Study Of SIgA-enhancing Anti-Porphyromonas Gingivalis FimA DNA Vaccine

Chronic periodontitis is one of the major causes of tooth loss in adults, affecting health and well being. In China, it has a high incidence. Now periodontitis is considered as a potential risk factor for the development of some systemic diseases and manifestations such as cerebro-vascular and cardio-vascular diseases. Since periodontal care is unable to restore once-damaged periodontal tissue to its original condition, damaged periodontal tissue remains continuously vulnerable to bacterial infection, and periodontal disease often recurs at a later time. Therefore, effort should be oriented toward effective prevention.Chronic periodontitis is a bacterially induced inflammation of the periodontium. Porphyromonas gingivalis has been considered as a key pathogen in chronic periodontitis and FimA (fimbrillin) is one of the important cell surface virulence factors involved in adherence and invasion of P. gingivalis to host cells and a potent inducer of proinflammatory cytokines contributing to loss of alveolar bone. The present study was to construct a IgA-enhancing DNA vaccine against Porphyromonas gingivalis FimA and investigate the protection provided by the vaccine against P. gingivalis-induced alveolar bone loss.To construct the eukaryotic co-expression plasmid pIRES-fimA:IL15, the fimA gene and IL-15 gene were inserted into plasmid pIRES by molecular cloning methods. fimA gene and IL-15 gene were connected by internal ribosome entry site (IRES), permiting expression of the two genes with no interference to each other. At the same time, plasmid pIRES-fimA was constructed. The recombinated plasmids were characterized by restricted endonuclease mapping, PCR and DNA sequencing. The plasmids were transfected into mammalian cell CHO using Lipofectamine 2000. The expression of FimA and IL-15 in CHO cells was verified by Western blot and ELISA.To investigate the immunogenicity, plasmid pIRES-fimA:IL15 and pIRES-fimA were immunized to BABL/c mice via nasal or intramucusal route. Serum IgG and salivary sIgA levels after immunization were analyzed by ELISA. The results showed that nasal immunization with plasmid pIRES-fimA:IL15 elicited significantly higher level of salivary sIgA response than with pIRES-fimA. When immunized via nasal route, IL-15 expressed by the plasmid may act as an adjuvent cytokine to enhance FimA-specific sIgA antibody response. Nasal immunization was capable of promoting Ag-specific immune responses in the oral region as well as systemic immunity. There is no significant difference of the serum IgG responses between nasal immunization mice and intramuscular immunization mice but nasal immunization elicited significantly higher level of salivary FimA-specific IgA responses compared with intramuscular immunization. So nasal immunization with pIRES-fimA:IL15 could elicit FimA-specific antibody responses in serum and oral region. IL-15 has a positive regulation effect to sIgA response. Nasal dropping may be an effective mucosal immunization route.The co-expression plasmid pIRES-fimA.TL15 was used as a DNA vaccine to investigate the protection against P.gingivalis-induced alveolar bone loss. After immunized with the vaccine, the rats were infected with P. gingivalis. PCR was performed to detect P. gingivalis in saliva. Alveolar bone loss(expressed by distance from the cenentoenamel junction to the crest of the alveolar bone) was measured directly after soft tissue around tested teeth was removed. The results showed that no P. gingivalis were detected in saliva of pIRES-fimA:IL15 and pIRES-fimA immunized groups at the end of the experiment and P.gingivalis-induced alveolar bone loss in sham-immunized rats was significantly greater than pIRES-fimA:IL15 and pIRES-fimA immunized rats. Alveolar bone loss in pIRES-fimA:IL15 immunized rats was significantly less than in pIRES-fimA immunized rats. The weight increase at the end of experiment among groups had no significant difference. The HE stain of the main internal organs showed no evident pathological manifestations. The results suggest that nasal immunization with pIRES-fimA:IL15 in rats provided more protection against P. gingivalis-induced alveolar bone loss.In conclusion, the study constructed a co-expression plasmid pIRES-fimA:IL15 harboring fimA gene and IL-15 gene. When immunized as a DNA vaccine against P.gingivalis FimA via nasal route, the plasmid could induce FimA-specific IgG in serum and sIgA response in saliva. Nasal immunization may be an effective mucosal immunization route. IL-15 expressed by the plasmid pIRES-fimA:IL15 enhanced FimA-specific sIgA antibody response. And the vaccine could provide effective protection against P.gingivalis-induced experimental periodontitis. Nasal immunization is an effective mucosal immunization and is acceptable and easy to be put in practice. The results provide foundation to develop an safe and effective gene vaccine against chronic periodontitis.