Microbes and Infection 16 (2014) 73e79www.elsevier.com/locate/micinf

Short communication

Investigations of TB vaccine-induced mucosal protection in mice

Azra Blazevic a,1, Christopher S. Eickhoff a,1, Jaime Stanley a,1, Mark R. Buller b,2,Jill Schriewer b,2, Eric M. Kettelson b,2, Daniel F. Hoft a,b,*,2

aDepartment of Internal Medicine, Saint Louis University, Doisy Research Center, 8th Floor, 1100 S. Grand Blvd., St. Louis, MO 63104, United StatesbDepartment of Molecular Microbiology and Immunology, Saint Louis University, Doisy Research Center, 7th Floor, 1100 S. Grand Blvd., St. Louis, MO 63104,

United States

Received 1 July 2013; accepted 28 September 2013

Available online 8 October 2013

Abstract

A better understanding of mucosal immunity is required to develop more protective vaccines against Mycobacterium tuberculosis. Wedeveloped a murine aerosol challenge model to investigate responses capable of protecting against mucosal infection. Mice received vacci-nations intranasally with CpG-adjuvanted antigen 85B (Ag85B/CpG) and/or Bacillus CalmetteeGuerin (BCG). Protection against aerosolchallenge with a recombinant GFP-expressing BCG was assessed. Mucosal prime/boost vaccinations with Ag85B/CpG and BCG were pro-tective, but did not prevent lung infection indicating more efficacious mucosal vaccines are needed. Our novel finding that protection correlatedwith increased airway dendritic cells early post-challenge could help guide the development of enhanced mucosal vaccines.� 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Mycobacterium tuberculosis (Mtb); BCG; Vaccination; Mucosal specific immunity

1. Introduction

One third of the world’s population is infected withMycobacterium tuberculosis (Mtb), the causative agent oftuberculosis (TB), leading to 1-2 million deaths annually [1].Although the Bacille CalmetteeGuerin (BCG) vaccine hasbeen used for prevention of TB since 1921 efficacy rates ofintradermal BCG vaccination are highly variable [2]. Meta-analyses suggest that BCG vaccination can reduce pulmo-nary TB by approximately 50% [3]. These partially protectiveeffects appear to wane within 10e15 years of infant BCGvaccination, and there is little evidence to support prolongedbenefits [4].

* Corresponding author. Division of Infectious Diseases, Allergy & Immu-

nology, Department of Internal Medicine, Saint Louis University, Doisy

Research Center, 8th Floor, 1100 S. Grand Blvd., St. Louis, MO 63104, United

States. Tel.: þ1 314 977 5500; fax: þ1 314 771 3816.

E-mail address: hoftdf@slu.edu (D.F. Hoft).1 Tel.: þ1 314 977 5500; fax: þ1 314 771 3816.2 Tel.: þ1 314 977 8853; fax: þ1 314 977 8717.

1286-4579/$ - see front matter � 2013 Institut Pasteur. Published by Elsevier Ma

http://dx.doi.org/10.1016/j.micinf.2013.09.006

Heterologous prime/boost vaccination strategies inducerobust T cell responses and may improve protection comparedto BCG alone [5e7]. Therefore, many new TB vaccine ap-proaches under development focus on “booster” vaccines toenhance and extend immunity acquired after primary BCGimmunization [8]. Another potentially useful strategy ismucosal vaccination. BCG is usually delivered intradermallywhich is not expected to induce optimal mucosal TB immunity.Regional immunity in the lung may be important for enhancedprotection at the site of initial infection, and intranasal (IN) orother mucosally delivered vaccines might induce Mtb specificmucosal immunity capable of preventing TB infection [9].

In the present work, we investigated whether prime/boostintranasal vaccinations with BCG and Ag85B/CpG can inducemucosal protection against primary mycobacterial infection ofthe lung better than a single dose of BCG given intranasally.We use a recombinant BCG expressing green fluorescentprotein (GFPrBCG) strain for aerosol challenge to allowsensitive detection of infection, and to maximize our abilityto identify at least partially protective mucosal immuneresponses that can be iteratively improved.

sson SAS. All rights reserved.

5.00E+02

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5.00E+06 Day 3 2 Wks 3 Wks 4 Wks 2 Mos 4 Mos 6 Mos

0.0E-01

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4.0E+03 Medium Mtb CF BCG Danish

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1.6E+04 Naïve BCG IN Day 3 BCG IN 2 Wks BCG IN 3 Wks BCG IN 4 Wks

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Fig. 1. BCG persistence and recruitment of mycobacterial-specific T cells into the lungs of mice after intranasal BCG vaccination (AeF). C57BL6/J mice were

sacrificed 3 days to 6 months after two administrations of 1 � 107 BCG on two consecutive days, and lung and spleen homogenates were plated on Middlebrook

agar and incubated at 37 �C for >3weeks to enumerate BCG (colony forming units, CFU) (A). C57BL6/J mice were immunized IN with 1 � 107 BCG two times

on two consecutive days and bronchoalveolar lavage (BAL) cells harvested 3 days, 2 weeks, 3 weeks and 4 weeks later for analysis by flow cytometry (BeE) and

74 A. Blazevic et al. / Microbes and Infection 16 (2014) 73e79

75A. Blazevic et al. / Microbes and Infection 16 (2014) 73e79

2. Materials and methods

2.1. Animals and organisms

Studies using six to eight week old NCI/Charles RiverLaboratory C57BL/6 mice were carried out in AAALACaccredited facilities, and approved by the Institutional AnimalCare and Use Committee of Saint Louis University (A3225-01). Mycobacterium bovis Bacillus CalmetteeGuerin (BCG)and GFPrBCG were grown, frozen and titered as previouslydescribed [10].

2.2. Immunizations

Prior to IN immunization with BCG and/or CpG-adjuvantedprotein (10 ml volume split between naris), mice were anes-thetized by intraperitoneal injection of ketamine (60mg/kg) andxylazine (5mg/kg). Based on previously published data [11] andupon our preliminary dose-optimization studies, mice received1 � 107 CFU of BCG Danish strain (Statens Serum Institut,Denmark) twice on two consecutive days and/or 10 mg Ag85B(provided by M.A. Horowitz [12]) mixed with 10 mg CpG 1826(Coley Pharmaceuticals).

2.3. Aerosol challenge and determination of BCGgrowth

Stock vials of GFPrBCG were thawed, sonicated (DigitalSonifier 450 sonicator) and diluted in 0.9% saline containing0.04% Tween-80 to a final concentration of w1 � 107 CFU/ml. Mice were challenged with aerosolized BCG using a nose-only inhalation exposure system (NOIES; CH Technologies)as described previously [13]. Animals were exposed to aero-solized BCG for 20 min with an air pressure of 20 psi, an airflow rate of 2.0 L/min and a BCG suspension flow rate of1.0 ml/min, followed by 5 min of air flow only. Growth ofBCG in tissues was evaluated 24 h to 6 months after aerosolchallenge. Lungs and spleens were homogenized in albumin-dextrose-catalase (ADC) supplemented Middlebrook 7H9media and plated on Middlebrook 7H10 agar containingoleic-acid-albumin-dextrose-catalase (OADC) enrichment �Kanamycin (30 mg/ml).

2.4. Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) was performed to collectcells for use in IFN-g ELISPOT and flow cytometric analyses.Briefly, mice were euthanized, and the trachea was exposedand cannulated. The lungs were lavaged three times with 1 mlPBS. Red blood cells were lysed in NH4Cl lysis buffer, thencells washed with PBS, before being resuspended in completemedia.

IFN-g ELISPOT (F). Shown in B are representative FACS plots of BAL cells from

numbers (AN) of BAL CD4þ T cells (C), BAL CD8þ T cells (D), and BAL CD

Additionally, BAL cells from naıve mice and IN BCG vaccinated mice were stimula

3, and 4 weeks post-vaccination (F). Bars represent mean values, and whiskers rep

For flow cytometric analysis, cells were stained using thefollowing antibodies: FITC-anti-CD40, PE-anti-MHCII,PerCP-anti-CD8, PE-Cy7-anti-CD11c, APC-anti-CD19 andPacific Blue-anti-CD4 (BD) and analyzed using an LSR IIFlow Cytometry unit (BD) and FlowJo7 software (TreeStar,Ashland, OR) [14].

2.5. Antigen-specific IFN-g ELISPOT responses

IFN-g production was measured by ELISPOT assay usingsplenocytes (5 � 105 cells/well) or cells obtained by BAL(5 � 104 cells/well). Cells were stimulated overnight withrecombinant Ag85B protein (10 mg/ml), Mtb culture filtrateproteins (Mtb CF, 10 mg/ml, Colorado State University), Mtbwhole cell lysate (MtbWL, 10 mg/ml) [15], live BCG (MOI of2.0, 0.2 and 0.02), or medium alone. Results are reported asnumbers of IFN-g spot-forming cells (SFC) per million cells.

2.6. Statistical analysis

ManneWhitney U tests and SpearmaneRank tests wereperformed using Statistica v6.0 software (Statsoft, Tulsa, OK).Probability values below 0.05 were considered significant.

3. Results

3.1. Intranasal BCG vaccination results in long-terminfection and induces potent CD4þ and CD8þ T cellresponses in the airways of mice

To investigate persistence of BCG after mucosal immuni-zation, mice were vaccinated IN with 1 � 107 BCG on twoconsecutive days (optimal immunogenic dose determined inpreliminary experiments; not shown) and kinetics of BCGpersistence in lungs and spleens were examined at varioustime points post-vaccination (day 3, week 2, week 3, week 4,month 2, month 4 and month 6). We observed peak BCG CFUin the lungs and spleens 2 weeks following IN infection(>5 � 106 CFU/mouse), though high numbers of BCG per-sisted throughout the first month of infection (>5 � 105 CFU/mouse). Importantly, low levels of BCG persisted in the lungs(250e450 CFU/mouse), for at least 6 months post-vaccination(Fig. 1A).

BAL cells are important for mucosal protection in thelungs, and responses by BAL cells serve as a potential sur-rogate marker for assessing new TB vaccines [16]. To examinethe kinetics of BCG-specific T cell responses in the lungs,mice were vaccinated intranasally with BCG, and BAL cellswere harvested at various time points for flow cytometry andIFN-g ELISPOT assays. We observed an increase of bothCD4þ and CD8þ T cells in the lungs of mice within the firstfour weeks following vaccination (Fig. 1B). The absolute

naıve and BCG vaccinated mice (2 and 4 weeks post immunization). Absolute

11cþ cells (E), were determined 3 days to 4 weeks post-BCG vaccination.

ted with Mtb culture filtrate (MtbCF) or live BCG in IFN-g ELISPOT assays 2,

resent standard errors.

Fig. 2. Mycobacterial growth and immune protection against aerosolized mycobacterial challenge after intranasal prime-boost vaccination (AeD). Naıve C57BL6/

J mice were infected by aerosol with GFPrBCG with a challenge dose ofw250 GFPrBCG CFU/mouse, sacrificed at day 0, 2 weeks and 4 weeks post-infection and

lung homogenates were plated on kanamycin selective 7H10 Middlebrook agar. BCG growth is reported as mycobacterial colony forming units (CFU) per animal.

Each time point represents data from of 3e4 mice per group (A). To demonstrate our ability to properly discriminate between BCG used for vaccination and BCG

used for challenge, wild type BCG (WT BCG) and GFP-expressing recombinant BCG (GFPrBCG) were plated on selective and nonselective (NS) 7H10 Mid-

dlebrook agar (with and without 30 mg/ml kanamycin, respectively). After incubation at 37 �C for w3 weeks, colonies were visualized under white and ultraviolet

(UV) light (B). The results of immunization and challenge experiments are shown in panel (C). C57BL6/J mice were vaccinated IN with 2 doses of a subunit

vaccine containing Ag85B/CpG, with 2 consecutive days of BCG intranasal vaccination alone, or with heterologous prime/boost combinations of BCG and Ag85B/

CpG (all mice received vaccinations or saline at time 0, week 2 and week 4) and subsequently challenged by aerosol with GFPrBCG two months after the initial

vaccination. Mice were sacrificed 4 weeks post aerosol challenge and lung homogenates were plated on kanamycin selective 7H10 Middlebrook agar. Colonies of

challenge organisms (GFPrBCG), growing on selective plates are reported as the average mycobacterial colony forming units (CFU) per animal. (Shown are

means � SE from 8 mice per group, *p < 0.05 compared with the control group by ManneWhitney U test.) BAL cells from naıve mice and mice vaccinated IN

with BCG and/or Ag85B/CpG (day 0, 2 weeks and 4 weeks) and subsequently challenged by aerosol with GFPrBCG two months after the initial vaccination were

harvested 2 weeks after aerosol challenge and stimulated overnight with Mtb antigens or live BCG in IFN-g ELISPOT assays (D). The results are presented as the

numbers of IFN-g spot forming cells per million BAL cells. Bars represent mean values, and whiskers represent standard errors.

76 A. Blazevic et al. / Microbes and Infection 16 (2014) 73e79

numbers (AN) of BAL CD4þ and CD8þ T cells increasedwithin 3 days post-immunization, peaked 2 weeks post-immunization, and remained increased through week 4(Fig. 1C and D). It is noteworthy that during the time period ofT cell influx into the lungs of BCG vaccinated mice (3 dayse4weeks post-vaccination), a log decrease in absolute number ofairway CD11cþ cells was observed (Fig. 1E). These CD11cþ

cells expressed other markers characteristic of dendritic cells(DC) including high levels of MHC-II and CD40 (not shown).

IFN-g responses produced by BAL cells after in vitrostimulation with MtbCF or BCG in ELISPOT assaysconfirmed that antigen-specific CD4þ and CD8þ T cells wererecruited into the lungs (Fig. 1F), and also demonstrated thatthe frequencies of TB-specific T cells among BAL cells were10 fold greater than the frequencies present among spleen cells(the numbers of Mtb culture filtrate- and live BCG-stimulated

IFN-g-producing SFC detectable in BAL were 3386 � 93 and1506 � 179/million cells, respectively; the matching resultsfor splenocytes were more than 10 fold lower with 268 � 32and 21 � 3/million cells, respectively).

3.2. Development of a sensitive challenge model systemfor detection of mucosal protection against initial lunginfection

As an initial screen for mucosal immune responses relevantfor protection against initial TB infection, we developed anaerosol BCG challenge model. We reasoned that if a mucosalvaccine does not protect against BCG mucosal challengeit would not protect against Mtb mucosal challenge. Todistinguish BCG persisting in the lungs of mice after BCGvaccination from mycobacteria establishing a new infection,

Table 1

Specific immune cell subsets detected in bronchoalveolar lavage cells 3 days

after aerosol challenge were compared to the mean protective responses in

lungs. This protection was determined based on number of GFPrBCG colony

forming units (CFU) counted 4 weeks post-aerosol challenge (log10 CFU

GFPrBCG for naıve mice minus log10 CFU GFPrBCG for immunized mice).

Comparison* Valid Spearman-rank test P-level

AN CD11cþ vs protective responses

in lungs

8 0.7381 0.036

AN CD11cþCD40þ vs protective

responses in lungs

8 0.7381 0.036

AN CD11cþ MHCIIþ vs protective

responses in lungs

8 0.7143 0.046

AN e Absolute number.

77A. Blazevic et al. / Microbes and Infection 16 (2014) 73e79

post-challenge naıve mice were challenged with aerosolizedGFPrBCG. Numbers of CFUs in the lungs of GFPrBCG-infected mice increased 3 fold from day 0e2 weeks andwere more than 10 fold higher 4 weeks post-infection(Fig. 2A). Homogenized lungs after wild type (WT) BCGvaccination and GFPrBCG challenge were plated on selective(kanamycin) and nonselective 7H10 Middlebrook agar. Asshown (Fig. 2B), GFPrBCG grows on both types of media andis fluorescent under ultraviolet lighting, whereas WT BCGdoes not grow on selective agar and is not fluorescent underultraviolet illumination.

3.3. Ag85B vaccinations with and without BCG primingpartially protect against mucosal infection

The protective efficacy of IN immunization against aerosolinfection was examined after immunizations with BCGalone, Ag85B/CpG alone, or various prime/boosting combi-nations of BCG and CpG � Ag85B. Groups of 8 mice werevaccinated IN three times, two weeks apart with BCG and/orCpG � Ag85B. Two months after the first immunization, micewere challenged with aerosolized GFPrBCG. The numbers ofGFPrBCG were assessed in the lungs one month post-challenge (Fig. 2C). All BCG and/or Ag85B/CpG vaccinatedmice were significantly protected compared to controls(P < 0.05). Although there was a trend for enhanced protec-tion, BCG priming followed by Ag85B/CpG boosting did notsignificantly enhance protective mucosal immunity. Similarresults were seen in additional experiments challenging mice7.5 months after immunization (data not shown).

Fig. 2D presents the results of IFN-g ELISPOT assaysconducted using BAL cells collected 2 weeks post-aerosolchallenge. Immunization with BCG � Ag85B inducedincreased IFN-g-producing T cells specific for Mtb secretedproteins and live BCG when compared to naıve mice, whileimmunization with Ag85B protein alone failed to showdifferences after Mtb secreted protein and live BCG stimu-lation compared with the control group. The numbers ofantigen-specific T cells recruited into the lungs and reactivewith Ag85B, Mtb CF and Mtb WL were 2e8 fold higherwhen BCG priming was followed by Ag85B/CpG boosting(Fig. 2D). However, IFN-g ELISPOT responses in BALcells stimulated with live BCG were similar between BCG/

Ag85B prime/boosted mice and mice vaccinated with BCGalone.

The failure to enhance overall responses to all antigensexpressed by the challenge strain may explain why the prime/boost vaccination protocol was not significantly more pro-tective than BCG vaccination alone. To assess potential bio-markers capable of guiding the development of more suc-cessful mucosal vaccine strategies, we studied correlationsbetween immune subsets recruited to the lungs early (day 3)after aerosol challenge, and the levels of protection measuredby CFUs of the challenge strain present 4 weeks post-aerosolchallenge. The numbers of CD4þ and CD8þ airway T cellspresent on day 3 post-challenge did not correlate with pro-tection, however, spearman rank tests confirmed significant(positive) correlations between the absolute numbers ofCD11cþ, CD11cþCD40þ and CD11cþMHC-IIþ dendriticcells in BAL on day 3 post-challenge and the mean protectivevalue in lungs (log10 GFPrBCG in lungs for naıve mice minuslog10 GFPrBCG in lungs for immunized mice) 4 weeks post-aerosol challenge (Table 1).

4. Discussion

BCG vaccination has not lowered the overall prevalence ofTB infection and disease. In fact, it is thought that currentBCG vaccines provide very limited to no protection againstmucosal TB infection [17e19]. Thus, the development of asensitive model to identify partially protective mucosal im-mune responses remains of great importance. We reasonedthat an attenuated BCG strain would not overwhelm subopti-mal mucosal immune responses allowing for their sensitivedetection, and that mucosal immune responses unable toprotect against BCG infection would not protect against morevirulent Mtb challenges. Therefore, we have developed asensitive model that can identify the mechanisms of immunityresponsible for partial mucosal protection to allow for theiterative optimization of future mucosal vaccination strategies.

This study presents evidence that BCG administeredintranasally to mice results in chronic infection and persists inhost lungs for months. In addition, we developed a GFPrBCGaerosol challenge model, which can be used to distinguishbetween BCG persistence after vaccination and infection afteraerosol challenge. Intranasal vaccinations with BCG and/orAg85B/CpG were partially protective against mucosal infec-tion. These IN vaccinations resulted in recruitment of bothCD4þ Th1 and CD8þ T cells into the airways of mice withintwo weeks of infection. Large granular CD11cþ DCs notice-ably decreased in frequency in the lungs two weeks post-infection. However, the number of DCs present in the air-ways three days post-aerosol challenge significantly correlatedwith levels of protection measured 4 weeks post-challenge.

Mucosal vaccination with soluble antigens has been shownto enhance protection against mucosally invasive intracellularpathogens [20]. For example, IN vaccination of mice withdimethyl-dioctadecyl-ammonium-bromide-adjuvanted Ag85induced protective immunity against virulent Mtb challenge[21]. Our results are consistent with these findings. Although

78 A. Blazevic et al. / Microbes and Infection 16 (2014) 73e79

Ag85B/CpG boosting of BCG immunity did not significantlyenhance protective mucosal immunity, future subunit vaccinesgiven IN may be useful in boosting immunity in BCG primedindividuals. Our new model will be useful in assessing thesepossibilities. In addition, the protection provided by Ag85B/CpG IN vaccination alone in our model is important becausesubunit vaccines in general are safer for mucosal delivery thanlive vaccines.

Induction of potent immune responses at the initial site ofmucosal TB infection in the lung is likely to be essential forinducing optimal protection. It has been demonstrated thatmucosal immunization with BCG induces strong type-1 im-mune responses in mice, associated with protection againstmycobacterial challenges [22]. Although it is known that BCGinduces CD4þ, CD8þ, CD1-restricted ab and gd T cell re-sponses, while soluble Ag85 protein likely induces only CD4þ

and some CD8þ T cell responses, very little is known aboutthe immune subsets important for vaccine-induced mucosalprotection [5,23]. It has been reported that mucosal vaccina-tion with a recombinant adenovirus induces better protectionagainst Mtb aerosolized challenges, and that the numbers ofTB specific CD4þ Th1 cells within the airways correlate withoptimal mucosal protection [24]. In our current study we didnot find a correlation between airway T cells and protection.This may be related to the time (3d post-challenge) we per-formed BAL which may have been too early to see maximal Tcell recruitment into the airways.

We observed a significant correlation between the numberof airway DCs 3d post-challenge and protection. This earlyDC correlation with protection has not been reported previ-ously. Infected DCs migrate from the lungs of Mtb-infectedmice into the mediastinal lymph nodes, where they activateboth memory and new antigen-specific T cells [25]. In addi-tion, early post-challenge, infected DCs could rapidly activateresident airway antigen specific T cells already present in theairways. Both of these early effects mediated by DCs couldpartially explain the novel correlation we have observed. MoreDCs present early post-challenge could result in more relevantT cells being recruited earlier to the lungs. This hypothesiswill need to be further studied. The relative loss of DCs inBAL samples later after mucosal challenge could be due to thedevelopment and recruitment of mycobacterial-specific T cellscapable of eliminating BCG-infected DCs.

Our newly developed GFPrBCG aerosol challenge modelhas a sensitive capacity for identification of immune responsesimportant for protective mucosal immunity against mycobac-terial infection. The use of BCG should not overwhelm partialimmunity, and can serve as an initial screen; providing theadvantages of limiting biohazardous exposure to laboratorypersonnel and serving as an initial milestone for the evaluationof mucosal vaccines. This model will allow us to more care-fully examine the efficacy of protection against infection infuture prime-boost immunization experiments because im-mune responses unable to protect against BCG could not beexpected to provide protection against virulent Mtb. Of course,mucosal vaccination strategies and immune subsets protectiveagainst aerosolized BCG will need to be further tested for the

capacity to protect against aerosolized Mtb challenge inorder to confirm mucosal protection against virulentMycobacterium.

Acknowledgments

We are grateful to Drs. Liana Tsenova and Gilla Kaplan(Rockefeller University, New York, NY) for their adviceswith mycobacterial aerosol challenge. This work was sup-ported by NIH R21 AI059362 (“Vaccine induced mucosalprotection against TB infection”) to D.F.H. Mtb whole celllysate was provided as part of NIH, NIAID Contract No.HSN266200400091C, entitled “Tuberculosis Vaccine Testingand Research Materials,” which was awarded to ColoradoState University. Michael O’Donnell (University of Iowa)kindly provided the GFPrBCG strain.

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