Katz J, Black K P, Michalek S M
Department of Oral Biology, School of Dentistry, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Infect Immun. 1999 Sep;67(9):4352-9. doi: 10.1128/IAI.67.9.4352-4359.1999.
Porphyromonas gingivalis, a gram-negative, black-pigmented anaerobe, is among the microorganisms implicated in the etiology of adult periodontal disease. This bacterium possesses a number of factors, including hemagglutinins, of potential importance in virulence. Several hemagglutinin genes have been identified, cloned, and expressed in Escherichia coli. The purpose of this study was to characterize host responses to purified recombinant hemagglutinin B (rHag B), using the conventional Fischer rat as the experimental animal model. The effectiveness of immunization with rHag B on protection against experimental periodontal bone loss following infection with P. gingivalis was also evaluated. Groups of rats were immunized by the subcutaneous route with rHag B in complete Freund's adjuvant, immunized with rHag B and orally infected with P. gingivalis, nonimmunized and noninfected, or orally infected with P. gingivalis only. Serum and saliva samples were collected throughout the experiment and evaluated for serum immunoglobulin G (IgG) and IgM and salivary IgA antibody activity by enzyme-linked immunosorbent assay. No salivary IgA anti-Hag B activity was detected in the various groups of rats. A slight serum IgM response similar to that seen in preimmune samples was observed. Serum IgG antibody activity to Hag B was detected only in samples from rats immunized with rHag B. This response was primarily of the IgG1 and IgG2a subclasses, followed by IgG2b and low levels of IgG2c. Supernatants from rHag B-stimulated splenic lymphoid cell cultures from immunized rats contained high levels of gamma interferon, followed by interleukin-2 (IL-2), IL-10, and then IL-4. These results are consistent with the induction of T helper type 1 (Th1)- and Th2-like responses. Western blot analysis of sera derived from rHag B-immunized rats reacted with trichloroacetic acid (TCA) precipitates of P. gingivalis 33277, 381, A7A1-28, and W50, revealing a 50-kDa band reflective of Hag B. However, sera derived from rats immunized with P. gingivalis whole cells or from rats infected with P. gingivalis only did not react with rHag B but did react with TCA precipitates of P. gingivalis strains. Finally, radiographic measurements of periodontal bone loss indicated that rats immunized with rHag B had less bone loss than those infected with P. gingivalis only. These results demonstrate the effectiveness of purified rHag B in inducing a protective immune response and support the potential usefulness of this component of P. gingivalis in the development of a vaccine against adult periodontitis.
牙龈卟啉单胞菌是一种革兰氏阴性、产黑色素的厌氧菌，是与成人牙周病病因相关的微生物之一。这种细菌具有多种因子，包括血凝素，这些因子在毒力方面可能具有重要意义。已鉴定、克隆了多个血凝素基因，并在大肠杆菌中进行了表达。本研究的目的是使用传统的Fischer大鼠作为实验动物模型，来表征宿主对纯化的重组血凝素B（rHag B）的反应。还评估了用rHag B免疫对预防牙龈卟啉单胞菌感染后实验性牙周骨丧失的有效性。将大鼠分组，分别皮下注射含完全弗氏佐剂的rHag B进行免疫、用rHag B免疫后再经口感染牙龈卟啉单胞菌、不免疫也不感染、或仅经口感染牙龈卟啉单胞菌。在整个实验过程中收集血清和唾液样本，并通过酶联免疫吸附测定法评估血清免疫球蛋白G（IgG）和IgM以及唾液IgA抗体活性。在各组大鼠中均未检测到唾液IgA抗Hag B活性。观察到有轻微的血清IgM反应，类似于免疫前样本中的反应。仅在经rHag B免疫的大鼠样本中检测到血清IgG对Hag B的抗体活性。这种反应主要是IgG1和IgG2a亚类，其次是IgG2b和低水平的IgG2c。来自免疫大鼠的rHag B刺激的脾淋巴细胞培养物的上清液中含有高水平的γ干扰素，其次是白细胞介素-2（IL-2）、IL-10，然后是IL-4。这些结果与1型辅助性T细胞（Th1）样和Th2样反应的诱导一致。对来自经rHag B免疫大鼠的血清进行的蛋白质印迹分析与牙龈卟啉单胞菌33277、381、A7A1 - 28和W50的三氯乙酸（TCA）沉淀物反应，显示出一条反映Hag B的50 kDa条带。然而，来自经牙龈卟啉单胞菌全细胞免疫的大鼠或仅感染牙龈卟啉单胞菌的大鼠的血清不与rHag B反应，但与牙龈卟啉单胞菌菌株的TCA沉淀物反应。最后，牙周骨丧失的影像学测量表明，用rHag B免疫的大鼠的骨丧失比仅感染牙龈卟啉单胞菌的大鼠少。这些结果证明了纯化的rHag B在诱导保护性免疫反应方面的有效性，并支持牙龈卟啉单胞菌的这一成分在开发抗成人牙周炎疫苗方面的潜在用途。
