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The Journal of Immunology, 2008, 181, 5618 -5626
Copyright © 2008 by The American Association of Immunologists, Inc.
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Airway Delivery of Soluble Mycobacterial Antigens Restores Protective Mucosal Immunity by Single Intramuscular Plasmid DNA Tuberculosis Vaccination: Role of Proinflammatory Signals in the Lung1

Mangalakumari Jeyanathan*, Jingyu Mu*, Kapilan Kugathasan*, Xizhong Zhang*, Daniela Damjanovic*, Cherrie Small*, Maziar Divangahi*, Basil J. Petrof{dagger}, Cory M. Hogaboam{ddagger} and Zhou Xing2,*

* Centre for Gene Therapeutics, M. G. DeGroote Institute for Infectious Disease Research, and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada; {dagger} Respiratory Division, Royal Victoria Hospital, Montreal, Quebec, Canada; and {ddagger} Department of Pathology, University of Michigan, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Protection by parenteral immunization with plasmid DNA vaccines against pulmonary tuberculosis (TB) is very modest. In this study, we have investigated the underlying mechanisms for the poor mucosal protective efficacy and the avenues and mechanisms to improve the efficacy of a single i.m. immunization with a monogenic plasmid DNA TB vaccine in a murine model. We show that i.m. DNA immunization fails to elicit accumulation of Ag-specific T cells in the airway lumen despite robust T cell responses in the spleen. Such systemically activated T cells cannot be rapidly mobilized into the airway lumen upon Mycobacterium tuberculosis exposure. However, airway deposition of low doses of soluble mycobacterial Ags in previously immunized mice effectively mobilizes the systemically activated T cells into the airway lumen. A fraction of such airway luminal T cells can persist in the airway lumen, undergo quick, robust expansion and activation and provide marked immune protection upon airway M. tuberculosis exposure. Airway mucosal deposition of soluble mycobacterial Ags was found to create a tissue microenvironment rich in proinflammatory molecules including chemokines and hence conducive to T cell recruitment. Thus, in vivo neutralization of MIP-1{alpha} or IFN-inducible protein-10 markedly inhibited the accumulation of Ag-specific T cells in the airway lumen. Our data suggest that immunoprotective efficacy on the mucosal surface by i.m. plasmid DNA immunization could be substantially improved by simple mucosal soluble Ag inoculation and restoration of mucosal luminal T cells. Our study holds implication for the future design of DNA vaccination strategies against intracellular infections.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Despite the longstanding effort to control tuberculosis (TB),3 TB continues to remain one of the leading causes of death globally (1). Control strategies for TB rely on prophylactic and therapeutic measures achieved through bacillus Calmette-Guérin (BCG) vaccination and chemotherapeutic drugs, respectively. BCG provides poor protection in countries where TB incidence is high (2, 3, 4). With this in mind, in the last 15 years, various TB vaccine platforms have been developed to offer an efficient stand-alone vaccine superior to BCG or more realistically a vaccine that could effectively boost BCG-triggered protective immunity (5). Among the leading TB vaccine platforms are genetic-vectored vaccines including both recombinant plasmid DNA and virus vectors that have been widely utilized to deliver M. tuberculosis Ag-coding genes for the purpose of vaccination (6, 7).

Compared with several other vaccine platforms, plasmid DNA vaccines have a number of advantages including the ease of their large-scale production, stability, and repeatability of injection (7, 8). Repeated i.m. immunization with plasmid DNA vaccine triggers potent T cell activation in the systemic compartments (7, 9). However, protection achieved by primary DNA immunization against pulmonary Mycobacterium tuberculosis challenge in various animal models is very modest and is far from being superior to BCG vaccination (7, 9, 10, 11, 12), although DNA vaccines have shown some promise when used in heterologous prime and boost vaccination regimens (11, 13, 14). There has been a lack of understanding of the mechanisms underlying the poor mucosal protective efficacy by parenteral DNA immunization. Enhanced understanding in this regard is required to develop effective plasmid DNA immunization strategies not only for TB but also for other mucosal intracellular infectious diseases.

Mounting evidence suggests that mucosal immunization at the site of pathogen entry is able to trigger better protection than parenteral immunization (8, 15, 16, 17, 18, 19, 20, 21). The work from us and others by using models of mucosal immunization has revealed that the persisting Ag-specific T cells on the surface of the cells or in the mucosa hold the key to robust immune protection (12, 15, 18, 22, 23). However, different from genetic viral vaccines, plasmid DNA vaccines cannot be used directly for efficient mucosal delivery due to their low transfection efficiency at the mucosa (7, 10, 24). Recent evidence further suggests that different from the intranasal route, i.m. recombinant viral immunization, similar to i.m. DNA immunization, provides little immune protection from pulmonary M. tuberculosis challenge (12, 22, 23). The lack of protection by parenteral viral genetic immunization was associated with the lack of respiratory luminal T cells despite the presence of Ag-specific T cells in the systemic tissue sites, whereas deposition of soluble mycobacterial protein into the respiratory lumen restored T cell responses and protection (23, 25). It has, however, remained to be addressed whether poor protective efficacy of parenteral plasmid DNA TB immunization is attributed to the lack of respiratory mucosal luminal T cells and, if so, what are the means and mechanisms to turn parenteral DNA immunization to be immunoprotective.

By using murine models of i.m. immunization with a plasmid DNA TB vaccine encoding both M. tuberculosis Ag85A Ag and GM-CSF in our current study, we have addressed: 1) whether i.m. DNA immunization is able to elicit T cell responses within the airway lumen; 2) whether intranasal administration of soluble M. tuberculosis Ags is able to mobilize the T cells systemically activated by i.m. immunization into the airway lumen; if so, 3) whether restored T cell responses within the airway lumen of i.m.-immunized hosts could translate to enhanced immune protection from M. tuberculosis challenge; and 4) what signals are required for T cell recruitment to the airway lumen following soluble M. tuberculosis Ag delivery.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Female BALB/c mice (6–8 wk old) were purchased from Harlan Laboratory and housed in a specific pathogen-free, level B facility. All experiments were conducted in accordance with the guidelines of the animal research ethics board of McMaster University.

i.m. immunization and intranasal Ag administration

The development and characterization of a bicistronic plasmid DNA vaccine coexpressing M. tuberculosis Ag85A and murine GM-CSF (pAg85A-GM-CSF) has been previously documented by us (9). Coexpression of GM-CSF as an immune adjuvant was found to markedly enhance the immunogenicity of Ag85A following a single i.m. injection aided by electroporation (9). Mice were immunized once i.m. with 50 µg of pAg85A/GM-CSF in conjunction with electroporation into the thigh quadriceps muscle as described by us (9). As controls, some mice were injected i.m. only with a plasmid DNA expressing GM-CSF (pGM-CSF) in conjunction with electroporation or PBS. Intranasal provocation was conducted on i.m. immunized mice by intranasal administration of 2.5 µg of Ag85 complex proteins in 25 µl of PBS once weekly for a total of 2 wk. As a control, one group of immunized mice received only PBS or 2.5 µg of OVA. A group of mice were immunized s.c with BCG (Connaught strain; 0.5 million CFUs) as a control for M. tuberculosis challenge experiments.

Pulmonary M. tuberculosis challenge

A virulent M. tuberculosis H37Rv strain (ATCC27294) and an avirulent M. tuberculosis H37Ra strain (ATCC 25177) were grown in Middlebrook 7H9 broth supplemented with Middlebrook oleic acid-albumin-dextrose-catalase enrichment (Invitrogen), 0.002% glycerol, and 0.05% Tween 80 for 10–15 days, then aliquoted, and stored in –70°C until needed (12, 22, 23). Before each use, M. tuberculosis bacilli were washed with PBS containing 0.05% Tween 80 twice and passed through a 27-gauge needle 10 times to disperse clumps. For the secondary Ag recall response study with M. tuberculosis H37Ra strain, 0.5 million CFUs of live bacilli were administered intratracheally route in 40 µl of PBS and infected mice were sacrificed 5 days postchallenge. For challenge with M. tuberculosis H37Rv strain, immunized and control mice were infected intranasally with 10,000 CFUs of M. tuberculosis. The level of bacterial burden was determined in the lung and spleen at the described time points by plating serial dilutions of tissue homogenates in triplicates onto Middlebrook 7H10 agar plates containing oleic acid-albumin-dextrose-catalase enrichment. Plates were incubated at 37°C for 17 days in semisealed plastic bags. Colonies were then counted, calculated, and presented as log10 CFU per organ as previously described (12, 22, 23).

In vivo chemokine neutralization

For T cell recruitment mechanism studies, rabbit anti-murine MIP-1{alpha} or IFN-inducible protein-10 (IP-10) serum or normal rabbit serum as control (200 µl/injection) was injected i.p into DNA-immunized mice at designated times before and after Ag85 complex administration.

Expression of inflammatory cytokines/chemokines

Gene expression in the lung was analyzed using the RNase protection assay (RPA). For RPA, total tissue RNA samples of lung tissues were isolated by using TRIzol reagent (Invitrogen) and subjected to RPA assay to assess cytokine mRNA levels as previously described (26, 27, 28). Briefly, 32P-labeled riboprobes were synthesized using a commercial mouse multiprobe kit (BD Pharmingen) containing templates against the following gene transcripts: inducible NO synthase, IL-18, KC, RANTES, MIP-1{alpha}, MIP-1β, MIP-2, MCP-1, and housekeeping genes L32 and GAPDH. The riboprobes were hybridized with each RNA sample overnight at 56°C according to the manufacturer’s instructions. The protected RNA fragments were separated using a 5% polyacrylamide gel.

For in vivo cytokine protein measurement, bronchoalveolar lavage (BAL) fluids were collected. IP-10 levels were determined by using chemokine multiplex kit (Millipore) as directed by the manufacturers. Standards and reference points were measured in triplicate, each sample was measured once, and blank values were subtracted from all readings. All assays were conducted directly in a 96-well filtration plate (Millipore) at room temperature and protected from light. Briefly, wells were prewetted with 100 µl of PBS containing 1% BSA; then beads (5000 beads per cytokine) together with a standard, sample, reference point, or blank were added in a final volume of 100 µl and incubated together at room temperature for 30 min with continuous shaking. Beads were washed three times with 100 µl of PBS containing 1% BSA and 0.05% Tween 20. A mixture of biotinylated Abs (50 µl/well) was added to the beads for a further 30-min incubation with continuous shaking. Beads were washed three times; then streptavidin-PE was added for 10 min. Beads were again washed three times and resuspended in 125 µl of PBS containing 1% BSA and 0.05% Tween 20. The fluorescence intensity of the beads was measured using the Luminex multianalyte technology (Luminex) (29). The levels of detection for IP-10 was 2.0 pg/well. The level of TNF-{alpha} in the BAL was measured by ELISA (R&D Systems).

Cell isolation

Mononuclear cells were isolated from bronchoalveolar lavage (BAL), lung, lymph nodes and spleen for intracellular cytokine staining (ICCS), Ag85A tetramer staining, and cytokine production following ex vivo Ag stimulation. Intra-airway luminal cells were removed from lung by exhaustive lavage as previously described (12, 22, 23). After BAL, lungs were perfused through the left ventricle with Hanks’ buffer to remove leukocytes from the pulmonary vasculature. The lungs were then cut into small pieces and digested with collagenase type 1 (Sigma-Aldrich) for 1 h at 37°C before crushing them through a 100-µm pore size filter. Splenocytes and cells from lymph nodes were isolated as previously described (12, 22, 23). Collected cells were enumerated on a hemocytometer after appropriate dilution in Turk’s counting buffer. All isolated cells were then resuspended in RPMI 1640 supplemented with 5% FBS and 1% penicillin and streptomycin.

Cell culture, ICCS, tetramer staining, and flow cytometry

Single-cell suspensions isolated from lung, spleen, and lymph nodes as described in Cell Isolation were cultured in a U-bottom 96-well plate at a concentration of 20 million cells/ml, and BAL cells were plated at a concentration of 0.5 million cells/ml. Cells were cultured and stimulated for ICCS, tetramer staining, and FACS as previously described (12, 22, 23). Briefly, cells were cultured in the presence of Golgi plug (5 µg/ml brefeldin A; BD Pharmingen) and with or without stimulation by Ag85A-specific CD4 (LTSELPGWLQANRHVKPTGS) or CD8 (MPVGGQSST) T cell peptides at a concentration of 1 µg/well for 5–6 h (22). Cells were then washed and blocked with CD16-CD32 in 0.5% BSA-PBS for 15 min on ice and immunostained with selected mAbs. Cells were then washed, permeabilized, and stained according to the manufacturer’s instructions (BD Pharmingen). The following fluorochrome-labeled Abs were used: CD8a-allophycocyanin-Cy7, CD4-PE-Cy7, IFN-{gamma}-allophycocyanin, and CD3-CyChrome (BD Pharmingen). For tetramer immunostaining and FACS, a tetramer for immunodominant Ag85A CD8 T cell peptide (MPVGGQSST) bound to the BALB/c MHC class I allele H-2Ld ordered from the MHC Tetramer Laboratory of Baylor College of Medicine was used (22, 23). Cells were then washed and blocked with CD16-CD32 in 0.5% BSA-PBS for 15 min on ice and stained with tetramer for 1 h in the dark at room temperature. Cells are then washed and stained with surface Abs. Stained cells were then run on a LSR II (BD Pharmingen), and 250,000 events were collected per sample and analyzed with FlowJo Software.

Data analysis

Statistical analysis was conducted to evaluate the significance between differences. For two-sample comparison, Student’s t test was used. For comparison between more than two groups, ANOVA was used; wherever applicable, a post hoc Fisher’s least significant difference (LSD) test was used for further pairwise comparison. A p value of <0.05 was regarded as statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lack of airway luminal T cells following i.m. DNA immunization

We have previously demonstrated that a single i.m. electroporated immunization with a plasmid DNA vaccine pAg85A-GM-CSF led to robust Ag85A-specific CD4 and CD8 T cell responses detected in the spleen, but the accomplished levels of protection from pulmonary M. tuberculosis challenge remained modest (9). The reasons underlying the general poor protection by i.m. DNA immunization have yet to be elucidated. To investigate the potential mechanisms, we examined the level of airway luminal T cells following i.m. pAg85A-GM-CSF immunization and compared this with T cell distribution in the lung interstitium and spleen, based on our enhanced understanding of the importance of airway luminal T cells for immunoprotection (23). Thus, BALB/c mice were immunized i.m. once with pAg85A-GM-CSF in conjunction with electroporation. Six weeks after immunization, mononuclear cells isolated from BAL, lung, and spleen were subjected to tetramer and ICCS to evaluate the Ag85A-specific T cell profile in these tissue compartments (Fig. 1A; DNA-PBS group). Although i.m. plasmid DNA vaccination stimulated potent CD4 and CD8 T cell responses in the spleen (Table I) as we have previously reported (9), we detected almost no T cell responses in the airway lumen (Fig. 1, B and D) and only small responses in the lung interstitium (Fig. 1, C and E). These data suggest the lack of airway luminal T cells to be the reason for poor immunoprotection by i.m. DNA immunization.


Figure 1
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  		FIGURE 1. Ag-specific T cell responses in the airway lumen and lung tissue. Mice were sacrificed 6 wk after immunization or treatment with DNA-protein, DNA-PBS, pGM-CSF/protein, or PBS-protein protocol (A), and mononuclear cells were isolated from BAL fluids (B, D, F, and G) and lung tissue (C and E) were subjected to tetramer (Tet) and ICCS and FACS. Data are presented as representative dot plots of percentage of CD4+IFN-{gamma}+, CD8+IFN-{gamma}+ or CD8+Tet+ T cells out of total CD4 or CD8 T cells (B and C) or as average absolute numbers of such T cells (D and G) or total CD3+ T cells (F) per BAL or per lung (E). Data are expressed as means ± SEM of four mice per group from two independent experiments (D and E) or of three mice (F and G). **, p ≤ 0.01; ***, p < 0.005 as compared with the corresponding DNA/PBS analyzed by Student’s t test (D and E) or compared with pGM-CSF-protein and PBS/protein groups analyzed by ANOVA (F and G).

 

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  		Table I. Ag-specific T cell responses in the spleena

 
Intranasal delivery of soluble Ag85 proteins mobilizes i.m. DNA immunization-activated T cells into the airway lumen

To investigate whether respiratory mucosal delivery of soluble M. tuberculosis Ags could elicit the influx into the airway luminal compartment of systemically distributed T cells activated by i.m. pAg85A-GM-CSF immunization, we administered a small amount of Ag85 complex proteins intranasally twice (once weekly for a total of 2 wk) starting at 4 wk post-i.m. immunization (Fig. 1A). These mice (DNA-protein) were then sacrificed 1 wk after the second delivery of Ag85 proteins, and Ag-specific T cells in BAL, lung, and spleen were analyzed by tetramer staining and ICCS and compared with those in DNA-PBS control mice. We found that airway exposure to soluble Ag85 proteins led to the remarkable increases in Ag-specific, tetramer+ CD8 or IFN-{gamma}-producing CD4 and CD8 T cells not only in the airway lumen but also in the lung interstitium (Fig. 1, B–E). In comparison, in the spleen there was relatively a decline in the number of IFN-{gamma}-producing CD4 and CD8 T cells in DNA-protein mice (Table I). We also found that two repeated administrations of Ag85 complex proteins were necessary given that one administration failed to mobilize systemically activated T cells into the airway of i.m.-immunized mice (data not shown). Furthermore, airway luminal T cell mobilization was Ag85A dependent or Ag specific given that exposure of DNA immunized mice to an irrelevant protein, OVA, failed to recruit systemically activated Ag85A-specific T cells into the airway lumen (data not shown). On the other hand, airway luminal T cell mobilization was systemic Ag85A-specific T cell priming dependent because airway exposure to Ag85 complex proteins alone in nonimmunized mice (PBS/protein) or in the mice injected i.m. only with GM-CSF-expressing plasmid DNA (pGM-CSF-protein) failed to elicit significant total T cell responses within the airway lumen, in sharp contrast to the DNA-protein group (Fig. 1F). As a result, Ag85A-specific tetramer+ or IFN-{gamma}+ T cells were absent in the airway of PBS-protein or pGM-CSF-protein mice (Fig. 1G). Airway exposure to Ag85 proteins alone in nonimmunized mice also failed to activate significant T cell responses in the spleen (data now shown). Together, these results suggest that airway luminal recruitment of Ag-specific T cells by airway mucosal Ag85 complex exposure was specific to DNA-immunized animals.

Experiments were next conducted to determine whether the T cells recruited in response to Ag85 complex protein exposure could persist within the airway lumen beyond 1 wk after the final administration of Ag85 Ag. To this end, pAg85A-GM-CSF i.m.-immunized mice were given two doses of Ag85 complex proteins intranasally and sacrificed at 4 wk after the second delivery of Ag85 proteins for examination of the persistence of Ag-specific T cells in the airway lumen (Fig. 2A). Significant Ag-specific IFN-{gamma}-producing CD4 and CD8 or tetramer+ CD8 T cells were still detected in the airway lumen of mice that were DNA immunized i.m. and exposed to Ag85 complex proteins (DNA-protein) by this time, in sharp contrast to very few T cells detected in the airway lumen of control mice (DNA-PBS; Fig. 2B). Compared with the number of T cells detected at 1 wk after the second Ag85 protein administration, an overall 20% of the initially detected airway luminal T cells was maintained by 4 wk after the second intranasal administration of Ag85 proteins. The amounts of Ag-specific T cells in the spleen were comparable at this time between DNA-protein and DNA-PBS groups (Fig. 2C). Together, the above data suggest that simple local deposition of Ag85 Ags is sufficient not only to mobilize the systemically activated T cells into but also to retain these cells within the airway lumen, with a relatively mild effect on splenic T cells.


Figure 2
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  		FIGURE 2. Persistence of Ag-specific T cells in the airway lumen. Mice were sacrificed 9 wk following immunization with DNA-PBS and DNA-protein protocols (A) and mononuclear cells isolated from BAL (B) and spleen (C) were subjected to tetramer (Tet), ICCS, and FACS. Data are expressed as the mean value ± SEM of absolute numbers of CD4+IFN-{gamma}+, CD8+IFN-{gamma}+ or CD8+Tet+ T cells per BAL or spleen from 3 mice per group. *, p < 0.05, **, p < 0.001 compared with the corresponding DNA-PBS control analyzed by using Student’s t test. The data are representative of two independent experiments.

 
Ag-elicited airway luminal T cells are capable of robust secondary recall responses upon pulmonary M. tuberculosis challenge

To examine the ability of airway luminal T cells elicited by Ag85 Ag exposure to respond to pulmonary mycobacterial exposure, mice were DNA immunized i.m. for 4 wk and treated intranasally with Ag85 complex proteins; 4 wk later, they were challenged via the airway with an attenuated strain of M. tuberculosis (H37Ra; DNA-protein-H37Ra; (Fig. 3A). A virulent M. tuberculosis was used as a surrogate to virulent M. tuberculosis H37Rv and could be handled conveniently outside of our P3 facility, thus permitting detailed immunological analysis. As controls, a group of mice that were DNA immunized but did not receive Ag85 complex proteins were also challenged with H37Ra (DNA-PBS-H37Ra), and another group of mice that were immunized and received intranasal Ag85 complex protein but not challenged with M. tuberculosis H37Ra (DNA-protein-PBS) were included. Five days after M. tuberculosis challenge, mice were sacrificed and airway luminal cells were collected and subjected to tetramer and ICCS analysis. As expected, the unchallenged DNA-protein mice (DNA-protein-PBS) had only small numbers of Ag-specific T cells in the airway lumen, whereas M. tuberculosis challenge hardly increased Ag-specific T cells in the airway lumen of the DNA-PBS-H37Ra group (Fig. 3, B and C). In sharp contrast, markedly increased numbers of tetramer+ or IFN-{gamma}-producing CD8 T cells were detected in the airway lumen of DNA/protein/H37Ra mice (Fig. 3, B and C). Such potent T cell recall responses were largely restricted to CD8 but not CD4 T cells. We also examined the splenocytes from these mice and found markedly increased Ag-specific IFN-{gamma} production by T cells isolated from DNA-protein-H37Ra mice (data not shown). Thus, these data suggest that i.m. DNA-immunized mice lack airway luminal T cells not only before but even after pulmonary mycobacterial challenge (DNA-PBS-H37Ra group), and this may explain well why normally i.m. DNA vaccination on its own provides poor immune protection. Furthermore, our data suggest that the airway luminal T cells elicited by mucosal delivery of soluble Ag85 proteins in i.m.-immunized mice (DNA-protein-H37Ra group) are capable of robust secondary recall responses upon pulmonary mycobacterial infection.


Figure 3
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  		FIGURE 3. Increased airway luminal T cells upon airway mycobacterial challenge. Mice were challenged intratracheally with M. tuberculosis H37Ra at 9 wk following immunization with DNA-PBS and DNA-protein protocols (A). A group of DNA-protein mice were left unchallenged as an additional control. Mice were then sacrificed 5 days post-M. tuberculosis challenge, and mononuclear cells isolated from BAL were subjected to tetramer (Tet), ICCS, and FACS. Data are presented as dot plots of percentage of CD4+IFN-{gamma}+, CD8+IFN-{gamma}+ or CD8+Tet+ T cells out of total CD4 or CD8 T cells (B) or as the mean value ± SEM of absolute numbers of CD4+IFN-{gamma}+, CD8+IFN-{gamma}+ or CD8+Tet+ T cells per BAL from 3 mice per group (C). *, p ≤ 0.05; ** p ≤ 0.01, compared with two other control groups analyzed by ANOVA.

 
Intranasal delivery of soluble Ag85 proteins enhances immune protection by i.m. DNA immunization from pulmonary M. tuberculosis challenge

To examine the immune protective potential of airway luminal T cells elicited by mucosal delivery of soluble Ag85 proteins in i.m. DNA-immunized mice, mice were immunized once i.m. in conjunction with electroporation and received Ag85 complex proteins intranasally at 4 wk as illustrated in Fig. 4A. These mice were then challenged via the airway with a virulent strain of M. tuberculosis (M. tuberculosis H37Rv) 1 wk after the second Ag85 Ag exposure (Fig. 4A; DNA-protein). As controls, a group of naive, i.m. plasmid DNA- or s.c BCG-immunized mice were included. The level of M. tuberculosis burden in the lung and the spleen was examined 4 wk postchallenge. As we have previously reported (9), i.m. DNA immunization on its own provided only a very small level of protection from M. tuberculosis challenge in both the lung and the spleen (Fig. 4, B and C). In contrast, the level of protection was significantly increased in the lung of DNA-protein-immunized mice, although it was not as good as that by BCG (Fig. 4B), and the level of protection was substantially enhanced in the spleen of these mice, comparable with that by BCG immunization (Fig. 4C).


Figure 4
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  		FIGURE 4. Enhanced immune protection by Ag-elicited airway luminal T cells. Mice were challenged via the airway with M. tuberculosis H37Rv at 6 wk following immunization with DNA/PBS and DNA/protein protocols (A). As controls, a group of mice were not immunized (naive) or immunized s.c with BCG vaccine. These mice were then sacrificed at 4 wk post-M. tuberculosis challenge (A). Lung (B) and spleen (C) from these mice were subjected to M. tuberculosis CFU assay. Data are expressed as mean values± SEM of 4–6 mice per group. *, p ≤ 0.05; **, p ≤ 0.01; ***, p < 0.005 compared with indicated groups analyzed by ANOVA and post hoc Fisher’s LSD tests.

 
To examine whether the persisting Ag-specific T cells elicited within the airway lumen by mucosal delivery of Ag85 Ags would retain their immune protective potential, mice were immunized once i.m. in conjunction with electroporation and received Ag85 complex proteins intranasally at 4 wk as illustrated in Fig. 5A. These mice were then challenged via the airway with M. tuberculosis H37Rv at 4 wk after the second Ag85 Ag exposure (Fig. 5A). The level of M. tuberculosis burden in the lung and the spleen was examined 4 wk postchallenge. Again, i.m. DNA immunization on its own provided only a modest level of protection from M. tuberculosis challenge both in the lung and the spleen (Fig. 5, B and C). In sharp contrast, DNA-protein-immunized mice continued to demonstrate a significant level of immune protection in the lung and spleen compared with unvaccinated and DNA-vaccinated animals and at this time point, the degree of such protection achieved by DNA-protein regimen was at least comparable with that by s.c BCG immunization (Fig. 5, B and C).


Figure 5
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  		FIGURE 5. Enhanced immune protection by Ag-elicited persisting airway luminal T cells. Mice were challenged via the airway with M. tuberculosis H37Rv at 9 wk following immunization with DNA-PBS and DNA-protein protocols (A). As controls, a group of mice were not immunized (naive) or immunized s.c with BCG vaccine. These mice were then sacrificed at 4 wk post-M. tuberculosis challenge (A). Lung (B) and spleen (C) from these mice were subjected to M. tuberculosis CFU assay. Data are expressed as mean values ± SEM of 4–6 mice per group. *, p ≤ 0.05; **, p ≤ 0.01 compared with indicated groups analyzed by ANOVA and post hoc Fisher’s LSD tests.

 
Lung inflammatory responses to respiratory mucosal delivery of Ag85 complex proteins

To begin understanding the mechanisms by which brief airway mucosal exposure to soluble Ag85 complex proteins mobilizes the Ag-specific T cells systemically activated by i.m. DNA vaccination, we first evaluated the level of production of proinflammatory cytokine TNF-{alpha} in the lung upon Ag85 protein delivery. Mice were DNA immunized i.m. and exposed intranasally twice to Ag85 proteins as illustrated in Fig. 6A. As controls, groups of mice that were naive or immunized but did not receive Ag85 proteins (DNA-PBS), or nonimmunized but received Ag85 protein treatment (naive-protein), were included. At 1 wk after the second Ag delivery, BAL fluids were collected and subjected to TNF-{alpha} ELISA (Fig. 6A). We observed a significant increase in the level of TNF-{alpha} in the lung of mice receiving Ag85 proteins, regardless of DNA immunization status (naïve-protein or DNA-protein) (Fig. 6B). In contrast, the level of TNF-{alpha} were minimal in the lung of DNA-PBS or naive mice (Fig. 6B). Because TNF-{alpha} is an alarm proinflammatory cytokine responsible for the secondary induction of chemokines (30), we next examined the profile of expression of inflammatory mediators, particularly chemokines, in the lung tissue of DNA-immunized mice on exposure to Ag85 complex proteins by using RPA. Indeed, DNA-protein-immunized mouse lung expressed heightened levels of mRNAs for a number of cytokines/chemokines (Fig. 6C). To verify whether there were correspondingly increased chemokine protein levels in the lung, we elected to measure the concentration of IP-10. We found that indeed in accordance with its ability to induce TNF-{alpha}, Ag85 complex treatment, regardless of DNA immunization status (naive-protein or DNA-protein), significantly enhanced the protein level of this chemokine in the lung, in contrast to much lower levels of IP-10 in the lung of DNA-OVA or naive mice (Fig. 6D). Despite up-regulated responses of proinflammatory cytokines and chemokines in the lung by exposure to mycobacterial Ag85 complex proteins, we found only a very mild non-Ag-specific inflammatory cellular infiltration in lung tissue of naive mice (data not shown), in keeping with the few T cells seen in the airway lumen (Fig. 1F). Taken together, these observations suggest that the proinflammatory signals, particularly chemokine responses, triggered by respiratory mucosal exposure to soluble mycobacterial Ags, may be involved in the airway luminal influx of the T cells systemically activated by i.m. DNA immunization.


Figure 6
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  		FIGURE 6. Proinflammatory responses in the lung to airway delivery of soluble mycobacterial Ags. Mice were sacrificed 6 wk following immunization with DNA-protein or DNA-OVA or DNA-PBS or following treatment with mycobacterial proteins (naïve-protein) or nothing (naive) (A). BAL samples obtained from these mice were subjected to TNF-{alpha} (B) or IP-10 (D) detection assay. Lung tissue RNA samples harvested from these mice were subjected to RPA for assessment of gene expression of proinflammatory mediators (C). L32 and GAPDH gene expressions were used as housekeeping controls. The data on TNF-{alpha} and IP-10 are expressed as mean values ± SEM of 2–3 mice/group. *, p ≤ 0.05; ***, p < 0.005 compared with DNA-PBS and naive control groups analyzed by ANOVA.

 
MIP-1{alpha} or IP-10 is required for Ag-triggered airway luminal accumulation of CD8 T cells systemically activated by i.m. DNA immunization

To further understand the action of chemokines in Ag85 protein-triggered T cell influx to the airway, we elected to address the role of MIP-1{alpha} and IP-10 in our model. Not only were these two chemokines induced by Ag85 protein exposure in the lung, they have been shown to play an important role in the recruitment of memory T cells, particularly CD8 T cells (31, 32, 33). To this end, mice were DNA-immunized once i.m. and treated with Ag85 complex proteins as illustrated in Fig. 7A and injected i.p with polyclonal neutralizing Abs against MIP-1{alpha} or IP-10 or with normal control serum 1 day before the first administration of Ag85 complex proteins (Fig. 7A). Such Ab treatment was repeated at days 5 and 8 after the first Ag85 complex protein delivery (Fig. 7A). Mice were sacrificed 1 wk following the second administration of Ag85 complex proteins. Analysis of the T cell profile was performed with BAL cells and splenocytes by means of ICCS and tetramer staining. The in vivo neutralization of either MIP-1{alpha} or IP-10 alone strongly abrogated Ag-specific T cell accumulation into the airway lumen by 92 and 78%, respectively, as compared with the control (Fig. 7, B and C). This led to markedly reduced absolute numbers of Ag85A tetramer+ or IFN-{gamma}-producing CD8 T cells in the airway lumen (Fig. 7C). This reduction of T cell accumulation in the airway lumen by chemokine blockade coincided with a relatively increased number of tetramer+ T cells in the spleen (Fig. 7, D and E). These results indicate that MIP-1{alpha} or IP-10 plays an important role in the recruitment of CD8 T cells triggered by airway mucosal deposition of soluble mycobacterial Ags.


Figure 7
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  		FIGURE 7. Role of chemokines in airway luminal accumulation of systemically activated CD8 T cells. Mice were immunized and treated as described and injected i.p. with polyclonal neutralizing Abs against MIP-1{alpha} or IP-10 or with normal control serum 1 day before the first administration of Ag85 complex proteins (A). Ab treatment was repeated at days 5 and 8 after the first Ag85 complex protein delivery (A). Mice were sacrificed 1 wk following the second administration of proteins. Analysis of the T cell profile was performed with BAL cells (B and C) and splenocytes (D and E) by means of ICCS or tetramer staining. Data are presented as dot plots of percentage of CD8+IFN-{gamma}+ or CD8+Tet+ T cells out of total CD8 T cells (B and D) or as the mean value ± SEM of absolute numbers of CD8+IFN-{gamma}+ or CD8+Tet+ T cells per BAL (C) or spleen (E) from 3 mice per group.*, p ≤ 0.05; ***, p < 0.005 compared with anti-IP-10 and anti-MIP-1{alpha} groups analyzed by ANOVA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have shown that i.m. DNA immunization fails to elicit the accumulation of airway luminal T cells whereas it induces potent T cell activation in the systemic lymphoid organs such as the spleen. Furthermore, pulmonary mycobacterial exposure is unable to mobilize such systemically activated T cells into the airway lumen at least within the first five days postmycobacterial challenge. We have further demonstrated that simply two repeated intranasal deliveries of a small amount of soluble mycobacterial Ags would be able to cause a robust influx of systemically activated T cells, particularly CD8 T cells, into the airway lumen following a single i.m. DNA immunization. Such airway luminal T cells could undergo secondary expansion and activation upon pulmonary M. tuberculosis exposure and confer much enhanced immune protection from M. tuberculosis challenge. The mucosal deposition of soluble mycobacterial Ags was found to elicit proinflammatory cytokine and chemokine responses in the lung, and both MIP-1{alpha} and IP-10 were found to play a critical role in T cell recruitment mediated by mycobacterial Ags.

i.m. plasmid DNA-based vaccination represents one of the promising immunization strategies and has been widely explored for a number of infectious agents including hepatitis B virus, HSV, HIV, malaria, influenza, and TB (7, 34, 35, 36, 37). DNA vaccines are well known for their capability to elicit potent systemic Ab and T cell responses (7). However, when it is applied for immunization against pulmonary TB or mucosal HIV infection, the level of protection has been in general considered to be poor or disappointing (9, 10, 11, 12, 38). The mechanisms for this phenomenon have remained to be fully understood, and whether or how such defect could be improved is yet to be addressed. Our current study suggests the lack of airway luminal T cells following i.m. DNA immunization to be the mechanism underlying poor respiratory mucosal protection as is the case for i.m. adenovirus-mediated immunization (25). We further found that pulmonary mycobacterial challenge at least within the initial 5 days could not create a microenvironment conducive to recruiting systemically activated T cells into the airway lumen. This may be explained by our current understanding that live mycobacteria have evolved to dampen host innate immune responses and that the doubling time for mycobacteria is lengthy (39, 40, 41). This contention is further supported by our observation that airway deposition of soluble mycobacterial proteins could effectively mobilize systemically activated T cells into the airway lumen by creating a tissue microenvironment rich in proinflammatory and T cell chemotactic factors. Our findings also suggest that whether Ag-specific T cells could be mobilized into the airway lumen within 5 days upon airway mycobacterial exposure holds the key to the ensuing immunoprotective efficacy. Early appearance of effector T cells within the airway lumen may play an important role in harnessing mycobacterial growth within infected cells at early stages of infection. Contrast to the potent effect of airway deposition of soluble mycobacterial proteins on airway luminal mobilization of Ag-specific T cells in i.m. DNA-immunized hosts (DNA-protein), we found that the mice treated with PBS-protein, pGM-CSF-protein, or DNA-OVA protocol resembled the DNA/PBS mice and had little T cell responses within the airway lumen. Similar to i.m. adenovirus-mediated immunization (12, 23), the inability of DNA-PBS protocol to elicit airway luminal T cells led to poor immunoprotection in the lung despite systemic T cell activation. These observations together suggest that the recruitment of systemically activated Ag-specific T cells into the airway lumen of DNA-protein mice is critically dependent not only on mucosal delivery of the specific mycobacterial Ag but also on parenteral immunization and robust systemic Ag-specific T cell priming. Together these findings point to the need to develop the strategies to improve airway luminal T cell accumulation following parenteral genetic TB immunization. Such knowledge also holds important implication for improving the efficacy of parenteral genetic immunization for other mucosal intracellular infectious diseases.

In our current study, we have identified a simple, safe, and effective way to restore airway luminal T cells and immunoprotection following i.m. DNA TB vaccination. This method involves only two repeated intranasal administrations of a small dose of soluble mycobacterial proteins. Although repeated intranasal administration of soluble mycobacterial proteins elicited an overt proinflammatory cytokine response in the lung regardless of immunization status, Ag-specific T cell recruitment occurred only to DNA-immunized animals, but not to nonimmunized mice or those receiving pGM-CSF plasmid DNA (nonvaccine DNA), indicating that such local soluble mycobacterial Ag exposure and cytokine responses by themselves even when an immune adjuvant is systemically present (GM-CSF) are still not adequate enough for robust T cell activation and airway luminal mobilization in the absence of potent systemic immunization. Although such protein deposition creates a proinflammatory response in the lung sufficient to draw systemically activated mycobacterial Ag-specific T cells into the airway lumen in DNA-immunized mice, it on its own does not cause immunopathology. This mode of soluble myocbacterial Ag provocation employed in our current study represents a significant simplification from the mode required for improving protection by i.m. viral vaccination (25). The delivery of soluble mycobacterial Ags markedly improves immunoprotection by only a single i.m. DNA injection in our study. Although our DNA immunization was facilitated by electroporation, our current findings do imply that airway mucosal soluble M. tuberculosis Ag delivery will also help cut down the number of repeated i.m. DNA immunizations that are conventionally required to fully activate adaptive immune components (7, 9, 10, 11, 12, 13).

Our current findings also indicate that the systemically activated and located T cells by i.m. DNA immunization are not defective in their immune protective capacity because when they are mobilized into the airway lumen they are able to render robust protection against pulmonary M. tuberculosis challenge. We have further demonstrated that as long as these T cells enter the airway lumen, they would be able to persist, retain their immunoprotective potential, and undergo quick expansion and activation upon mycobacterial exposure, regardless of markedly reduced numbers of such T cells, particularly CD8 T cells, within the airway lumen with time. We have also previously noticed a much greater retraction of Ag85A-specific CD8 T cell populations relative to CD4 T cells within the airway lumen after intranasal Ag85 complex administrations in a viral immunization model (25). This could be due to differential mechanisms for the maintenance of memory CD4 and CD8 T cell subsets in the airway. Our result that increased numbers of airway luminal T cells by mucosal deposition of soluble mycobacterial Ags coincide with a decreased number of Ag-specific T cells in the spleen suggests that the spleen is one of the systemic supplies of such T cells. At this point, although the mechanisms underlying the long-term maintenance of airway luminal Ag85A-specific CD8 T cells in our model remain to be fully understood, the previous studies conducted in respiratory virus infection models have suggested that both de novo proliferation and continuing recruitment may play a role (42, 43, 44, 45, 46). We have further noticed in our study that upon secondary mycobacterial infection, the major expanded and activated T cells are of the CD8, but not CD4, subset. The majority of the CD8 T cells recruited into the airway lumen of DNA/protein group within the initial 5 days upon secondary mycobacterial infection were Ag specific (tetramer+), and the size of tetramer+ CD8 T cell population was always greater than IFN-{gamma}-producing CD8 T cells, suggesting that not all Ag-specific T cells were activated. We have often observed such difference in tetramer+ and IFN-{gamma}+ CD8 T cells in other studies (22, 23, 25). However, the percentage of IFN-{gamma}+ CD8 T cells relative to tetramer+ cells in the DNA-protein group was much higher than in DNA-PBS group, suggesting a higher level of activation of Ag-specific T cells in the former. Such readiness of Ag-specific T cells in the airway lumen early during the course of mycobacterial infection may play a critical role in the effectiveness of immune protection. The early readiness of protective T cells at the site of infection contrasts the kinetics of routine T cell activation triggered by a natural mycobacterial infection, which usually entails 2–3 wk to be full-blown (47). Overall our data suggest that CD8 T cells represent a primary immunoprotective effector in our model. This information holds implication for helping the future design of TB vaccines for immunization in HIV hosts.

We identified via our study that both CC MIP-1{alpha} and CXC IP-10 chemokines are involved in T cell recruitment triggered by intranasal Ag85 complex protein delivery. These two chemokines have previously been reported to play an important role in Th1 and cytotoxic T cell recruitment (31, 32, 33). Circulating memory T cells have been found to express higher levels of receptors for such chemokines (32, 48). Our findings further suggest that regardless of certain functional redundancy, these chemokines cannot compensate entirely for the lack of each other. Thus, blockade of either MIP-1{alpha} or IP-10 resulted in markedly diminished T cells within the airway lumen. Such decreased numbers of T cells in the airway were temporally associated with increased Ag-specific T cells in the spleen, again supporting our notion that the spleen is a systemic supply site.

In conclusion, our data demonstrate for the first time that the poor protection by i.m. DNA TB immunization is due to lack of airway luminal T cells before and upon pulmonary mycobacterial infection and that such lack of airway luminal T cells and protection could be effectively corrected by brief airway mucosal deposition of soluble mycobacterial Ags, which creates a tissue microenvironment conducive to the recruitment of systemically activated T cells following i.m. plasmid DNA vaccination. We believe that these findings will help guide the development of future DNA immunization strategies not only for TB but also for other mucosal intracellular infections.


   Acknowledgments
 
The authors are indebted to Dr. Marcel Behr for providing M. tuberculosis H37Ra organisms; Susanna Goncharova for assistance in conducting the Luminex assay; and Christopher R. Shaler for assistance in graphic preparation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study is supported by funds from the Canadian Institutes for Health Research. Back

2 Address correspondence and reprint requests to Dr. Zhou Xing, Room 4012-MDCL, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada. E-mail address: xingz{at}mcmaster.ca Back

3 Abbreviations used in this paper: TB, tuberculosis; BCG, bacillus Calmette-Guérin; IP-10, IFN-inducible protein-10; RPA, RNase protection assay; BAL, bronchoalveolar lavage; ICCS, intracellular cytokine staining; LSD, least significant difference. Back

Received for publication April 2, 2008. Accepted for publication August 8, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Dye, C., S. Scheele, P. Dolin, V. Pathania, M. C. Raviglione. 1999. Consensus statement: global burden of tuberculosis: estimated incidence, prevalence, and mortality by country: WHO Global Surveillance and Monitoring Project. J. Am. Med. Assoc. 282: 677-686. [Abstract/Free Full Text]
  2. Brewer, T. F.. 2000. Preventing tuberculosis with bacillus Calmette-Guérin vaccine: a meta-analysis of the literature. Clin. Infect. Dis. 31: (Suppl. 3):S64-S67. [Medline]
  3. Colditz, G. A., T. F. Brewer, C. S. Berkey, M. E. Wilson, E. Burdick, H. V. Fineberg, F. Mosteller. 1994. Efficacy of BCG vaccine in the prevention of tuberculosis: meta-analysis of the published literature. J. Am. Med. Assoc. 271: 698-702. [Abstract/Free Full Text]
  4. Fine, P. E.. 1995. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 346: 1339-1345. [Medline]
  5. Xing, Z., T. J. Carters. 2007. Heterologous boost vaccines for bacillus Calmette-Guérin prime immunization against tuberculosis. Expert Rev. Vaccines 6: 539-546. [Medline]
  6. Xing, Z., M. Santosuosso, S. McCormick, T. C. Yang, J. Millar, M. Hitt, Y. Wan, J. Bramson, H. M. Vordermeier. 2005. Recent advances in the development of adenovirus- and poxvirus-vectored tuberculosis vaccines. Curr. Gene Ther. 5: 485-492. [Medline]
  7. Huygen, K.. 2005. Plasmid DNA vaccination. Microbes Infect. 7: 932-938. [Medline]
  8. Wang, J., Z. Xing. 2002. Tuberculosis vaccines: the past, present and future. Expert Rev. Vaccines 1: 341-354. [Medline]
  9. Zhang, X., M. Divangahi, P. Ngai, M. Santosuosso, J. Millar, A. Zganiacz, J. Wang, J. Bramson, Z. Xing. 2007. Intramuscular immunization with a monogenic plasmid DNA tuberculosis vaccine: enhanced immunogenicity by electroporation and co-expression of GM-CSF transgene. Vaccine 25: 1342-1352. [Medline]
  10. Huygen, K., J. Content, O. Denis, D. L. Montgomery, A. M. Yawman, R. R. Deck, C. M. DeWitt, I. M. Orme, S. Baldwin, C. D'Souza, et al 1996. Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat. Med. 2: 893-898. [Medline]
  11. Tanghe, A., S. D'Souza, V. Rosseels, O. Denis, T. H. Ottenhoff, W. Dalemans, C. Wheeler, K. Huygen. 2001. Improved immunogenicity and protective efficacy of a tuberculosis DNA vaccine encoding Ag85 by protein boosting. Infect. Immun. 69: 3041-3047. [Abstract/Free Full Text]
  12. Wang, J., L. Thorson, R. W. Stokes, M. Santosuosso, K. Huygen, A. Zganiacz, M. Hitt, Z. Xing. 2004. Single mucosal, but not parenteral, immunization with recombinant adenoviral-based vaccine provides potent protection from pulmonary tuberculosis. J. Immunol. 173: 6357-6365. [Abstract/Free Full Text]
  13. Gilbert, S. C., V. S. Moorthy, L. Andrews, A. A. Pathan, S. J. McConkey, J. M. Vuola, S. M. Keating, T. Berthoud, D. Webster, H. McShane, A. V. Hill. 2006. Synergistic DNA-MVA prime-boost vaccination regimes for malaria and tuberculosis. Vaccine 24: 4554-4561. [Medline]
  14. McShane, H., R. Brookes, S. C. Gilbert, A. V. Hill. 2001. Enhanced immunogenicity of CD4+ T-cell responses and protective efficacy of a DNA-modified vaccinia virus Ankara prime-boost vaccination regimen for murine tuberculosis. Infect. Immun. 69: 681-686. [Abstract/Free Full Text]
  15. Dietrich, J., C. Andersen, R. Rappuoli, T. M. Doherty, C. G. Jensen, P. Andersen. 2006. Mucosal administration of Ag85B-ESAT-6 protects against infection with Mycobacterium tuberculosis and boosts prior bacillus Calmette-Guérin immunity. J. Immunol. 177: 6353-6360. [Abstract/Free Full Text]
  16. Foxwell, A. R., J. M. Kyd, A. W. Cripps. 2003. Mucosal immunization against respiratory bacterial pathogens. Expert Rev. Vaccines 2: 551-560. [Medline]
  17. Giri, P. K., S. B. Sable, I. Verma, G. K. Khuller. 2005. Comparative evaluation of intranasal and subcutaneous route of immunization for development of mucosal vaccine against experimental tuberculosis. FEMS Immunol. Med. Microbiol. 45: 87-93. [Medline]
  18. Kohama, H., M. Umemura, Y. Okamoto, A. Yahagi, H. Goga, T. Harakuni, G. Matsuzaki, T. Arakawa. 2007. Mucosal immunization with recombinant heparin-binding haemagglutinin adhesin suppresses extrapulmonary dissemination of Mycobacterium bovis bacillus Calmette-Guérin (BCG) in infected mice. Vaccine 26: 924-932. [Medline]
  19. Oliveira, M. L., A. P. Areas, P. L. Ho. 2007. Intranasal vaccines for protection against respiratory and systemic bacterial infections. Expert Rev. Vaccines 6: 419-429. [Medline]
  20. Santosuosso, M., S. McCormick, Z. Xing. 2005. Adenoviral vectors for mucosal vaccination against infectious diseases. Viral Immunol. 18: 283-291. [Medline]
  21. Xing, Z., B. D. Lichty. 2006. Use of recombinant virus-vectored tuberculosis vaccines for respiratory mucosal immunization. Tuberculosis 86: 211-217. [Medline]
  22. Santosuosso, M., S. McCormick, X. Zhang, A. Zganiacz, Z. Xing. 2006. Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis. Infect. Immun. 74: 4634-4643. [Abstract/Free Full Text]
  23. Santosuosso, M., X. Zhang, S. McCormick, J. Wang, M. Hitt, Z. Xing. 2005. Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen. J. Immunol. 174: 7986-7994. [Abstract/Free Full Text]
  24. Bivas-Benita, M., T. H. Ottenhoff, H. E. Junginger, G. Borchard. 2005. Pulmonary DNA vaccination: concepts, possibilities and perspectives. J. Controlled Release 107: 1-29. [Medline]
  25. Santosuosso, M., S. McCormick, E. Roediger, X. Zhang, A. Zganiacz, B. D. Lichty, Z. Xing. 2007. Mucosal luminal manipulation of T cell geography switches on protective efficacy by otherwise ineffective parenteral genetic immunization. J. Immunol. 178: 2387-2395. [Abstract/Free Full Text]
  26. Divangahi, M., T. Yang, K. Kugathasan, S. McCormick, S. Takenaka, G. Gaschler, A. Ashkar, M. Stampfli, J. Gauldie, J. Bramson, et al 2007. Critical negative regulation of type 1 T cell immunity and immunopathology by signaling adaptor DAP12 during intracellular infection. J. Immunol. 179: 4015-4026. [Abstract/Free Full Text]
  27. Divangahi, M., A. Demoule, G. Danialou, L. Yahiaoui, W. Bao, Z. Xing, B. J. Petrof. 2007. Impact of IL-10 on diaphragmatic cytokine expression and contractility during Pseudomonas infection. Am. J. Respir. Cell Mol. Biol. 36: 504-512. [Abstract/Free Full Text]
  28. Demoule, A., M. Divangahi, L. Yahiaoui, G. Danialou, D. Gvozdic, K. Labbe, W. Bao, B. J. Petrof. 2006. Endotoxin triggers nuclear factor-{kappa}B-dependent up-regulation of multiple proinflammatory genes in the diaphragm. Am. J. Respir. Crit. Care Med. 174: 646-653. [Abstract/Free Full Text]
  29. Fernandez, S., A. Gillgrass, C. Kaushic. 2007. Differential responses of murine vaginal and uterine epithelial cells prior to and following herpes simplex virus type 2 (HSV-2) infection. Am. J. Reprod. Immunol. 57: 367-377. [Medline]
  30. Roach, D. R., A. G. Bean, C. Demangel, M. P. France, H. Briscoe, W. J. Britton. 2002. TNF regulates chemokine induction essential for cell recruitment, granuloma formation, and clearance of mycobacterial infection. J. Immunol. 168: 4620-4627. [Abstract/Free Full Text]
  31. Taub, D. D., D. L. Longo, W. J. Murphy. 1996. Human interferon-inducible protein-10 induces mononuclear cell infiltration in mice and promotes the migration of human T lymphocytes into the peripheral tissues and human peripheral blood lymphocytes-SCID mice. Blood 87: 1423-1431. [Abstract/Free Full Text]
  32. Qin, S., J. B. Rottman, P. Myers, N. Kassam, M. Weinblatt, M. Loetscher, A. E. Koch, B. Moser, C. R. Mackay. 1998. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J. Clin. Invest. 101: 746-754. [Medline]
  33. Krathwohl, M. D., J. L. Anderson. 2006. Chemokine CXCL10 (IP-10) is sufficient to trigger an immune response to injected antigens in a mouse model. Vaccine 24: 2987-2993. [Medline]
  34. Wang, S., J. S. Kennedy, K. West, D. C. Montefiori, S. Coley, J. Lawrence, S. Shen, S. Green, A. L. Rothman, F. A. Ennis, et al 2008. Cross-subtype antibody and cellular immune responses induced by a polyvalent DNA prime-protein boost HIV-1 vaccine in healthy human volunteers. Vaccine 26: 1098-1110. [Medline]
  35. Pol, S., M. L. Michel. 2006. Therapeutic vaccination in chronic hepatitis B virus carriers. Expert Rev. Vaccines 5: 707-716. [Medline]
  36. Dunachie, S. J., M. Walther, J. E. Epstein, S. Keating, T. Berthoud, L. Andrews, R. F. Andersen, P. Bejon, N. Goonetilleke, I. Poulton, et al 2006. A DNA prime-modified vaccinia virus ankara boost vaccine encoding thrombospondin-related adhesion protein but not circumsporozoite protein partially protects healthy malaria-naive adults against Plasmodium falciparum sporozoite challenge. Infect. Immun. 74: 5933-5942. [Abstract/Free Full Text]
  37. Drape, R. J., M. D. Macklin, L. J. Barr, S. Jones, J. R. Haynes, H. J. Dean. 2006. Epidermal DNA vaccine for influenza is immunogenic in humans. Vaccine 24: 4475-4481. [Medline]
  38. Shiver, J. W., T. M. Fu, L. Chen, D. R. Casimiro, M. E. Davies, R. K. Evans, Z. Q. Zhang, A. J. Simon, W. L. Trigona, S. A. Dubey, et al 2002. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity. Nature 415: 331-335. [Medline]
  39. Geijtenbeek, T. B., S. J. Van Vliet, E. A. Koppel, M. Sanchez-Hernandez, C. M. Vandenbroucke-Grauls, B. Appelmelk, Y. Van Kooyk. 2003. Mycobacteria target DC-SIGN to suppress dendritic cell function. J. Exp. Med. 197: 7-17. [Abstract/Free Full Text]
  40. Sundaramurthy, V., J. Pieters. 2007. Interactions of pathogenic mycobacteria with host macrophages. Microbes Infect. 9: 1671-1679. [Medline]
  41. Banaiee, N., E. Z. Kincaid, U. Buchwald, W. R. Jacobs, Jr, J. D. Ernst. 2006. Potent inhibition of macrophage responses to IFN-{gamma} by live virulent Mycobacterium tuberculosis is independent of mature mycobacterial lipoproteins but dependent on TLR2. J. Immunol. 176: 3019-3027. [Abstract/Free Full Text]
  42. Hogan, R. J., L. S. Cauley, K. H. Ely, T. Cookenham, A. D. Roberts, J. W. Brennan, S. Monard, D. L. Woodland. 2002. Long-term maintenance of virus-specific effector memory CD8+ T cells in the lung airways depends on proliferation. J. Immunol. 169: 4976-4981. [Abstract/Free Full Text]
  43. Kohlmeier, J. E., S. C. Miller, D. L. Woodland. 2007. Cutting edge: antigen is not required for the activation and maintenance of virus-specific memory CD8+ T cells in the lung airways. J. Immunol. 178: 4721-4725. [Abstract/Free Full Text]
  44. Kohlmeier, J. E., D. L. Woodland. 2006. Memory T cell recruitment to the lung airways. Curr. Opin. Immunol. 18: 357-362. [Medline]
  45. Zammit, D. J., D. L. Turner, K. D. Klonowski, L. Lefrancois, L. S. Cauley. 2006. Residual antigen presentation after influenza virus infection affects CD8 T cell activation and migration. Immunity 24: 439-449. [Medline]
  46. Moyron-Quiroz, J. E., J. Rangel-Moreno, L. Hartson, K. Kusser, M. P. Tighe, K. D. Klonowski, L. Lefrancois, L. S. Cauley, A. G. Harmsen, F. E. Lund, T. D. Randall. 2006. Persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs. Immunity 25: 643-654. [Medline]
  47. Wolf, A. J., L. Desvignes, B. Linas, N. Banaiee, T. Tamura, K. Takatsu, J. D. Ernst. 2008. Initiation of the adaptive immune response to Mycobacterium tuberculosis depends on antigen production in the local lymph node, not the lungs. J. Exp. Med. 205: 105-115. [Abstract/Free Full Text]
  48. Bonecchi, R., G. Bianchi, P. P. Bordignon, D. D'Ambrosio, R. Lang, A. Borsatti, S. Sozzani, P. Allavena, P. A. Gray, A. Mantovani, F. Sinigaglia. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187: 129-134. [Abstract/Free Full Text]



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