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The Journal of Immunology, 2003, 171: 1602-1609.
Copyright © 2003 by The American Association of Immunologists

Enhanced Immunogenicity and Protective Efficacy Against Mycobacterium tuberculosis of Bacille Calmette-Guérin Vaccine Using Mucosal Administration and Boosting with a Recombinant Modified Vaccinia Virus Ankara1

Nilu P. Goonetilleke2, Helen McShane, Carolyn M. Hannan, Richard J. Anderson, Roger H. Brookes and Adrian V. S. Hill

Nuffield Department of Clinical Medicine, Oxford University, John Radcliffe Hospital, Oxford, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Heterologous prime-boost immunization strategies can evoke powerful T cell immune responses and may be of value in developing an improved tuberculosis vaccine. We show that recombinant modified vaccinia virus Ankara, expressing Mycobacterium tuberculosis Ag 85A (M.85A), strongly boosts bacille Calmette-Guérin (BCG)-induced Ag 85A specific CD4+ and CD8+ T cell responses in mice. A comparison of intranasal (i.n.) and parenteral immunization of BCG showed that while both routes elicited comparable T cell responses in the spleen, only i.n. delivery elicited specific T cell responses in the lung lymph nodes, and these responses were further boosted by i.n. delivery of M.85A. Following aerosol challenge with M. tuberculosis, i.n. boosting of BCG with either BCG or M.85A afforded unprecedented levels of protection in both the lungs (2.5 log) and spleens (1.5 log) compared with naive controls. Protection in the lung correlated with the induction of Ag 85A-specific, IFN-{gamma}-secreting T cells in lung lymph nodes. These findings support further evaluation of mucosally targeted prime-boost vaccination approaches for tuberculosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Tuberculosis is caused by the respiratory pathogen Mycobacterium tuberculosis and kills ~2 million people each year, predominantly in the developing world (http://www.who.int/gtb/publications/globrep01/index.html). The only licensed vaccine against M. tuberculosis, bacille Calmette-Guérin (BCG)3 (1), is an attenuated strain of Mycobacterium bovis, which in developing countries is typically administered intradermally as a single dose to newborn infants. Review of many studies suggests that BCG vaccination is protective against childhood meningeal tuberculosis and systemic forms of the disease. However, protective efficacy is variable (ranging from 0–80%) (2) against adult pulmonary disease, the major global cause of tuberculosis mortality, and wanes with time (3). The basis of the variability is uncertain. Recent evidence suggests that exposure to high levels of environmental mycobacteria may diminish the protective effects of BCG (4). Even so, 80% of infants throughout the world receive BCG each year (http://www.who.int/inf-fs/en/fact104.html). This implies that trials of future tuberculosis vaccine candidates will probably be performed in BCG-vaccinated communities. Moreover, these vaccines should aim to protect where BCG fails, namely, against adult pulmonary tuberculosis.

The development of a new vaccine against tuberculosis has been aided by increased understanding of the immune responses induced following M. tuberculosis infection. Approximately 90% of people infected with this pathogen do not develop active disease, indicating that the host protective response can normally control bacterial growth. Protection from tuberculosis is associated with the maintenance of a strong cell-mediated response to infection involving both CD4+ and CD8+ T cells and the ability to respond with Th1-type cytokines, particularly IFN-{gamma} (5, 6). BCG vaccination induces IFN-{gamma}-secreting T cells, predominately of the CD4+ T cell phenotype, and many that cross-react with M. tuberculosis proteins (7). Recent studies suggest that BCG delivered parenterally may fail to induce T cell immune responses in the lung mucosa, which may be critical for protection against pulmonary disease (8, 9). These studies show that long-lived T cell populations in the mucosa are best achieved by mucosal delivery of vaccines. They support M. tuberculosis challenge studies in macaques and guinea pigs that have shown aerosol administration of BCG to be more protective than parenteral (p.) vaccination (10, 11). BCG has also been given as an aerosol to healthy adults and children and repeatedly to patients with metastatic lung disease (12, 13, 14). Respiratory function tests and postmortem examination of cancer patients administered BCG by aerosol indicated no significant pulmonary dysfunction or disseminated BCG infection (13, 14).

More recent strategies to induce enhanced T cell responses in tuberculosis vaccine research have harnessed recombinant DNA technology, using plasmid, bacterial, or viral vectors and recombinant protein to express M. tuberculosis Ags. Much attention has focused on the highly immunogenic Ag 85 complex. This is a family of proteins comprising Ags 85A, 85B, and 85C secreted by M. tuberculosis, BCG, and many other species of mycobacteria (15). Parenteral vaccination with either 85A or B as a protein with adjuvant or expressed by a plasmid or a viral vector has not achieved levels of protection greater than a single BCG immunization in animal models (16, 17).

We have previously shown that DNA85A vaccination boosted with a modified vaccinia virus Ankara (MVA) expressing Ag 85A (Ag85A) induced high frequencies of Ag85A-specific, IFN-{gamma}-secreting T cells and afforded protection equivalent to BCG following M. tuberculosis challenge (18). Horwitz et al. (19) have recently developed a recombinant vaccine that elicited greater protection than low dose BCG in a guinea pig model. This vaccine is a recombinant BCG that overexpresses Ag 85B, suggesting that the inclusion of BCG may facilitate the design of future and more efficacious vaccines against tuberculosis.

In this study we investigate whether in a mouse M. tuberculosis aerosol challenge model, BCG-induced protection can be improved by mucosal lung delivery and/or boosting with a second dose of BCG or M.85A.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and immunizations

Eight- to 10-wk-old female BALB/c (H-2d) mice (Harlan Orlac, Blackthorn U.K.) were used in these studies. Generation of M.85A has been previously reported (18). Parenteral immunizations were given in the ear (25 µl/ear) or the left hind-footpad (50 µl). Intranasal (i.n.) immunizations were performed under light anesthesia (halothane), administered as a 100-µl bolus over 5 s. Immediately following i.n. BCG vaccination (Pasteur 1173P2D2) at doses of either 107 or 105 CFU, 1% of the total CFU delivered could be cultured from the lungs. In all the studies described, the MVA dose given was 106 PFU.

Cell culture and peptides

All cells were cultured in {alpha}-MEM (Life Technologies, Paisley, U.K.) supplemented with 10% FBS (Sigma-Aldrich, Watford, U.K.), 0.05 mM 2-ME (Life Technologies), 10 µM HEPES buffer (Life Technologies), 20 mM L-glutamine (Life Technologies), and 100 µg/ml penicillin/streptomycin (Life Technologies). The sequences of peptides used in assays are -P11-EWYDQSGLSV VMPVGGQSSF (aa 102–121) and P15-TFLTSELPGW LQANRHVKPT (aa 142–161) from Ag85A (GenBank accession no. CAA 17868), and PB9-SYIPSAEKI (aa 372–80) from circumsporozoite protein from Plasmodium berghei (GenBank accession no. AAA29541; Research Genetics, Huntsville, AL).

ELISPOT assays

Specific IFN-{gamma} secretion by spleen, lung, or lymph node cell suspensions was assayed by ELISPOT following 18- to 20-h incubation of cells with Ag (20). Isolation of lymphocytes from perfused lungs has been described previously (21). The concentration of Ags used were purified protein derivative (PPD) at 10 µg/ml (SSI, Copenhagen, Denmark), Ag85A culture filtrate protein at 10 µg/ml (22), and individual peptides at 2 µg/ml. Results are expressed as the mean spot-forming cells per million cells (SFC/106 cells) ± SEM.

51Cr release assays

Cells were stimulated with 2 µg/ml of peptide P11 and cultured for 7 days. Cells were fed 10 U/ml Lymphocult-T (Biotest, Dreieich, Germany) on day 3, harvested, then incubated for 4 h with 51Cr-loaded P815 cells (TIB-64; American Type Culture Collection, Manassas, VA). Specific lysis was calculated as follows: 100 x [(experimental radioactivity - spontaneous radioactivity)/(total radioactivity - spontaneous radioactivity)]. Results are expressed as the mean percent specific lysis ± SEM.

M. tuberculosis aerosol challenge

M. tuberculosis strain H37Rv (Trudeau Mycobacterial Culture Collection 102) was grown in Dubos medium at 37°C for 21–28 days. The solution was centrifuged, suspended in a tryptic soy broth-glycerol, and stored at -70°C after titration. Stock solutions were sonicated before use.

Four weeks after the second immunization, mice were challenged by aerosol with M. tuberculosis using a modified Henderson apparatus, which was enclosed within a category III isolator (23). Mice were individually restrained to allow exposure of only the nose during challenge (ADG Developments, Hitchin, Hertfordshire, U.K.). Deposition in the lungs was estimated 24 h after challenge to be 250 CFU/lung. Six weeks after challenge, bacterial loads in the lungs and spleens were calculated by plating 10-fold serial dilutions of organ homogenates on both Middlebrook 7H11 agar and thiophene-2-carboxylic acid hydrazide (TCH; 5 µg/ml)-supplemented Middlebrook plates. TCH (Sigma-Aldrich, Poole, U.K.) at this concentration prevents BCG growth (24). Protection results are expressed as log10(mean CFU) ± SEM (n = 9–12).

Statistical analysis

The SPSS statistics package (SPSS, Chicago, IL) was used for all analysis. Student’s t tests were used to compare the bacterial loads of different vaccinated groups following challenge. Linear regression analysis or Student’s t tests were performed to compare T cell responses between groups in ELISPOT and chromium release assays.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ag 85A is immunodominant following BCG vaccination

The magnitude of PPD- and 85A-specific T cell responses induced by p. BCG vaccination was compared over the course of 1 year (Fig. 1). The level of response to 85A protein ranged between 20–60% of the overall PPD response. PPD and 85A protein T cell responses were abrogated by CD4+ T cell depletion, but were not affected by CD8+ T cell depletion (data not shown). Responses to Ag85A class I- and II-restricted epitopes (P11 and P15, respectively) (25) were also assayed. T cell responses specific for the major CD4+ epitope in Ag85A, the P15 peptide, accounted for the majority of the T cell response to the whole protein at all time points. T cells specific for the CD8+ epitope were not detected in either ELISPOT or chromium release assays performed on cultured splenocytes (data not shown).



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  		FIGURE 1. Ag 85A is immunodominant following BCG immunization. BALB/c mice (n = 3–6) were immunized with 5 x 105 CFU of BCG in the footpad. At the weeks indicated, spleens were assayed by ex vivo ELISPOT for responses to PPD, 85A protein, and 85A-CD4+ (P15) and 85A-CD8+ (P11) peptides. Responses are expressed as the mean number of IFN-{gamma}-secreting SFC per 106 cells ± SEM.

 
Intranasal BCG immunization induces strong T cell responses in spleen, lung, and lung lymph nodes (LLN)

The frequency and localization of IFN-{gamma} secreting T cells induced by either p. or i.n. delivered BCG were compared. Intranasal BCG and p. BCG vaccination produced comparable levels of PPD- and Ag85A peptide-specific CD4+ T cell responses in the spleen (Fig. 2A), while p. BCG immunization in the ear produced specific IFN-{gamma} secretion in the draining facial lymph nodes (FLN), intranasal delivery produced responses exclusively in the LLN, comprising cells pooled from the paratracheal and mediastinal lymph nodes (Fig. 2, B and C). No responses were detected in the inguinal lymph nodes, which drain the lower flanks, following either route of immunization (data not shown). Parenteral BCG immunization induced strong specific T cell responses in the lung (~300 SFC/106 to PPD stimulation), but i.n. BCG induced a 5-fold higher response (Fig. 2D). The total number of cells isolated from the lungs did not significantly differ between naive mice and mice given BCG by either route (data not shown). The specific lung immune responses induced by the different routes of vaccination paralleled the pattern observed in the LLN (Fig. 2C). This suggests that T cell responses in lung are compartmentalized and dependent on the route of vaccination. In summary, while both i.n. and p. BCG produce strong splenic T cell responses, i.n. BCG elicits strong long-lived T cell immunity in the LLN and significantly higher specific responses in the lung parenchyma.



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  		FIGURE 2. Intranasal BCG immunization induces higher frequencies of specific T cells in the lung compartment than p. BCG (p-BCG) immunization. BALB/c mice were immunized with 107 CFU of BCG either i.n. or p. in the ear. Twelve weeks later, spleens (A), FLN (B), draining LLN (C), and lungs (D) were assayed by ELISPOT for responses to PPD and the 85A-CD4+ and 85A-CD8+ epitopes. Spleens and lungs were assayed individually. Results for these organs are the mean ± SEM of two independent experiments with four mice per group. For the lymph node assays, nine mice were divided into three subgroups from which nodes were pooled. Results are expressed as the mean of the three subgroups ± SEM. *, Significant differences (p < 0.05) by Student’s t tests in the induction of T cell responses by the different routes of vaccination.

 
M.85A boosts both CD4+ and CD8+ T cell responses induced by high dose BCG vaccination

We examined whether we could specifically boost the strong Ag85A T cell response induced by BCG with MVA expressing 85A (M.85A). Mice were boosted with M.85A 14 wk after p. BCG vaccination. Parenteral boosting with M.85A doubled the PPD response and increased the Ag85A-specific CD4+ and CD8+ T cell responses 4- and 5-fold, respectively, in the spleen (Fig. 3A). Immune responses in the FLN showed a similar pattern of boosting in PPD and Ag85A CD4+ and CD8+ peptide assays (data not shown). To determine whether BCG was specifically priming the Ag85A CD8+ T cell response, parallel experiments were conducted in which mice were immunized with BCG and boosted with an irrelevant MVA expressing a malarial P. berghei circumsporozoite protein (M.CS) Ag (Fig. 3B). The frequency of CS-specific CD8+ T cells was not significantly different between BCG/M.CS and M.CS-only groups, suggesting that the Ag85A CD8+ T cell response (while undetectable following the BCG immunization) is specifically boosted by M.85A. Conversely, M.CS immunization did not affect the frequency of 85A-specific T cell responses induced by BCG (data not shown).



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  		FIGURE 3. M.85A specifically boosts both 85A CD4+ and CD8+ T cell responses induced by BCG. BALB/c mice were p. immunized with 107 CFU of BCG. Fourteen weeks later, mice were p. boosted (25 µl/ear) with 106 PFU of M.85A (A) or 106 PFU of M.CS (B). M.85A- or BCG-only controls were included. Ten days after boost, spleens were assayed by IFN-{gamma} ELISPOT. Results are the mean ± SEM of two experiments with three or four animals per group. Mice were immunized i.n. with 107 CFU of BCG and after 14 wk were boosted i.n. or p. with 106 PFU of M.85A. Ten days later, spleens (C) and LLN (D) were assayed by ELISPOT. Spleens were assayed individually, and results shown are representative of three experiments. For LLN, results are expressed as the mean ± SEM of LLN pooled from three or four mice from three experiments. *, p < 0.05, as determined by Student’s t test comparisons between groups receiving BCG or M.85A only. Mice were immunized p. with 107 CFU of BCG and after 14 wk were boosted i.n. or p. (25 µl/ear) with 106 PFU of M.85A or M.CS. Ten days later LLN (E) were assayed by IFN-{gamma} ELISPOT. LLN were pooled from three or four mice. Results show 85A-CD4+-specific T cell responses in two independent experiments.

 
Similar to the wholly parenteral BCG/M.85A prime-boost regimen described above, parenteral M.85A boosting of i.n. BCG-immunized mice significantly increased the level of PPD and Ag85A CD4+ and CD8+ T cell responses in the spleen (Fig. 3C). Intranasal M.85A boosting of i.n. BCG immunized mice produced a 3- to 5-fold increase in the level of Ag85A CD4+ T cells in the LLN (Fig. 3D), but did not significantly increase the Ag85A-specific T cell response in the spleen (Fig. 3C).

Parenteral BCG immunization, although a poor inducer of specific T cell responses in the LLN, did elicit specific T cell responses in the lung parenchyma (Fig. 2, C and D). We therefore examined whether T cells induced in the lung compartment by p. BCG immunization could be boosted by i.n. M.85A vaccination. Intranasal M.85A boosting did not significantly increase T cell responses in the spleen (data not shown). However, i.n. M.85A immunization did boost p. BCG-induced 85A CD4+ T cell responses in the LLN from background levels to >125 SFC/106 cells (Fig. 3E).

M.85A boosts 85A-specific CD4+, but not CD8+, T cell responses following low dose BCG vaccination

M.85A boosting of 85A-specific T cells induced by vaccination with lower doses of BCG was investigated. Mice were immunized i.n. with BCG doses ranging from 102 to 106 CFU. Eight weeks later, groups were boosted either i.n. or p. with M.85A (Fig. 4, A and B). No PPD-specific T cell response was detected following vaccination with 102 and 103 CFU BCG (data not shown). T cell responses to PPD were detected after vaccination with 104 CFU BCG, but M.85A immunization did significantly increase 85A-specific CD4+ or CD8+ T cell responses (data not shown). The 85A CD4+ T cell response was significantly boosted following vaccination with 105 and 106 CFU of BCG, but no boosting of the P11-specific CD8+ T cell response was observed (compare with Fig. 3C). This suggests that lower doses of BCG either do not induce or inadequately induce 85A-specific CD8+ T cells that can be expanded by subsequent M.85A vaccination.



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  		FIGURE 4. BCG induces 85A-specific CD8+ T cells following high, but not low, dose vaccination. BALB/c mice were vaccinated i.n. with 105 (A) or 106 (B) CFU of BCG. After 8 wk, mice were boosted either i.n. or p. with M.85A (106 PFU). Ten days later, spleens were assayed for responses to PPD and 85A-CD4+ and 85A-CD8+ peptides by IFN-{gamma} ELISPOT. Spleens were assayed individually, and results are expressed as the mean ± SEM (SFC per 106 cells). *, p < 0.05, as determined by Student’s t test comparison to i.n. BCG- and p. M.85A-only groups.

 
Two doses of BCG afford greater protection than one in BALB/c mice

We tested whether the higher levels of specific IFN-{gamma} produced by BCG/M.85A vaccination and the induction of specific T cells in the LLN produced by i.n. immunization impacted on the levels of protection against M. tuberculosis in an aerosol challenge model (Fig. 4). There was no significant difference in the number of mycobacterial colonies on plates with or without TCH, indicating no detectable BCG in the lungs or spleens 6 wk after challenge (data not shown). Compared with naive controls, significant protection was conferred in the lung, but not the spleen, by a single parenteral low dose of BCG administered 22 wk before challenge in BALB/c mice (lung, p < 0.01; spleen, p = 0.84). In previous studies BCG immunization 4 wk before aerosol challenge yielded highly significant protection in both spleen and lungs (data not shown), indicating that the protective effect of BCG in spleen diminishes with time (26). When mice were immunized with a second dose of BCG, protection in both lungs and spleens was highly significant compared with that in naive controls (lung, p < 0.001; spleen, p = 0.001) and that in the 1x p. BCG group (lung, p = 0.022; spleen, p = 0.001; Fig. 5, A and B).



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  		FIGURE 5. Protection of BALB/c mice against aerosol M. tuberculosis challenge is afforded by BCG boosting regimens. Mice were immunized with BCG given either i.n. (105 CFU) or p. in the footpad (5 x 105 CFU). Twenty-two weeks later, groups were boosted either i.n. or p. (footpad) with BCG or M.85A (106 PFU). Four weeks following the second immunization, mice (n = 9–12) were challenged with M. tuberculosis H37Rv (~250 CFU deposited into the lung). After 6 wk, lungs and spleens were cultured on Middlebrook agar plates containing TCH (5 µg/ml). Results are shown for lung and spleens in A and B, respectively. Results are expressed as the mean (log10CFU) ± SEM. *, Significant differences (p < 0.05) by Student’s t tests in bacterial load between the various groups clustered by arrows. The origin of the arrow (circle) designates the reference group with which statistical comparisons were made.

 
Intranasal BCG offers greater protection than intradermal BCG in BALB/c mice

A single i.n. BCG immunization (0.5 log CFU lower than the parenteral dose) resulted in significant protection in the lung compared with the naive control group (p < 0.001), but this was not significantly different from the 1x p. BCG group (p = 0.065) (Fig. 5A). Numbers of bacilli in the spleen were similar in the i.n. BCG, p. BCG, and naive control groups (all comparisons, p > 0.5; Fig. 5B). When a second i.n. BCG immunization was given, protection in both lung and spleen was significantly improved compared with the naive, 1x i.n. BCG, and 1x p. BCG groups (all comparisons: lung, p < 0.001; spleen, p < 0.001). Two doses of i.n. BCG also showed significantly greater protection in the lung than immunization with 2x p. BCG (p < 0.001).

M.85A boosting of BCG provides comparable protection to two BCG immunizations and 2.5 log protection over naive group

Boosting i.n. BCG, either i.n. or p. with M.85A, produced significantly higher levels of protection against challenge compared with the 1x i.n. BCG group (Fig. 5). Protection in the i.n. M.85A-boosted group was striking, showing the same levels of protection as the 2x i.n. BCG group (lung, 2.5 logs; spleen, 1.5 logs). Compared with the i.n. BCG group, the mice boosted parenterally with M.85A did not show increased protection in the lung (p = 0.494; Fig. 5A). However, the level of protection in the spleen was significantly more than that in the 1x i.n. BCG group (p < 0.05) and was comparable to that in the 2x i.n. BCG and i.n. BCG/i.n. M.85A groups (Fig. 5B).

Protection in the lung correlates with detection of T cell responses in the LLN

On the day of challenge (4 wk after second immunization), four mice from each group were sacrificed for immunogenicity studies. Only the 2x i.n. BCG and i.n. BCG/i.n. M.85A groups had detectable T cell responses to the 85A CD4+ T cell epitope in their LLN (Fig. 6A). These groups showed the highest levels of protection in both lungs and spleen following challenge, which indicates that Ag85A-specific T cell responses in the LLN correlate better with protection in the lung than splenic immune responses. No T cell response was detected in the LLN to the 85A CD8+ P11 peptide in any group (Fig. 6A), most likely because the BCG dose used in the challenge experiment was too low to induce a specific CD8+ T cell response (Fig. 4).



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  		FIGURE 6. Protection against aerosol M. tuberculosis correlates with the presence of specific IFN-{gamma}-secreting CD4+ T cells in the LLN. Additional mice (n = 4) were sacrificed at the time of challenge, and pooled LLN and spleens were assayed for T cell responses to PPD, 85A-CD4+ and 85A-CD8+ peptides in IFN-{gamma} ELISPOT (A and B). The results are expressed as SFC per 106 cells. In B, * indicates a significant differences (p < 0.05) by Student’s t test in SFC frequency between the groups clustered by arrows. The origin of the arrow (circle) designates the reference group with which statistical comparisons were made. Splenocytes from each mouse were cultured with 85A-CD8+ peptide and assayed in a 51Cr release assay (C). Results are expressed as the percentage of specific lysis ± SEM. *, p < 0.05, as determined by linear regression analysis comparison of all groups.

 
Some differences were observed between groups in the splenic assays. While all groups produced comparable responses in ELISPOT to PPD (data not shown), the Ag85A CD4+ T cell response to P15 in the i.n. BCG/i.n. M.85A and i.n. BCG/p. M.85A groups were increased 2- and 3-fold, respectively, over that in the 1x i.n. BCG group (Fig. 6B). Responses to the P11 CD8+ T cell epitope were detectable at low levels in the i.n. BCG/p. M.85A group in ELISPOT (data not shown). In 51Cr release assays performed on cultured splenocytes, only this group produced significant levels of specific lysis (30%) of P11-pulsed targets (Fig. 6C). Again, because of the lower BCG dose used in the challenge experiment, the detection of 85A-specific CD8+ T cells in the spleen was most likely induced by M.85A vaccination alone.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study the BALB/c mouse model was used to examine two strategies to increase BCG-mediated protection against M. tuberculosis infection: 1) boosting BCG-induced T cell responses with either M.85A or a second dose of BCG, and 2) eliciting Ag85A-specific mucosal T cell responses by i.n. immunization.

Recombinant MVA effectively induces both CD4+ and CD8+ IFN-{gamma}-secreting T cells to foreign Ags, including Ag85A, and is particularly effective as a boosting vector (18, 20, 27). Nonrecombinant MVA has been administered, without significant side effects, to >100,000 people as part of the smallpox eradication program (28). A recombinant MVA encoding malaria Ags has been administered safely to >200 volunteers (A. V. S. Hill et al., unpublished observations). In this study M.85A immunization boosted Ag85A-specific CD4+ and CD8+ T cell pools induced by BCG, demonstrating that a recombinant virus can also boost T cell responses induced by a bacterial prime. CD4+ T cells have a clear role in controlling progression to tuberculosis disease in man (29). While Ag85A-specific CD8+ T cell lines have been derived from BCG vaccinees and healthy M. tuberculosis-exposed individuals, the exact role of CD8+ T cells in human tuberculosis remains to be defined. (30). A recent report suggests that in mice, CD8+ T cells protect more against reactivation tuberculosis than CD4+ T cells (31). This implies that vaccination regimens that can effectively boost low level M. tuberculosis-specific CD8+ T as well as CD4+ T cell responses, such as MVA, may be particularly effective in maintaining long term protection from disease.

The protective effects of one vs two doses of BCG given either p. or i.n. were compared. In BALB/c mice, regardless of the route of delivery, two immunizations were more protective than one in lungs and spleens. In humans, repeated intradermal BCG immunization did not provide any greater protection against pulmonary tuberculosis regardless of age at time of boost or time between BCG immunizations in Malawi (32). However, in the region examined, a single BCG immunization conferred no protection against pulmonary tuberculosis. Multiple BCG doses or indeed boosting with M.85A may still provide some benefit in a population in which a single BCG immunization is partially protective.

We investigated whether protection from pulmonary tuberculosis could be improved through eliciting lung-localized T cell populations following mucosal delivery of tuberculosis vaccines. We showed that while i.n. BCG was as effective as p. BCG in inducing peripheral T cell responses, the induction of responses in lymph nodes is highly dependent on the route of immunization. Intranasal delivery of BCG elicited robust responses in the LLN that could be further boosted by i.n. immunization with M.85A. When these vaccination regimens were tested in an aerosol M. tuberculosis challenge, a single dose of i.n. BCG gave the same level of protection as p. BCG. In mice that were immunized intranasally with BCG and again boosted i.n. with either a second BCG dose or M.85A, the levels of protection in the lungs and spleens were both significantly higher than those with a single BCG immunization. To our knowledge this level of protection (2.5 logs) has not previously been reported in murine vaccination studies.

At the time of challenge only the i.n. prime/i.n. boost vaccination regimens produced detectable responses in LLN and the highest levels of protection. The level of splenic T responses did not correlate with the strong protection observed in these groups. These data suggest that the frequency of Ag-specific, IFN-{gamma}-secreting T cells in the LLN may constitute a better indicator of protection in the lung than systemic responses against pulmonary tuberculosis. We suggest two explanations for this observation. Firstly, in mice that have been infected aerogenically with M. tuberculosis, bacilli disseminate first to the LLN, possibly via dendritic cells, and then to the periphery (33, 34). The induction of M. tuberculosis-specific T cells follows dissemination of bacilli. The responses are induced first in the LLN (but not in other LN compartments) and later in the spleen (33). The strong protection observed in both lungs and spleens in the i.n. primed/i.n. boosted groups may have resulted from the microbicidal actions of resident M. tuberculosis-specific T cells in the LLN limiting the dissemination of bacilli back to the lungs and periphery. Whether M. tuberculosis dissemination from the lung parenchyma to the LLN also occurs in humans is unknown, but in both symptomatic and asymptomatic individuals pathology is often found in the hilar lymph nodes. Alternatively, we showed a correlation between the frequency of 85A-specific T cells in the lung and LLN, implying that Ag-specific T cells in LLN may reflect lymphatic drainage of protective T cell populations resident in the lung (35, 36). Other murine studies have shown a correlation between an influx of IFN-{gamma}-secreting CD4+ and CD8+ T cells into the lung during acute infection and protection from tuberculosis (37, 38, 39). In humans, Condos et al. (40) showed that the broncheo-alveolar lavage of patients with less clinically advanced pulmonary tuberculosis contained higher levels of IFN-{gamma}-secreting lymphocytes than patients with advanced tuberculosis who conversely had increased numbers of neutrophils in their broncheo-alveolar lavage.

The acute M. tuberculosis challenge model used here did not in itself distinguish between the utility of BCG and M.85A as boosting agents after BCG priming. Previous studies have shown that BCG is still recoverable from the lung, albeit in very low numbers, 1 mo after lung immunization (41). At this time point, histological analysis showed an ongoing inflammatory response in the lungs, and the numbers of macrophages, neutrophils, NK cells, and antimicrobial mediators such as IL-12 and TNF-{alpha} in lung lavage were elevated (41, 42). The level of protection observed following challenge of the 2x i.n. BCG group could be due in part to ongoing innate immune reactions induced by the residual BCG in the lungs. In contrast, it is unlikely that innate immune mechanisms contributed to the protection observed in the i.n. BCG/i.n. M.85A group. Unlike BCG, the MVA virus does not persist in rodent lungs (43). In cotton rats that are highly susceptible to vaccinia virus infection, MVA could not be detected in lungs 5 days after i.n. immunization, and no pathology was observed. Similarly, i.n. immunization of BALB/c mice with 5 x 107 PFU (1.5 logs greater that the vaccination dose in this study) of nonrecombinant MVA did not provide any protection from challenge with the lung pathogens, respiratory syncytial virus or parainfluenza virus (44, 45). In those studies, groups of mice vaccinated with recombinant MVA expressing respiratory syncytial virus or parainfluenza virus proteins were protected following i.n. challenge 1 mo after vaccination.

While the i.n. prime/i.n. boost vaccination regimen was highly protective in this study, was i.n. delivery of the vaccines necessary for both the prime and the boost? A single i.n. BCG vaccination induced significantly higher T cell responses in the lung than p. BCG. Even so, the frequency of T cells in the lung elicited by p. BCG vaccination was sufficient to afford protection equivalent to that of i.n. BCG in the lungs. A similar observation was recently reported by Palendira et al. (46). These results conflict with earlier studies in other animal models in which aerosolized BCG afforded better protection than parenterally delivered BCG (10, 11). In these early studies the time between vaccination and challenge was relatively short, and as described above, any ongoing inflammatory reaction in the lung may have contributed to increased levels of protection. In our study and that by Palendira et al. (46), BCG was eliminated from the lung by resting the animals after BCG vaccination (22 wk) or by antibiotic treatment. Palendira et al. (46) also showed that regardless of the route of BCG vaccination, the recruitment of IFN-{gamma}-secreting CD4+ T cells to the lung following aerogenic M. tuberculosis challenge was similar. This suggests, in this model at least, that following a single immunization of BCG, protection is independent of route of vaccination. Studies in viral systems have similarly shown that following a single i.n. immunization, T cell responses first develop in the LLN and then extravasate stochastically from the blood to organs such as spleens and lungs (9, 36, 47). When the animals were challenged i.n., the presence of Ag drew T cells back to the lung (9, 36, 46, 48). Applying these observations to the prime-boost regimen in this study, an i.n. M.85A boost of BCG-immunized mice will express Ag85A in the lung and not only expand Ag85A-specific T cells resident in the respiratory compartment, but also pull in memory T cells from the periphery (49). In support of this, we have observed that i.n. boosting with M.85A of p. BCG-immunized mice strongly increases Ag85A CD4+ T cell responses in the LLN (9). This view is not in conflict with the wealth of vaccinology publications showing that mucosal vaccination affords longer lasting and greater protection than parenteral vaccination from mucosal pathogens (reviewed in Ref. 50). In the majority of these studies vaccination regimens have involved repeated mucosal vaccination, each time not only boosting, but also targeting, memory T cells to the mucosal tissue.

These data suggest that the induction of T cell responses in the respiratory compartment is important in protection from pulmonary disease. Even so, strong peripheral T cell responses remain central to protection in the spleen. Parenteral M.85A boosting of mice immunized with i.n. BCG increased the level of protection in the spleen, but not the lung. No Ag85A-specific immune responses were detected in the i.n. BCG/p. M.85A group in the LLN. However, splenic immune responses in this group were significantly higher than those in other groups. Parenteral immunization of recombinant MVA has been previously reported to elicit strong lytic T cells responses in the periphery, but not the mucosa (9). Together this suggests that p. boosting of BCG-immunized mice with M.85A expanded peripheral CD4+ T cell populations that, following challenge, better controlled the levels of M. tuberculosis bacilli that had disseminated from the LLN. In humans, p. BCG vaccination is protective against systemic forms of disease, and p. BCG vaccination in rhesus macaques appears to protect from hemogenous spread of bacilli (2, 51). In summary, while LLN T cell responses may correlate with protection in the lung, specific T cell responses in the spleen may constitute a good indicator of protection from systemic forms of disease.

In the clinic, regardless of the route of delivery, M.85A boosting may be of more benefit than multiple BCG vaccination for several reasons. Increasing evidence suggests that exposure to environmental mycobacteria has a negative effect on BCG-induced protection (4). Unlike BCG and as outlined above, the strong immunogenicity and protective capacity of M.85A occurs without any viral replication and therefore is very unlikely to be compromised by exposure to environmental mycobacteria (52). In addition, MVA has been given without adverse effects to severely immunocompromised primates, suggesting that it would carry less risk in HIV-positive individuals than subsequent vaccinations with live BCG (53). We show here that following a BCG immunization M.85A expands the low level, Ag85A-specific CD8+ T cell response as well as CD4+ T cell responses. We are investigating vaccination regimens in mice and macaques to further augment the induction of 85A-specific CD8+ T cells following vaccination at lower BCG doses. The ability to boost CD8+ T cell responses with recombinant viruses should provide an advantage, particularly for long term protection, over multiple BCG immunizations and possibly over other subunit vaccination regimens that induce T cell responses that are predominately CD4+ restricted. We have now begun clinical studies to investigate the potential of a BCG/M.85A prime-boost immunization to improve protection against pulmonary tuberculosis.


   Acknowledgments
 
We thank Ernie Gould (NERC, Oxford, U.K.) for the category III facilities used. We acknowledge the kind gifts of the vaccine BCG 1173P2 from Micheline Lagranderie (Laboratoire du BCG, Institut Pasteur, Paris, France) and Ag85A protein from Kris Huygen (Mycobacteriele Immunologie Institut Pasteur, Brussels, Belgium).


   Footnotes
 
1 This work was supported by the Wellcome Trust. A.V.S.H is a Wellcome Trust Principal Fellow. Back

2 Address correspondence and reprint requests to Nilu P. Goonetilleke, Nuffield Department of Clinical Medicine, Oxford University, John Radcliffe Hospital, Headington, U.K. OX3 9DU. E-mail address: nilu.goonetilleke{at}ndm.ox.ac.uk Back

3 Abbreviations used in this paper: BCG, bacille Calmette-Guérin; MVA, modified vaccinia virus Ankara; CSP, circumsporozoite protein; FLN, facial lymph nodes; i.n., intranasal(ly); LLN, lung lymph nodes; p., parenteral(ly); PPD, purified protein derivative; P11, peptide 11; P15, peptide 15; SFC, spot-forming cells; TCH, thiophene-2-carboxylic acid hydrazide. Back

Received for publication October 31, 2002. Accepted for publication May 1, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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