Attenuated Bordetella pertussis protects against highly pathogenic influenza A viruses by dampening the cytokine storm

Abstract

The threat of a pandemic spread of highly virulent influenza A viruses currently represents a top global public health problem. Mass vaccination remains the most effective way to combat influenza virus. However, current vaccination strategies face the challenge to meet the demands in a pandemic situation. In a mouse model of severe influenza virus-induced pneumonitis, we observed that prior nasal administration of an attenuated strain of Bordetella pertussis (BPZE1) provided effective and sustained protection against lethal challenge with two different influenza A virus subtypes. In contrast to most cross-protective effects reported so far, the protective window offered upon nasal treatment with BPZE1 lasted up to at least 12 weeks, suggesting a unique mechanism(s) involved in the protection. No significant differences in viral loads were observed between BPZE1-treated and control mice, indicating that the cross-protective mechanism(s) does not directly target the viral particles and/or infected cells. This was further confirmed by the absence of cross-reactive antibodies and T cells in serum transfer and in vitro restimulation experiments, respectively. Instead, compared to infected control mice, BPZE1-treated animals displayed markedly reduced lung inflammation and tissue damage, decreased neutrophil infiltration, and strong suppression of the production of major proinflammatory mediators in their bronchoalveolar fluids (BALFs). Our findings thus indicate that protection against influenza virus-induced severe pneumonitis can be achieved through attenuation of exaggerated cytokine-mediated inflammation. Furthermore, nasal treatment with live attenuated B. pertussis offers a potential alternative to conventional approaches in the fight against one of the most frightening current global public health threats.

FIG. 1.

Protection rates of BPZE1-treated mice against lethal challenge with H3N2 influenza A virus. Mice were i.n. administered once with 5 × 10⁶ CFU of BPZE1 and lethally challenged with 2 LD₅₀ of mouse-adapted H3N2 influenza A virus. The survival rates were compared to those of infected control mice (solid lozenge). Body weights were monitored daily, and mice were sacrificed when the body weight loss exceeded 20% of the original body weight. (A and B) Viral challenge was performed at either 3 weeks (solid square) or 6 weeks (solid triangle) after BPZE1 treatment. (C) Viral challenge was performed at 12 weeks post-BPZE1 treatment (solid triangle). (D) Mice were i.n. administered with live (solid triangle) or dead (solid square) BPZE1 bacteria and challenged 6 weeks later with H3N2 virus. Ten animals per group were monitored.

FIG. 2.

Effect of the bacterial dose on BPZE1 protective efficacy against lethal challenge with H3N2 virus. Survival rate (A) and average body weight changes (B) of mice i.n. administered once with 5 × 10⁵ (open square), 5 × 10⁶ (solid square), or 5 × 10⁷ (open circle) CFU of live BPZE1 bacteria were compared with those of nontreated infected mice (solid lozenge). Lethal viral challenge (2 LD₅₀) with mouse-adapted H3N2 virus was performed 6 weeks post-BPZE1 treatment. Mice were sacrificed when body weight loss exceeded 20% of the original body weight. Ten animals per group were monitored. (C) Lung colonization profiles of BPZE1 bacteria upon i.n. administration of 5 × 10⁵ (open square), 5 × 10⁶ (solid square), or 5 × 10⁷ (open circle) CFU. Four animals per group per time point were individually processed.

FIG. 3.

Booster effect. Mice were i.n. administered twice at a 4-week interval with 5 × 10⁶ CFU of live BPZE1 bacteria (solid square) and lethally (2 LD₅₀) challenged with H3N2 virus 2 weeks after the last BPZE1 treatment. Survival rate (A) and body weight loss (B) were compared to those of nontreated infected animals (solid lozenge). Mice were sacrificed when body weight loss exceeded 20% of the original body weight. Ten animals per group were assessed.

FIG. 4.

Protective efficacy of BPZE1 against H1N1 A/PR8/34 influenza virus. (A) Mice were i.n. administered three times with 5 × 10⁶ CFU of live BPZE1 bacteria and challenged 2 weeks after the last BPZE1 treatment with 4 LD₅₀ of H1N1 A/PR8/34 virus (solid triangle). The survival rate was compared to that of nontreated infected animals (solid lozenge). Ten animals per group were assessed. (B and C) Naïve mice were i.n. infected with 2 LD₅₀ (open lozenge) or 10 LD₅₀ (solid lozenge) of H1N1 A/PR8/34 virus. Survival rates (B) and body weight losses (C) were monitored over time. Ten animals per group were used.

FIG. 5.

Lung histology, cellular infiltrates, and CD3⁺ T-cell population in the lungs of BPZE1-treated mice. Mice were i.n. administered once with 5 × 10⁶ CFU of live BPZE1 bacteria and lethally challenged (2 LD₅₀) 6 weeks later with mouse-adapted H3N2 virus. (A) Lung histology. At 3 days post-viral challenge, infected control mice displayed pulmonary edema (black arrow) and necrotizing bronchitis with necrotic cell debris (open arrows). A total of 40 fields were analyzed per group (>5 fields/section, 2 sections/mouse, and 4 mice/group). (B) Cellular infiltrates. Nontreated (a and b) or BPZE1-treated (c and d) mice were left uninfected (a and c) or were challenged with H3N2 virus (b and d), and the macrophage, neutrophil, and lymphocyte counts in their BALF samples were determined 3 days postchallenge. Four animals per time point per group were individually assessed. (C) Analysis of the CD3⁺ T-cell population. Nontreated (a, c, and e) and BPZE1-treated (b, d, and f) mice were left uninfected (a and b) or were challenged with H3N2 virus (c to f). Three (c and d) or five (e and f) days after viral challenge, the CD3⁺ T-cell population in the mouse lungs was analyzed. Four animals per time point and per group were individually assessed. Results are expressed as the means ± SD of the percentages of CD3⁺ T cells in the total lung cell population. **, P ≤ 0.01; ***, P ≤ 0.001.

FIG. 6.

Cross-reactive and neutralizing antibodies. Mice were i.n. administered twice at a 4-week interval with 5 × 10⁶ CFU of live BPZE1 or were i.p. injected with heat-inactivated (HI) H3N2 virus. Serum and BALF samples were collected 2 weeks after the last administration. An ELISA was performed using purified HI-H3N2 viral particles as coating antigens, and the serum and BALF samples from naive or BPZE1- or HI-H3N2-immunized mice were used as primary antibody. IgG (A) and IgA (B) antibody responses were measured. Results are expressed as the optical density at 490 nm (OD_490nm). a, naïve serum (dilution, 1:40); b, anti-BPZE1 serum (dilution, 1:40); c, anti-H3N2 serum (dilution, 1:1,000); d, naïve BALFs (neat); e, anti-BPZE1 BALFs (neat); f, anti-H3N2 BALFs (neat). (C) In vitro neutralization assay. Twofold serial dilutions of the serum (starting at 1:2) or neat BALF samples from BPZE1-immunized mice were incubated with H3N2 influenza virus before MDCK cells were infected, and the cytopathic effect (CPE) was observed 72 h later. Anti-H3N2 immune serum was used as a positive control. Serum or BALF samples from naïve and HI-H3N2-immunized mice were pooled, while those from mice treated with BPZE1 were tested individually. Serum dilution is the highest dilution for which total protection was observed. nd, not determined; *, P ≤ 0.05; ***, P ≤ 0.001.

FIG. 7.

Cross-protective antibodies, cross-reactive T cells, and viral loads. (A) Passive transfer of immune serum. Naive (open triangle), anti-H3N2 (solid circle), or anti-BPZE1 (open circle) immune sera were i.p. injected into naive mice 1 day prior to lethal challenge (2 LD₅₀) with mouse-adapted H3N2 virus. Survival rates were compared to those of nontreated infected mice (solid triangle). Ten animals per group were assessed. (B) Proliferation assay. Pooled spleens from 6 BPZE1-treated (gray bar), HI-H3N2-immunized (black bar), or naive (open bar) animals were stimulated with either BPZE1 lysate or HI-H3N2 viral particles as indicated, and [³H]thymidine incorporation was determined. Results are expressed as the stimulation index (SI) corresponding to the ratio between the mean of [³H]thymidine uptake in the presence of test antigen and the mean of [³H]thymidine uptake in the absence of test antigen. An SI value of >2 was considered positive. Each sample was assayed in quadruplicate. (C) IFN-γ ELISPOT assay. Individual spleens from 6 BPZE1-treated (gray bar) or untreated (open bar) animals were stimulated with either BPZE1 lysate or HI-H3N2 viral particles as indicated, and IFN-γ ELISPOT assays were performed. Results are expressed as the means ± SD of the number of positive spots per 2 × 10⁵ cells. Results are representative of two independent experiments. *, P ≤ 0.05; ***, P ≤ 0.001. (D) Quantification of the viral load in the lungs of protected and nonprotected mice. Mice were i.n. administered with 5 × 10⁶ CFU of live BPZE1 bacteria and lethally challenged 6 weeks later with mouse-adapted H3N2 virus (3.5 × 10⁵ PFU). At 3 and 5 days post-viral challenge, the viral loads in the lungs of BPZE1-treated (a) and nontreated (b) mice were measured. Five animals per group per time point were assayed.

FIG. 8.

Pro- and anti-inflammatory cytokine and chemokine profiles. Adult BALB/c mice were i.n. administered once or twice at a 4-week interval with 5 × 10⁶ CFU of live BPZE1 bacteria and were lethally challenged with 2 LD₅₀ of mouse-adapted H3N2 virus at 6 and 4 weeks after the last BPZE1 administration, respectively. At 3 days post-viral challenge, 5 mice per group were sacrificed, and BALF specimens were collected. Ten inflammation-related cytokines and chemokines in the individual BALF samples were measured. (A) Cytokine levels in naïve (black bar) or nontreated infected (open bar) mice. (B) Cytokine levels in nontreated infected mice (black bar) or in mice treated once (open bar) or twice (gray bar) with BPZE1 prior to challenge with H3N2 virus. Results are expressed in pg/ml. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.