Recombinant protein chimeras of the KAS peptide and KgpA1 domain of the RgpA–Kgp proteinase-adhesin complex are effective as prophylactic vaccines in experimental periodontitis

We have previously identified antigens from the RgpA–Kgp complex that protect against P. gingivalis-induced lesions and alveolar bone resorption.25,31,39 We hypothesised that a recombinant chimera of the most effective antigens; a Lys-gingipain active site peptide (KAS) and a recombinant Kgp-A1 adhesin sequence may exhibit enhanced protection against P. gingivalis induced disease by neutralising both proteolytic and adhesin functions of the virulence factor (Supplementary Figure 1). On the basis of epitope mapping and structural modelling of the Kgp proteinase and adhesin domain25,31 two chimera proteins were produced; KAS1-sA1 representing the minimal immunogenic sequences of KAS (Kgp-cat, 432–454) and KgpA1 (759–989) and KAS2-A1, which extended the minimal immunogenic sequences of KAS (Kgp-cat, 433–468) and KgpA1 (751–1056) (Supplementary Figure 2).

Immunogenicity studies showed that KAS1-sA1 and KAS2-A1 antisera recognised KAS2 peptide, KgpA1(751–1056) and FK-P. gingivalis W50 cells at comparable or higher levels when compared with antisera generated to KAS2-DT, KgpA1(759–989), KgpA1(751–1056) and FK-P. gingivalis W50 cells (Figure 1). Only the recombinant chimera proteins were found to recognise all three (KAS2, KgpA1 and P. gingivalis W50 cells) antigens (Figure 1a). Western blot analysis showed that the KAS1-sA1 and KAS2-A1 antisera recognised the Kgp proteinase and adhesin domains (Figures 1b–d). Epitope mapping of the lysine proteinase active site sequence (433–468) identified that the antigenic epitopes for both KAS1 and KAS2 were around the His(444) catalytic residue30,39 (Supplementary Figure 3). For KAS1 the major epitopes were 444HGSETAWADPL454>434TGVSFANYTA443 and for KAS2 447ETAWADPLL455>434TGVSFANYTA443=456TTSQLKAL463. These antigenic epitopes matched those induced by the KAS2-DT conjugate indicating that the KAS epitope in KAS1-sA1 and KAS2-A1 remained antigenic (Supplementary Figure 3).

Figure 1 Characterisation of the antigenicity of recombinant chimera proteins KAS1-sA1 and KAS2-A1. (a) KAS1-sA1 and KAS2-A1 antisera were used to probe KAS2 peptide, recombinant A1 (rA1 751–1056) and formalin killed P. gingivalis W50 cells coated plates in an ELISA and the response compared to antisera raised to formalin killed P. gingivalis W50 cells (FK-W50), recombinant proteins sA1 (759–989), A1 (751–1056), KAS2 peptide-DT conjugate and PBS/IFA in an ELISA. Antibody responses are expressed as the ELISA titre OD415 obtained minus double the background level, with each titre representing the mean±s.d. of three values. (b) SDS-PAGE gel of; lanes: 1, P. gingivalis whole-cell lysate; 2, RgpA–Kgp complex; 3, KAS2-A1; and 4, KAS1-sA1. Molecular mass markers (Pharmacia) are indicated in kDa. (c) Western blot analysis using KAS2-A1 antisera to probe; lanes: 1, P. gingivalis whole-cell lysate; 2, RgpA–Kgp complex; 3, KAS2-A1; and 4, KAS1-sA1. (d) Western blot analysis using KAS1-sA1 antisera to probe; lanes: 1, P. gingivalis whole-cell lysate; 2, RgpA–Kgp complex; 3, KAS2-A1; and 4, KAS1-sA1. Full size image

The recombinant proteins KAS1-sA1, KAS2-A1, KgpA1(759–989), KgpA1(751–1056); FK-P. gingivalis W50 and the RgpA–Kgp complex were used to immunise BALB/c mice to determine their efficacy as prophylactic vaccines against P. gingivalis-induced alveolar bone resorption. Mice immunised with all of the recombinant antigens, RgpA–Kgp complex and FK-W50 cells exhibited significantly (P<0.001) less bone loss compared with that of the PBS control group (Figure 2a). Both KAS2-A1 and KAS1-sA1 immunised mice exhibited significantly less alveolar bone resorption than the mice immunised with the RgpA–Kgp complex. KAS2-A1 provided a greater level of protection (P<0.01) than KgpA1(759–989), KgpA1(751–1056) or FK-P. gingivalis W50.

Figure 2 Recombinant chimera proteins KAS1-sA1 and KAS2-A1 protected against P. gingivalis-induced bone loss in a mouse prophylactic vaccine periodontitis model. (a) Mice were immunised with KAS1-sA1, KAS2-A1, RgpA–Kgp complex, formalin killed P. gingivalis W50 cells (FK-W50), recombinant proteins rA1 (759–989), rA1 (751–1056) or adjuvant alone (PBS, IFA). After the second immunisation mice were pre-treated with antibiotics and orally challenged with a total of eight doses of 1.0×1010 P. gingivalis W50 or treated with PG buffer containing 2% CMC alone (non-challenged group as N-C). P. gingivalis-induced bone resorption was determined as described in the Materials and Methods section for each group (n=12), and the data are expressed as the mean±s.d. in mm2 and were analysed using a one-way ANOVA and Dunnetts T3 post hoc test. * and ** indicates data that are significantly different (P<0.01, P<0.001, respectively) from the data for P. gingivalis challenged group. Serum antibody subclass responses of immunised mice in the periodontitis model, (b) post 1° and 2° immunisation and pre intra-oral challenge and (c) post intra-oral challenge with live P. gingivalis cells. Antisera was used to probe formalin killed P. gingivalis strain W50 as the absorbed antigen in an ELISA. Antibody responses IgG (black bars), IgG1 (grey bars), IgG2a (white bars), IgG2b (horisontal stripped bars), IgG3 (diagonal stripped bars), are expressed as the ELISA titre (log 2) obtained minus double the background level, with each titre representing the mean±s.d. of three values. * indicates IgG subclass significantly higher (P<0.05) than other IgG subclasses in that group. Full size image

Prior to intra-oral challenge with P. gingivalis each mouse group immunised with one of the immunogens had a high IgG antibody titre to FK-P. gingivalis W50 cells with a predominant IgG1 antibody subclass and only weakly immunoreactive IgG2a, IgG2b and IgG3 responses (Figure 2b). Post intra-oral challenge with P. gingivalis W50 each of the protective immunogens retained a predominant and significantly (P<0.05) higher IgG1 antibody subclass response to P. gingivalis (Figure 2c). However, the PBS/IFA immunised group had a predominant and significantly (P<0.05) higher IgG2b antibody subclass response to P. gingivalis (Figure 2c). Based on the above data the KAS2-A1 chimera was chosen as the lead vaccine candidate to be used in subsequent studies.

KAS2-A1 chimera prophylactic vaccine confers protection against P. gingivalis-induced bone resorption at low antigen doses

The KAS2-A1 chimera was formulated with aluminium hydroxide (alum) and BALB/c mice were immunised with 50 μg, 5.0 μg or 0.5 μg per dose (being adult human equivalent doses of 12, 1.2 and 0.12 mg40) (Figure 3). The KAS2-A1 formulated in alum at all doses trialed conferred significant (P<0.01) protection against P. gingivalis-induced bone loss (Figure 3a). There was no significant difference in the level of bone loss for the positive intervention (amoxicillin-treated), positive vaccination (RgpA–Kgp complex/IFA) and all of the KAS2-A1/alum immunised groups (Figure 3a).

Figure 3 Low antigen doses of recombinant chimera protein KAS2-A1 protect against P. gingivalis-induced bone loss in a mouse periodontitis model. (a) Mice were immunised with KAS2-A1 at 50, 5.0 and 0.5 μg doses in alum, RgpA–Kgp complex/IFA, or adjuvant alone (PBS, alum). After the second immunisation mice were pre-treated with antibiotics and orally challenged with a total of eight doses of 1.0×1010 P. gingivalis W50 or treated with PG buffer containing 2% CMC alone (non-challenged group as N-C). One group of mice received amoxicillin (500 μg/ml drinking water) from the first oral challenge to the end of the experiment. P. gingivalis-induced bone resorption was determined as described in the Materials and Methods section for each group (n=12), and the data are expressed as the mean±s.d. in mm2 and were analysed using a one-way ANOVA and Dunnett’s T3 post-hoc test. ## indicates data that are significantly different (P<0.01) from the data for P. gingivalis inoculated group. (b) Serum antibody subclass responses of immunised mice in the periodontitis model. Antisera was used to probe formalin killed P. gingivalis strain W50 as the absorbed antigen in an ELISA. Antibody responses IgG (blue bars), IgG1 (green bars), IgG2a (yellow bars), IgG2b (orange bars), IgG3 (grey bars), IgM (brown bars), IgA (white bars) are expressed as the ELISA titre (log 2) obtained minus double the background level, with each titre representing the mean±s.d. of three values. * indicates IgG subclass significantly higher (P<0.05) than other IgG subclasses in that group. (c) IFNγ and IL-4 producing submandibular lymph nodes (SMLN) T-cells and gingival tissue (gingiva) lymphocytes isolated from immunised mice in the periodontitis model. Cytokine producing cells were determined by ELISPOT using FK-P. gingivalis W50 as the stimulating antigen. Cytokine response expressed as cytokine producing cells/106 obtained minus the background level, with data representing the mean±standard deviation of three values. * indicates which cytokine is significantly higher (P<0.05) than other in SMLN or gingival tissue for that group. Full size image

Mice immunised with KAS2-A1/alum at each dose or the RgpA–Kgp complex/IFA had a high IgG titre to FK-P. gingivalis W50 cells with a predominant IgG1 subclass (Figure 3b). The PBS/alum immunised group and the infected control groups had a predominant and significantly (P<0.05) higher IgG2b antibody subclass response to P. gingivalis (Figure 3b).

To determine the cellular interferon (IFN)-γ and IL-4 cytokine response for each group following P. gingivalis oral infection, submandibular lymph node (SMLN) T cells and gingival tissue lymphocytes were isolated and the number of P. gingivalis-specific cytokine producing cells determined by ELISPOT (Figure 3c). The PBS/alum treated and P. gingivalis-infected control groups had significantly (P<0.05) higher numbers of IFNγ secreting SMLN T cells and gingival lymphocytes. Mice immunised with KAS2-A1/alum at all doses had significantly (P<0.05) higher numbers of IL-4 secreting SMLN T cells and gingival lymphocytes, characteristic of a Th2 predominant response.

Protective efficacy of the KAS2-A1 chimera in a therapeutic vaccination periodontitis model

To develop a therapeutic vaccination model we first conducted a time course study to determine the timing of a distinct P. gingivalis-specific immune response post intra-oral challenge (infection). Figure 4a shows that only challenge with viable P. gingivalis W50 cells, and not formalin-killed (FK) P. gingivalis cells, induced significant (P<0.05) bone loss. Infiltrating gingival CD4+ T cells were significantly (P<0.05) higher in numbers on days 8, 17, 28 and 56 in the viable P. gingivalis infected group compared with the uninfected control and the FK-P. gingivalis challenged group (Figure 4b). A significantly (P<0.05) higher number of P. gingivalis-specific IFNγ and IL-17 secreting T cells and P. gingivalis-specific serum antibody responses were found on days, 17, 28 and 56 for the viable P. gingivalis infected group compared with the uninfected control and the FK-P. gingivalis challenged group (Figures 4c–f). The time course study showed that only challenge with viable P. gingivalis cells induced bone loss and that an antigen-specific immune response was established from day 17 and onwards after the first oral challenge. Hence, for the therapeutic vaccination model we chose day 19 as an appropriate treatment/vaccination time point.

Figure 4 Time course analysis of the P. gingivalis-induced bone loss and development of a P. gingivalis-specific proinflammatory T-cell response. BALB/c mice were pretreated with antibiotics and then intra-orally challenged with a total of four doses of 1.0×1010 viable P. gingivalis W50 cells, formalin-killed P. gingivalis W50 cells (FK-P. gingivalis) or treated with PG buffer containing 2% CMC alone (non-challenged group N-C) on days 0, 2, 4, 6. On days 8, 17, 28 and 56 mice were killed and; (a) P. gingivalis-induced bone resorption was determined as described in the Materials and Methods section for each group (n=12), and the data are expressed as the mean±s.d. in mm2 and were analysed using a one-way ANOVA and Dunnetts T3 post hoc test. * indicates data that are significantly different (P<0.05) from the data for negative control group; (b) the number of gingival CD4+ T cells (TCRβ+CD4+) in the lymphocyte infiltrate were determined using flow cytometry using lymphocytes isolated from gingival tissue. Data are expressed as the mean±standard deviation and were analysed using Student's t-test. * indicates data that are significantly different (P<0.05) from the data for negative control group; (c,d) IFNγ and IL-17-producing submandibular lymph nodes (SMLN) T-cells were determined by ELISPOT using FK-P. gingivalis W50 as the absorbed antigen. Cytokine response expressed as spot forming cells per 106 obtained minus the background level, with data representing the mean±s.d. of three values. * indicates which cytokine is significantly higher (P<0.05) from the data for negative control group; (e) P. gingivalis-specific serum antibody subclass responses were determined by ELISA using formalin killed P. gingivalis strain W50 as the absorbed antigen. * indicates IgG subclass significantly higher (P<0.05) than other IgG subclasses in that group; (f) P. gingivalis-specific antibody producing submandibular lymph nodes (SMLN) B-cells were determined by ELISPOT on day 56 using FK-P. gingivalis W50 as the stimulating antigen. Antibody response expressed as spot forming cells/106 obtained minus the background level, with data representing the mean±s.d. of three values. * indicates Ig subclass significantly higher (P<0.05) than other Ig subclasses in that group. Full size image

To test the KAS2-A1 chimera as a therapeutic vaccine BALB/c mice were immunised on day 19 and 40, post oral infection with P. gingivalis, using the same antigen doses and alum formulation as described above. KAS2-A1-induced significant (P<0.01) protection against P. gingivalis-induced bone resorption in the therapeutic vaccination model (Figure 5a). There was no significant difference in the level of bone loss for the positive intervention (amoxicillin-treated), positive vaccination (RgpA–Kgp complex/IFA) and all of the KAS2-A1/alum immunised groups (Figure 5a).

Figure 5 The recombinant chimera KAS2-A1 protects against P. gingivalis-induced bone loss in a therapeutic vaccination periodontitis model. (a) BALB/c mice were pre-treated with antibiotics and then orally challenged with a total of four doses of 1.0×1010 viable P. gingivalis W50 cells or treated with PG buffer containing 2% CMC alone (non-challenged group N-C) on days 0, 2, 4, 6. On day 19 after the first oral challenge, mice were immunised with KAS2-A1 at 50, 5.0 and 0.5 μg doses in alum, the RgpA–Kgp complex/IFA, or adjuvant alone (PBS, alum). A group was also treated with amoxicillin (500 μg/ml drinking water). Mice received a second immunisation (day 40) and then killed in day 62. (a) P. gingivalis-induced bone resorption was determined as described in the Materials and Methods section for each group (n=12), and the data are expressed as the mean±s.d. in mm2 and were analysed using a one-way ANOVA and Dunnetts T3 post hoc test. ## indicates data that are significantly different (P<0.01) from the data for P. gingivalis challenged group. (b) Serum antibody subclass responses of immunised mice in the periodontitis model. Antisera was used to probe formalin killed P. gingivalis strain W50 as the absorbed antigen in an ELISA. Antibody responses are expressed as the ELISA titre (log 2) obtained minus double the background level, with each titre representing the mean±s.d. of three values. * indicates IgG subclass significantly higher (P<0.05) than other IgG subclasses in that group. (c) IFNγ and IL-4 producing submandibular lymph nodes (SMLN) T cells and gingival tissue (gingiva) lymphocytes isolated from immunised mice in the periodontitis model. Cytokine-producing cells were determined by ELISPOT using FK-P. gingivalis W50 as the stimulating antigen. Cytokine response expressed as cytokine producing cells/106 obtained minus the background level, with data representing the mean±s.d. of three values. * indicates which cytokine is significantly higher (P<0.05) than other in SMLN or gingival tissue for that group. (d) P. gingivalis cells enumerated in half maxillae from immunised mice in the periodontitis model by real time PCR, using P. gingivalis-specific and Universal bacteria 16S rRNA forward and reverse primers. Data are expressed as the percentage of P. gingivalis cells of the total bacterial cells recovered per half maxilla mean±s.d. (n=5) and were analysed using a Student's t-test. * indicates data that are significantly different (P<0.05) from the value for the uninoculated group. Full size image

Mice immunised with KAS2-A1/alum or the RgpA–Kgp complex/IFA had a high IgG antibody titre to FK-P. gingivalis cells with a predominant IgG1 antibody subclass response (Figure 5b). The PBS/alum immunised group and the infected control groups had a predominant and significantly (P<0.05) higher IgG2b antibody subclass response to P. gingivalis (Figure 5b). Mice immunised with KAS2-A1/alum at 50 and 5.0 μg doses had significantly (P<0.05) higher numbers of IL-4 secreting SMLN T cells and gingival lymphocytes, whereas, the PBS/alum immunised group and P. gingivalis infected control group had significantly (P<0.05) higher numbers of IFN-γ secreting SMLN T cells (PBS/alum only) and gingival lymphocytes (Figure 5c).

DNA was extracted from half maxillae of each of the groups and the P. gingivalis level expressed as a percentage of total bacterial cell numbers recovered. All of the KAS2-A1 immunised and RgpA–Kgp complex groups had significantly (P<0.05) lower levels of P. gingivalis as a percentage of total bacterial cells compared with the PBS/alum and infected control groups (Figure 5d).

KAS2-A1 chimera provides protection against alveolar bone resorption in mice orally challenged with P. gingivalis/T. denticola/T. forsythia

As P. gingivalis is found in subgingival plaque with T. denticola and T. forsythia we investigated whether the KAS2-A1 chimera would protect against challenge and superinfection with all three species (Figure 6). The P. gingivalis/T. denticola/T. forsythia challenged animals exhibited a slightly higher level of bone resorption when compared with the animals challenged with P. gingivalis alone at the higher dose confirming bacterial synergy in bone loss with these species.41 KAS2-A1 formulated in alum conferred significant (P<0.01) protection against P. gingivalis/T. denticola/T. forsythia-induced bone loss (Figure 6a). KAS2-A1 immunised animals had a predominant P. gingivalis-specific IgG1 (Figure 6b) and IL-4 secreting T-cell response (Figure 6c). For both P. gingivalis/T. denticola/T. forsythia and P. gingivalis infected control groups, only a P. gingivalis-specific antibody, IgG2b dominant (Figure 6b), and IFNγ secreting T-cell response (Figure 6c) was detected. No significant immune responses were observed towards T. forsythia or T. denticola.

Figure 6 The recombinant chimera protein KAS2-A1 formulated in alum protects against bacteria-induced bone loss in a multi-pathogen superinfection periodontitis model. (a) Mice were immunised with KAS2-A1 at 50 μg in alum. After the second immunisation mice were pretreated with antibiotics and orally challenged with a total of four doses of 1.0×1010 total cells of P. gingivalis W50 or 1.0×1010 total cells of P. gingivalis W50/T. denticola/T. forsythia or treated with PG buffer containing 2% CMC alone (non-challenged group N-C). Bacteria-induced bone resorption was determined as described in the Materials and Methods section for each group (n=12), and the data are expressed as the mean±standard deviation in mm2 and were analysed using a one-way ANOVA and Dunnetts T3 post-hoc test. * indicates data that are significantly different (P<0.01) from the data for P. gingivalis or P. gingivalis W50/T. denticola/T. forsythia challenged groups. (b) Serum antibody subclass responses of immunised mice in the multi-pathogen periodontitis model. Antisera was used to probe formalin killed P. gingivalis strain W50 as the absorbed antigen in an ELISA and antibody responses are expressed as the ELISA titre (log 2) obtained minus double the background level, with each titre representing the mean±standard deviation of three values. * indicates IgG subclass significantly higher (P<0.01) than other IgG subclasses in that group. (c) IFNγ and IL-4 producing submandibular lymph node (SMLN) T-cells isolated from each group in the multi-pathogen periodontitis model. Cytokine producing cells were determined by ELISPOT using FK-P. gingivalis W50 as the stimulating antigen. Cytokine response expressed as cytokine producing cells/106 obtained minus the background level, with data representing the mean±standard deviation of three values. * indicates which cytokine is significantly higher (P<0.05) than other for that group. (d) the per cent of gingival CD4+ T cells [TCRβ+CD4+] that were CD25+ or CD69+ was determined by flow cytometry using lymphocytes isolated from gingival tissue. Data are expressed as the mean±standard deviation and were analysed using Student's t-test. * indicates data that are significantly different (P<0.05) from the data for negative control group. Full size image

Isolation and phenotyping of gingival CD69+ (early activated) and CD25+ (activated/antigen stimulated) CD4+ T cells showed that KAS2-A1 immunised animals had a significantly higher population of CD25+ but not CD69+, CD4+ gingival T cells compared with the non-infected (NC) animals. The P. gingivalis/T. denticola/T. forsythia and P. gingivalis infected control groups had a significantly higher population of CD25+ and CD69+, CD4+ gingival T cells compared with the non-infected and KAS2-A1 immunised animals. Taken together, the results indicate that the KAS2-A1 immunised/infected groups had developed a Th2 (IL-4 secreting) antigen-stimulated gingival T-cell population, whereas the P. gingivalis/T. denticola/T. forsythia and P. gingivalis alone infected, control groups exhibited a Th1 (INFγ secreting) early and antigen activated gingival T-cell population.

KAS2-A1 chimera protection against P. gingivalis-induced bone loss is antibody mediated

To investigate whether KAS2-A1-specific T cells or B cells mediate protection in the mouse periodontitis model, donor mice (Ly5.1 C57Bl6 congenic mice) were immunised with KAS2-A1 chimera or ovalbumin (antigen control) and IgG1 and IgG2 expressing B cells and CD4+ T cells harvested and adoptively transferred into recipient mice (Ly5.2 C57Bl6 congenic mice). The day following the adoptive transfer mice were orally infected with P. gingivalis and bone loss determined. Mice that received CD4+ T cells, IgG2 expressing B cells from KAS2-A1 immunised mice or B cells from ovalbumin immunised mice developed P. gingivalis-induced bone loss at a similar level to that in the infected control group and had a predominant IgG2a/b response to P. gingivalis (Figures 7a and b). However, mice that received IgG1 expressing B cells from KAS2-A1 immunised mice did not develop P. gingivalis-induced bone loss and had similar alveolar bone levels to that in the non-infected (NC) control group and a predominant IgG1 response to P. gingivalis. Cellular phenotyping of the gingival and SMLN lymphocytes showed that mice which received IgG1 expressing B cells from KAS2-A1 immunised mice had a higher percentage of CD19+/CD138+ plasma B cells in SMLNs as well as being the only group to exhibit an increase in these cells in the gingival lymphocyte population (Supplementary Figure 4). These data indicate that B cells and not CD4+ T cells are pivotal for protection against P. gingivalis-induced bone loss and that antigen-specific IgG1, and not IgG2, is the critical IgG subclass that mediates protection.

Figure 7 KAS2-A1 chimera protective efficacy is antibody mediate. (a) Adoptive transfer of IgG1 and IgG2 expressing B cells and CD4+ T cells from KAS2-A1 immunised mice in the periodontitis model. Donor mice (Ly5.1 C57Bl6 congenic mice) were immunised with KAS2-A1/ISCOMATRIX adjuvant (day 0) and IgG1 expressing B cells; IgG2 expressing B cells and CD4+ T cells isolated (day 30) from spleen and lymph nodes using magnetic bead separation and cells resuspended in PBS containing 0.5 μg of KAS2-A1/200 μl. Recipient mice (Ly5.2 C57Bl6 congenic mice) were inject (day 30) i.v. with 1×106 cells; IgG1 or IgG2 expressing B cells or CD4+ T cells from KAS2-A1 immunised mice or B cells from ovalbumin immunised mice to act as an antigen-specific B cell control. The recipient mice had been pre-treated with antibiotics and were orally challenged (day 31) with a total of four doses of 1.0×1010 total cells of P. gingivalis W50 or treated with PG buffer containing 2% CMC alone (non-challenged group N-C). Bacteria-induced bone resorption was determined as described in the Materials and Methods section for each group (n=12), and the data are expressed as the mean±standard deviation in mm2 and were analysed using a one-way ANOVA and Dunnetts T3 post-hoc test. * indicates data that are significantly different (P<0.01) from the data for P. gingivalis infected control group. (b) Serum antibody subclass responses of donor mice in the periodontitis model. Antisera was used to probe formalin killed P. gingivalis strain W50 as the absorbed antigen in an ELISA and antibody responses are expressed as the ELISA titre (log 2) obtained minus double the background level, with each titre representing the mean±standard deviation of three values. * indicates IgG subclass significantly higher (P<0.05) than other IgG subclasses in that group. (c) Purified KAS2-A1 polyclonal antibodies administered by oral topical or intraperitoneal injection protect mice against P. gingivalis-induced bone loss in the periodontitis model. Purified KAS2-A1 rabbit polyclonal antibodies were injected (i.p. 500μg/mouse) one day prior oral challenge with P. gingivalis or applied intra-orally (topical, 2×250μg/application) 15 min pre- and post- each oral challenge. Bacteria-induced bone resorption was determined as described in the Materials and Methods section for each group (n=12), and the data are expressed as the mean±s.d. in mm2 and were analysed using a one-way ANOVA and Dunnetts T3 post hoc test. # indicates data that are significantly different (P<0.05) from the data for P. gingivalis infected control group. Full size image

To investigate whether KAS2-A1-specific antibodies when passively administered were able to facilitate protection against bone loss in the periodontitis model, rabbits were immunised with KAS2-A1 and polyclonal antibodies (pAb) purified using protein A/G columns. The antigenicity of the purified pAb was confirmed by ELISA and Western blot analysis (Supplementary Figure 5) and used prophylactically or therapeutically in the respective periodontitis models. In one treatment group, BALB/c mice were injected (i.p.) with KAS2-A1-pAb (500 μg per mouse) one day prior to (prophylactic model) or on day 19 post (therapeutic model) oral challenge with P. gingivalis to evaluate whether systemic administration could mediate protection. In a second treatment group the KAS2-A1-pAb was applied intra orally pre and post oral challenge with P. gingivalis, (250 μg per application, prophylactic model; or 4×500 μg doses from day 19, therapeutic model) to evaluate whether topical application of antibodies would provide protection. Both systemic and topical application of the KAS2-A1-pAb, but not control pAb, when used prophylactically or therapeutically significantly reduced P. gingivalis-induced bone resorption (Figure 7c; Supplementary Figure 6).

KAS2-A1-pAb neutralisation of gingipain activities and binding to P. gingivalis

Several studies have shown that the Lys-X and Arg-X proteolytic activity, binding to oral epithelial cells, host matrix and blood proteins and co-aggregation with T. denticola are activities by which gingipains contribute to P. gingivalis virulence.24,29,41–47 We investigated the ability of purified KAS2-A1-pAb compared with a non-specific (NS, PBS/alum)-pAb to inhibit each of these gingipain activities associated with the bacterium’s pathogenicity (Figure 8). The KAS2-A1-pAb inhibited P. gingivalis whole-cell Lys-X proteolytic activity within 30 min and the level of inhibition slightly increased with incubation time (Figure 8a). Interestingly, the KAS2-A1-pAb also inhibited P. gingivalis whole-cell Arg-X proteolytic activity after 4 h of incubation and this increased at the 24 h time point. The ability to inhibit both Lys-X and Arg-X proteolytic activities can be attributed to the sequence similarity of the Kgp and Rgp active sites and that the minimal antigenic sequence for KAS2 (identified in Supplementary Figure 3) also occurs in the Arg-proteinases; RgpA and RgpB. KAS2-A1-pAb was also found to significantly inhibit P. gingivalis whole cells from binding to oral (OKF6-TERT2) epithelial cells and from co-aggregation with T. denticola (Figures 8b,c). The antibodies also significantly inhibited RgpA–Kgp binding to the host proteins; fibronectin, fibrinogen and haemoglobin (Figure 8d).

Figure 8 Purified KAS2-A1 pAb inhibit gingipain activities. (a) KAS2-A1 pAb inhibition of Lys-X and Arg-X proteinase activity of P. gingivalis whole cells. P. gingivalis W50 whole cells (1×108/ml) in TC150 buffer containing 2 mmol/l L-cysteine was preincubated (0.5, 4 and 24 h) with purified KAS2-A1 pAb or non-specific (NS) pAb (100 μg/ml) and whole cell Arg-X and Lys-X proteinase activity determined. Data are expressed as the mean±s.d. of the per cent inhibition of P. gingivalis W50 whole cells incubated for equivalent times in the absence of antibodies and were analysed using a Student’s T test. * indicates data that are significantly different (P<0.01) from the data for the NS-pAb group. (b) KAS2-A1 pAb inhibition of P. gingivalis whole-cell binding to oral epithelial cells. FITC-labelled P. gingivalis W50 cells (1×108/ml) in EMEM media were preincubated (60 min) with purified KAS2-A1 pAb or non-specific (NS) pAb (100 μg/ml) and then incubated (90 min) with a confluent monolayer of oral epithelial cells at a bacteria to cell ratio of 20:1. The binding of P. gingivalis W50 to oral epithelial cells was determined by flow cytometry and the data expressed as the mean±s.d. of the per cent inhibition of P. gingivalis W50 whole cells incubated for equivalent times in the absence of antibodies and were analysed using a Student’s T test. * indicates data that are significantly different (P<0.01) from the data for the NS-pAb group. (c) KAS2-A1 pAb inhibition of the co-aggregation of P. gingivalis and T. denticola whole cells. AlexaFluor 555-labelled P. gingivalis W50 cells (5×107/ml) in EMEM media were pre-incubated (60 min) with purified KAS2-A1 pAb or non-specific (NS) pAb (250 μg/ml) and then incubated (60 min) with AlexaFluor 647-labelled T. denticola cells (5×107/ml) and the level of coaggregation determined by flow cytometry. Data expressed as the mean±s.d. of the per cent inhibition of P. gingivalis W50 and T. denticola co-aggregation incubated for equivalent times in the absence of antibodies and were analysed using a Student’s T test. * indicates data that are significantly different (P<0.01) from the data for the NS-pAb group. (d) KAS2-A1 pAb inhibition of the RgpA–Kgp complex binding to host proteins; fibronectin, fibrinogen and haemoglobin. RgpA–Kgp complex (0.5 μg/ml) was incubated (3 h) with fibronectin, fibrinogen and haemoglobin precoated ELISA plates in the presence of KAS2-A1 pAb or non-specific (NS) pAb (5 μg/ml) in 0.1 mol/l PBS containing 1 mmol/l TLCK. The per cent of inhibition of binding was determined from the binding of RgpA–Kgp complex in the absence of antibody. Data expressed as the mean±s.d. of the per cent inhibition and were analysed using a Student’s T test. * indicates data that are significantly different (P<0.01) from the data for the NS-pAb group. Full size image

Further, the KAS2-A1-pAb was found to recognise all of thirteen P. gingivalis strains representing the major laboratory strains, serotypes A-D43,48 and clinical isolates (from different regions of the World) tested at a similar antibody titre when compared with RgpA–Kgp complex antibodies (Supplementary Figure 7). These data suggest that the KAS2-A1 chimera should produce antibodies that would cross-react with all currently known P. gingivalis strains.