The Research Of RgpA Gene Vaccine Prevents Peri-implantitis

Background:Peri-implantitis is an important complication of implant, which is characterized by soft tissue inflammation, bleeding, suppuration and bone loss. The lesion is associated with the presence of bacterial plaque which contains a large variety of Gram-negative anaerobic bacterias, Porphyromonas gingivalis (P.gingivalis, P.g), Actinobacillus actinomycetemcomitans (Aa). There are richly large amounts of densely packed inflammatory cells (lymphocytes, neutrophils, macrophages and plasma cells) in gingival crevicular fluid (GCF) At last, craterlike bone defect that surrounds the contaminated implant will be found. Many methods have been applied for the treatment of peri-implantitis including surgical intervention, the use of locally or systemically administered antimicrobial agents, ultrasonic therapy, laser therapy. However, there are limitations about the treatments.Gonzalez et al. using mice as models demonstrated that vaccination with P.gingivalis results in protection against experimental periodontal inflammation.However, various studies showed vaccination with bacterial cell performed less protection than vaccination with antigen-encoding plasmid DNA. The DNA gene immunization can induce humoral and cellular immune responses against a variety of pathogens, including bacteria, viruses, and tumor cells.Porphyromonas gingivalis, a Gram-negative anaerobic bacterium, possesses multiple pathogenic factors such as gingipain proteases, fimbriae, lipopolysaccharides (LPS), hemagglutinins. Among these, gingipains, a group of cysteine proteases, are major factor in the bacteria’s attack on the periodontal region. Gingipains consist of Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp), which are encoding by rgpA, rgpB and kgp. They are believed to play an important role in the pathogenesis of periodontitis, degrading the host defense mechanisms, resulting in tissue destruction and alveolar bone resorption.It has been reported that immunization of mice with rgpA DNA vaccine elicited high levels of specific IgGs against P.gingivalis. However, this method hasn’t been used in the preventive treatment about lesions of peri-implant. In this experiment, gene vaccine will be used to immune dogs to detect its function in preventing and treating peri-implantitis.Objective:To construct rgpA DNA vaccine with whole gene of Pg ATCC33277as template, and evaluated the effect of immunization with this vaccine to consider its function on the progression of experimental peri-implantitis.Methods:rgpA gene was combined by PCR using P.gingivalis ATCC33277whole gene as templante, then it was connected to the vector pVAXl.6dogs were divided into groups A, B and C, and24threaded cylindrical implants were randomly implanted into the extraction fosses of the bilateral mandibular premolar areas of the dogs. Animals of group A were immunized with pVAX1-rgpA, while group C with normal saline and group B with heat-killed P.gingivalis at2weeks intervals, and3times in all.2weeks after the last immunization (base line), cotton sutures infiltrated with Porphyromonas gingivalis were bound around the implants necks at one side of the animal madible. The peri-implant probing depth (PPD), bleeding on probing(BOP) and x-ray radiographies were measured at0,2,4and6 weeks after base line. Serum and saliva were collected before the first immunization and2weeks after the last immunization. Serum IgG and saliva secretory immune globulin A (IgA) were detected by enzyme-linked immunosorbent assay (Elisa).At last, animals were sacrificed, and tissue slices were burnished into50um slice, which were dyed with methylene blue, and be prepared for histology observation. Vertical and horizontal alveolar bone losses (VBL and HBL) around implants were measured.Results:1. Targeted plasmid pVAX1-rgpA was successfully constructed.2. Compared with the antibodies before immunization, group A and B showed higher titres (P<0.05).2weeks after the last immunization, group A and B showed higher titres of serum IgG and salivary slgA antibodies than group C (P<0.05). Although animals in group A showed higher antibodies than group B, there was no significant difference between group A and group B (P>0.05).3.4weeks after base line, PPD value of C group performed worse than group A and B (P<0.05). However, no significant difference was found between three groups at6weeks after base line (P>0.05).No significant difference about BOP was found among the three groups during the experiment (P>0.05).4. Much inflammtary cells and bacterium were found around the implants, especially around the screw threads. Group A showed lower bone loss compared with the group B and group C.Conclusions:1. Immunization with plasmid pVAX1-rgpA could enhance immunity and successively reduce soft tissue inflammation and bone loss in experimental peri-implantitis. 2. Immunization with P.gingivalis whole cells could also produce specific antibodies, but it failed to prevent bone loss successfully. A DNA-based immunization strategy may be an effective way to attenuate peri-implantitis induced by P.gingivalis.