Autoimmune responses to heat-shock protein 60 (HSP60) contribute to the progression of atherosclerosis, whereas immunization with HSP60 may induce atheroprotective responses. We assessed the capacity of an atheroprotective vaccine that targeted a recombinant HSP60 from Porphyromonas gingivalis (rGroEL) to induce a protective mucosal immune response. Female apolipoprotein E-deficient spontaneously hyperlipidemic (Apoe(shl)) mice received sublingual delivery of rGroEL prior to P. gingivalis 381 injection. The animals were euthanized 16 weeks later. Sublingual immunization with rGroEL induced significant rGroEL-specific serum IgG responses. Antigen-specific cells isolated from spleen produced significantly high levels of IL-10 and IFN-γ after antigen re-stimulation in vitro. Flow cytometric analysis indicated that the frequencies of both IL-10(+) and IFN-γ(+) CD4(+) Foxp3(+) cells increased significantly in submandibular glands (SMG). Furthermore, sublingual immunization with rGroEL significantly reduced atherosclerosis lesion formation in the aortic sinus and decreased serum CRP, MCP-1, and ox-LDL levels. These findings suggest that sublingual immunization with rGroEL is associated with the increase of IFNγ(+) or IL-10(+) Foxp3(+) cells in SMG and a systemic humoral response, which could be an effective strategy for the prevention of naturally occurring or P. gingivalis-accelerated atherosclerosis.

The oral cavity is dominated by both mucosal and systemic immune systems, due to the presence of immunoglobulin (Ig)A antibodies (Abs) in saliva and IgG Abs in gingival crevicular fluid. Transmucosally administered vaccines allowing the development of both systemic and local immune responses are thus optimal for preventing oral infections. We assessed the capacity of a mucosal vaccine targeting recombinant HSP60 from Porphyromonas gingivalis (GroEL) to induce protective mucosal immune responses. As the basic experiment, optimal concentrations of GroEL as antigen (Ag) and synthetic oligonucleotides containing unmethylated CpG dinucleotide (CpG ODN) as a mucosal adjuvant for sublingual immunization were examined in mice. GroEL‐specific immune responses were observed in sublingual immunization with a combination of GroEL plus cholera toxin or GroEL plus CpG ODN. The optimal concentrations of GroEL and CpG ODN as antigen and adjuvant in sublingual immunization were 3 and 25 μg per mouse, respectively. These results suggest that the sublingual administration of GroEL plus CpG ODN is effective for inducing mucosal and systemic Ag‐specific Ab responses.

Periodontal disease is one of the most prevalent chronic inflammation of oral cavity, and has been reported to be associated with atherosclerosis. In this study, we assessed the potential of orally or nasally administered recombinant HSP60 from Porphyromonas gingivalis (rGroEL) for prevention of atherosclerosis accelerated by P. gingivalis. Apolipoprotein E deficient spontaneously hyperlipidemic (Apoe shl ) mice were orally or nasally immunized with GroEL and then challenged intravenously with P. gingivalis 381. Atherosclerotic lesions in the proximal aorta of each animal were analyzed histomorphometrically, and the serum concentrations of rGroEL-specific antibodies and cytokines were determined.Although antibody titer sufficient for taking oral or nasal immunity of GroEL under adjuvant-free condition was not obtained, the antibody titers significantly rose by booster effect of P. gingivalis infection. Furthermore, mice given rGroEL orally or nasally followed by P. gingivalis-challenge possessed significant reduction of atherosclerotic plaque accumulation in aortic sinus and lowered the serum MCP-1 and ox-LDL levels compared to nonimmunized, P. gingivalis-challenged mice. These results suggest that oral or nasal immunization with rGroEL could be an effective vaccine for prevention of atherosclerosis accelerated by P. gingivalis.

Periodontitis is associated with an increased risk of atherosclerosis, and accumulating evidence suggests a positive association between anti-heat shock protein 60 autoantibodies and atherosclerosis in humans. Heat shock proteins of the GroEL or HSP60 class are highly conserved proteins essential to all living organisms. In this study, we examined the effects of immunization with recombinant HSP60 from Porphyromonas gingivalis on antibody responses and the development of atherosclerosis.Atherosclerosis was examined in BALB/c mice fed a high-fat diet or a regular chow diet following subcutaneous immunization with GroEL or intravenous injection with P. gingivalis. The proximal aorta lesion area, serum levels of anti-GroEL antibodies, CRP and MCP-1 levels, expression of HSPs, and inflammatory mediator expression in aorta were measured.Early atherosclerotic lesion area was substantially lower in HFD-fed mice immunized with GroEL compared to P. gingivalis-challenged mice, although significant atherosclerotic lesions, serum immunoglobulin G responses to GroEL, and HSP60 were detected in GroEL-immunized mice. Immunohistochemical staining confirmed strong HSP60 expression in the vascular wall of HFD-fed mice compared to RD-fed mice. Although immunization of the HFD-fed mice with GroEL slightly enhanced serum MCP-1 secretion as well as CD40/40L and LOX-1 expression in the aorta, it did not affect serum CRP levels.These results suggest that immune response cross-reactivity to bacterial HSPs, including periodontal pathogens, with arterial endothelial cells expressing HSP60 are not associated with atherosclerosis severity caused by P. gingivalis challenge. This may explain why antibody responses to bacterial HSPs are an unlikely major risk factor for coronary artery disease.