Anti-atherosclerotic vaccination against Porphyromonas gingivalis as a potential comparator of statin in mice

P. gingivalis and pitavastatin

P. gingivalis strain ATCC 53978 was cultured with tryptic soy broth containing haemin and vitamin K (Becton Dickinson, Tokyo Chemical Industry Co., Ltd, Sigma-Aldrich) in an anaerobic chamber with 80% N2, 10% H2, and 10% CO2. The bacteria concentration was standardized using a spectrophotometer at OD 650 nm (0.1 of P. gingivalis = 10¹⁰ bacteria/ml). Vaccine was produced by heat-shocking of P. gingivalis solution through sonicating three times for 15 s each. Pitavastatin was dissolved in DMSO with PBS dilution (1:1 ratio) by sonicating for 1 min to improve the solubility. A total of 10⁹ CFUs of live P. gingivalis were suspended in 100 μL PBS with 2% carboxymethyl cellulose (Sigma-Aldrich) for animal studies.

Mouse and diet

Experimental procedures, including housing and care, were conducted according to the regulation of the National Institute of Health and approved by the Ethics Committee and the Institutional Animal Care and Use Committee of Yonsei University, College of Medicine (IACUC Approval number, 2017-0167). Eight-week-old ApoE −/− male mice were purchased from Jackson Laboratories, and housed at 23 ∼24 °C in a 12-hour light/dark cycle. APOE -/- mice were fed by 21gm% Western diet powder (D12079B, Research Diet) for 8 weeks and monitored daily. The power analysis was used to calculate the minimum number N at a significance level of 0.01 and power of 0.8. The minimum number N was 4.245 (n = 4).

Pre-check of vaccination in mice

Mice were nasally immunized with 10 ug of vaccine with 1 ug of Cholera toxin every 2 weeks for 5 weeks, from week 11 to week 15. Serum and mucosal wash samples were collected at week 16 following a previous study (Namikoshi et al., 2003). Serum was obtained through a tail vein, and tear-wash samples were obtained by lavaging with 10 μl of PBS per eye. Saliva was obtained by intraperitoneal injection of pilocarpine (500 mg/kg body weight; Sigma-Aldrich) into the mice. Fecal extracts were obtained by dissolving the feces in PBS containing 0.1% sodium azide. The feces were mixed by vortexing and centrifuged, and the supernatants were collected for assays.

Successful vaccination was determined using the collected body samples in PBS by reacting IgG and IgA in ELISA plates (Falcon, Franklin Lakes, NJ) overnight at 4 °C. ELISA plate coated with sonicated whole cell extracts of P. gingivalis ATCC 53978(10 μg/ml) were blocked with 1% BSA (Sigma-Aldrich) in PBS. Samples were then reacted with HRP-conjugated goat anti-mouse IgG or IgA Ab (Southern Biotechnology Associates) in each well, followed by incubation overnight at 4 °C. Color signals were generated by adding tetramethylbenzidine solution (Life Technologies) until the addition of stopping solution (0.5 N HCl). Then, the signals were measured at 450 nm on an ELISA reader (Molecular Devices). Endpoint titers of Ag-specific Ab were expressed as reciprocal log2 titers of the last dilution that showed >0.1 absorbance over the background levels.

Bacterial challenging of mice with anti-atherosclerotic effects

A total of 32 male APOE-/-mice (8-week old) were subjected to Western diet for 8 weeks, in order to induce atherosclerosis in a physiological manner (Fig. 1). After the period of adaptation, the mice were randomly and equally grouped to undergo four treatment conditions (i.e., no treatment, pitavastatin, vaccine, or pitavastatin with vaccine). The vaccination was conducted through nasal immunization and confirmed by a Pg-specific humoral immune reaction. Then, half of the mice in each group were orally injected with P. gingivalis for the next 5 weeks while the other half remained uninfected, generating a total of eight groups (n = 4/group). Pitavastatin (30 mg/kg/week) was injected three times (10 mg/mL/injection) every week for 5 weeks. P. gingivalis was administrated through the oral route five times per week for 5 weeks at a dose of 10⁹CFU. The mice were euthanized with carbon dioxide gas, and the aortas were harvested for analysis. After cleaning the adventitia, the aorta was cut open longitudinally under a dissecting microscope to be analyzed entirely. The en face aortas were imaged by Oil Red O staining and HP Printer scanning. According to the method of a previous study (Lin et al., 2015), the images were quantitatively analyzed to determine the size of atherosclerotic plaque using Image J software (NIH, MD, US). When damage occurred during the process of staining and analyzing the tissue, it was excluded from the results; and a total of seven animals were excluded.

Statistical analyses

Analyses were performed using SPSS 23.0 (Systat software, Chicago, IL). One-way ANOVA with Tukey multiple comparisons test was performed to compare the groups. P values less than 0.05 were considered significant.

Antibody reactions to body samples as working validation of vaccination

The atherosclerosis model was produced by subjecting ApoE-/- male to either normal or Western diet (WD), and the formation of atherosclerotic plaques was determined by Oil Red O staining with quantitative image analysis. The normal diet group started to show plaque formation at 20 weeks in contrast to 4 weeks in the WD group, and the plaque sizes of both groups grew over time. The WD production (D12079B) was used by refereeing the previous studies to profile microbiome in the gut (Chan et al., 2016) and to analyze atherosclerotic burden in the disease models using the WD product (Hutter et al., 2015).

Successful vaccination was determined based on antigen formation of the collected body samples. ELISA assays were used to determine IgG reaction to serum as well as IgA reaction to fecal, saliva, and tear. The nasal immunization of mice with vaccine and cholera toxin with or without PTV resulted in successful formation of antigens in the corresponding samples compared to the groups without vaccination (Fig. 2). These results indicated that the components and methods are effective in approaching vaccination.

Anti-atherosclerotic effect of vaccine in comparison with PTV

The effect of vaccination to prevent atherosclerosis against repeated infection of P. gingivalis was compared with the treatment and co-treatment of PTV (Fig. 3). Atherosclerosis was induced in APOE -/- mice in physiological fashion by 8-week Western diet, and vaccination and PTV treatment were conducted prior to P. gingivalis. The infected mice with no treatment served as a control (Group 2) for comparison with the other groups, since this control represented the model of oral infection by P. gingivalis in a causative relation with the atherosclerosis progress. The effective prevention by vaccination (Group 6) against P. gingivalis infection was suggested by Oil Red O staining (Fig. 3A) with quantification of atherosclerotic plaques (Fig. 3B) in the en face of each aorta. Regarding the quantitative image analysis of the lesion area (% of total area) after Oil Red O staining, Group 1 (Pg-uninfected with no treatment) exhibited 37.07 ± 2.42% out of the total area compared to 92.64 ± 16.71% of Group 2 (Pg-infected mice with no treatment). This result confirmed that the disease model of infection was successfully produced by Western diet and Pg infection, as indicated by the more than two-fold difference in the formation of atherosclerotic plaques between the two groups.

Group 6 (vaccination) and Group 4 (PTV treatment) showed comparable anti-atherosclerotic effects, as shown by 16.18 ± 1.52% and 19.59 ± 1.83% out of the total area, respectively. However, the co-treatment group (Group 8), with 15.72 ± 2.82% (of the total area), did not show significant synergistic effects compared to our expectation. The uninfected groups did not exhibit meaningful differences in the response, indicating a key role of P. gingivalis infection in establishing the disease model.