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A New Recombinant Bacille Calmette-Guérin Vaccine Safely Induces Significantly Enhanced Tuberculosis-Specific Immunity in Human Volunteers

  1. Daniel F. Hoft1,
  2. Azra Blazevic1,
  3. Getahun Abate1,
  4. Willem A. Hanekom5,
  5. Gilla Kaplan2,
  6. Jorge H. Soler2,
  7. Frank Weichold3,
  8. Larry Geiter3,
  9. Jerald C. Sadoff3 and
  10. Marcus A. Horwitz4
  1. 1Division of Immunobiology Departments of Internal Medicine and Molecular Biology, Saint Louis University Medical Center, and Center for Vaccine Development, Saint Louis, Missouri
  2. 2Laboratory of Mycobacterial Immunity and Pathogenesis, Public Health Research Institute, University of Medicine and Dentistry of New Jersey
  3. 3Aeras Global TB Vaccine Foundation, Rockville Pike, Maryland
  4. 4Departments of Internal Medicine and of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles
  5. 5South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa
  1. Reprints or correspondence: Dr. Daniel F. Hoft, Div. of Immunobiology, Depts. of Internal Medicine and Molecular Microbiology, Saint Louis University Medical Center, Edward A. Doisy Research Center, Rm. 807, 1100 S. Grand Blvd., St. Louis, MO 63104 (hoftdf{at}slu.edu).
 
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Abstract

Background. One strategy for improving anti-tuberculosis (TB) vaccination involves the use of recombinant bacille Calmette-Guérin (rBCG) overexpressing protective TB antigens. rBCG30, which overexpresses the Mycobacterium tuberculosis secreted antigen Ag85b, was the first rBCG shown to induce significantly greater protection against TB in animals than parental BCG.

Methods. We report here the first double-blind phase 1 trial of rBCG30 in 35 adults randomized to receive either rBCG30 or parental Tice BCG intradermally. Clinical reactogenicity was assessed, and state-of-the-art immunological assays were used to study Ag85b-specific immune responses induced by both vaccines.

Results. Similar clinical reactogenicity occurred with both vaccines. rBCG30 induced significantly increased Ag85b-specific T cell lymphoproliferation, interferon (IFN)-γ secretion, IFN-γ enzyme-linked immunospot responses, and direct ex vivo intracellular IFN-γ responses. Additional flow cytometry studies measuring carboxyfluorescein succinimidyl ester dilution and intracellular cytokine production demonstrated that rBCG30 significantly enhanced the population of Ag85b-specific CD4+ and CD8+ T cells capable of concurrent expansion and effector function. More importantly, rBCG30 significantly increased the number of Ag85b-specific T cells capable of inhibiting intracellular mycobacteria.

Conclusions. These results provide proof of principal that rBCG can safely enhance human TB immunity and support further development of rBCG overexpressing Ag85b for TB vaccination.

One-third of the world's population is infected with Mycobacterium tuberculosis, and 2 million deaths due to tuberculosis (TB) occur annually [1]. These staggering statistics persist despite the availability of a TB vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG), for >75 years. New vaccines are urgently needed to reduce this immense burden of TB disease. One potential approach for improving TB vaccination is the generation of recombinant BCG (rBCG), which may work better than standard BCG strains by overexpressing key M. tuberculosis antigens, immunoenhancers, and/or proteins that promote phagosomal escape and potent CD8+ T cell stimulation [2–8]. Furthermore, rBCG is an attractive option because of the extensive clinical experience, known immunogenicity protective against severe TB disease, and relative safety profile of standard BCG strains. However, rBCG TB vaccines have not been studied in humans for the purpose of demonstrating the safety and enhanced immunogenicity of this approach.

M. tuberculosis is an intracellular pathogen that replicates in host mononuclear phagocytes [9, 10]. Bacillary proteins secreted intracellularly are early targets of TB immunity [9–11]. Immunization of guinea pigs with purified M. tuberculosis extracellular proteins, including the 30-kDa major secretory protein (a mycolyltransferase known as “α-antigen” and “Ag85b” [12, 13]), induces substantial protection against aerosol challenge with highly virulent M. tuberculosis [8]. Furthermore, M. tuberculosis-secreted proteins [14] and DNA encoding secreted antigens [15] also induce TB immunity in mice. Although previous vaccinations in animals induced significant levels of protection, the protection was never superior and was usually less than that induced by BCG vaccination. Horwitz et al. [2] and Horwitz and Harth [3] generated rBCG overexpressing the 30-kDa Ag85b of M. tuberculosis (rBCG30) in 2 distinct BCG strains; these rBCG30 vaccines were the first new TB vaccines capable of inducing protective immunity in guinea pigs significantly better than the nonrecombinant BCG.

On the basis of these promising results, the Aeras Global TB Vaccine Foundation initiated the clinical development of rBCG30 as its first model TB vaccine candidate. We report here the initial clinical and detailed immunological testing of rBCG30 in purified protein derivative (PPD)-negative (by QuantiFERON assay; Cellestis) adult volunteers. rBCG30 was as safe as nonrecombinant BCG and induced significantly increased Ag85b-specific immunity in multiple relevant subsets of mycobacteria-specific immune responses.

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Methods

BCG vaccinations. After informed consent was obtained, 35 PPD-negative and HIV-negative healthy adults were randomized in a double-blind fashion to receive ∼5 × 105 cfu of either rBCG30 (Korean Institute of Tuberculosis, Aeras investigational new drug, lot 200303; parental Tice BCG strain used to construct rBCG30) or parental nonrecombinant Tice BCG (Organon Teknika) intradermally. Volunteers were recruited, vaccinated, and followed up at Saint Louis University (n = 20) or Piedmont Medical Research Associates, Winston-Salem, North Carolina (n = 15). Fresh samples were studied in the Saint Louis University cohort (10 samples per group), and frozen peripheral blood mononuclear cells (PBMCs) were studied from both sites. The protocol was approved by Saint Louis University and Aeras Institutional Review Boards.

Antigen-specific lymphoproliferation. Heparinized blood was diluted 1:10 with RPMI 1640 containing optimal doses of control and mycobacterial antigens and incubated for 7 days at 37°C in 5% CO2 before thymidine incorporation measurements were obtained. Tetanus toxoid control was used at 5 µg/mL (Statens Serum Institut, Copenhagen, Denmark). Recombinant Ag85b (rAg85b) protein (prepared in the laboratory of M.A.H.) was used at 5 µg/mL. Live Tice BCG was thawed, and 100,000 cfu was added per 200 µL of diluted blood.

Antigen-specific interferon (IFN)-γ secretion. Heparinized blood diluted 10-fold with RPMI 1640 alone or with optimal doses of antigens was incubated at 37°C in 5% CO2 for 4 days. Secreted IFN-γ was measured by ELISA, as described elsewhere [16].

Detection of antigen-specific intracellular IFN-γ responses directly ex vivo. Antigen-specific IFN-γ-producing CD4+ and CD8+ T cells were identified by whole blood intracellular cytokine assay, as described elsewhere [17]. Heparinized blood was incubated with anti-CD28 and anti-CD49d alone (negative control) or with rAg85b protein (0.5 mg/mL). After 12 h at 37°C (with brefeldin A added for the last 5 h), red blood cells were lysed and white blood cells fixed with BD FACS Lysing Solution (BD Biosciences) before cryopreservation. For analysis, cells were thawed, permeabilized, and stained with antibodies for flow cytometry (FACSCalibur and CellQuest software; BD Biosciences), as described elsewhere [17].

Antigen-specific IFN-γ enzyme-linked immunospot (ELISPOT) responses. IFN-γ-producing cells were identified by ELISPOT assay using ImmunoSpot plates (Cellular Technology) and IFN-γ-specific antibodies (BD Pharmingen). PBMCs (0.2 × 105–1 × 105 cells/well) were stimulated with Ag85b peptide pools (1 µg/mL), rAg85b protein (5 µg/mL), live BCG (MOI of 0.5) or medium alone for 24 h at 37°C in 5% CO2. Spots were identified by use of a CTL analyzer and ImmunoSpot software (version 3.2; CTL).

Simultaneous detection of antigen-specific proliferation and IFN-γ production. PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) and either expanded with optimal doses of Ag85b peptide pools, rAg85b protein, or live BCG or rested in medium for 7 days at 37°C in 5% CO2. Then cells were incubated with 50 ng/mL phorbol myristate acetate (PMA; Sigma), 750 ng/mL ionomycin (Sigma) and 0.7 µL/mL GolgiStop (BD Biosciences) for 2 h before CD3, CD4, and CD8 staining and permeabilization with Cytofix/Cytoperm (BD Biosciences), followed by staining for intracellular IFN-γ. Data were acquired with a FACSCalibur flow cytometer and analyzed using CellQuest and FlowJo (Tree Star). Absolute numbers of effector CD4+ and CD8+ T cells (defined as both CFSElow and IFN-γ+) were calculated by multiplying the total number of viable cells recovered by the T cell subset percentages detected.

Mycobacterial growth inhibition assay. Inhibitory responses were studied as described elsewhere [18, 19]. Adherent monocytes were incubated without antibiotics for 6 days to allow macrophage differentiation. Concurrently, PBMCs were expanded with 5 µg/mL rAg85b protein or Tice BCG (MOI of 0.5) for 7 days. One day before the addition of expanded T cells, target macrophages were infected with BCG (MOI of 3). Extracellular mycobacteria were removed, and effector cells were added at a ratio of 12.5:1 for 72 h at 37°C. Saponin lysates were prepared, and the numbers of colony-forming units of released bacilli were determined. The percentage of inhibition was calculated as 100 − [100 × (experimental colony-forming units/control colony-forming units)]. Negative percentages were assumed to represent 0% inhibition, and 4 outliers (baseline response greater than the mean plus 1 SD of all baseline responses) from the 20 volunteers available for study were excluded.

Statistics. Friedman repeated-measures analysis of variance (ANOVA) was used to study overall increases after vaccination, the Wilcoxon matched-pairs test was used to identify significantly increased postvaccination compared with matched prevaccination responses, and the Mann-Whitney U test was used to compare responses between groups. McNemar's test and Fisher's exact test were used to compare paired and unpaired categorical data, respectively. Analyses were completed with Statistica (Statsoft).

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Results

Reactogenicity. No serious adverse events were encountered. Overall, nonserious adverse events were similar between groups (table 1). Expected local reactions at BCG intradermal vaccination sites occurred (papules followed by ulcers draining for days to weeks). Mean and median ulcer size and drainage duration were similar between the rBCG30 and Tice groups. A trend toward increased pain/erythema/swelling at injection sites occurred in rBCG30 recipients, and significantly more rBCG30 volunteers experienced moderately severe erythema at vaccination sites during the first 2 weeks after vaccination (12/18 vs. 2/17; P < .05, 2-tailed Fisher's exact test). However, similar events were reported after 2 weeks, and all lesions healed by 3 months after vaccination.

Lymphoproliferative responses. Lymphoproliferative responses induced by live Tice BCG and rAg85b protein were studied before (day 0) and after (days 56, 112, and 252) vaccination (figure 1). Both vaccines induced significant increases in BCG-specific lymphoproliferation detected on day 56 after vaccination (P < .03, Wilcoxon matched-pairs test; n = 10 per group). Only rBCG30 recipients (and not Tice recipients) developed significant increases in Ag85b-specific lymphoproliferation (P < .01, Friedman ANOVA including day 0, 56, 112, and 252 time points; n = 10 per group). These results demonstrate the immunogenicity of both vaccines, which induced significant increases in lymphoproliferation specific for antigens expressed by live BCG; however, only rBCG30 induced significantly increased lymphoproliferation specific for the Ag85b antigen overexpressed in rBCG30.

Figure 1
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Figure 1

Ag85b- and bacille Calmette-Guérin (BCG)-specific whole blood lymphoproliferative responses before and after vaccination. Heparinized whole blood harvested before vaccination and on days 56, 112, and 252 after vaccination was diluted 10-fold with RPMI 1640 and stimulated for 7 days with optimal doses of recombinant Ag85b protein and live Tice strain BCG. Shown are the median values (points), interquartile ranges (boxes), and nonoutlier ranges (whiskers). Ag85b- and BCG-specific postvaccination responses in the recombinant BCG30 (rBCG30) vaccination group were significantly increased (P < .01, Friedman analysis of variance). *P < .03 for before vs. after vaccination (Wilcoxon matched-pairs test).

Detection of IFN-γ-producing effector T cells directly ex vivo. We measured mycobacteria-specific IFN-γ responses by intracellular cytokine staining after short-term stimulation. This assay quantitates antigen-specific effector T cells and allows for simultaneous detection of CD4+ and CD8+ T cells. Figure 2 presents the percentages of CD4+ (figure 2A) and CD8+ (figure 2B) T cells producing intracellular IFN-γ after stimulation with purified Ag85b protein. Only rBCG30 induced significantly increased Ag85b-specific CD4+ and CD8+ T cell responses. By Friedman ANOVA, overall IFN-γ-producing CD4+ T cells were significantly increased in rBCG30 recipients after vaccination (P < .02, analyzing days 0, 56, 112, and 252). In addition, Ag85b postvaccination responses on day 56 in rBCG30 recipients were significantly greater than day 0 responses by Wilcoxon matched-pairs testing (P < .02). Furthermore, rBCG30-induced CD4+ T cell responses were significantly greater than Tice-induced responses on days 56 and 252 after vaccination (P < .05, Mann-Whitney U test). Ag85b-specific CD8+ T cell responses were significantly increased only in rBCG30 recipients on day 252 after vaccination (P < .05, Wilcoxon matched-pairs test). Both the Tice and rBCG30 groups developed significant increases in BCG-specific CD4+ and CD8+ IFN-γ-producing T cells after vaccination (P < .05, Friedman ANOVA including days 0, 56, 112, and 252). By Wilcoxon matched-pairs testing, BCG-specific CD4+ IFN-γ-producing T cells were significantly increased in both the Tice and rBCG30 groups on days 56 and 112 after vaccination (P < .04), and BCG-specific CD8+ IFN-γ-producing T cells were significantly increased in both the Tice and rBCG30 groups on day 112 after vaccination (P < .03). Overall, these results demonstrate that both vaccines induced BCG-specific responses, whereas only rBCG30 vaccination induced significant Ag85b-specific responses.

Figure 2
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Figure 2

Detection of Ag85b-specific interferon (IFN)-γ-producing T cells directly ex vivo by flow cytometry. Heparinized whole blood harvested before vaccination and on days 56, 112, and 252 after vaccination was stimulated for 12 h with recombinant Ag85b protein (brefeldin A was added for the last 5 h of incubation) before being frozen in liquid nitrogen. Matching sets of pre- and postvaccination samples from individual volunteers were thawed, processed for staining of T cell surface markers and intracellular IFN-γ, and then analyzed by flow cytometry. The percentages of CD4+ (A) and CD8+ (B) T cells positive for intracellular IFN-γ staining are presented. Shown are the median values (points), interquartile ranges (boxes), and nonoutlier ranges (whiskers). Postvaccination IFN-γ+CD4+ T cell responses in the recombinant bacille Calmette-Guérin (rBCG) 30 group were significantly increased (P < .02, Friedman analysis of variance). *P < .05 for pre- vs. postvaccination responses (Wilcoxon matched-pairs test); **P < .05 for rBCG30 vs. Tice vaccination groups (Mann-Whitney U test).

Partial mapping of major Ag85b-specific T cell epitopes. We generated 55 individual 15mer peptides overlapping by 10 aa and spanning the entire Ag85b sequence (M. tuberculosis gene Rv1886c). Epitope-mapping investigations of immunodominant Ag85b-specific T cell epitopes were initiated using 3 separate pools of these peptides to stimulate IFN-γ ELISPOT responses in thawed PBMCs. Each Ag85b peptide pool contained 19 peptides: peptide pool (PP) 1 covered the amino-terminal 1–105 aa, PP2 covered the central 91–195 aa, and PP3 covered the carboxy-terminal 181–285 aa. Figure 3 demonstrates that only rBCG30 recipients developed significant increases in Ag85b-specific responses after vaccination (days 7 and 56). rBCG30-induced increases in Ag85b-specific IFN-γ responses were stimulated with PP1 (P < .01, Friedman ANOVA including results from days 0, 7, and 56). In addition, rBCG30-induced PP1-specific responses were significantly greater than day 0 responses on both days 7 and 56 after vaccination (P < .01, Wilcoxon matched-pairs test). Furthermore, rBCG30 day 7 PP1 responses were significantly greater than Tice day 7 PP1 responses (P < .05, Mann-Whitney U test). Only PP1 stimulations detected vaccine-induced responses in rBCG30 recipients. This suggests that the amino-terminal Ag85b sequence contains immunodominant epitopes. Table 2 presents the proportions of positive IFN-γ ELISPOT responses (more than or equal to the mean plus 1 SD of matching baseline responses). Only rBCG30 recipients developed significantly increased positive responses after vaccination, and only after stimulation with PP1. On day 7 after vaccination, 10 of 16 rBCG30 recipients had PP1-positive responses, compared with only 1 of 16 on day 0 (P < .01, McNemar's test) and only 3 of 15 Tice recipients with positive responses on day 7 (P < .03, Fisher's exact test). On day 56 after vaccination, 8 of 17 rBCG30 recipients had PP1-positive responses, compared with only 1 of 16 on day 0 (P < .05, McNemar's test).

Figure 3
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Figure 3

Peptide epitope mapping of Ag85b-specific interferon (IFN)-γ-producing T cell responses. Frozen peripheral blood mononuclear cells (PBMCs) harvested before vaccination and on days 7 and 56 after vaccination were stimulated with pools of peptides representing the entire Ag85b protein sequence (15mer peptides overlapping by 10 aa) in IFN-γ enzyme-linked immunospot (ELISPOT) assays. Peptide pools 1, 2, and 3 correspond to the initial amino-terminal third, middle third, and carboxy-terminal third of the Ag85b molecule, respectively. Shown in panel A are the median values of IFN-γ spot-forming cells (points), interquartile ranges (boxes), and nonoutlier ranges (whiskers). Postvaccination peptide pool 1-specific IFN-γ-producing T cell responses in the recombinant bacille Calmette-Guérin (rBCG) 30 group were significantly increased (P < .01, Friedman analysis of variance). *P < .01 for pre- vs. postvaccination responses (Wilcoxon matched-pairs test); **P < .05 for rBCG30 vs. Tice vaccination groups (Mann-Whitney U test).

Detection of IFN-γ responses in vaccine-induced memory T cells capable of antigen-specific expansion. Long-term protection requires memory T cells that can both expand and produce effector functions in response to a later infectious challenge [20–22]. To determine whether Tice and/or rBCG30 vaccinations induced memory T cells capable of both expansion and IFN-γ effector responses, we developed a flow-based assay that measures CFSE dilution to track lymphoproliferation coupled with intracellular staining to detect IFN-γ effector responses. With this assay, we studied frozen PBMCs harvested from days 0 and 56 after vaccination in 12 volunteers (5 Tice and 7 rBCG30 recipients). Figure 4A and 4B present CD4+ and CD8+ T cell responses, respectively. Both Ag85b PP1 and whole recombinant protein induced significantly increased numbers of CD4+ T cells to proliferate (i.e., to become CFSElow) and produce IFN-γ in PBMCs from rBCG30 recipients on day 56 after vaccination (P < .05, Wilcoxon matched-pairs test). In addition, Ag85b PP1-induced CD8+ T cell responses were significantly increased in rBCG30 recipients after vaccination (P < .05, Wilcoxon matched-pairs test). Ag85b-specific responses were not increased after vaccination in Tice recipients. These results indicate that rBCG30 uniquely induced increases in the number of Ag85b-specific memory CD4+ and CD8+ T cells capable of both antigen-specific expansion and IFN-γ effector function.

Figure 4
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Figure 4

Detection of Ag85b-specific memory T cells capable of both expansion and interferon (IFN)-γ effector function. Matching pre- and postvaccination frozen peripheral blood mononuclear cells from individual volunteers were thawed, labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE), stimulated with Ag85b peptide pool 1 or recombinant protein for 7 days, and then stimulated with phorbol myristate acetate/ionomycin for 2 h in the presence of GolgiStop (BD Biosciences) to maximize detection of intracellular IFN-γ before surface and intracellular staining for flow cytometry. Presented are the absolute nos. (AN) of CD4+ (A) and CD8+ (B) T cells that both proliferated (became CFSElow) and produced detectable intracellular IFN-γ. Shown are the median values (points), interquartile ranges (boxes), and nonoutlier ranges (whiskers). *P < .05 for pre- vs. postvaccination responses (Wilcoxon matched-pairs test). d, day; rBCG, recombinant bacille Calmette-Guérin.

Detection of vaccine-induced T cell responses capable of inhibiting intracellular mycobacteria. Mycobacteria predominantly replicate within macrophages in vivo, and T cells capable of inhibiting intracellular replication are critical for protective TB immunity. We have developed an assay that measures T cell-mediated inhibition of intracellular mycobacteria [18, 19]. Figure 5 presents T cell-mediated inhibitory responses by Ag85b protein-expanded PBMCs (n = 9 for rBCG30 and n = 7 for Tice). rBCG30 recipients demonstrated progressive increases in Ag85b-specific inhibitory responses, which were significantly increased on day 112 after vaccination (P < .05, Wilcoxon matched-pairs test) and were significantly greater than day 112 Tice responses (P < .05, Mann-Whitney U test). Similarly increased inhibitory effects were mediated by T cells expanded with live BCG in both groups after vaccination (data not shown).

Figure 5
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Figure 5

Ag85b-specific inhibitory T cell responses. Peripheral blood mononuclear cells harvested before vaccination and on days 56 and 112 after vaccination were stimulated with recombinant Ag85b protein for 7 days, and then Ag85b-specific expanded T cells were cocultured with bacille Calmette-Guérin (BCG)-infected autologous macrophages for 3 days. After coculture, mammalian membranes were disrupted by saponin lysis, and released viable colony-forming units of BCG were enumerated on Middlebrook agar plates. The percentage of inhibition mediated by Ag85b-specific T cells compared with medium-rested T cells is graphically presented. Shown are the median values (points), interquartile ranges (boxes), and nonoutlier ranges (whiskers). *P < .05 for pre- vs. postvaccination responses (Wilcoxon matched-pairs test); **P < .05 for recombinant (r) BCG30 vs. Tice vaccination groups (Mann-Whitney U test).

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Discussion

Our results provide proof of principle that rBCG can safely induce enhanced immunity in humans. rBCG30 was the first rBCG shown to induce significantly better protection against aerosolized TB challenge in the stringent guinea pig model of pulmonary TB [2, 3]. The present results extend these observations, providing the first demonstration in humans that rBCG30 vaccination is not only well-tolerated but also capable of inducing significantly enhanced Ag85b-specific immunity compared with the parental Tice BCG. Our results should encourage continued work on rBCG vaccines currently in differing stages of clinical development.

We carefully evaluated local and systemic reactions after Tice and rBCG30 intradermal vaccination and found that both had similar clinical effects (table 1). Except for increased injection site erythema early after rBCG30 vaccination, local responses to Tice and rBCG30 were very similar. rBCG30 recipients tended to report more injection site pain and swelling early after vaccination, but these differences were not statistically significant. Local effects after 2 weeks and systemic adverse events after vaccination were not different (data not shown). These results demonstrate that intradermal rBCG30 vaccination was as well-tolerated as nonrecombinant BCG.

Table 1
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Table 1

Demographics and clinical reactogenicity.

Human responses to BCG vaccination are complex, involving numerous components of immunity. We therefore compared a number of immune functions induced by rBCG30 and wild-type BCG—including antigen-specific T cell expansion capacity, IFN-γ secretion, phenotypes of CD4+ and CD8+ T cells consistent with effector and memory T cell populations, and direct inhibitory effects of vaccine-induced T cells on intracellular mycobacteria—and we demonstrated that rBCG30 significantly induced all of these responses, which are predicted to be critical for protective TB immunity. Our finding that humans develop enhanced Ag85b-specific responses after immunization with rBCG30 but not parental wild-type BCG mirrors previous results in guinea pigs, in which enhanced cutaneous delayed-type hypersensitivity and antibody responses developed after rBCG30 but not BCG vaccination [2, 3, 23]. For both hosts, these differences in immune responses likely reflect the low levels of Ag85b produced by standard BCG strains compared with rBCG30 [2, 3].

CD4+ Th1 cells produce IFN-γ, interleukin-2, and tumor necrosis factor-α, all cytokines involved in the activation of intracellular microbicidal activities as well as the initiation and maintenance of protective granulomatous inflammation [24, 25]. CD4+ Th1 cells are of predominant importance for protection in animal models of TB [26]. In humans, natural genetic polymorphisms resulting in deficient IFN-γ signaling result in extreme susceptibility to overwhelming disseminated mycobacterial infections [27]. We demonstrated here that rBCG30 induced significantly increased Ag85b-specific CD4+ Th1 cell responses in humans (figures 1 and 2A), which are predicted to be important for protective TB immunity.

CD8+ T cells can recognize and destroy target cells infected with intracellular pathogens, including M. tuberculosis. Peptides are presented to CD8+ T cells by MHC class I molecules, which generally bind to peptide fragments synthesized in the cytoplasm of the cell. Therefore, intracellular pathogens that escape phagosomal compartments and replicate freely within cytoplasm are potent stimulators of CD8+ T cell responses, and protective immunity against these pathogens generally depends more on these responses. However, M. tuberculosis prevents phagosome-lysome fusion, preferring to replicate within the original phagosomal/endosomal compartments it infects. These intracellular compartments favor presentation of MHC class II-restricted peptide antigens to CD4+ T cells, which have classically been considered the more important T cell subset for protective TB immunity. Despite this dogma, it has recently been shown that CD8+ T cells do contribute to protective TB immunity and may be particularly important for the prevention of TB reactivation [28–33]. Peptide antigens not synthesized in cytoplasmic locations can be presented by activated dendritic cells to CD8+ T cells via a mechanism known as “cross-presentation” [34–36]. In addition, M. tuberculosis-specific CD8+ cytolytic T cells can recognize mycobacterial lipid antigens presented by nonclassical MHC class Ib restriction elements [37–39]. Furthermore, human CD8+ T cells produce granulysin, a component of their cytolytic granules, which has been shown to have direct microbicidal effects against both extracellular and intracellular mycobacteria [40]. In the present study, we demonstrated that rBCG30 significantly enhanced Ag85b-specific CD8+ T cell responses in humans (figure 2B), which are predicted to contribute to protective TB immunity.

Two subpopulations of memory T cells have been identified, described as central and effector memory T cells [20, 41, 42]. Central memory T cells reside within lymph nodes and spleen (at least in part because of the expression of the lymph node homing molecule CCR7), have the capacity for long-term homeostatic proliferation in the absence of residual antigen, and expand robustly in response to new antigenic challenge before differentiating into effector T cells with protective functions. On the other hand, effector memory T cells do not express CCR7 and reside predominantly within peripheral tissues. Effector memory T cells rapidly provide protective cytokine and cytolytic responses but have reduced lymphoproliferative capacity and may depend on the persistence of at least low levels of antigen. Induction of both memory T cell populations may be necessary for optimal long-term vaccine-induced protection. IFN-γ production stimulated by mycobacterial antigens directly ex vivo can be used to assess the relative functional levels of effector memory T cell responses. Identification of cells with the capacity for both expansion and effector function can assess the relative functional levels of central memory T cell responses. Our combined results (figures 2–4 and table 2) suggest that rBCG30 induced significant increases in the number of both effector and central memory Ag85b-specific T cells, which are predicted to be important for protective TB immunity. Future studies will need to investigate whether these putative central and peripheral effector memory populations can be distinguished in human blood by the presence or absence of CCR7 expression, respectively.

Table 2
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Table 2

Proportions of positive interferon-γ enzyme-linked immunospot responses.

Mycobacteria replicate within macrophages, and defects in cell-mediated immunity result in increased susceptibility to these intracellular pathogens. Therefore, we predict that T cells capable of inhibiting intracellular mycobacterial growth are important for in vivo protection against TB, and immune-mediated inhibition of mycobacterial growth would more directly correlate with protective TB immunity than other immunological responses. For these reasons, we have developed an in vitro assay capable of measuring T cell-mediated inhibition of intracellular mycobacterial growth [18, 19]. In the present study, we found that rBCG30 but not Tice BCG induced significant increases in Ag85b-specific inhibitory T cell responses (figure 5). These results further indicate that vaccinations with rBCG can enhance responses relevant for protective immunity. However, only future phase 3 efficacy trials can prove whether rBCG30 or other rBCG vaccines can enhance protection against TB infection and disease.

rBCG30 is the first TB vaccine intended as a replacement for BCG, the current vaccine used worldwide. Other vaccines currently in clinical development are intended primarily as booster vaccines for BCG-immunized people. Only one such vaccine, MVA85A, has been tested in humans and the results reported. MVA85A is a modified vaccinia virus expressing Ag85a, a close relative of Ag85b, the antigen overexpressed in rBCG30. In humans, MVA85A induced significantly enhanced Ag85a-specific IFN-γ-secreting T cells [43]. Although such a response has generally been thought to indicate important immune induction, in a recent study increased Ag85a-specific IFN-γ responses produced by CD4+ T cells alone did not result in enhanced protection [44]. The capacity of T cells to both proliferate and mediate effector functions or to mediate inhibitory effects against intracellular mycobacteria after MVA85A vaccination has not been reported. It would be of interest to compare preclinical studies completed with the Ag85a and Ag85b vaccines. Boosting BCG with MVA85A was not more efficacious than administering BCG alone in the guinea pig model of pulmonary TB [45]. In contrast, boosting BCG with purified Ag85b in adjuvant significantly enhanced protective immunity in guinea pigs compared with BCG alone [23], and the use of rBCG30 overexpressing Ag85b also significantly enhanced protection in guinea pigs compared with wild-type BCG [2, 3]. In future trials, it will be important to make direct comparisons between the levels of memory T cells capable of proliferation, IFN-γ production, and inhibition of intracellular mycobacteria induced by these different experimental TB vaccines.

In summary, we have shown that rBCG30 is safe and induces significantly increased CD4+ Th1 and CD8+ T cell immunity, central and effector memory T cell subsets, and inhibitory T cells specific for the overexpressed Ag85b protective antigen. rBCG30 is thus the first TB vaccine to achieve 3 key developmental milestones. First, it demonstrated superior efficacy in the guinea pig model of TB. Second, it is safe and well-tolerated in humans. Third, it has the capacity to induce relevant TB immunity in humans by several criteria. This provides optimism that rBCG30 and/or similar vaccines modeled on it will be safe and provide superior protection against TB in humans.

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Acknowledgments

We thank the volunteers who participated in this trial for their magnanimity and the nurses from the Saint Louis University Vaccine Center for their tireless devotion to their work. We thank Barbara Jane Dillon for technical assistance with protein purification.

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Footnotes

  • Potential conflicts of interest: none reported.

  • Financial support: Aeras Global TB Vaccine Foundation; National Institutes of Health (grant NO1-AI-25464 to the Saint Louis University Vaccine and Treatment Evaluation Unit, grant RO1-AI-48391 to D.F.H., and grant RO1-AI-31338 to M.A.H.).

  • Received February 18, 2008.
  • Accepted May 13, 2008.
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References

  1. ↵
    World Health Organization. Global tuburculosis control: world report 2001. [21 August 2008]. Document WHO/CDS/TB/2001.287. Available at: http://www.who.int/docstore/gtb/publications/globrep01/index.html.
  2. ↵
    1. Horwitz MA,
    2. Harth G,
    3. Dillon BJ,
    4. Maslesa-Galic' S
    . Recombinant bacillus Calmette-Guerin (BCG) vaccines expressing the Mycobacterium tuberculosis 30-kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. Proc Natl Acad Sci USA 2000;97:13853-8.
    Abstract/FREE Full Text
  3. ↵
    1. Horwitz MA,
    2. Harth G
    . A new vaccine against tuberculosis affords greater survival after challenge than the current vaccine in the guinea pig model of pulmonary tuberculosis. Infect Immun 2003;71:1672-9.
    Abstract/FREE Full Text
    1. O'Donnell MA,
    2. Aldovini A,
    3. Duda RB,
    4. et al
    . Recombinant Mycobacterium bovis BCG secreting functional interleukin-2 enhances gamma interferon production by splenocytes. Infect Immun 1994;62:2508-14.
    Abstract/FREE Full Text
    1. Murray PJ,
    2. Aldovini A,
    3. Young RA
    . Manipulation and potentiation of antimycobacterial immunity using recombinant bacille Calmette-Guérin strains that secrete cytokines. Proc Natl Acad Sci USA 1996;93:934-9.
    Abstract/FREE Full Text
    1. Young SL,
    2. O'Donnell MA,
    3. Buchan GS
    . IL-2-secreting recombinant bacillus Calmette Guerin can overcome a type 2 immune response and corticosteroid-induced immunosuppression to elicit a type 1 immune response. Int Immunol 2002;14:793-800.
    Abstract/FREE Full Text
    1. Conradt P,
    2. Hess J,
    3. Kaufmann SH
    . Cytolytic T-cell responses to human dendritic cells and macrophages infected with Mycobacterium bovis BCG and recombinant BCG secreting listeriolysin. Microbes Infect 1999;1:753-64.
    CrossRefMedlineWeb of Science
  8. ↵
    1. Grode L,
    2. Seiler P,
    3. Baumann S,
    4. et al
    . Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin. J Clin Invest 2005;115:2472-9.
    CrossRefMedlineWeb of Science
  9. ↵
    1. Pal PG,
    2. Horwitz MA
    . Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect Immun 1992;60:4781-92.
    Abstract/FREE Full Text
  10. ↵
    1. Horwitz MA,
    2. Lee BE,
    3. Dillon BJ,
    4. Harth G
    . Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc Natl Acad Sci USA 1995;92:1530-4.
    Abstract/FREE Full Text
  11. ↵
    1. Blander SJ,
    2. Horwitz MA
    . Vaccination with the major secretory protein of Legionella pneumophila induces cell-mediated and protective immunity in a guinea pig model of Legionnaires' disease. J Exp Med 1989;169:691-705.
    Abstract/FREE Full Text
  12. ↵
    1. Wiker HG,
    2. Harboe M
    . The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol Rev 1992;56:648-61.
    Abstract/FREE Full Text
  13. ↵
    1. Belisle JT,
    2. Varalakshmi DV,
    3. Sievert T,
    4. Takayama K,
    5. Brennan PJ,
    6. Besra GS
    . Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 1997;276:1420-2.
    Abstract/FREE Full Text
  14. ↵
    1. Andersen P
    . Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect Immun 1994;62:2536-44.
    Abstract/FREE Full Text
  15. ↵
    1. Huygen K,
    2. Content J,
    3. Denis O,
    4. et al
    . Immunogenicity and protective efficacy of a tuberculosis DNA vaccine. Nat Med 1996;2:893-8.
    CrossRefMedlineWeb of Science
  16. ↵
    1. Hoft DF,
    2. Brown R,
    3. Roodman S
    . Bacille Calmette-Guérin vaccination enhances human γδ T cell responsiveness to mycobacteria suggestive of a memory-like phenotype. J Immunol 1998;161:1045-54.
    Abstract/FREE Full Text
  17. ↵
    1. Hanekom WA,
    2. Hughes J,
    3. Mavinkurve M,
    4. et al
    . Novel application of a whole blood intracellular cytokine detection assay to quantitate specific T-cell frequency in field studies. J Immunol Methods 2004;291:185-95.
    CrossRefMedlineWeb of Science
  18. ↵
    1. Hoft DF,
    2. Worku S,
    3. Kampmann B,
    4. et al
    . Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. J Infect Dis 2002;186:1448-57.
    Abstract/FREE Full Text
  19. ↵
    1. Worku S,
    2. Hoft DF
    . Differential effects of control and antigen-specific T cells on intracellular mycobacterial growth. Infect Immun 2003;71:1763-73.
    Abstract/FREE Full Text
  20. ↵
    1. Sallusto F,
    2. Lenig D,
    3. Forster R,
    4. Lipp M,
    5. Lanzavecchia A
    . Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 1999;401:708-12.
    CrossRefMedline
    1. Lauvau G,
    2. Vijh S,
    3. Kong P,
    4. et al
    . Priming of memory but not effector CD8+ T cells by a killed bacterial vaccine. Science 2001;294:1735-9.
    Abstract/FREE Full Text
  22. ↵
    1. Migueles SA,
    2. Laborico AC,
    3. Shupert WL,
    4. et al
    . HIV-specific CD8+ T cell proliferation is coupled to perforin expression and is maintained in nonprogressors. Nat Immunol 2002;3:1061-8.
    CrossRefMedlineWeb of Science
  23. ↵
    1. Horwitz MA,
    2. Harth G,
    3. Dillon BJ,
    4. Maslesa-Galic S
    . Enhancing the protective efficacy of Mycobacterium bovis BCG vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein. Infect Immun 2005;73:4676-83.
    Abstract/FREE Full Text
  24. ↵
    1. Mosmann TR,
    2. Cherwinski H,
    3. Bond MW,
    4. Giedlin MA,
    5. Coffman RL
    . Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986;136:2348-57.
    Abstract
  25. ↵
    1. Abbas AK,
    2. Murphy KM,
    3. Sher A
    . Functional diversity of helper T lymphocytes. Nature 1996;383:787-93.
    CrossRefMedline
  26. ↵
    1. Wangoo A,
    2. Sparer T,
    3. Brown IN,
    4. et al
    . Contribution of Th1 and Th2 cells to protection and pathology in experimental models of granulomatous lung disease. J Immunol 2001;166:3432-9.
    Abstract/FREE Full Text
  27. ↵
    1. Newport MJ,
    2. Huxley CM,
    3. Huston S,
    4. et al
    . A mutation in the IFN-gamma receptor gene and susceptibility to mycobacterial infection. N Engl J Med 1996;335:1941-9.
    CrossRefMedlineWeb of Science
  28. ↵
    1. Flynn JL,
    2. Goldstein MM,
    3. Triebold KJ,
    4. Koller B,
    5. Bloom BR
    . Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc Natl Acad Sci USA 1992;89:12013-7.
    Abstract/FREE Full Text
    1. Bloom BR,
    2. Flynn J,
    3. McDonough K,
    4. Kress Y,
    5. Chan J
    . Experimental approaches to mechanisms of protection and pathogenesis in M. tuberculosis infection. Immunobiology 1994;191:526-36.
    MedlineWeb of Science
    1. Ladel CH,
    2. Daugelat S,
    3. Kaufmann SH
    . Immune response to Mycobacterium bovis bacille Calmette Guérin infection in major histocompatibility complex class I- and II-deficient knock-out mice: contribution of CD4 and CD8 T cells to acquired resistance. Eur J Immunol 1995;25:377-84.
    MedlineWeb of Science
    1. Feng CG,
    2. Britton WJ
    . CD4+ and CD8+ T cells mediate adoptive immunity to aerosol infection of Mycobacterium bovis bacillus Calmette-Guérin. J Infect Dis 2000;181:1846-9.
    Abstract/FREE Full Text
    1. Cho S,
    2. Mehra V,
    3. Thoma-Uszynski S,
    4. et al
    . Antimicrobial activity of MHC class I-restricted CD8+ T cells in human tuberculosis. Proc Natl Acad Sci USA 2000;97:12210-5.
    Abstract/FREE Full Text
  33. ↵
    1. van Pinxteren LA,
    2. Cassidy JP,
    3. Smedegaard BH,
    4. Agger EM,
    5. Andersen P
    . Control of latent Mycobacterium tuberculosis infection is dependent on CD8+ T cells. Eur J Immunol 2000;30:3689-98.
    CrossRefMedlineWeb of Science
  34. ↵
    1. Cresswell P,
    2. Ackerman AL,
    3. Giodini A,
    4. Peaper DR,
    5. Wearsch PA
    . Mechanisms of MHC class I-restricted antigen processing and cross-presentation. Immunol Rev 2005;207:145-57.
    CrossRefMedlineWeb of Science
    1. Rock KL,
    2. Shen L
    . Cross-presentation: underlying mechanisms and role in immune surveillance. Immunol Rev 2005;207:166-83.
    CrossRefMedlineWeb of Science
  36. ↵
    1. Bevan MJ
    . Cross-priming. Nat Immunol 2006;7:363-5.
    CrossRefMedlineWeb of Science
  37. ↵
    1. Lewinsohn DM,
    2. Alderson MR,
    3. Briden AL,
    4. Riddell SR,
    5. Reed SG,
    6. Grabstein KH
    . Characterization of human CD8+ T cells reactive with Mycobacterium tuberculosis-infected antigen-presenting cells. J Exp Med 1998;187:1633-40.
    Abstract/FREE Full Text
    1. Lalvani A,
    2. Brooks R,
    3. Wilkinson R,
    4. et al
    . Human cytolytic and IFN-γ secreting CD8+ T lymphocytes specific for Mycobacterium tuberculosis. Proc Natl Acad Sci USA 1998;95:270-5.
    Abstract/FREE Full Text
  39. ↵
    1. Lewinsohn DM,
    2. Briden AL,
    3. Reed SG,
    4. Grabstein KH,
    5. Alderson MR
    . Mycobacterium tuberculosis-reative CD8+ T lynphocytes: the realtive contribution of classical versus nonclassical HLA restriction. J Immunol 2000;165:925-30.
    Abstract/FREE Full Text
  40. ↵
    1. Stenger S,
    2. Hanson DA,
    3. Teitlbaum R,
    4. et al
    . An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998;282:121-5.
    Abstract/FREE Full Text
  41. ↵
    1. Campbell JJ,
    2. Murphy KE,
    3. Kunkel EJ,
    4. et al
    . CCR7 expression and memory T cell diversity in humans. J Immunol 2001;166:877-84.
    Abstract/FREE Full Text
  42. ↵
    1. Geginat J,
    2. Sallusto F,
    3. Lanzavecchia A
    . Cytokine-driven proliferation and differentiation of human naive, central memory, and effector memory CD4+ T cells. J Exp Med 2001;194:1711-9.
    Abstract/FREE Full Text
  43. ↵
    1. McShane H,
    2. Pathan AA,
    3. Sander CR,
    4. et al
    . Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat Med 2004;10:1240-4.
    CrossRefMedlineWeb of Science
  44. ↵
    1. Majlessi L,
    2. Simsova M,
    3. Jarvis Z,
    4. et al
    . An increase in antimycobacterial Th1-cell responses by prime-boost protocols of immunization does not enhance protection against tuberculosis. Infect Immun 2006;74:2128-37.
    Abstract/FREE Full Text
  45. ↵
    1. Williams A,
    2. Hatch GJ,
    3. Clark SO,
    4. et al
    . Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis. Tuberculosis (Edinb) 2005;85:29-38.
    CrossRefMedline
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  1. J Infect Dis. (2008) 198 (10): 1491-1501. doi: 10.1086/592450
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