RESUMEN
Porphyromonas gingivalis is a Gram-negative bacterium strongly associated with chronic periodontitis, an inflammatory oral disease. A major virulence factor common to all characterized strains of P. gingivalis is the RgpA-Kgp proteinase-adhesin complexes (RgpA-Kgp complexes). In this study, we investigated T cell proliferative and cytokine responses to the RgpA-Kgp complexes and identified T cell epitopes in BALB/c mice utilizing Pepscan methodology. T cell proliferative responses were found to be predominantly directed toward the proteinase catalytic domains. Eleven T cell epitopes were identified using RgpA-Kgp-primed lymph node T cells (IL-4 dominant) and 21 using an RgpA-Kgp-specific T cell line (IFN-gamma dominant), with 5 T cell epitopes, including the immunodominant epitope peptide 22, common to both T cell populations. Peptide 22 ((439)ANYTAHGSETAWADP(453)) from the Kgp proteinase catalytic domain induced a Th2 cytokine response in mice, and peptide 22-primed T cells had a Th2 cytokine profile when stimulated with the RgpA-Kgp complexes. Truncation and alanine scanning of peptide 22 identified the minimum epitope ((442)TAHGSETAWA(451)), and residues His(444), Glu(447), and Trp(450) as critical for T cell proliferation. With a view to vaccine development, peptide 22 was incorporated into a synthetic peptide polymer. Peptide 22 polymer induced strong T cell proliferation and crossreactivity to native RgpA-Kgp complexes. In conclusion, we have identified a major T cell epitope of P. gingivalis and established that antigenicity of the T cell epitope is retained when delivered as a peptide polymer. The strategies employed here may have potential in the development of a synthetic peptide vaccine for P. gingivalis.
Asunto(s)
Adhesinas Bacterianas/inmunología , Cisteína Endopeptidasas/inmunología , Porphyromonas gingivalis/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/microbiología , Secuencia de Aminoácidos , Animales , Dominio Catalítico/inmunología , Línea Celular , Epítopos de Linfocito T/inmunología , Cisteína-Endopeptidasas Gingipaínas , Interleucina-4/biosíntesis , Ganglios Linfáticos/enzimología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/microbiología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Subgrupos de Linfocitos T/enzimologíaRESUMEN
Porphyromonas gingivalis is a pathogen associated with chronic periodontitis, an inflammatory disease of the supporting tissues of the teeth. A major virulence factor for P. gingivalis is the RgpA-Kgp proteinase-adhesin complex. We have prepared the following recombinant proteins corresponding to domains/regions of the RgpA-Kgp complex; rRgpA(cat), rRgpA(A1), rRgpA(A2), rRgpA(A4), rRgpA(A1)(784-1009), rRgpA(A1)(911-1009), rKgp(A1) and rKgp(A1)(759-989). The ability of each recombinant protein to attenuate P. gingivalis infection, when used as a vaccine, in the murine lesion model was determined. All of the recombinant adhesin domains were found to significantly attenuate P. gingivalis infection with the most effective being rKgp(A1) and rKgp(A1)(759-989) followed by rRgpA(A1), rRgpA(A1)(784-1009) and rRgpA(A1)(911-1009). The predominant antibody isotype was IgG1. The A1 adhesins, which gave the best protection, contain specific motifs implicated in binding to host tissue. Immunisation with rRgpA(cat) had no effect on P. gingivalis infection. As well as detecting the Kgp(A1) adhesin in a Western blot, the rKgp(A1) and rKgp(A1)(759-989) antisera were also immunoreactive with the A1 and A3 adhesins of RgpA and HagA. Flow cytometric analysis indicated that rKgp(A1) and rRgpA(A1) antisera recognised native antigen on P. gingivalis whole cells. Furthermore, the rKgp(A1) and rKgp(A1)(759-989) antisera exhibited a similar immunoreactive profile with outer membrane preparations of all P. gingivalis serotypes and clinical isolates tested. The recombinant A1 adhesin therefore has potential in the development of a P. gingivalis vaccine.