JOURNAL OF VIROLOGY, July 2010, p. 7105–7113 Vol. 84, No. 140022-538X/10/$12.00 doi:10.1128/JVI.02542-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Attenuated Bordetella pertussis Protects against Highly PathogenicInfluenza A Viruses by Dampening the Cytokine Storm�

Rui Li,1,2 Annabelle Lim,1,2 Meng Chee Phoon,1 Teluguakula Narasaraju,1 Jowin K. W. Ng,1,2

Wee Peng Poh,3 Meng Kwoon Sim,4 Vincent T. Chow,1 Camille Locht,5,6,7,8

and Sylvie Alonso1,2*Department of Microbiology1 and Immunology Programme,2 Yong Loo Lin School of Medicine, National University of Singapore,

CeLS Building no. 03-05, 28 Medical Drive, Singapore 117597, Singapore; Department of Physiology, National University ofSingapore, Singapore 117597, Singapore3; Department of Pharmacology, National University of Singapore, Singapore 117597,

Singapore4; Inserm, U1019, F-59019 Lille, France5; CNRS UMR8204, F-59019 Lille, France6; Universite Lille Nord deFrance, F-59000 Lille, France7; and Institut Pasteur de Lille, F-59019 Lille, France8

Received 4 December 2009/Accepted 19 April 2010

The threat of a pandemic spread of highly virulent influenza A viruses currently represents a top globalpublic 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 mousemodel of severe influenza virus-induced pneumonitis, we observed that prior nasal administration of anattenuated strain of Bordetella pertussis (BPZE1) provided effective and sustained protection against lethalchallenge with two different influenza A virus subtypes. In contrast to most cross-protective effects reported sofar, the protective window offered upon nasal treatment with BPZE1 lasted up to at least 12 weeks, suggestinga unique mechanism(s) involved in the protection. No significant differences in viral loads were observedbetween BPZE1-treated and control mice, indicating that the cross-protective mechanism(s) does not directlytarget the viral particles and/or infected cells. This was further confirmed by the absence of cross-reactiveantibodies and T cells in serum transfer and in vitro restimulation experiments, respectively. Instead, com-pared to infected control mice, BPZE1-treated animals displayed markedly reduced lung inflammation andtissue damage, decreased neutrophil infiltration, and strong suppression of the production of major proin-flammatory mediators in their bronchoalveolar fluids (BALFs). Our findings thus indicate that protectionagainst influenza virus-induced severe pneumonitis can be achieved through attenuation of exaggeratedcytokine-mediated inflammation. Furthermore, nasal treatment with live attenuated B. pertussis offers apotential alternative to conventional approaches in the fight against one of the most frightening current globalpublic health threats.

Influenza virus pandemics are unpredictable but recurringevents that can have severe consequences on societies world-wide. In the 20th century, three novel influenza virus strainsemerged, causing the 1918, 1957, and 1968 pandemics, themost devastating being the 1918 Spanish flu that led to anestimated 50 million deaths (47). The recent spread of highlypathogenic avian influenza (HPAI) H5N1 virus across parts ofAsia, Europe, and the Middle East, with an overall fatality rateof over 60% for humans, as well as the rapid pandemic spreadof a novel influenza A virus of the H1N1 subtype, has causedworldwide concern about a potential remake of the 1918 di-saster (8).

Severe complications arising from pandemic influenza orHPAI H5N1 viruses are associated with rapid, massive inflam-matory cell infiltration, resulting in acute respiratory distress,and reactive hemophagocytosis with multiple organ involve-ment. Both the 1918 Spanish influenza virus and HPAI H5N1induce a cytokine storm characterized by an exaggerated pro-

duction of inflammatory cytokines and chemokines in the se-rum and lungs caused by uncontrolled activation of the host’sinnate immune system. This triggers massive pulmonaryedema, primary and/or secondary pneumonia, and alveolarhemorrhage with acute bronchopneumonia (4, 12, 24, 27, 37,40, 43, 44).

The relationship between mortality, viral load, and immu-nopathology during influenza virus infection remains elusiveand somewhat controversial. Some studies suggest that severelung immunopathology is a direct consequence of a high viralload that the host is unable to resolve (12, 13), whereas othershave reported that influenza virus-induced mortality is not adirect function of viral burden but a consequence of immune-mediated pathology (9, 11). Moreover, the picture is furthercomplicated by the fact that different highly virulent influenzaA viruses may induce distinct pathological signatures and leadto different courses of acute respiratory distress syndrome,refuting the hypothesis of a single, universal cytokine stormunderlying all fatal influenza virus diseases (16).

Currently, vaccination remains the cornerstone of influenzavirus prevention. However, due to constant antigenic drift andshift of the two major viral surface proteins hemagglutinin(HA) and neuraminidase (NA) (7), influenza virus vaccinesmust be reformulated each year in order to match the circu-lating subtypes (41). The potential emergence of an influenza

* Corresponding author. Mailing address: Department of Microbi-ology, Yong Loo Lin School of Medicine, National University of Sin-gapore, CeLS Building no. 03-05, 28 Medical Drive, Singapore 117597,Singapore. Phone: (65)6516-3541. Fax: (65)6778-2684. E-mail: micas@nus.edu.sg.

� Published ahead of print on 5 May 2010.

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virus pandemic at any time, combined with limited vaccinesupplies, has rendered global vaccination strategies difficult.Therefore, a universal influenza virus vaccine that can provideprotection against different variants or strains and thus notrequire frequent updates is highly desirable.

Here, we report that nasal administration of a recently de-veloped live attenuated Bordetella pertussis vaccine strain,named BPZE1 (35), provides effective and sustained protec-tion against lethal challenge with mouse-adapted H3N2 orH1N1 (A/PR/8/34) influenza A viruses. We demonstrate thatthe protective mechanism(s) does not target the viral particlesor the infected host cells but controls the influenza virus-mediated inflammation by dampening the cytokine storm. AsBPZE1 has recently entered phase I safety trials with humans(http://www.child-innovac.org), our observations support thepotential application of this vaccine strain as a universal pro-phylactic treatment against highly pathogenic influenza A vi-ruses.

MATERIALS AND METHODS

Bacterial and viral strains and growth conditions. B. pertussis BPZE1 is astreptomycin-resistant Tohama I derivative deleted of the dermonecrotic(DNT)-encoding gene, producing inactivated pertussis toxin (PT) and back-ground levels of tracheal cytotoxin (TCT) (35). BPZE1 bacteria were grown at37°C for 72 h on Bordet-Gengou (BG) agar (Difco, Detroit, MI) supplementedwith 1% glycerol, 10% defibrinated sheep blood, and 100 �g/ml streptomycin(Sigma Chemical, St. Louis, MO). Liquid cultures were performed as describedpreviously (33) with Stainer-Scholte (SS) medium containing 1 g/liter heptakis(2,6-di-o-methyl) �-cyclodextrin (Sigma). When appropriate, heat inactivation ofthe bacteria was performed at 95°C for 1 h.

Mouse-adapted A/Aichi/2/68 (H3N2) virus (passage 10) was obtained as de-scribed previously (36). H1N1 A/PR/8/34 virus was purchased from the ATCC(no. VR-95) and amplified in egg following ATCC’s recommendations. Whereindicated, heat inactivation of H3N2 virus was performed at 56°C for 30 min.

Determination of the viral titers. Mouse lungs were harvested and homoge-nized using mechanical disruption (Omni homogenizer) and tested for the pres-ence of viable virus by a 50% tissue culture infectious dose (TCID50) assay usinga modified method reported by the WHO (46). Briefly, 90% confluent Madin-Darby canine kidney (MDCK) cells in 96-well plates were inoculated with 100 �lof 10-fold serially diluted lung homogenates. Plates were incubated at 35°C in ahumidified incubator (5% CO2) for 3 days. The TCID50 was determined by areduction in cytopathic effect (CPE) of 50%, and the log TCID50/lung wasderived. Five mice per group per time point were individually assessed.

In vitro neutralization assay. MDCK cells (5 � 104) were seeded in 96-wellflat-bottom plates and incubated at 37°C in a 5% CO2 atmosphere for 24 h.Twenty-five microliters of 2-fold serial dilutions of heat-treated (56°C, 30 min)sera or neat bronchoalveolar lavage fluids (BALFs) was mixed with equal vol-umes of 102 TCID50s of virus and incubated for 1 h at 37°C in 5% CO2. Theantibody-virus mixtures were then transferred to a MDCK cell monolayer thathad been washed twice with serum-free Dulbecco modified Eagle medium(DMEM) (Gibco) and incubated for 1 h at 37°C in 5% CO2. Three days later, thecells were observed for CPE, and the neutralizing antibody titer was read as thehighest dilution of serum that inhibited virus growth and prevented CPE. Eachdilution was assayed in six to eight replicates, and each neutralization assay wasrepeated twice independently.

Intranasal infections. All the animal experiments were carried out under theguidelines of the Institutional Animal Care and Use Committee, National Uni-versity of Singapore. Six- to eight-week-old female BALB/c mice were kept underspecific-pathogen-free conditions in individual ventilated cages. For BPZE1treatment, sedated mice were intranasally (i.n.) administered once or twice (asindicated) with 5 � 105, 5 � 106, or 5 � 107 CFU (as indicated) of live or dead(heat-inactivated) BPZE1 bacteria in 20 �l sterile phosphate-buffered saline(PBS) supplemented with 0.05% Tween 80 (PBST) (Sigma) as previously de-scribed (20). For influenza virus infection, sedated mice were i.n. administeredwith 2 50% lethal doses (LD50) of mouse-adapted H3N2, and 2, 4, or 10 LD50 (asindicated) of H1N1 A/PR/8/34 in sterile PBS supplemented with penicillin andstreptomycin. Ten mice per group were used to determine the survival rates

based on body weight loss, and the mice were euthanized when body weight lossexceeded 20% of the original body weight.

Lung colonization profiles. Four adult female BALB/c mice were adminis-tered i.n. with 5 � 105, 5 � 106, or 5 � 107 CFU of live BPZE1. At the indicatedtime points, four animals per group and per time point were euthanized; theirlungs were individually harvested and homogenized as described previously (20).Appropriate dilutions were plated for colony counting.

Passive transfer experiment. High-titer anti-B. pertussis immune sera weregenerated in 10 adult BALB/c mice nasally infected twice at a 4-week intervalwith live BPZE1 bacteria. Another group of 10 adult naïve BALB/c mice wereinjected intraperitoneally (i.p.) with 105.5 TCID50s of heat-inactivated humanA/Aichi/2/68 (H3N2) virus (HI-H3N2) in complete Freund’s adjuvant andboosted with the same amount of HI-H3N2 virus in incomplete Freund’s adju-vant 2 weeks later. Immune serum samples from each mouse group were col-lected 2 weeks after the boost and pooled, and the anti-B. pertussis and anti-influenza virus antibody titers were measured by enzyme-linked immunosorbentassay (ELISA). In addition, HI-H3N2 serum was tested for the presence ofneutralizing antibodies by neutralization assay. The immune serum samples werefilter sterilized, heat treated at 56°C for 30 min, and stored at �80°C until furtheruse. Serum samples from naïve control mice were also collected.

Six- to 8-week-old recipient BALB/c mice were i.p. injected with 200 �l ofnaïve, anti-BPZE1 or anti-H3N2 immune serum 1 day prior to lethal challengewith mouse-adapted H3N2 virus. Body weight losses were monitored to deter-mine the survival rates. Ten mice per group were assayed.

Histopathologic examination. The mouse lungs were harvested and fixed in10% formalin in PBS, embedded in paraffin, sectioned, and stained with hema-toxylin and eosin (H&E). Observations were made using an inverted light mi-croscope at 10� and 40� objectives.

Cellular infiltrates in bronchoalveolar lavage fluids. Individual BALF sampleswere recovered by injecting 1 ml of sterile PBS into the lungs of sacrificedanimals and performing one lavage step. BALFs were then centrifuged, and thesupernatant was removed and stored at �80°C for cytokine detection. Cells wereresuspended, spotted onto a glass slide using a Cytospin device (Thermo Shan-don), and stained using a modified Wright staining procedure (3). Results wereexpressed as the percentage of each cell type in the total cell population. A totalof 500 cells were considered per slide. Four mice per group were individuallyassessed.

FACS analysis. Single-cell suspensions were prepared by digesting the mouselungs at 37°C for 15 min in 2 ml digestion buffer containing 0.5 mg/ml Liberase(Roche) in RPMI with 1% fetal calf serum (FCS) and 2 U/ml DNase I (Qiagen)and centrifuging them on Ficoll-Paque Plus (GE) for 20 min at 600 � g at roomtemperature. Cells were collected and washed twice with sterile fluorescence-activated cell sorter (FACS) buffer (2% FCS, 5 mM EDTA in PBS). Cells (106)were stained with fluorescein isothiocyanate (FITC)-labeled anti-mouse CD3antibody (eBioscience) and analyzed on a CyAn ADP cytometer (Dako). Fivemice per group per time point were individually assessed.

Cytokine and chemokine analysis. Cytokine and chemokine levels in theBALF supernatants were measured using a multiplex cytokine detection kit(Bioplex; Bio-Rad) according to the manufacturer’s instructions. The sampleswere analyzed using a Bio-Plex instrument (Bio-Rad). Granulocyte-macrophagecolony-stimulating factor (GM-CSF), KC (interleukin-8 [IL-8]), IL-1�, IL-6,IL-12p70, gamma interferon (IFN-�), tumor necrosis factor alpha (TNF-�),monocyte chemoattractant protein 1 (MCP-1), and IL-10 were assayed. In ad-dition, transforming growth factor � (TGF-�) levels were measured using ahuman/mouse TGF-�1 ELISA kit (eBioscience) according to the manufacturer’sinstructions.

Antibody detection. The presence of antibodies in the serum and BALF sam-ples were measured by ELISA. Ninety-six-well microtiter plates (Costar; Corn-ing) were coated overnight at 4°C with 100 �l of 0.1 M carbonate buffer (pH 9.6)containing 5 �g/ml of heat-inactivated H3N2 viral particles. After blocking with2% bovine serum albumin (BSA) in PBS containing 0.1% Tween 20, 100 �l ofserum diluted at 1:40 (anti-BPZE serum) or 1:1,000 (anti-H3N2 serum) or 50 �lof undiluted BALFs was added to the wells. The plates were incubated at 37°Cfor 1 h, rinsed in PBS-0.1% Tween 20, and incubated at 37°C for 1 h with 50 �lof horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H � L) oranti-mouse IgA secondary antibodies (Sigma) diluted 1:3,000 or 1:2,000, respec-tively. The reaction was then developed using o-phenylenediamine dihydrochlo-ride substrate (Sigma) at room temperature for 30 min in the dark and stoppedby the addition of 1 M sulfuric acid. The absorbance at 490 nm was measured byan ELISA plate reader (Tecan Sunrise).

T-cell proliferation assay. Lymphocyte proliferation was measured by incor-poration of tritiated [3H]thymidine as described previously (25). Briefly, spleensfrom naïve, HI-H3N2- and BPZE1-immunized mice (6 mice per group) were

7106 LI ET AL. J. VIROL.

pooled, and single-cell suspensions were prepared and centrifuged on Ficoll-Paque Plus (GE) for 20 min at 600 � g at room temperature. The splenocyteswere seeded in 96-well round-bottom plates (Nunc) at a density of 2 � 105

cells/well in 100 �l medium (RPMI 640 supplemented with 10% FCS, 5 � 10�5

M �-mercaptoethanol, 2 mM L-glutamine, 10 mM HEPES, 200 U/ml penicillin,200 �g/ml streptomycin). Medium (100 �l) containing 20 �g/ml of BPZE1whole-cell lysate or 105 TCID50s of HI-H3N2 (test antigen) was added to thesplenocytes. Noninfected egg amniotic fluid (100 �l) and medium containing 5�g/ml concanavalin A (conA) (100 �l) were used as mock and vitality controls,respectively. After 3 days of incubation at 37°C in a 5% CO2 atmosphere, thecells were pulsed with 0.4 �Ci [3H]thymidine in 20 �l RPMI complete medium.After incubation for 18 h, cells were harvested and washed and the incorporatedradioactivity was measured in a TopCount NXT microplate scintillation andluminescence counter (PerkinElmer). Results are expressed as the stimulationindex (SI) corresponding to the ratio between the mean of [3H]thymidine uptakein the presence of test antigen and the mean of [3H]thymidine uptake in theabsence of test antigen. An SI value of �2 was considered positive. Each samplewas assayed in quadruplicate.

IFN-� ELISPOT assay. The frequency of antigen-specific IFN-�-producingsplenocytes was determined by an enzyme-linked immunospot (ELISPOT) assayusing a mouse ELISPOT set (BD PharMingen) according to the manufacturer’sinstructions. Briefly, single-cell suspensions of individual spleens from naïve andBPZE1-treated mice were plated in 96-well microplates (Millipore, Bedford,MA) precoated with 100 �l of 5 �g/ml anti-IFN-� antibody in sterile PBSovernight at 4°C, washed three times, and blocked for 2 h at room temperaturewith RPMI 1640 containing 10% FCS. Cells were then incubated with 20 �g/mlof BPZE1 whole-cell lysate, with 105 TCID50s HI-H3N2, or with 5 �g/ml conAfor 12 to 20 h at 37°C in a 5% CO2 atmosphere. The plates were then washed,and biotin-conjugated anti-mouse IFN-� antibody was added for 2 h at roomtemperature. After the wells were washed, streptavidin-HRP conjugate wasadded and incubated at room temperature for 1 h. The wells were washed againand developed with a 3-amino-9-ethyl-carbazole (AEC) substrate solution untilspots were visible. After drying, spot-forming cell numbers were counted byBioreader 4000 (Biosystem). Six animals per group were individually assayed.

Statistical analysis. Unless otherwise stated, in the figures, bars representmeans standard deviations (SD) and averages were compared using a bidi-rectional unpaired Student t test with a 5% significance level (�, P � 0.05; ��, P �

0.01; and ���, P � 0.001).

RESULTS

A single nasal treatment with B. pertussis BPZE1 protectsagainst lethal challenge with mouse-adapted H3N2 virus.Adult BALB/c mice were nasally inoculated once with 5 � 106

CFU of live B. pertussis BPZE1 and challenged 3 or 6 weekslater with 2 LD50 of mouse-adapted H3N2 virus, obtained after10 successive lung-to-lung passages of the A/Aichi/2/68(H3N2) virus (36). Survival rates (Fig. 1A) and body weightchanges (Fig. 1B) indicated that the mice challenged 6 weeksafter nasal BPZE1 treatment were significantly protected(60% protection rate), whereas mice challenged at 3 weekspost-BPZE1 treatment were not protected. A protection rateof 60% was still observed when the viral challenge was per-formed 12 weeks post-BPZE1 treatment (Fig. 1C). In contrast,nasal administration of the same amount of heat-killed bacte-ria did not provide any significant protection against H3N2lethal challenge (Fig. 1D).

To further explore the protective efficacy of live BPZE1bacteria, 10-fold serially diluted BPZE1 suspensions were ad-ministered nasally to the mice prior to lethal challenge withH3N2 virus performed 6 weeks later. The mouse group whichreceived 5 � 105 CFU of BPZE1 was not significantly pro-tected, whereas mice administered with 5 � 106 CFU and 5 �107 CFU were significantly protected, with 50% and 62.5%survival rates, respectively (Fig. 2A). Interestingly, the animalsimmunized with the highest bacterial dose (5 � 107 CFU)displayed significantly smaller body weight losses than the an-

imal group administered with 5 � 106 CFU (Fig. 2B). Concur-rently, the lung colonization profiles of BPZE1 were deter-mined for each bacterial dose and showed that the bacterialload present in the lungs during the colonization period wasdirectly dependent on the initial bacterial dose administered(Fig. 2C).

Taken together, these results indicate that the protectiveefficacy of BPZE1 against lethal challenge with H3N2 virusdepends on the bacterial dose administered and that the abilityof the bacteria to effectively colonize the mouse respiratorytract is necessary to prime the protective mechanisms.

Booster effect. The effect of a second nasal BPZE1 treat-ment was addressed. Live BPZE1 bacteria were nasally admin-istered twice at a 4-week interval prior to lethal challenge withmouse-adapted H3N2 virus performed 2 weeks after the lastBPZE1 treatment. A survival rate of 100% was obtained (Fig.3A), with minimal body weight changes, in contrast to theinfected control mice (Fig. 3B). These data demonstrate that asecond nasal administration of BPZE1 not only enhances theprotective efficacy but also shortens the time necessary to trig-ger the protective mechanism(s).

BPZE1 protects against H1N1 virus. The protective poten-tial of BPZE1 against a different influenza A virus subtype wasexplored. Whereas a single nasal administration of 5 � 106

CFU of live BPZE1 bacteria did not confer any significantprotection against lethal challenge with H1N1 A/PR/8/34 virus(data not shown), three consecutive administrations of liveBPZE1 provided about 50% protection (Fig. 4A). These ob-servations indicate that BPZE1 is able to protect against dif-

FIG. 1. Protection rates of BPZE1-treated mice against lethal chal-lenge with H3N2 influenza A virus. Mice were i.n. administered oncewith 5 � 106 CFU of BPZE1 and lethally challenged with 2 LD50 ofmouse-adapted H3N2 influenza A virus. The survival rates were com-pared to those of infected control mice (solid lozenge). Body weightswere monitored daily, and mice were sacrificed when the body weightloss exceeded 20% of the original body weight. (A and B) Viral chal-lenge was performed at either 3 weeks (solid square) or 6 weeks (solidtriangle) after BPZE1 treatment. (C) Viral challenge was performed at12 weeks post-BPZE1 treatment (solid triangle). (D) Mice were i.n.administered with live (solid triangle) or dead (solid square) BPZE1bacteria and challenged 6 weeks later with H3N2 virus. Ten animalsper group were monitored.

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ferent influenza A virus subtypes, although with variable effi-cacy. In this experiment, an H1N1 dose of 4 LD50 wasadministered to the mice, whereas H3N2 challenge was per-formed with a dose of 2 LD50. One could thus argue that thedifferential protective efficacy of BPZE1 against the two virussubtypes may be due to the different lethal doses used for thechallenge. However, we found that the survival rates, times ofdeath, and body weight losses of naïve mice infected witheither 2 LD50 or 10 LD50 of H1N1 A/PR8/34 virus were com-parable (Fig. 4B&C), indicating that a higher initial viral dosedoes not worsen the disease severity, based on survival rate,time of death, and body weight loss. Therefore, the differentialability of nasal BPZE1 treatment to protect against H1N1 orH3N2 subtypes is likely not attributable to the different lethaldoses used for the viral challenge but rather suggests differ-ences in the virulence mechanisms developed by both virussubtypes, as recently reported (16).

BPZE1 treatment protects mice from influenza virus-in-duced immunopathology and lymphocyte depletion. Lung im-munopathology was examined by histology of lung sectionsfrom infected BPZE1-treated and nontreated animals. As ex-pected and as described previously (36), the infected non-treated mice displayed signs of severe bronchopneumonia andinterstitial pneumonitis, characterized by necrosis of the bron-chiolar epithelium and the presence of necrotic debris in the

bronchioles and alveoli, as well as significant pulmonary em-physema and moderate edema (Fig. 5A). In contrast, only mildinflammation, minimal airway and alveolar damage, and mildperivascular or peribronchiolar damage, together with minimaledema, were observed with the lungs of the protected BPZE1-treated mice.

The cell populations present in the BALF samples recoveredfrom protected and nonprotected animals were also examined.Whereas the total numbers of cells present in the BALF spec-imens from both animal groups were comparable (11.8 � 105

versus 16.1 � 105), a significantly higher number of macro-phages but a lower number of neutrophils were found in theinfected BPZE1-treated mice than in the nontreated mice 3days after viral challenge (Fig. 5B).

Lymphocyte depletion has been reported for mice infectedwith highly pathogenic H1N1 (1918) and H5N1 influenza vi-ruses (24, 29, 44), as well as for mice infected with the mouse-adapted H3N2 virus strain used in this study (36). The lym-phocyte populations present in the lungs of protected andnonprotected mice were thus compared. At 3 days post-viralchallenge, the percentages of CD3� cells in the infected con-trol and BPZE1-treated mice were comparable to those foundin the animals before challenge (Fig. 5C). However, significantCD3� T-cell depletion was observed with the infected controlanimals at 5 days post-viral challenge. In contrast, the T-cellpopulation remained constant before and after challenge inthe protected BPZE1-treated animals, indicating that nasaltreatment with live BPZE1 prevented influenza virus-inducedlymphocyte depletion.

B. pertussis-specific adaptive immunity is not involved inprotection against influenza A virus. The presence of cross-reactive and virus-neutralizing antibodies and T cells in theBPZE1-treated animals was examined. A BLAST search failedto identify any matching epitopes between B. pertussis andH3N2 or H1N1 influenza A viruses (data not shown).

Moreover, the serum and BALF samples from BPZE1-treated mice did not react with whole H3N2 viral antigens in anELISA (Fig. 6A and B) and did not neutralize virus infectivityin vitro (Fig. 6C). Consistently, high-titer anti-BPZE1 immunesera did not confer any significant protection against H3N2lethal challenge in an in vivo passive transfer experiment,whereas immune sera raised against heat-inactivated H3N2

FIG. 2. Effect of the bacterial dose on BPZE1 protective efficacy against lethal challenge with H3N2 virus. Survival rate (A) and average bodyweight 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 BPZE1bacteria were compared with those of nontreated infected mice (solid lozenge). Lethal viral challenge (2 LD50) with mouse-adapted H3N2 viruswas performed 6 weeks post-BPZE1 treatment. Mice were sacrificed when body weight loss exceeded 20% of the original body weight. Ten animalsper group were monitored. (C) Lung colonization profiles of BPZE1 bacteria upon i.n. administration of 5 � 105 (open square), 5 � 106 (solidsquare), 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 a4-week interval with 5 � 106 CFU of live BPZE1 bacteria (solidsquare) and lethally (2 LD50) challenged with H3N2 virus 2 weeks afterthe last BPZE1 treatment. Survival rate (A) and body weight loss(B) were compared to those of nontreated infected animals (solidlozenge). Mice were sacrificed when body weight loss exceeded 20% ofthe original body weight. Ten animals per group were assessed.

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virus provided 100% protection (Fig. 7A). Finally, prolifera-tion and IFN-� ELISPOT assays revealed that splenocytesfrom BPZE1-treated mice did not proliferate and did not pro-duce IFN-� upon stimulation with H3N2 viral particles,whereas strong stimulation and IFN-� production were seenupon stimulation with BPZE1 extracts (Fig. 7B and C). Alltogether, these data indicated that B. pertussis-specific adaptiveimmunity is unlikely to mediate the cross-protection againstinfluenza A viruses.

The viral load is not significantly reduced in BPZE1-treatedmice. To further characterize BPZE1-induced protectionagainst influenza A viruses, the viral loads in the lungs of miceeither untreated or treated with BPZE1 were quantified andlethally challenged with mouse-adapted H3N2 virus. No sig-nificant differences in the viral loads were observed betweenthe two groups at 3 and 5 days postchallenge (Fig. 7D). Thisresult indicates that the BPZE1-induced protective mechanismdoes not directly target the virus particles and/or infected host

cells and demonstrates that effective protection against influ-enza A virus-induced pathology and mortality can be achievedwithout affecting the virus titer in the lungs.

The production of major proinflammatory cytokines andchemokines is dampened in the protected BPZE1-treatedmice. The levels of 8 major proinflammatory cytokines andchemokines as well as TGF-� and IL-10 in the BALF samplesfrom BPZE1-treated and untreated mice were measured be-fore and 3 days after lethal challenge with H3N2. A significantupregulation of 6 out of the 8 proinflammatory cytokines/chemokines was observed with the untreated infected micecompared to that in the levels measured for noninfected ani-mals (Fig. 8A). IFN-� and IL-12p70 production was un-changed in the infected mice compared to that in the controlgroup. Interestingly, the level of TGF-� was markedly reducedin the infected mice, whereas IL-10 levels were higher thanthose in the controls (Fig. 8A). In mice treated once withBPZE1, the levels of 3 out of 8 proinflammatory cytokines and

FIG. 4. Protective efficacy of BPZE1 against H1N1 A/PR8/34 influenza virus. (A) Mice were i.n. administered three times with 5 � 106 CFUof live BPZE1 bacteria and challenged 2 weeks after the last BPZE1 treatment with 4 LD50 of H1N1 A/PR8/34 virus (solid triangle). The survivalrate 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) weremonitored 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 oncewith 5 � 106 CFU of live BPZE1 bacteria and lethally challenged (2 LD50) 6 weeks later with mouse-adapted H3N2 virus. (A) Lung histology. At3 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 themacrophage, neutrophil, and lymphocyte counts in their BALF samples were determined 3 days postchallenge. Four animals per time point pergroup were individually assessed. (C) Analysis of the CD3� T-cell population. Nontreated (a, c, and e) and BPZE1-treated (b, d, and f) mice wereleft 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-cellpopulation in the mouse lungs was analyzed. Four animals per time point and per group were individually assessed. Results are expressed as themeans SD of the percentages of CD3� T cells in the total lung cell population. **, P � 0.01; ***, P � 0.001.

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chemokines, namely, IL-1�, IL-6, and GM-CSF, were signifi-cantly lower than those measured in the untreated infectedcontrols (Fig. 8B). In mice treated twice with BPZE1, theproduction of all 8 proinflammatory cytokines and chemokineswas either strongly reduced or completely suppressed com-pared to that in the nontreated infected control group (Fig.8B). Thus, nasal treatment with live BPZE1 prior to viralchallenge suppresses the influenza virus-induced cytokinestorm, strongly suggesting a correlation between protectionand reduced lung inflammation.

Interestingly, no significant difference in the levels of TGF-�between BPZE1-treated mice and the infected controls (Fig.8B) was detected, whereas IL-10 production was reduced inthe BPZE1-treated animals.

DISCUSSION

Severe respiratory disease and immunopathology resultingin high case fatality rates are hallmarks of highly pathogenicinfluenza virus infections in humans as well as in other mam-mals. The cytokine storm, characterized by excessive levels ofchemokines and cytokines in the serum and lungs, has beenlinked to fatal outcome in humans (4, 12, 37, 44) and in ex-perimental animals infected with reconstructed 1918 H1N1and H5N1 influenza viruses (24, 27, 40, 43). Furthermore,histological and pathological indicators strongly suggest a keyrole for an excessive host response in mediating at least someof the extreme pathology associated with highly pathogenicinfluenza viruses. Thus, although cytokine production may beimportant for viral clearance, cytokine inflammatory proper-ties may also lead to tissue damage (28). In this study, we foundthat administration of the live attenuated B. pertussis BPZE1strain protects mice against lethal challenge with influenza Avirus by strongly decreasing lung immunopathology and reduc-ing the production of major proinflammatory cytokines andchemokines, without affecting the viral load. These findingsthus indicate a direct link between protective efficacy and re-duction of the cytokine-mediated inflammation and stronglyargue for an important role of the overproduction of proin-

flammatory cytokines in influenza virus disease severity andmortality.

These conclusions are in line with those of a recent study inwhich the intratracheal administration of the sphingosine an-alog AAL-R significantly reduced lung immunopathologycaused by influenza virus by decreasing the release of cytokinesand chemokines known to contribute to the cytokine stormeffect, while no change in lung viral titers was observed (31).However, other studies have suggested that the severe immu-nopathology observed with infected animals is a consequenceof the inability to resolve a high viral load in the respiratorytract (12, 13). Moreover, reduced inflammatory cell infiltrationand pulmonary damage but delayed viral clearance were ob-served with macrophage inflammatory protein 1� (MIP-1�)gene knockout mice (9). Likewise, CCR2 (primary receptor forMCP-1)-deficient mice displayed reduced mortality, with de-creased pulmonary cell infiltration and tissue damage, but witha significantly elevated viral burden compared to control mice(11). The relationship between viral load, immunopathology,and disease severity appears rather complex and involves avariety of host factors which play different roles during influ-enza virus infection and whose disruption or inactivation leadsto different outcomes.

The reduced production of proinflammatory cytokines andchemokines in the respiratory tract of the protected BPZE1-treated animals likely impacted on cellular infiltration andimmune cell activation. Lower neutrophil counts were ob-served with BALF samples from the protected animals, con-sistent with lower levels of KC and TNF-�, two cytokines thatare involved in the recruitment and activation of neutrophils inthe infected tissues (17, 26, 28). Neutrophils can elicit numer-ous responses in the presence of IFN-�, including increasedoxidative burst and the induction of antigen presentation, andchemokine production (14, 15). Therefore, although increasednumbers of neutrophils in the lungs contribute to the inhibition ofvirus replication as part of the host innate immune response, theymay also play a role in the enhanced immunopathology inducedupon infection with highly pathogenic influenza viruses.

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 orwere i.p. injected with heat-inactivated (HI) H3N2 virus. Serum and BALF samples were collected 2 weeks after the last administration. An ELISAwas performed using purified HI-H3N2 viral particles as coating antigens, and the serum and BALF samples from naive or BPZE1- orHI-H3N2-immunized mice were used as primary antibody. IgG (A) and IgA (B) antibody responses were measured. Results are expressed as theoptical density at 490 nm (OD490nm). 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 theserum (starting at 1:2) or neat BALF samples from BPZE1-immunized mice were incubated with H3N2 influenza virus before MDCK cells wereinfected, and the cytopathic effect (CPE) was observed 72 h later. Anti-H3N2 immune serum was used as a positive control. Serum or BALFsamples from naïve and HI-H3N2-immunized mice were pooled, while those from mice treated with BPZE1 were tested individually. Serumdilution is the highest dilution for which total protection was observed. nd, not determined; *, P � 0.05; ***, P � 0.001.

7110 LI ET AL. J. VIROL.

Likewise, the complete suppression of IL-12 and GM-CSFobserved with the protected BPZE1-treated mice likely im-paired activation, differentiation, and recruitment of variousimmune cells, including macrophages, dendritic cells (DC),cytotoxic CD8� T cells, and natural killer (NK) cells whoseactivation may be involved in immunopathology upon releaseof inflammatory mediators (28).

Interestingly, a higher number of macrophages were ob-served with the BALF samples from protected BPZE1-treatedmice. Alveolar macrophages (AMs) constitute the predomi-nant macrophage population recovered in BALFs (23) anddisplay suppressive effects on the inflammatory reaction byregulating T-cell function (42) and by suppressing dendriticcell maturation (5, 21) and migration to the mesenteric lymphnodes (23). Whether the increased numbers of AMs present in

the BPZE1-treated animals upon influenza virus challengecontributes to the control of lung inflammation warrants fur-ther investigation.

In addition, treatment with BPZE1 prevented influenza vi-rus-induced CD3� T-cell depletion. Lymphocyte depletionduring highly pathogenic influenza virus infection has previ-ously been reported (24, 29, 36, 44), and apoptosis has beenproposed as a potential mechanism (19, 29, 44). Since weobserved no difference in viral loads between protected andnonprotected animals, virus-induced lymphocyte apoptosisdoes not likely result from a direct cytotoxic effect of the virusitself. Instead, consistent with previous studies of H5N1-in-fected humans and mice (30, 44), lymphocyte apoptosis may beattributed to cytokine dysregulation and overactivation of thehost immune response. In particular, TNF-� and related TNF-superfamily members, including TNF-related apoptosis-induc-ing ligand (TRAIL), are known to induce T-cell apoptosis (40,45). Consistently, lower levels of TNF-� were measured in theBALF samples from protected BPZE1-treated mice upon in-fluenza virus challenge, thus possibly translating into dimin-ished T-cell apoptosis.

Nonspecific protection against influenza viruses has previ-ously been reported. Nasal administration of Autographa cali-fornica nuclear polyhedrosis baculovirus (AcNPV) protectedmice from lethal H1N1 A/PR/8/34 influenza virus challenge(1). Reduced immunopathology, together with lower levels ofIL-6 production but also with reduced viral loads, was observedwith the protected mice compared to the control mice. Inanother report, prophylactic intranasal treatment with chitinmicroparticles enhanced the local accumulation of NK cellsand suppressed hyperinduction of cytokines, resulting in pro-

FIG. 7. Cross-protective antibodies, cross-reactive T cells, and viralloads. (A) Passive transfer of immune serum. Naive (open triangle),anti-H3N2 (solid circle), or anti-BPZE1 (open circle) immune serawere i.p. injected into naive mice 1 day prior to lethal challenge (2 LD50)with mouse-adapted H3N2 virus. Survival rates were compared tothose of nontreated infected mice (solid triangle). Ten animals pergroup were assessed. (B) Proliferation assay. Pooled spleens from 6BPZE1-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 wasdetermined. Results are expressed as the stimulation index (SI) cor-responding to the ratio between the mean of [3H]thymidine uptake inthe presence of test antigen and the mean of [3H]thymidine uptake inthe absence of test antigen. An SI value of �2 was considered positive.Each sample was assayed in quadruplicate. (C) IFN-� 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-� ELISPOT assays wereperformed. Results are expressed as the means SD of the number ofpositive spots per 2 � 105 cells. Results are representative of twoindependent experiments. *, P � 0.05; ***, P � 0.001. (D) Quantifi-cation of the viral load in the lungs of protected and nonprotectedmice. Mice were i.n. administered with 5 � 106 CFU of live BPZE1bacteria and lethally challenged 6 weeks later with mouse-adaptedH3N2 virus (3.5 � 105 PFU). At 3 and 5 days post-viral challenge, theviral loads in the lungs of BPZE1-treated (a) and nontreated (b) micewere measured. Five animals per group per time point were assayed.

FIG. 8. Pro- and anti-inflammatory cytokine and chemokine pro-files. Adult BALB/c mice were i.n. administered once or twice at a4-week interval with 5 � 106 CFU of live BPZE1 bacteria and werelethally challenged with 2 LD50 of mouse-adapted H3N2 virus at 6 and4 weeks after the last BPZE1 administration, respectively. At 3 dayspost-viral challenge, 5 mice per group were sacrificed, and BALFspecimens were collected. Ten inflammation-related cytokines andchemokines in the individual BALF samples were measured. (A) Cy-tokine levels in naïve (black bar) or nontreated infected (open bar)mice. (B) Cytokine levels in nontreated infected mice (black bar) or inmice treated once (open bar) or twice (gray bar) with BPZE1 prior tochallenge with H3N2 virus. Results are expressed in pg/ml. *, P � 0.05;**, P � 0.01; ***, P � 0.001.

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tection against infection with HPAI virus (22). However, inboth studies, the prophylactic treatment needed to be per-formed less than a few days prior to the virus challenge inorder to be protective, resulting in a transient, short-termdownregulation of the host inflammatory response. In contrast,BPZE1-induced protection takes more than 3 weeks to beeffective and lasts up to at least 12 weeks posttreatment. Theprotective mechanism(s) primed upon nasal treatment withBPZE1 thus appears to be rather unique and likely does notinvolve fast-induced short-lived innate immune cells such asNK, DC, or macrophages.

The lack of T- and B-cell cross-reactivity between B. pertussisand influenza A virus, as well as the inability to confer protec-tion by passive transfer of antiserum from BPZE1-treatedmice, further demonstrates that B. pertussis-specific adaptiveimmunity is not involved. In addition, the observation that livebut not heat-killed BPZE1 bacteria induce protection indicatesthat bacterial lung colonization, i.e., a prolonged exposure tothe host immune system, is necessary. This hypothesis is fur-ther supported by the fact that the protection rate is bacte-rial dose dependent, which directly correlates with lung col-onization efficacy. Moreover, we showed that a secondBPZE1 treatment shortened the time necessary to induceprotection and enhanced the protection rate, implying thatsome memory has been triggered upon first exposure toBPZE1 and can be boosted by a second encounter withBPZE1 bacteria.

Many activities of B. pertussis virulence factors are dedicatedto immunomodulation in order to suppress, subvert, and evadethe host defense system (6). The immune response to B. per-tussis is initiated and controlled through Toll-like receptor 4(TLR-4) signaling, inducing the production of the anti-inflam-matory cytokine IL-10 to inhibit inflammatory responses andlimit pathology in the airways (18). Filamentous hemagglutinin(FHA), the major B. pertussis adhesin, can stimulate IL-10production and inhibit TLR-induced IL-12 production, result-ing in the development of IL-10-secreting Tr1 cells (32). How-ever, we found no difference in the production levels of TGF-�and reduced levels of IL-10 in the BPZE1-treated mice com-pared to those in the infected control animals, thus exclud-ing the potential involvement of FHA-mediated inductionof Tr1 cells as a mechanism of BPZE1-mediated protectionagainst influenza A virus. The involvement of other types ofregulatory mechanisms is thus likely at play and is beinginvestigated.

In conclusion, our study demonstrates the anti-inflammatoryproperties of the B. pertussis BPZE1 strain and supports thepotential use of this bacterial agent as a novel, highly effectiveprophylactic agent, with long-lasting effects, against severe andlethal pneumonitis induced by H3N2 and H1N1 influenza Aviruses. In addition, attenuated B. pertussis is particularly welladapted for the nasal delivery of heterologous vaccine antigensand represents an attractive mucosal vaccine delivery system(2, 10, 20, 34, 38, 39). Constructing BPZE1 derivatives produc-ing viral antigens, thereby combining anti-inflammatory prop-erties with the ability to induce adaptive anti-influenza virusimmune responses, may thus constitute an interesting strategyfor future development.

ACKNOWLEDGMENTS

We gratefully thank K. Tan (Department of Microbiology, NationalUniversity of Singapore) for his critical reading of and useful com-ments on the manuscript.

This work was supported by the National Medical Research Council(Individual Research Grants no. NMRC/0962/2005 and NMRC/1135/2007) and the Department of Microbiology and Immunology Pro-gramme, National University of Singapore (start-up grant no. R-182-000-122-731).

We declare that we have no conflicts of interest.

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