抗UspA2血清能阻断O35E分离株与HEp - 2细胞的结合。UspA1还能结合纤连蛋白，似乎在细菌黏附中起作用。然而，纯化的UspA2不结合纤连蛋白，但对玻连蛋白有亲和力。两种蛋白均能在小鼠体内诱导针对同源和异源疾病分离株的杀菌抗体。最后，用每种蛋白免疫小鼠，随后用同源或异源分离株进行肺部攻击，与 mock - 免疫小鼠相比，这些小鼠清除细菌的速度更快。这些结果表明UspA1和UspA2发挥不同的毒力功能，且二者都是有前景的疫苗候选物。
