Functional characteristics of antibodies induced by Arg-gingipain (HRgpA) and Lys-gingipain (Kgp) from Porphyromonas gingivalis

Arginine-specific gingipain (HRgpA) and lysine-specific gingipain (Kgp), en zymes produced by Porphyromonas gingivalis, may be candidates for an anti-P . gingivalis vaccine. The purpose of our study was to determine whether HRg pA 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 immunog lobulin G (IgG) fractions were isolated from preimmune and immune sera. Fun ctional Characteristics of the antibodies were assessed by determining anti body titers by enzyme-linked immunosorbent assay (ELISA), generating Wester n immunoblots, and measuring antibody enhancement of P. gingivalis opsoniza tion, phagocytosis and killing by polymorphonuclear leukocytes (PMN) of int act cells of strains of P. gingivalis representative of the four serotypes. Strains studied included 33277 (serotype A), A7A1-28 (serotype B), W50 (se rotype C) and 381 (serotype D). Both HRgpA and Kgp induced high titers of I gG antibody. Anti-HRgpA and anti-Kgp bound to both HRgpA and Kgp demonstrat ing a large proportion of shared antigenic epitopes. The two antibodies bou nd equally well to all four P. gingivalis serotypes with titers ranging fro m 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 identic al. Both antibodies detected components in HRgpA at 27, 35 and 45 kDa and i n 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 mas s components in the cell sonicates of P. gingivalis. Both proteins induced antibodies that significantly enhanced opsonization as assessed by chemilum inescence, with values ranging from 130 mV to 375 mV for anti-HRgpA IgG and from 240 mV to 375 mV for anti-Kgp IgG. Both antibodies significantly enha nced PMN-mediated bacterial killing of the four P. gingivalis serotypes, al though the percentage of killing varied among the serotypes (24-81% for ant i-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.