Intranasal vaccination with pneumococcal surface protein A and interleukin-12 augments antibody-mediated opsonization and protective immunity against Streptococcus pneumoniae infection

Abstract

Streptococcus pneumoniae is a major pathogen in humans that enters the host primarily through the respiratory tract. Targeting mucosal surfaces directly may therefore be an optimal approach for vaccination to prevent bacterial colonization and invasive disease. We have previously demonstrated the effectiveness of interleukin-12 (IL-12) delivered intransally (i.n.) as an antiviral respiratory adjuvant. In this study, we examined the effects of i.n. IL-12 treatment on induction of protective humoral immunity against S. pneumoniae. Immunization i.n. with pneumococcal surface protein A (PspA) and IL-12 resulted in enhanced lung IL-10 mRNA expression and marked augmentation of respiratory and systemic immunoglobulin G1 (IgG1), IgG2a, and IgA antibody levels compared to those in animals receiving PspA alone. In addition, i.n. vaccination with PspA and IL-12 provided increased protection against nasopharyngeal carriage. Flow cytometric analysis revealed a threefold increase in antibody-mediated, complement-independent opsonic activity in the sera of PspA- and IL-12-treated animals, which was mainly contributed by IgG2a and, to a lesser extent, IgA. Passive transfer of these immune sera conferred complete protection from death upon systemic pneumococcal challenge. These findings demonstrate the effectiveness of combining PspA and IL-12 at mucosal sites to achieve optimal antibody-mediated opsonization and killing of S. pneumoniae.

FIG. 1

Effects of IL-12 administered i.n. on systemic antibody responses to PspA. BALB/c mice were immunized i.n. on day 0 with PspA, treated i.n. with either IL-12 or PBS vehicle on days 0, 1, 2 and 3, and boosted with PspA on days 14 and 28. On day 28, the mice also received IL-12 or PBS vehicle. Serum anti-PspA antibody levels on day 35 were determined by isotype-specific ELISA using PspA-coated microtiter plates. Each line represents binding of antibody from an individual mouse (four mice per group). Differences in binding between mice immunized with PspA and IL-12 and those immunized with PspA and PBS vehicle were significant (P < 0.05) for total antibody (Ab) and IgG1 and (P < 0.1 for IgA and IgG2a). O.D., optical density.

FIG. 2

Effects of IL-12 administered i.n. on respiratory antibody (Ab) responses to PspA. BALB/c mice were immunized as described in the legend to Fig. 1 and sacrificed on day 35, and BAL fluid was assayed by ELISA for anti-PspA antibody levels. Each line represents binding of antibody from an individual mouse (four mice per group). Differences in binding between mice immunized with PspA and IL-12 and those immunized with PspA and PBS vehicle were significant (P < 0.05 for total Ab and P < 0.1 for other antibody isotypes). O.D., optical density.

FIG. 3

(A) Effect of immune serum on phagocytosis of S. pneumoniae by J774A.1 cells. S. pneumoniae type 3 was opsonized with immune serum from BALB/c mice vaccinated with PspA plus PBS (open circles) or PspA plus IL-12 (solid triangles). Phagocytosis was performed with the J774A.1 macrophage cell line. The percentage of macrophages containing fluorescently labeled bacteria was used as a measure of phagocytic activity. Each condition was tested in duplicate, and the mean values are shown. Three independent experiments gave identical results. (B) UPC10 IgG2a and MOPC315 IgA inhibit the opsonic activity of immune serum from IL-12-treated mice. J774A.1 cells were incubated for 1 h at 4°C with 100 μg of MOPC315 IgA or UPC10 IgG2a and then further incubated for 1 h at 4°C with a 1:4 dilution of immune serum. The results are shown as the mean percentage of macrophages containing fluorescent bacteria ± the standard error of the mean.

FIG. 4

Protective immunity induced by PspA vaccination. Protection against S. pneumoniae was assessed by passive transfer of pooled serum to naïve BALB/c mice (eight recipient mice per group). Sera were collected on day 35 after i.n. immunization with PspA plus IL-12 (solid triangles), PspA plus PBS (open circles), or PBS vehicle only (open diamonds). Pooled sera were given i.p. simultaneously with a bacterial challenge dose of 4.7 × 10⁴ CFU of S. pneumoniae type 3. The mice were monitored daily for mortality. The differences in survival between mice immunized with PspA and IL-12 and those immunized with PspA alone were significant (P < 0.002).