Nakagawa T, Sims T, Fan Q, Potempa J, Travis J, Houston L, Page R C
Department of Periodontics, School of Dentistry, University of Washington, Seattle 98195-7480, USA.
Oral Microbiol Immunol. 2001 Aug;16(4):202-11. doi: 10.1034/j.1399-302x.2001.160402.x.
Arginine-specific gingipain (HRgpA) and lysine-specific gingipain (Kgp), enzymes produced by Porphyromonas gingivalis, may be candidates for an anti-P. gingivalis vaccine. The purpose of our study was to determine whether HRgpA and Kgp have opsonic target sites and whether these sites are available and accessible on intact P. gingivalis cells. Rabbits were used to generate polyclonal antibodies to both proteins. Animals were immunized and immunoglobulin G (IgG) fractions were isolated from preimmune and immune sera. Functional characteristics of the antibodies were assessed by determining antibody titers by enzyme-linked immunosorbent assay (ELISA), generating Western immunoblots, and measuring antibody enhancement of P. gingivalis opsonization, phagocytosis and killing by polymorphonuclear leukocytes (PMN) of intact cells of strains of P. gingivalis representative of the four serotypes. Strains studied included 33277 (serotype A), A7A1-28 (serotype B), W50 (serotype C) and 381 (serotype D). Both HRgpA and Kgp induced high titers of IgG antibody. Anti-HRgpA and anti-Kgp bound to both HRgpA and Kgp demonstrating a large proportion of shared antigenic epitopes. The two antibodies bound equally well to all four P. gingivalis serotypes with titers ranging from 77 to 205 ELISA units when compared to preimmune IgG set at 1 ELISA unit. The immunoblot patterns of binding of the two antibodies to HRgpA and Kgp and to sonicates of the four P. gingivalis serotypes were virtually identical. Both antibodies detected components in HRgpA at 27, 35 and 45 kDa and in Kgp at 27, 32, 35, 40 and 55 kDa. The antibodies also detected components at or near these same positions in addition to multiple high molecular mass components in the cell sonicates of P. gingivalis. Both proteins induced antibodies that significantly enhanced opsonization as assessed by chemiluminescence, with values ranging from 130 mV to 375 mV for anti-HRgpA IgG and from 240 mV to 475 mV for anti-Kgp IgG. Both antibodies significantly enhanced PMN-mediated bacterial killing of the four P. gingivalis serotypes, although the percentage of killing varied among the serotypes (24-81% for anti-HRgpA and 37-89% for anti-Kgp). Thus, both HRgpA and Kgp express opsonic target sites and induce high titers of antibodies that opsonize and enhance killing of all four serotypes of P. gingivalis. These two proteins appear to be potential candidate antigens for an anti-P. gingivalis vaccine.
精氨酸特异性牙龈蛋白酶（HRgpA）和赖氨酸特异性牙龈蛋白酶（Kgp）是牙龈卟啉单胞菌产生的酶，可能是抗牙龈卟啉单胞菌疫苗的候选物。我们研究的目的是确定HRgpA和Kgp是否具有调理素靶位点，以及这些位点在完整的牙龈卟啉单胞菌细胞上是否可用且可及。用兔子制备针对这两种蛋白质的多克隆抗体。对动物进行免疫，并从免疫前和免疫血清中分离免疫球蛋白G（IgG）组分。通过酶联免疫吸附测定（ELISA）测定抗体效价、进行Western免疫印迹以及测量抗体对牙龈卟啉单胞菌调理作用的增强、吞噬作用以及代表四种血清型的牙龈卟啉单胞菌菌株完整细胞被多形核白细胞（PMN）杀伤的情况，来评估抗体的功能特性。所研究的菌株包括33277（血清型A）、A7A1 - 28（血清型B）、W50（血清型C）和381（血清型D）。HRgpA和Kgp均诱导产生高滴度的IgG抗体。抗HRgpA和抗Kgp与HRgpA和Kgp均结合，表明存在很大比例的共享抗原表位。与设定为1个ELISA单位的免疫前IgG相比，这两种抗体与所有四种牙龈卟啉单胞菌血清型的结合同样良好，效价范围为77至205个ELISA单位。这两种抗体与HRgpA和Kgp以及四种牙龈卟啉单胞菌血清型超声裂解物的结合免疫印迹模式几乎相同。两种抗体均检测到HRgpA中27、35和45 kDa的组分以及Kgp中27、32、35、40和55 kDa的组分。除了牙龈卟啉单胞菌细胞超声裂解物中的多个高分子量组分外，抗体还在这些相同位置或其附近检测到组分。通过化学发光评估，两种蛋白质诱导产生的抗体均显著增强了调理作用，抗HRgpA IgG的值范围为130 mV至375 mV，抗Kgp IgG的值范围为240 mV至475 mV。两种抗体均显著增强了PMN介导的对四种牙龈卟啉单胞菌血清型的细菌杀伤作用，尽管不同血清型的杀伤百分比有所不同（抗HRgpA为24 - 81%；抗Kgp为37 - 89%）。因此，HRgpA和Kgp均表达调理素靶位点，并诱导产生高滴度的抗体，这些抗体可调理并增强对所有四种牙龈卟啉单胞菌血清型的杀伤作用。这两种蛋白质似乎是抗牙龈卟啉单胞菌疫苗的潜在候选抗原。
Oral Microbiol Immunol. 2000-6
FEMS Immunol Med Microbiol. 2006-7
J Periodontol. 2003-10
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