Attenuated Bordetella pertussis vaccine protects against respiratory syncytial virus disease via an IL-17-dependent mechanism

Abstract

Rationale: We attenuated virulent Bordetella pertussis by genetically eliminating or detoxifying three major toxins. This strain, named BPZE1, is being developed as a possible live nasal vaccine for the prevention of whooping cough. It is immunogenic and safe when given intranasally in adult volunteers.

Objectives: Before testing in human infants, we wished to examine the potential effect of BPZE1 on a common pediatric infection (respiratory syncytial virus [RSV]) in a preclinical model.

Methods: BPZE1 was administered before or after RSV administration in adult or neonatal mice. Pathogen replication, inflammation, immune cell recruitment, and cytokine responses were measured.

Measurements and main results: BPZE1 alone did not cause overt disease, but induced efflux of neutrophils into the airway lumen and production of IL-10 and IL-17 by mucosal CD4(+) T cells. Given intranasally before RSV infection, BPZE1 markedly attenuated RSV, preventing weight loss, reducing viral load, and attenuating lung cell recruitment. Given neonatally, BPZE1 also protected against RSV-induced weight loss even through to adulthood. Furthermore, it markedly increased IL-17 production by CD4(+) T cells and natural killer cells and recruited regulatory cells and neutrophils after virus challenge. Administration of anti-IL-17 antibodies ablated the protective effect of BPZE1 on RSV disease.

Conclusions: Rather than enhancing RSV disease, BPZE1 protected against viral infection, modified viral responses, and enhanced natural mucosal resistance. Prevention of RSV infection by BPZE1 seems in part to be caused by induction of IL-17. Clinical trial registered with www.clinicaltrials.gov (NCT 01188512).

Trial registration: ClinicalTrials.gov NCT01188512.

Figures

Figure 1.

Lung cellular response to BPZE1 infection. Mice were treated intranasally with phosphate-buffered saline (naive) or BPZE1 and analyzed 2, 7, and 14 days after infection. (A) Total number of cells recruited to the airways shows no significant change (P > 0.05). (B) BPZE1 infection induces a transient polymorphonuclear (PMN) cell efflux into the airway (open bars) P < 0.001 as compared with naive (two-way analysis of variance) with corresponding decline in the proportion of macrophages (black bars), whereas the proportion of lymphocytes (hatched bars) does not change significantly over time; this is mirrored in cell numbers. (C) Lymphocyte populations in the lung are similar 14 days after phosphate-buffered saline (naive, open bars) or BPZE1 (black bars) inoculation. (D) Intracellular cytokine staining of lung cells from BPZE1-infected mice stimulated with phorbol myristate acetate/ionomycin shows a transient boost of IL-10 and IL-17 production by CD4+ T cells on Day 7 (black bars Day 7; gray bars Day 14) (*P < 0.05, ***P < 0.001 as compared with naive; two-way analysis of variance; similar data in two experiments, six animals per group). BAL = bronchoalveolar lavage.

Figure 2.

Interactions between respiratory syncytial virus (RSV), BPZE1, and virulent Bordetella pertussis (BpSM) infections. (A) Mice were infected with RSV 14 days before BPZE1 (RSV/BPZE1, solid diamonds) and virulent B. pertussis (RSV/BpSM, black stars) or inoculated with phosphate-buffered saline (PBS) before BPZE1 (PBS/BPZE1, open diamonds) or BpSM (PBS/BpSM, black crosses), and the CFU were determined in the lungs by blood agar culture at the indicated time points. (B–D) Mice were infected with RSV or treated with PBS 14 days before BPZE1 or BpSM infection and analyzed on Day 7. (B) Total numbers of bronchoalveolar lavage (BAL) cell influx after bacterial infection. (C) Numbers of lymphocytes in BAL. (D) Numbers of polymorphonuclear (PMN) in BAL. *P < 0.05, **P < 0.01 compared with naive; °P < 0.05, °°P < 0.01 compared with BPZE1/RSV, Mann-Whitney test; similar data in two experiments, six animals per group.

Figure 3.

BPZE1 infection prior to respiratory syncytial virus (RSV) attenuates viral infection. Mice were treated with phosphate-buffered saline (PBS) (naive, open squares), infected with BPZE1 and then inoculated with PBS (BPZE1/PBS, open diamonds), treated with PBS and then infected with RSV (PBS/RSV, solid triangles), or infected with BPZE1 before RSV challenge (BPZE1/RSV, solid circles, dotted lines). RSV challenge 14 days after BPZE1 or PBS inoculation = Day 0 on the graphs. (A) Weight patterns after RSV or PBS challenge, showing that BPZE1 protects against RSV-induced weight loss. (B) Number of L gene copies 3, 4, and 5 days after RSV or PBS inoculation shows that BPZE1 reduces subsequent RSV replication (*P < 0.05 as compared with BPZE1/RSV, Mann-Whitney). (C) Prior BPZE1 inoculation reduces the total bronchoalveolar lavage (BAL) cell response to RSV infection and (D) lymphocyte numbers in BAL and (E) enhances the polymorphonuclear (PMN) response to RSV infection. *P < 0.05, ***P < 0.001 as compared with naive (two-way analysis of variance); °P < 0.05, °°°P < 0.001 as compared with BPZE1/RSV (two-way analysis of variance); the data shown are representative of three experiments, six animals per group.

Figure 4.

BPZE1 prior to respiratory syncytial virus (RSV) infection boosts IL-10 and IL-17 production by CD4+ cells. Mice were treated with phosphate-buffered saline (PBS) (naive, open bars), infected with BPZE1, and inoculated with PBS (BPZE1, black bars), treated with PBS and infected with RSV (RSV, gray bars), or infected with BPZE1 before RSV challenge (BPZE1/RSV, hatched bars). Bronchoalveolar lavage (BAL) and lung lymphocytes were analyzed by flow cytometry for intracellular cytokine production after phorbol myristate acetate/ionomycin stimulation on Days 4 and 8 after RSV infection. IL-10 single and IL-10/IFN-γ double-producing CD4+ T cells in (A) BAL on Day 4, (B) lung on Day 4, (C) lung on Day 8. (D) IFN-γ production by CD4+ and CD8+ T cells in lung on Day 8. (E) IL-17–producing CD4+ T cells in lung on Day 8. (F) Example of fluorescence-activated cell sorter plots for IFN-γ, IL-10, IL-17, and IL-4 intracellular staining in CD4+ lung cells of RSV- and BPZE1/RSV-treated mice. *P < 0.05, **P < 0.01, ***P < 0.001 as compared with naive; °P < 0.05, °°P < 0.01, as compared with BPZE1/RSV; the data shown are representative of three experiments, six animals per group.

Figure 5.

Priming of neonatal mice with BPZE1 induces IL-17 production and protects against adult respiratory syncytial virus (RSV) challenge. Neonatal mice (2–5 d old) were primed with phosphate-buffered saline (PBS/RSV, solid triangles, black bars) or BPZE1 (BPZE1/RSV, solid circles, white bars) and challenged with RSV in adulthood (age 8 wk). (A) Weight loss after RSV challenge. (B) CD4+ T cells producing IFN- γ, IL-17, or both. (C) Natural killer (NK) cells (DX5+CD3−) producing IFN-γ, IL-17, or both. Foxp3+ regulatory T cells (Tregs) in airways bronchoalveolar lavage (BAL) (D) and lung (E). *P < 0.05, **P < 0.01, ***P < 0.001 as compared with PBS/RSV; the data shown are representative of three experiments, four to six animals per group. (F–H) Depletion of IL-17 in vivo. Neonate mice were primed with BPZE1 or PBS and challenged 8 weeks later with RSV. On Days −1, 1, 3, 5, and 7, mice received an anti–IL-17 or control IgG2a antibody intraperitoneally (PBS/RSV/IgG2a = RSV+ control antibody, solid triangles, black bars; PBS/RSV/aIL-17 = RSV+ anti-IL-17 antibody, open triangles, gray bars; BPZE1/RSV/IgG2a = BPZE1+ RSV+ control antibody, solid circles, white bars; BPZE1/RSV/aIL-17 = BPZE1+ RSV+ anti–IL-17 antibody, open circles, hatched bars). (F) Weight loss after adult RSV challenge. IL-17 single or IL-17 IFN-γ double-producing (G) CD4+ T cells and (H) NK cells. (I) IFN-γ/IL-10 double-producing T cells in BAL. *P < 0.05, **P < 0.01, ***P < 0.001 compared with PBS/RSV/IgG2a; the data shown are representative of three experiments, five to six animals per group.