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Arg-gingipain A DNA Vaccine Prevents Alveolar Bone Loss in Mice

¹ Department of Microbiology and
² Oral Health Science Center, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba, 261-8502, Japan

View larger version (9K): [in this window] [in a new window]   Figure 1. Induction of P. gingivalis-specific serum IgG and salivary sIgA antibodies in mice immunized with rgpA DNA vaccine via abdominal skin by Gene Gun (2.5 µg/mouse) and mice immunized nasally with the rgpA-HVJ envelope vector (20 µg/mouse). rgpA DNA vaccine was injected a total of 7 times as indicated by arrows. (A) Serum IgG titers of mice against sonicates of P. gingivalis were determined on days 0, 7, 14, 28, 35, 42, and 49 after primary immunization (N = 15). Serial dilutions were used to measure endpoint titers (> 0.15). Results are means ± standard deviations of log2 ELISA antibody titers. Variation in endpoint titers was within a two-fold serial dilution. *p < 0.01, against naïve mice; **p < 0.01 against Gene Gun-immunized mice by one-way ANOVA, followed by the Student-Newman-Keuls test. (B) Salivary sIgA titers against sonicates of P. gingivalis were determined on days 0, 7, 14, 28, 35, 42, and 49 after primary immunization. Serial dilutions were used to measure endpoint titers (> 0.15). Results are means ± standard deviations of log2 ELISA antibody titers (N = 15). Variation in endpoint titer was within a two-fold serial dilution. *p < 0.01 against naïve mice and Gene Gun-immunized mice by one-way ANOVA, followed by the Student-Newman-Keuls test.

View larger version (27K): [in this window] [in a new window]   Figure 2. Specificity of salivary sIgA against RgpA domains. Recombinant catalytic domain of RgpA (RgpA Cat, lane 1), 44-kDa adhesion hemagglutinin domain (HGRP 44, lane 2), and 15-, 17-, and 27-kDa hemagglutinin domains (HGRP 15–27, lane 3) with histidine tags were separated by SDS-PAGE and transferred to PVDF membrane. Blotted membrane was blocked with phosphate-buffered saline containing 3% bovine serum albumin, reacted with peroxidase-conjugated anti-mouse IgA and developed. Blotted lanes 1, 2, and 3 were probed with saliva from mice immunized nasally with rgpA-HVJ envelope vector.

View larger version (11K): [in this window] [in a new window]   Figure 3. P. gingivalis-specific IgG subclass responses against sonicates of P. gingivalis. Immunization groups of BALB/c mice immunized with rgpA DNA vaccine via abdominal skin by Gene Gun (2.5 µg/mouse) or immunized nasally with rgpA-HVJ envelope vector (20 µg/mouse). IgG subclass responses were assessed on day 49. Results are means ± standard deviations of log2 ELISA antibody titers (N = 15). Variation in endpoint titers was within a two-fold serial dilution. One-way ANOVA for repeated measurements was used for inter-group comparisons. The Student-Newman-Keuls test was used for multiple comparisons.

View larger version (12K): [in this window] [in a new window]   Figure 4. Levels of alveolar bone loss elicited following P. gingivalis oral challenge. BALB/c mice immunized with rgpA DNA vaccine via abdominal skin by Gene Gun (2.5 µg/mouse), mice immunized nasally with rgpA-HVJ envelope vector (20 µg/mouse); control group consisted of age-matched, non-vaccinated mice and pVAX1 immunized mice orally challenged with P. gingivalis ATCC 33277 (Pg). At 42 days after oral challenge, all mice were killed. Linear measurements (n = 14 sites) were obtained from the mean of the maxillary molars in each mouse, and pooled linear measurements (in millimeters) were calculated for each group (N = 20 for non-infected naïve mice, P. gingivalis-infected naïve mice, and P. gingivalis-infected HVJ envelope vector-immunized mice; N = 15 for P. gingivalis-infected Gene Gun-immunized mice; N = 15 for pVAX1 immunized with HVJ envelope vector). Data are shown as mean ± standard deviation. One-way ANOVA for repeated measurements was used for inter-group comparisons. The Student-Newman-Keuls test was used for multiple comparisons (*p < 0.01). CEJ = cemento-enamel junction, ABC = alveolar bone crest.