Nasal immunization with a fusion protein consisting of the hemagglutinin A antigenic region and the maltose-binding protein elicits CD11c(+) CD8(+) dendritic cells for induced long-term protective immunity

Abstract

We assessed the efficacy of a fusion protein consisting of the 25-kDa antigenic region of Porphyromonas gingivalis hemagglutinin A and the Escherichia coli maltose-binding protein (25k-hagA-MBP) as a nasal vaccine for the prevention of oral infection with P. gingivalis. Nasal immunization with 25k-hagA-MBP induced high levels of 25k-hagA-specific serum IgG, serum IgA, and salivary IgA antibodies in a Toll-like receptor 4 (TLR4)-dependent manner. These antibody responses were maintained for at least 1 year after immunization. Analysis of cytokine responses showed that nasal administration of 25k-hagA-MBP induced antigen-specific CD4(+) T cells producing interleukin 4 (IL-4) and IL-5, but not gamma interferon (IFN-γ), in the spleen and cervical lymph nodes (CLNs). Furthermore, increased numbers of CD11c(+) CD8α(+), but not CD11c(+) CD11b(+) or CD11c(+) B220(+), dendritic cells with upregulated expression of CD80, CD86, CD40, and major histocompatibility complex class II (MHC II) molecules were noted in the spleen, CLNs, and nasopharynx-associated lymphoreticular tissues (NALT). Interestingly, when 25k-hagA-MBP or cholera toxin (CT) was given intranasally to enable examination of their presence in neuronal tissues, the amounts of 25k-hagA-MBP were significantly lower than those of CT. Importantly, mice given 25k-hagA-MBP nasally showed a significant reduction in alveolar bone loss caused by oral infection with P. gingivalis, even 1 year after the immunization. These results suggest that 25k-hagA-MBP administered nasally would be an effective and safe mucosal vaccine against P. gingivalis infection and may be an important tool for the prevention of chronic periodontitis in humans.

FIG. 1.

25k-hagA-specific IgG and IgA antibody responses in serum. Groups of mice were immunized nasally with either 20 μg of 25k-hagA, 20 μg of 25k-hagA-MBP, or 20 μg of 25k-hagA plus 1 μg of CT on days 0, 7, and 14. (A) Time course of 25k-hagA-specific IgG and IgA antibodies induced by nasal 25k-hagA-MBP. (B and C) One week after the final immunization, serum samples were collected in order to compare antibody titers between 25k-hagA-MBP and 25k-hagA plus CT, as well as IgG subclass responses. (B) Comparison of antibody responses in mice given 25k-hagA, 25k-hagA-MBP, or 25k-hagA plus CT. (C) IgG subclass responses in mice given 25k-hagA or 25k-hagA-MBP. Results are expressed as means ± SE for 4 to 6 mice per group in a total of three experiments. The P values for the comparison of antibody titers with 25k-hagA-MBP versus 25k-hagA were <0.05 at all times except day 7. ND, not detectable.

FIG. 2.

25k-hagA-specific IgA antibody response in saliva. Groups of mice were nasally immunized with either 25k-hagA, 25k-hagA-MBP, or 25k-hagA plus CT as described in the legend to Fig. 1. (A) Saliva samples were collected 7 days after the final immunization and were assessed for 25k-hagA-specific IgA (left) and total-IgA (right) antibody responses. (B) The time course of 25k-hagA-specific IgA antibodies induced by nasal 25k-hagA-MBP was also examined. Results are expressed as means ± SE from 4 to 6 mice per group in a total of three experiments. The P values for the comparison of IgA antibody titers with 25k-hagA-MBP versus 25k-hagA were <0.05 at all times except days 7 and 280. ND, not detectable.

FIG. 3.

25k-hagA-specific serum IgG and IgA and salivary IgA antibody responses in TLR4^−/− mice. Groups of TLR4^−/− or TLR4^+/+ mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig. 1. Serum and saliva samples were assessed for 25k-hagA-specific serum IgG and IgA and salivary IgA antibody responses. Results are expressed as means ± SE from 5 mice per group. *, P < 0.05 for comparison with TLR4^−/− mice. ND, not detectable.

FIG. 4.

25k-hagA-specific CD4⁺ T cell responses in the spleen and CLNs. Groups of mice were immunized nasally with 25k-hagA-MBP as described in the legend to Fig. 1. CD4⁺ T cells were isolated from the spleens or CLNs of nonimmunized or immunized mice and were cultured with 25k-hagA in the presence of splenic feeder cells. (A) To measure cell proliferation, 1.0 μCi of [³H]thymidine was added to the culture 18 h before harvesting, and incorporated radioactivity was measured by scintillation counting. (B) In order to analyze cytokine synthesis, culture supernatants were harvested, and the levels of secreted IL-4 and IL-5 were assessed by cytokine-specific ELISA. IFN-γ was not detectable. The results are representative of three separate experiments with 4 to 6 mice in each group/experiment. *, P < 0.05 for comparison with cells from nonimmunized mice.

FIG. 5.

Comparison of the proportions of CD11c⁺ DCs in various lymphoid tissues. Mice were immunized nasally with 25k-hagA, MBP, or 25k-hagA-MBP as described in the legend to Fig. 1. DC-enriched cell populations isolated from the spleen, CLNs, and NALT were stained with fluorescent-dye-conjugated monoclonal antibodies and were then subjected to flow cytometry. The results are representative of three separate experiments. The percentage of cells contained in the boxed region is given in each panel.

FIG. 6.

Antibodies induced by nasal immunization with 25k-hagA-MBP reduced P. gingivalis-induced alveolar bone loss. Groups of mice were immunized nasally with either 25k-hagA, MBP, 25k-hagA plus CT, 25k-hagA-MBP, or PBS, as described in the legend to Fig. 1. (A and B) Seven days (A) or 1 year (B) after immunization, immunized mice were inoculated orally with 10⁹ CFU of P. gingivalis in 2% carboxymethylcellulose, as described in Materials and Methods. Control mice were sham-infected mice inoculated with 2% carboxymethylcellulose only. The distance (in micrometers) from the CEJ to the ABC was measured at 14 predetermined sites in defleshed maxilla and was totaled for each mouse. *, P < 0.05 for comparison with nonimmunized mice. (C and D) Furthermore, 25k-hagA-specific or P. gingivalis-specific serum IgG and IgA (C) and salivary IgA (D) antibody responses before and after infection with P. gingivalis were examined. The results are expressed as means ± SE for 6 mice per group.

FIG. 7.

Accumulation of 25k-hagA-MBP or CT in neuronal tissues after nasal challenge with acridinium ester-labeled 25k-hagA-MBP (10 μg or 20 μg) or acridinium ester-labeled CT (1 μg or 5 μg). The results are expressed as the mean relative light units for a particular tissue ± SE and are from two separate experiments with 5 mice per group. *, P < 0.05 for comparison with mice given 1 μg and 5 μg of CT.