Respiratory FimA-Specific Secretory IgA Antibodies Upregulated by DC-Targeting Nasal Double DNA Adjuvant Are Essential for Elimination of Porphyromonas gingivalis

Abstract

Our previous studies showed that a combination of a DNA plasmid encoding Flt3 ligand (pFL) and CpG oligodeoxynucleotides 1826 (CpG ODN) (FL/CpG) as a nasal adjuvant provoked antigen-specific immune responses. In this study, we investigated the efficacy of a nasal vaccine consisting of FimA as the structural subunit of Porphyromonas gingivalis (P. gingivalis) fimbriae and FL/CpG for the induction of FimA-specific antibody (Ab) responses and their protective roles against nasal and lung infection by P. gingivalis, a keystone pathogen in the etiology of periodontal disease. C57BL/6 mice were nasally immunized with recombinant FimA (rFimA) plus FL/CpG three times at weekly intervals. As a control, mice were given nasal rFimA alone. Nasal washes (NWs) and bronchoalveolar lavage fluid (BALF) of mice given nasal rFimA plus FL/CpG resulted in increased levels of rFimA-specific secretory IgA (SIgA) and IgG Ab responses when compared with those in controls. Significantly increased numbers of CD8- or CD11b-expressing mature-type dendritic cells (DCs) were detected in the respiratory inductive and effector tissues of mice given rFimA plus FL/CpG. Additionally, significantly upregulated Th1/Th2-type cytokine responses by rFimA-stimulated CD4⁺ T cells were noted in the respiratory effector tissues. When mice were challenged with live P. gingivalis via the nasal route, mice immunized nasally with rFimA plus FL/CpG inhibited P. gingivalis colonization in the nasal cavities and lungs. In contrast, controls failed to show protection. Of interest, when IgA-deficient mice given nasal rFimA plus FL/CpG were challenged with nasal P. gingivalis, the inhibition of bacterial colonization in the respiratory tracts was not seen. Taken together, these results show that nasal FL/CpG effectively enhanced DCs and provided balanced Th1- and Th2-type cytokine response-mediated rFimA-specific IgA protective immunity in the respiratory tract against P. gingivalis. A nasal administration with rFimA and FL/CpG could be a candidate for potent mucosal vaccines for the elimination of inhaled P. gingivalis in periodontal patients.

Keywords: Porphyromonas gingivalis; dendritic cells (DCs); double DNA adjuvant; mucosal IgA; nasal vaccine; recombinant FimA (rFimA).

Figure 1

P. gingivalis rFimA-specific Ab responses in the external secretions. C57BL/6 (6–8 weeks) mice were nasally immunized three times at weekly intervals with rFimA (10 µg) plus pFL (50 µg) and CpG ODN (10 µg) (filled bars), or rFimA (10 µg) alone (open bars). Seven days after the last immunization, the levels of rFimA-specific IgA and IgG Abs in NWs (A) and BALF (B), were determined by rFimA-specific ELISA. The values shown are the mean ± SE (n = 20). *p < 0.05 when compared with mice given rFimA alone.

Figure 2

Antibody forming cells in the respiratory tracts, Mice were nasally immunized as described in Figure 1 legend. Seven days after the last immunization, mononuclear cells were isolated from NALT (A), NPs (B), CLNs (C), lungs (D), and MeLNs (E), and were then subjected to ELISPOT assays to enumerate the numbers of Ag-specific IgG and IgA AFCs. The values shown are the mean ± SE (n = 20). *p < 0.05 and **p < 0.01 when compared with mice given rFimA alone.

Figure 3

P. gingivalis rFimA-specific Ab responses in plasma and spleens. Mice were nasally immunized as described in Figure 1 legend. Seven days after the last immunization, the levels of rFimA-specific IgA, IgG, and IgG subclass Abs in plasma (A, C) were determined by rFimA-specific ELISA. In some experiments, mononuclear cells were isolated from spleen (B), and were then subjected to ELISPOT assays to enumerate the numbers of Ag-specific IgG and IgA AFCs. The values shown are the mean ± SE (n = 20). *p < 0.05 when compared with mice given rFimA alone.

Figure 4

Aggregation of live P. gingivalis cells by SIgA Abs. Enriched SIgA Abs from NWs (A) and BALF (B) of mice given nasal rFimA plus FL/CpG (filled circles) or rFimA alone (open circles) were serially diluted with PBS. The serial-diluted IgA-enriched solutions (500 µl) from NWs or BALF were added to 3 × 10⁸ P. gingivalis cells (500 µl) in cuvettes for 5 min. The optical density of the mixture was measured by a spectrophotometer (OD 600 nm). The values shown are the means ± SE (n = 5). *p < 0.05, when compared with mice given rFimA alone.

Figure 5

Clearance of P. gingivalis cells in the upper and lower respiratory tracts. IgA^+/+ and IgA^−/− mice given nasal FL/CpG ODN-based rFimA vaccine (filled circles or triangles). As a control, mice were nasally immunized with rFimA alone (open circles). One week after the last immunization, all groups of mice were nasally challenged with P. gingivalis cells (1 × 10⁸ cfu). Two days after the bacterial challenge, NWs (A) and BALF (B) samples were collected. The samples (100 µl) were cultured anaerobically on kanamycin-supplemented blood agar plates to enumerate the recovered CFU. The values shown are the means ± SE (n = 10). Each line represents the median log₁₀ CFU/mouse. *p < 0.05 when compared with mice given rFimA alone.