Recombinant 40-kDa Outer Membrane Protein Research Articles

Association of the FcγRIIB-nt645+25A/G polymorphism with the expression level of the FcγRIIb receptor, the antibody response to Porphyromonas gingivalis and the severity of periodontitis

Human FcγRIIb is an immunoglobulin G (IgG) receptor that inhibits the activation of B lymphocytes through cross-linking with the B-cell receptor via immune complexes. This function acts as a negative regulator of antibody production. Our previous studies have demonstrated the gene polymorphisms in FcγRIIb to be associated with periodontitis. In this study, we presented a polymorphism--FcγRIIB-nt645+25A/G (rs2125685)--in intron 4 and analyzed its functional relevance to periodontitis. We examined whether the FcγRIIB-nt645+25A/G polymorphism is associated with periodontal parameters, the IgG response to the periodontopathic bacterium Porphyromonas gingivalis and/or the expression level of FcγRIIb on peripheral B lymphocytes. Thirty-two patients with chronic periodontitis were genotyped with nested PCR and by direct sequencing of genome DNA. The levels of serum IgG and of specific IgG subclasses for P. gingivalis sonicate and for the recombinant 40-kDa outer membrane protein (OMP) were determined. The expression levels of FcγRIIb on peripheral B lymphocytes from 19 healthy donors were measured by flow cytometry. Patients with the FcγRIIB-nt645+25AA genotype showed significantly higher mean clinical attachment levels compared to patients with the FcγRIIB-nt645+25GG genotype (p = 0.003) and a significantly lower IgG response to P. gingivalis sonicate and to the 40-kDa OMP. The expression levels of FcγRIIb protein on the cell surface in peripheral B lymphocytes were higher in healthy donors with the FcγRIIB-nt645+25AA genotype than in those with the FcγRIIB-nt645+25GG genotype (p = 0.03). The higher expression levels of FcγRIIb in subjects with the FcγRIIB-nt645+25AA genotype may induce a lower level of production of IgG against P. gingivalis and therefore more severe periodontitis.

The Trans‐Chromosomic Mouse‐Derived Human Monoclonal Antibody Promotes Phagocytosis of Porphyromonas gingivalis by Neutrophils

As a safe immunotherapeutic approach, human monoclonal antibody (hMAb) may be effective in clearing periodontopathic bacteria. The trans-chromosomic (TC) technology has recently been applied to construction of the TC mouse, which enables us to incorporate entire human chromosome fragments containing immunoglobulin (Ig) gene cluster. The aim of this study is to establish TC mouse-derived hMAb, and to test the in vitro opsonophagocytic activity. Human Ig-producing TC mouse was immunized by recombinant 40-kDa outer membrane protein (r40-kDa OMP) of Porphyromonas gingivalis 381, and the spleen cells were fused with the mouse myeloma cell line. The specificity of antir40- kDa OMP hMAb was evaluated with the enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance assays. Flow cytometric analyses were performed to assess the opsonophagocytic activity. We successfully constructed 99 IgG isotype clones (IgG1: 84; IgG2: 11; IgG4: four clones), which were specifically reactive with r40-kDa OMP. The anti-r40-kDa OMP IgG1 hMAbs promoted phagocytosis of P. gingivalis by neutrophils. Futhermore, an increased opsonophagocytic activitity of anti-r40-kDa OMP IgG1 hMAbs was observed not only in P. gingivalis 381, but also in the W50, W83, and Su63 strains. Our results document the TC mouse-derived hMAb to promote neutrophil phagocytosis of P. gingivalis, suggesting an immunotherapeutic option for clearance of P. gingivalis.

Identification of the functional domain in a coaggregation factor from Porphyromonas gingivalis

Porphyromonas gingivalis has been implicated as a major pathogen in periodontal diseases. The coaggregation factor plays an important role in colonization of P. gingivalis in the subgingival area through aggregation with other extant oral microorganisms. We previously succeeded in gene cloning of the 40-kDa outer membrane protein (OMP) from P. gingivalis and identified the 40-kDa OMP as one of coaggregation factors. The mouse monoclonal antibody Pg-omp-A2, which inhibited the coaggregation activity of P gingivalis, was successfully constructed. In order to develop a useful immunotherapy, it is essential to understand the functional domain expressing aggregation activity. The availability of recombinant 40-kDa OMP and the identification of the antigenic determinant recognized by Pg-omp-A2 will allow for the determination of the functional domain of the coaggregation factor.In this study, we used a phage-displayed epitope mapping system to examine the functional domain expressing aggregation activity. Peptide-displayed phage clones were isolated by biopanning using Pg-omp-A2 and determined DNA nucleotide sequence and then predicted the amino acid sequence of epitope. The amino acid sequence of epitope region was similar to the region of 96IALDQTLGIP105 in 40-kDa OMP. Next, a series of 6 peptides (designated peptide A to F) in 40-kDa OMP was designed and chemically synthesized, and then we examined their immuno-reactivity by Pg-omp-A2. Pg-omp-A2 recognized the peptide C that contained the IALDQTLGIP amino acid sequence. Furthermore, the peptide C reduced the inhibitory activity of Pg-omp-A2 against the aggregation of P. gingivalis vesicles with Streptococcus gordonii. These findings suggest that the aggregation-associated domain of P gingivalis 40-kDa coaggregation factor may be IALDQTLGIP.

Cancer vaccine design: a novel bacterial adjuvant for peptide-specific CTL induction.

The recent identification of tumor Ags as potential vaccines has prompted the search for efficient adjuvants and delivery systems, especially in the case of peptide-based vaccination protocols. Here, we investigated the adjuvant potential of the recombinant 40-kDa outer membrane protein of Klebsiella pneumoniae (P40) for specific CTL induction. We studied the CTL response induced in HLA-A*0201/K(b) transgenic mice immunized with peptides derived from two melanoma-associated differentiation Ags, the HLA-A*0201-restricted decapeptide Melan-A(26--35) substituted at position 2 and the K(b)-restricted tyrosinase-related protein 2(181--188) T cell epitope. We found that both peptides are able to generate a specific CTL response when mixed with the protein in the absence of conventional adjuvant. This CTL response is a function of the amount of P40 used for immunization. Moreover, the CTL response generated against the tyrosinase-related protein 2(181-188) peptide in presence of P40 is associated with tumor protection in two different experimental models and is independent of the presence of CD4(+) T lymphocytes. Thus, the recombinant bacterial protein P40 functions as a potent immunological adjuvant for specific CTL induction.

A human monoclonal antibody which inhibits the coaggregation activity of Porphyromonas gingivalis.

A B-cell line producing a human monoclonal antibody (HuMAb) against a recombinant 40-kDa outer membrane protein (OMP) of Porphyromonas gingivalis was constructed by in vivo immunization of a severe combined immunodeficiency C.B.-17/Icr mouse, which had been injected with human peripheral blood lymphocytes, with recombinant 40-kDa OMP and subsequent Epstein-Barr virus immortalization of B cells isolated from the spleen of the mouse. This HuMAb inhibited coaggregation between P. gingivalis vesicles and Actinomyces naeslundii cells.

Inhibition of a porphyromonas gingivalis colonizing factor between actinomyces viscosus ATCC 19246 by monoclonal antibodies against recombinant 40-kDa outer-membrane protein

1. 1. Porphyromonas gingivalis, an important pathogen in human periodontal disease, aggregates with Actinomyces viscosus ATCC 19246. 2. 2. Monoclonal antibodies (mAbs) against purified recombinant 40-kDa outer-membrane protein (r40-kDa OMP) of P. gingivalis 381 inhibited its coaggregation with A. viscosus ATCC 19246 in a dose-dependent manner. 3. 3. Five mAb clones against r40-kDa OMP were selected. The isotype of the five was IgG1. 4. 4. Pg-ompA2 inhibited the coaggregation of several strains of P. gingivalis with A. viscosus ATCC 19246 cells.

Complement-Mediated Killing ofPorphyromonas gingivalis381 by the Immunoglobulin G Induced by Recombinant 40-kDa Outer Membrane Protein

Porphyromonas gingivalishas been implicated as an important pathogen in severe adult periodontitis. We have previously cloned a 40-kDa outer membrane protein fromP. gingivalis381 and succeeded in producing sufficient quantities of the recombinant protein (r40-kDa OMP). r40-kDa OMP has been the subject of considerable interest to us as a possible vaccine candidate. To understand the role of anti-r40-kDa OMP antibody in the host defense mechanisms againstP. gingivalis,we examined the involvement of a rabbit antibody against r40-kDa OMP (r40-kDa OMP Ab) to anin vitrocomplement-mediated bactericidal assay forP. gingivalis381. By measuring the absorbance values in order to assay the surviving bacteria, we found significant anti-P. gingivalisactivity of r40-kDa OMP Ab when guinea pig complement was present. Using affinity-purified immunoglobulin G of r40-kDa OMP Ab (IgG-r40-kDa OMP), we demonstrated that the IgG contributed to anti-P. gingivalisactivity in the antibody–complement system. This was effected by measuring the incorporation of tritiated thymidine into newly synthesized nucleic acids. Finally, we confirmed the cell lysis ofP. gingivalis381 exposed to IgG-r40-kDa OMP in the presence of complement sources in a radioactive bactericidal assay using bacteria labeled with [14C]sodium acetate. Assembling the data from experiments using component-deficient complements, we concluded that IgG-r40-kDa OMP was related to the killing ofP. gingivalis381 by mediation in the complement activated through both the classical and the alternative pathways.

Role of Porphyromonas gingivalis 40-kDa outer membrane protein in the aggregation of P. gingivalis vesicles and Actinomyces viscosus

Porphyromonas gingivalis, an important pathogen in periodontitis, produces extracellular vesicles that aggregate with Actinomyces viscosus cells. A 40-kDa outer membrane protein (OMP)-coding gene from P. gingivalis was cloned and the protein was found to be localized in these vesicles. The recombinant 40-kDa OMP did not show aggregation activity. However, affinity-purified antibody against the recombinant protein significantly inhibited aggregation of P. gingivalis vesicles with A. viscosus cells. The antibody also inhibited cellular coaggregation of several strains of P. gingivalis with A. viscosus cells, but not with other periodontal pathogens. Moreover, aggregation of A. viscosus cells with P. gingivalis vesicles was inhibited in a dose-dependent manner by pre-treatment of the A. viscosus cells with the recombinant protein. These findings suggest that the 40-kDa OMP may be an important aggregation factor of P. gingivalis.