Controlled inflammatory responses in the lungs are associated with protection elicited by a pneumococcal surface protein A-based vaccine against a lethal respiratory challenge with Streptococcus pneumoniae in mice

Abstract

Streptococcus pneumoniae is a pathogen of great importance worldwide. We have previously described the efficacy of a nasal vaccine composed of the pneumococcal surface protein A and the whole-cell pertussis vaccine as an adjuvant against a pneumococcal invasive challenge in mice. Spread of bacteria to the bloodstream was probably prevented by the high levels of systemic antibodies induced by the vaccine, but bacteria were only cleared from the lungs 3 weeks later, indicating that local immune responses may contribute to survival. Here we show that a strict control of inflammatory responses in lungs of vaccinated mice occurs even in the presence of high numbers of pneumococci. This response was characterized by a sharp peak of neutrophils and lymphocytes with a simultaneous decrease in macrophages in the respiratory mucosa at 12 h postchallenge. Secretion of interleukin-6 (IL-6) and gamma interferon (IFN-γ) was reduced at 24 h postchallenge, and the induction of tumor necrosis factor alpha (TNF-α) secretion, observed in the first hours postchallenge, was completely abolished at 24 h. Before challenge and at 12 h postchallenge, vaccinated mice displayed higher numbers of CD4(+) T, CD8(+) T, and B lymphocytes in the lungs. However, protection still occurs in the absence of each of these cells during the challenge, indicating that other effectors may be related to the prevention of lung injuries in this model. High levels of mucosal anti-PspA antibodies were maintained in vaccinated mice during the challenge, suggesting an important role in protection.

Fig 1

Pneumococcal loads in the lungs and blood of mice after challenge with the ATCC 6303 strain. Lungs (A) or blood (B) from nonimmunized BALB/c mice (Non) or mice immunized with wP or PspA5-wP were collected at different time points after the intranasal challenge. CFU were determined in four mice per group after plating the samples in blood agar. Circles represent each animal, and lines represent the mean for each group. # and *, significantly lower numbers of cells compared with those at the same time point in nonimmunized mice or mice immunized with wP, respectively. In panel A, P = 0.03 for all time points except at 36 h, when no significant difference was observed when mice immunized with PspA5-wP were compared with mice inoculated with wP. In panel B, P = 0.02 for the time points shown in the graph.

Fig 2

Infiltration of cells in the respiratory tracts of mice after challenge with the ATCC 6303 strain. BALF samples were collected from nonimmunized BALB/c mice (Non) or mice immunized with wP or PspA5-wP at different time points after the intranasal challenge, and slides were prepared with 4 × 10⁴ cells. Numbers of total infiltrated cells (A), lymphocytes (B), neutrophils (C), or macrophages (D) are expressed as means for 4 mice per group with standard deviations. *, significantly higher numbers of cells compared with those at the same time point in nonimmunized mice; ○, significantly higher numbers of cells compared with those at the same time point in mice immunized with wP; #, significantly lower numbers of cells compared with those at the same time point in nonimmunized mice; ●, significantly lower numbers of cells compared with those at the same time point in mice immunized with wP. Statistical analyses were performed with the Mann-Whiney U test, and P ≤ 0.05 for all the comparisons marked.

Fig 3

Cytokine secretion in the respiratory tracts of mice after challenge with the ATCC 6303 strain. BALF samples were collected from nonimmunized BALB/c mice (Non) or mice immunized with wP or PspA5-wP at different time points after the intranasal challenge. Cells were removed by centrifugation, and cytokines were measured in the supernatants by sandwich ELISA against IL-6 (A), IFN-γ (B), TNF-α (C), and IL-17 (D). Results are represented as means for 4 animals per group with the standard deviations. *, significantly higher cytokine levels compared with those at the same time point in nonimmunized mice; #, significantly lower cytokine levels compared with those at the same time point in nonimmunized mice; ●, significantly lower cytokine levels compared with those at the same time point in mice immunized with wP. Statistical analyses were performed with the Mann-Whiney U test, and P ≤ 0.05 for all the comparisons marked.

Fig 4

Antigen-specific secretion of IL-17 by spleen and lung cells. Spleen (A) and lung (B) cells were obtained from immunized BALB/c mice at 12 h after challenge with the ATCC 6303 strain, and 5 × 10⁶ cells were incubated in the absence or presence of PspA5 for 72 h. Detection of IL-17 was performed by sandwich ELISA. Results are represented as the means from 5 animals per group with standard deviations. *, significantly higher levels of IL-17 compared with those in the indicated groups. Statistical analyses were performed with the Mann-Whiney U test; P < 0.05 in panel A and P ≤ 0.01 in panel B.

Fig 5

Characterization of the lymphocytes present in the respiratory tracts of mice before and after challenge with the ATCC 6303 strain. BALF samples were collected before or 12 h after the challenge, and 1 × 10⁶ cells were incubated with APC-conjugated anti-mouse CD4 (upper panels), PerCP-conjugated anti-mouse CD8 (middle panels), or PE-conjugated anti-B220 (lower panels). Samples were analyzed by flow cytometry, and results for 10,000 gated lymphocytes are represented as the means of total cells recovered (from groups of 3 to 5 mice) with standard deviations. Asterisks represent significantly different numbers of lymphocytes compared with those in the indicated groups. Statistical analyses were performed by one-way ANOVA with Tukey's posttest. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results are representative of two independent experiments.

Fig 6

Survival of mice subjected to depletion of specific lymphocytes after challenge with the ATCC 6303 pneumococcal strain. BALB/c mice immunized with PspA5-wP were subjected to the depletion of CD4⁺ T, CD8⁺ T, or B lymphocytes by the administration of specific monoclonal antibodies. One control group was immunized with the same formulation and received nonspecific rat IgG, whereas the other group was immunized only with wP. Mice were challenged, and survival was followed for 21 days. Circles represent each animal and, lines represent the mean of the group. **, significantly different survival compared with that of the group immunized with wP, using the Fisher exact test (P = 0.002). Results are representative of two independent experiments.

Fig 7

Evaluation of anti-PspA5 antibodies in the respiratory tracts of mice immunized with the different vaccine formulations. BALF samples were collected 21 days after the last immunization (left panels) or 12 h after the challenge (right panels). Anti-PspA5 IgG (upper panels) or IgA (lower panels) were detected through ELISA. Concentrations of antibodies (means for 6 animals) with standard deviations are shown. *, significant differences from the indicated groups. Statistical analyses were performed using the Mann-Whitney U test (*, P ≤ 0.05; **, P < 0.01). Results are representative of two independent experiments.

Fig 8

Binding of antibodies recovered from the respiratory mucosae of mice to pneumococcal surface and complement deposition. BALF samples were collected 21 days after the last immunization, and pools (from 3 mice) were incubated with the ATCC 6303 strain. Samples were incubated with FITC-conjugated anti-mouse IgA, IgG, IgG1, or IgG2a (upper and middle panels). For complement deposition, normal mouse sera were added to the samples, followed by the incubation with FITC-conjugated anti-mouse C3 (lower panel). Results were analyzed by flow cytometry with 10,000 events gated. BALF from nonimmunized animals (gray areas) or from mice immunized with wP (dotted black lines), PspA5 (solid heavy black lines), or PspA5-wP (solid black lines) were tested. The median fluorescence intensity is indicated for each sample. Data are representative of two experiments using different pools.

Fig 9

Absence of antibody binding to the pneumococcal surface of 6303^PspA− mutants. BALF samples from mice immunized with PspA5-wP were collected 21 days after the last immunization, and pools (from 3 mice) were incubated with different mutants derived from the ATCC 6303 strain or the wild-type strain. BALF from nonimmunized mice was used as control. Samples were incubated with FITC-conjugated anti-mouse IgA or IgG, and the results were analyzed by flow cytometry with 10,000 events gated. The median fluorescence intensity is indicated for each sample. Data are representative of two experiments using different pools.