Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly⁺ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C–deficient hly⁺ rBCG (ΔureC hly⁺ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. ΔureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of ΔureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell–mediated immunity.

Mycobacterium bovis bacille Calmette-Guérin (BCG) represents the most widely used viable vaccine for tuberculosis, with over 3 billion doses administered (1). General agreement exists that BCG can protect against, or at least ameliorate, severe forms of systemic tuberculosis in children, particularly meningitis. However, BCG’s protective value against adult pulmonary tuberculosis ranges from little to none. Although approximately 95% of all tuberculosis cases occur in the developing world, several countries of eastern Europe, including Russia, have witnessed increasing incidences of tuberculosis in recent years, with an estimated 9 million new tuberculosis cases globally in 2002 (2). Worldwide estimated mortality ranges from 1.6 to 2.2 million lives per year, depending on whether the half-million individuals suffering coinfection with HIV and Mycobacterium tuberculosis, are added to the toll of HIV or tuberculosis.

Recent field studies identified a novel genotype family of M. tuberculosis, Beijing, or W, which is increasingly spreading over the globe. It has been proposed that under the selective pressure of mass BCG vaccination and broad-scale administration of tuberculostatics, the population structure of M. tuberculosis is changing in favor of the selection of conserved genotypes such as Beijing with higher survival fitness for these conditions (3–5). This is particularly worrisome since this genotype family comprises a high proportion of multidrug-resistant strains, which are rapidly expanding globally (6, 7). In light of increasing incidences of multidrug-resistant M. tuberculosis strains, the urgent need for a more efficacious tuberculosis vaccine is no longer restricted to the developing world.

The preferred intraphagosomal location of M. tuberculosis and BCG in professional APCs — mostly macrophages and DCs — dictates trafficking of their antigens through the MHC II pathway, which results in preferential stimulation of CD4 T cells (8). Yet, MHC I–restricted CD8 T cells are known to contribute to immunity to M. tuberculosis (8). Recent evidence suggests that M. tuberculosis induces apoptosis in infected cells, which in turn results in the formation of vesicles carrying mycobacterial antigens that can be taken up by bystander APC (9). This process, termed cross-priming, not only promotes MHC I antigen processing and CD8 T cell stimulation but also bypasses immunosuppressive mechanisms imposed on the infected cell (10). BCG only induces weak apoptosis and CD8 T cell stimulation (9).

To improve access of mycobacterial antigens to the MHC I pathway, which would result in better CD8 T cell stimulation, we constructed an rBCG strain that secretes listeriolysin (Hly) of Listeria monocytogenes (11). The Hly enables L. monocytogenes to escape from the phagosomes of infected host cells (12). Hly has an acidic pH optimum of greater than 5.8 (13) while BCG modifies the phagosomal pH to remain neutral (14). To improve the pH milieu for Hly activity, a urease C-deficient (ureC-deficient) Hly-secreting BCG–vaccine strain was constructed (15). Mycobacterial ureC plays a role in pH neutralization of the phagosome and thus inhibits maturation of the phagolysosome (14, 16). Here, we demonstrate that Hly-secreting recombinant BCG (hly⁺ rBCG) and ureC–deficient hly⁺ rBCG (ΔureC hly⁺ rBCG) mutant strains induce more efficacious protection against M. tuberculosis than does the parental BCG strain in a low-to-medium dose aerosol-infection mouse model.

Generation and characterization of ΔureC hly+ rBCG. In order to generate a live vaccine that provides improved protection against M. tuberculosis, we equipped BCG with the membrane-perforating Hly of L. monocytogenes (hly⁺ rBCG) (Figure 1A). To improve the pH milieu for Hly activity, a ΔureC hly⁺ rBCG strain that lacked ureC activity was constructed. As predicted, hemolytic activities of supernatants from hly⁺ rBCG and ΔureC hly⁺ rBCG cultures against sheep red blood cells were considerably more intense than those seen with parental BCG (Figure 1B). To show whether absence of ureC allowed phagosomal acidification, the pH of mycobacterial phagosomes was determined by ratiometric analysis of dye-labeled phagosomes containing parental or ΔureC hly⁺ rBCG organisms. Macrophages were infected with DID-stained BCG strains. Phase contrast with fluorescence overlay is shown in Figure 1, C and E, while parts D and F show the same cells with pH pseudocolor. As expected, parental BCG faced an almost neutral pH (Figure 1, C and D) whereas Hly produced by ΔureC hly⁺ rBCG faced an acidic milieu between pH 4.5 and pH 5.5 (Figure 1, E and F). Similar effects were observed with parental BCG or ΔureC hly⁺ rBCG covalently coupled to the pH-sensitive dye Cypher5 (data not shown). While parental BCG showed a weak fluorescence signal over the 60-minute recording period, ΔureC hly⁺ rBCG gained strong fluorescence intensity after uptake by macrophages, and bacteria outside of cells did not show changes in fluorescent signal.

Figure 1

Hemolytic activity of and phagosomal acidification by ΔureC hly⁺ rBCG. (A) RT-PCR analysis of BCG-strains for Hly-secretion. Secretion of Hly was analyzed by RT-PCR using RNA from parental BCG (lane 1), rBCG ΔureC (lane 2), hly⁺ rBCG (lane 3), and ΔureC hly⁺ rBCG (lane 4). Shown is 1 out of 3 representative experiments. (B) Hemolysis by hly⁺ rBCG and ΔureC hly⁺ rBCG but not parental BCG. Sheep red blood cells were incubated with hly⁺ rBCG (open triangles), ΔureC hly⁺ rBCG (closed triangles), parental BCG (closed squares), or L. monocytogenes (open diamonds), or remained untreated (open circles). At indicated time points, aliquots were taken and release of hemoglobin was determined by optical absorption as measurement for lysis of red blood cells. Shown is 1 representative experiment of 4. (C–F) Primary murine macrophages were infected with parental BCG (C and D) or ΔureC hly⁺ rBCG (E and F) for 2.5 hours. Images on the left show BCG stained with DID (signal shown in green). Images on the right show a pseudocolor representation of the fluorescence ratio in the blue and green channels with the Lyso Sensor Yellow/Blue dye. Images are merged with a black-and-white phase contrast image. Note that not all bacteria in each batch were stained due to the poor solubility of DID and its high affinity to the hydrophobic cell wall. Scale bar: 20 μm.

Superior vaccine efficacy of hly+ rBCG and ΔureC hly ⁺ rBCG against M. tuberculosis laboratory strain H37Rv and clinical isolate of the Beijing/W genotype family. BALB/c mice were vaccinated (one single i.p. injection of 10⁶ CFU) with hly⁺ rBCG, ΔureC hly⁺ rBCG, or parental BCG and aerosol challenged 120 days after vaccination with M. tuberculosis H37Rv microorganisms. At this time point, BCG had been completely eradicated in vaccinated mice (verified by CFU analysis, data not shown). Vaccination with hly⁺ rBCG and parental BCG considerably reduced M. tuberculosis load in the lungs of aerosol-challenged mice as compared with naive controls (Figure 2A). In the early (day 30) to intermediate (day 90) phase of infection, the course of M. tuberculosis H37Rv infection did not significantly differ between the 2 vaccinated groups. However, at late stages (day 150) of infection, the M. tuberculosis H37Rv loads in the lung were significantly (P = 0.016) reduced by 1 order of magnitude in hly⁺ rBCG–vaccinated mice as compared with mice in the parental BCG group.

Figure 2

Greater protection against M. tuberculosis aerosol challenge by vaccination with hly⁺ rBCG⁺ and ΔureC hly⁺ rBCG. (A) BALB/c mice were vaccinated with hly⁺ rBCG (triangles) or parental BCG (circles) or left naive (squares). At day 120 after vaccination, mice were aerosol challenged with 200 CFU of M. tuberculosis H37Rv. P = 0.016 (Mann-Whitney U test) for hly⁺ rBCG compared with parental BCG at day 150 after infection (p.i.). Shown is 1 representative experiment of 2. The mean of results from 7 mice is shown (± SEM). (B and C) BALB/c mice were vaccinated with ΔureC hly⁺ rBCG (diamonds) or parental BCG (circles) or left naive (squares). At day 120 after vaccination, mice were aerosol challenged with 30 CFU (B) and 200 CFU (C) of M. tuberculosis H37Rv. M. tuberculosis load in lung was determined at the indicated time points. Shown are 2 representative experiments. *P = 0.0012 and **P = 0.001 (Mann-Whitney U test) for ΔureC hly⁺ rBCG compared with parental BCG at day 90 p.i. The mean of results for 7 mice is shown (± SEM).

Although both ΔureC hly⁺ rBCG and parental BCG protected against tuberculosis, protection evoked by ΔureC hly⁺ rBCG was substantially more successful than that evoked by parental BCG (Figure 2, B and C). This was true for infection with medium doses (200 CFU; Figure 2C) and low doses (30 CFU; Figure 2B) of M. tuberculosis. In the experiment shown in Figure 2B, protection led to an almost 1,000-fold reduction of the M. tuberculosis load in lungs as compared with naive mice at day 200 after challenge. Also, at earlier time points, enhanced protection was seen with ΔureC hly⁺ rBCG as compared with parental BCG (Figure 2B).

An equally striking improvement was noted after challenge with the clinical genotype strain M. tuberculosis Beijing/W (Figure 3, A and B). The ΔureC hly⁺ rBCG strain significantly (P < 0.001 at day 200) protected against the Beijing/W genotype strain in both lung and spleen over the entire 200-day period of observation (P < 0.05). Compared with vaccination with parental BCG, which virtually failed to protect against M. tuberculosis Beijing/W in this experiment, vaccination with ΔureC hly⁺ rBCG reduced CFU in the lung by more than 100-fold.

Figure 3

Greater protection against M. tuberculosis Beijing/W by vaccination with ΔureC hly⁺ rBCG. BALB/c mice were vaccinated with ΔureC hly⁺ rBCG (diamonds) or parental BCG (circles) or left naive (squares). At day 120 after vaccination, mice were aerosol challenged with 200 CFU of M. tuberculosis Beijing/W. M. tuberculosis load in lung was determined at the indicated time points. Upper panel: lung, P < 0.001 (Mann-Whitney U test) for ΔureC hly⁺ rBCG compared with parental BCG at day 200 p.i.; and P < 0.05 (Friedman test) for the whole observation period. Lower panel: spleen, P < 0.001 (Mann Whitney U test) for ΔureC hly⁺ rBCG compared with parental BCG at day 200 p.i. Shown is 1 representative experiment of 2. The mean of results for 7 mice is shown (± SEM).

In vivo safety of hly+ rBCG and ΔureC hly+ rBCG. So that we could analyze the safety of the new rBCG vaccine strains in vivo at high doses, SCID mice were vaccinated i.v. with approximately 10⁸ CFU (Figure 4A). At this high dose, SCID mice vaccinated with hly⁺ rBCG survived significantly longer (mean survival time, 65 days; P = 0.001) than did SCID mice vaccinated with parental BCG (mean survival time, < 25 days). The ΔureC hly⁺ rBCG–vaccinated mice survived even longer (mean survival time, 80 days; P < 0.001) than mice vaccinated with parental BCG. Thus, in the SCID mouse model, virulence of the rBCG strains was substantially reduced as compared with parental BCG. In further support of the high safety of hly⁺ rBCG vaccine candidates, CFUs in spleen and lung did not differ among BALB/c wild-type mice vaccinated with the 3 different BCG strains, and all 3 strains were cleared by a similar kinetics (data not shown). Similarly, CFU in spleen and lung of RAG1^–/– mutant mice after vaccination with hly⁺ rBCG and ΔureC hly⁺ rBCG did not significantly differ from CFU of mice vaccinated with parental BCG (Figure 4B). Consistent with these findings, histological analysis of vaccinated mice did not reveal any alterations in gross pathology in lung, spleen, and liver of either immunocompetent BALB/c mice or RAG1^–/– mutant mice (data not shown).

Figure 4

High safety profile of hly⁺ rBCG and ΔureC hly⁺ rBCG vaccines. (A) SCID immune-deficient mutant mice were vaccinated i.v. with 0.5 – 1 × 10⁸ CFU of hly⁺ rBCG (diamonds), ΔureC hly⁺ rBCG (triangles), and parental BCG (squares) to determine survival rate. P = 0.001 for hly⁺ rBCG compared with parental BCG, and P < 0.001 for ΔureC hly⁺ rBCG compared with parental BCG. (B) RAG1–/– immunodeficient mutant mice were vaccinated i.v. with 0.5 – 1 × 10⁶ CFU of hly⁺ rBCG (diamonds), ΔureC hly⁺ rBCG (triangles), and parental BCG (squares) to determine bacterial load in lung and spleen. Differences were not statistically significant (P = 0.7 for lung and P = 0.5 for spleen; Mann-Whitney U test). Shown is 1 representative experiment of each of 2 similar experiments.

Antigen translocation and apoptosis of host cells infected with hly+ rBCG. We wanted to gain deeper insight into the biological mechanisms responsible for superior vaccine efficacy of ΔureC hly⁺ rBCG. Two mechanisms can be envisaged, both depending on the capacity of Hly to perforate the phagosomal membrane. These mechanisms are not mutually exclusive. First, perforation of the membrane surrounding the mycobacterial phagosome facilitates translocation and subsequent MHC I loading of mycobacterial antigens (11, 17). Second, membrane perforation promotes leakage of phagolysosomal proteases such as cathepsins into the cytosol, which activate caspase cascade-inducing apoptotic cell death (11, 18–20). Apoptosis in turn results in cross-priming of mycobacterial antigens (9). Due to the immunosuppressive activities of mycobacteria (10), cross-priming would not only promote MHC I, but would also promote MHC II–restricted T cell stimulation. As shown in Figure 5, A–E, mycobacterial antigens were detected in the cytoplasm of macrophages infected with ΔureC hly⁺ rBCG; the antigens remained more concentrated in the phagosome of cells infected with parental BCG. Release of BCG from the phagosome of ΔureC hly⁺ rBCG–infected macrophages is indicated by arrows in Figure 5E. When we analyzed the density of the immunogold signal in the cytoplasm of infected cells, we found that ΔureC hly⁺ rBCG–infected macrophages had significantly (P = 0.026) more mycobacterial antigen in the cytoplasm than did macrophages infected with parental BCG (7.0 ± 3.7 gold particles/μm² for ΔureC hly⁺ rBCG vs. 3.5 ± 2.3 particles/μm² for parental BCG). Noninfected macrophages showed negligible background staining of 0.14 ± 0.07 particles/μm². Moreover, ΔureC hly⁺ rBCG induced profound apoptosis in both murine (P < 0.001) and human macrophages (P < 0.05) (Figure 6, A and B, respectively). In contrast, parental BCG marginally increased apoptosis above background levels. The hly⁺ rBCG–induced macrophage apoptosis was further increased by the absence of the ureC gene in ΔureC hly⁺ rBCG, presumably due to the optimized pH for Hly activity. These results were supported by TUNEL staining of infected macrophages (Figure 6, C–E). We concluded that cytosolic MHC I loading and, to a greater extent, cross-priming contribute to improved vaccine efficacy of hly⁺ rBCG.

Figure 5

Antigen presence in the cytosol. (A–D) Bone marrow macrophages infected for 24 hours with ΔureC hly⁺ rBCG (A and B) and parental BCG (C and D). The images are projections of 2 confocal planes from specimens stained for BCG (green), F-actin (red), and DNA (blue) (overlay in A and C). Material stained with an antibody raised against BCG is dispersed throughout the cytoplasm in cells infected with ΔureC hly⁺ rBCG while BCG material is exclusively located in bacteria within cells infected with parental BCG. Scale bar in A–D: 10 μm. (E and F) Fine structural analysis of mycobacterial localization in bone marrow macrophages 24 hours after infection with ΔureC hly⁺ rBCG (E) and parental BCG (F). Panel E shows strong staining of a bacterium in a phagosome; additional staining for mycobacterial material was found in the cytoplasm (indicated by an arrow). However, cytoplasmic staining was greatly reduced in cells infected with parental BCG (F). Quantification of the density of immunogold signal in the cytoplasm of infected cells: parental BCG, 3.5 ± 2.3 gold particles/μm²; ΔureC hly⁺ rBCG, 7.0 ± 3.7 gold particles/μm²; P = 0.022 (Mann-Whitney U test). Scale bar in E and F: 1 μm.

Figure 6

Increased apoptosis of infected macrophages. Primary human (A) and murine (B) macrophages were infected with hly⁺ rBCG (filled triangles), ΔureC hly⁺ rBCG (circles), parental BCG (open triangles), or ΔureC rBCG (open squares), or left uninfected (filled squares). After different periods of time, apoptotic cell death was determined by measurement of DNA-fragmentation according to ref. 43. Statistical analyses resulted in values of P < 0.05 in A and P < 0.001 in B for ΔureC hly⁺ rBCG compared with parental BCG over the whole observation period. Shown is 1 out of 3 independent experiments each. (C–E) After 24 hours of infection with ΔureC hly⁺ rBCG (C), about 45% of bone marrow macrophages underwent apoptosis as revealed by a positive TUNEL assay while only 6% of BCG-infected macrophages (D) were apoptotic. Scale bar: 40 μm. (E) Quantification of the TUNEL assay: blue, BCG positive; pink, BCG negative.

The present report describes superior protection against tuberculosis by vaccination of mice with hly⁺ rBCG of L. monocytogenes as compared with vaccination with parental BCG. Murine aerosol-challenge models with 2 different M. tuberculosis strains, the laboratory strain H37Rv and a clinical isolate of the Beijing/W genotype family, revealed that hly⁺ rBCG and, to a greater extent, ΔureC hly⁺ rBCG vaccine strains were significantly more efficacious in inducing protective immunity against tuberculosis than parental BCG.

The ΔureC rBCG strain used in our experiments was originally engineered by allelic exchange (15). ureC is involved in the maintenance of neutral intraphagosomal pH within BCG-containing vacuoles (21). Hence, lack of this enzyme allows phagosomal acidification, thereby creating an optimal pH milieu for cytolytic functions of Hly. In addition, absence of ureC has been suggested as promoting MHC II trafficking to the macrophage cell surface (22). Hly is a major virulence factor of L. monocytogenes. By perforating the phagosomal membrane, it facilitates egression of L. monocytogenes into the cytosol. The acidic pH optimum of Hly restricts its activity in the maturing phagosome. However, BCG arrests phagosome maturation at an early stage and prohibits pH acidification. This obstacle of maturation arrest could be overcome, at least partially, by the removal of ureC. In the absence of ureC, the phagosomal pH of infected macrophages first decreases, and full activity of Hly is achieved only at optimal (acidic) pH. It is possible that the pH gradient subsequently breaks down due to proton influx through Hly-mediated pores. We concluded that Hly secreted by ΔureC hly⁺ rBCG was more active than that secreted by hly⁺ rBCG because of an optimized phagosomal pH milieu for Hly.

BCG vaccination protects newborns against conventional M. tuberculosis isolates (1, 7, 23, 24) but has apparently lost vaccine efficacy against newly emerging isolates, notably of the M. tuberculosis Beijing/W genotype family (3, 24–28). In experimental animal models, BCG confers only weak protection against the clinical isolate Beijing/W (29). Consistent with this observation, we found low to absent protection against M. tuberculosis Beijing/W in BCG-vaccinated mice. In contrast, ΔureC hly⁺ rBCG not only induced more potent protection against the laboratory strain M. tuberculosis H37Rv but also showed greater protection against M. tuberculosis Beijing/W.

With respect to the mechanisms underlying improved protection by hly⁺ rBCG, 2 explanations, which are not mutually exclusive, are conceivable. First, the hly⁺ rBCG vaccine strains may have enhanced cytosolic antigen loading of MHC I molecules in infected APCs to prime CD8 T cells (17). Consistent with this notion, we detected a higher abundance of mycobacterial antigens in the cytoplasm of macrophages infected with ΔureC hly⁺ rBCG as compared with those infected with parental BCG. Second, perforation of the phagosomal membrane by Hly may promote cross-priming by induction of apoptosis within the infected macrophage (9). It remains to be clarified whether M. tuberculosis preferentially induces or inhibits apoptosis (9, 30, 31), and it is likely that the outcome of apoptosis regulation depends on specific conditions. Additionally, we assume that ΔureC hly⁺ rBCG induces apoptosis via a different mechanism than does M. tuberculosis. Hly from L. monocytogenes has been shown to induce apoptotic cell death in different cell types (19, 20, 32). It is likely that membrane perforation by Hly secreted by L. monocytogenes or by ΔureC hly⁺ rBCG facilitates delivery of phagolysosomal proteases into the cytosol (32), where cathepsins have been shown to induce apoptosis via the mitochondrial pathway (18). Although first experiments with a macrophage cell line did not reveal cytolytic activity of hly⁺ rBCG⁺ by lactate dehydrogenase–release assay (33), we found significant induction of apoptotic cell death by measuring DNA-fragmentation of primary macrophages. Apoptosis of infected cells promotes cross-priming of mycobacterial antigens via uptake of apoptotic vesicles by DCs and therefore improves cell-mediated immune responses (9). Transfer of antigens from infected cells to potent APCs such as DCs may represent an alternative pathway involving infection-mediated apoptosis of host cells, by which CD8 T cells recognize antigens of the phagosomally secluded tubercle bacillus (9). Recent experiments demonstrated that vesicles containing mycobacterial antigens derived from macrophages infected with BCG are capable of adoptively transferring protection against M. tuberculosis to mice (our unpublished observations).

In sum, we assume that improved vaccine efficacy of ΔureC hly⁺ rBCG as compared with hly⁺ rBCG and parental BCG is best explained by efficient perforation of phagosomal membranes by Hly. Hly meets an acidic pH optimum in the phagosome due to absent ureC activity. Perforation promotes antigen translocation into the cytoplasm and facilitates cross-priming through increased apoptosis. We consider the latter mechanism of major importance for augmented vaccine efficacy of ΔureC hly⁺ rBCG.

Concerning in vivo safety of the ΔureC hly⁺ rBCG and hly⁺ rBCG vaccines, we observed even lower virulence as compared with that of parental BCG in immunodeficient SCID mice, which is most likely due to reduced intracellular persistence of both rBCG strains in infected host cells (34, 35). Consistent with our finding that ΔureC hly⁺ rBCG was highly attenuated in SCID mice, ΔureC rBCG has already shown a slight decrease in multiplication and persistence in the lung of vaccinated mice compared with parental BCG (36). This is in agreement with the observation that ureC-deficient BCG is significantly attenuated in its capacity to block MHC class II expression (22). Both for safety and for vaccine efficacy reasons, we selected ΔureC hly⁺ rBCG for good manufacturing practices (GMP) production and clinical trials after licensing to Vakzine Projekt Management GmbH (Hannover, Germany).

A strategy to improve antigen display by BCG has previously been reported (37). Increased vaccine efficacy of the 2 rBCG strains is based on different mechanisms — elevated antigen secretion by the rBCG strain of Horwitz et al. (37) and altered intracellular behavior of the rBCG strain described here. A combination of both strains, therefore, may be considered for replacing conventional BCG for vaccination of newborns. Recently, several subunit vaccine candidates have been described, which can be used to boost immune responses induced by BCG prime (38–42). It is tempting to assume that a combination of priming with improved BCG such as ΔureC hly⁺ rBCG and boosting with the most efficacious subunit vaccine would provide more powerful intervention measures against one of the major threats among infectious diseases, tuberculosis.

The authors thank Beatrix Fauler for immunostainings of ultrathin cryosections and Silke Bandermann for excellent technical assistance. The BCG Pasteur 1173P2 strain was kindly provided by B. Gicquel. We thank D. Russell for providing unpublished data on the impact of ureC on pH neutralization. The paper received financial support from European Union FP5 (QLK2-1999-01093) and FP6 (LSHP-CT-2003-503367) as well as from the German Science Foundation Priority Programme Novel Vaccination Strategies (SPP 1089, KA 573/4-2).

Leander Grode’s present address is: Vakzine-Projekt Management GmbH, Braunschweig, Germany.

Peter Seiler’s present address is: Bayer HealthCare AG, Wuppertal, Germany.

Jürgen Hess’s present address is: responsif GmbH, Erlangen, Germany.

Leander Grode, Peter Seiler, and Sven Baumann contributed equally to this work.

Nonstandard abbreviations used: BCG, Mycobacterium bovis bacille Calmette-Guérin; Hly, listeriolysin; rBCG, recombinant BCG; hly⁺ rBCG, Hly-secreting rBCG; ΔureC hly⁺ rBCG, ureC-deficient hly⁺ rBCG; ureC, urease C.

Conflict of interest: L. Grode, S.H.E. Kaufmann, and J. Hess are the inventors of the rBCG strains, and they have exclusively licensed them through the Max Planck Society to Vakzine Projekt Management GmbH (VPM; Hannover, Germany). S.H.E. Kaufmann reports having received research grant support from VPM, and L. Grode is now an employee of VPM.

1. Fine, PE. BCG: the challenge continues [review]. Scand. J. Infect. Dis. 2001. 33:243-245.
   View this article via: PubMed Google Scholar
2. Fine, P.E.M., Carneiro, I.A.M., Milstien, J.B., and Clements, M. 1999. Issues relating to the use of BCG in immunisation programmes. A discussion document. WHO. Geneva, Switzerland. WHO/V&B/99.23. http://www.who.int/vaccines-documents/DocsPDF99/www9943.pdf.
3. Glynn, JR, Whiteley, J, Bifani, PJ, Kremer, K, van Soolingen, D. Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg. Infect. Dis. 2002. 8:843-849.
   View this article via: PubMed Google Scholar
4. Manca, C, et al. Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis. Infect. Immun. 2004. 72:5511-5514.
   View this article via: PubMed Google Scholar
5. Reed, MB, et al. A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature. 2004. 431:84-87.
   View this article via: PubMed Google Scholar
6. Munsiff, SS, et al. Persistence of a highly resistant strain of tuberculosis in New York City during 1990-1999. J. Infect. Dis. 2003. 188:356-363.
   View this article via: PubMed Google Scholar
7. Espinal, MA. The global situation of MDR-TB [review]. Tuberculosis (Edinb). 2003. 83:44-51.
   View this article via: PubMed Google Scholar
8. Kaufmann, SHE, McKnight, AJ. Annulling a dangerous liaison: vaccination strategies against AIDS and tuberculosis. Nat. Med. 2005. 11(4 Suppl.):S33-S44.
   View this article via: PubMed Google Scholar
9. Schaible, UE, et al. Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis. Nat. Med. 2003. 9:1039-1046.
   View this article via: PubMed Google Scholar
10. Pancholi, P, Mirza, A, Bhardwaj, N, Steinman, RM. Sequestration from immune Cd4+ T-Cells of mycobacteria growing in human macrophages. Science. 1993. 260:984-986.
    View this article via: PubMed Google Scholar
11. Mandal, M, Lee, KD. Listeriolysin O-liposome-mediated cytosolic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency, activity, and tumor protection. Biochim Biophys Acta. 2002. 1563:7-17.
    View this article via: PubMed Google Scholar
12. Portnoy, DA, Chakraborty, T, Goebel, W, Cossart, P. Molecular determinants of Listeria monocytogenes pathogenesis [review]. Infect. Immun. 1992. 60:1263-1267.
    View this article via: PubMed Google Scholar
13. Glomski, IJ, Gedde, MM, Tsang, AW, Swanson, JA, Portnoy, DA. The Listeria monocytogenes hemolysin has an acidic pH optimum to compartmentalize activity and prevent damage to infected host cells. J. Cell Biol. 2002. 156:1029-1038.
    View this article via: PubMed Google Scholar
14. Honer zu Bentrup, K, Russell, DG. Mycobacterial persistence: adaptation to a changing environment [review]. Trends Microbiol. 2001. 9:597-605.
    View this article via: PubMed Google Scholar
15. Reyrat, JM, Berthet, FX, Gicquel, B. The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guerin. Proc. Natl. Acad. Sci. U. S. A. 1995. 92:8768-8772.
    View this article via: PubMed Google Scholar
16. Schaible, UE, Sturgill-Koszycki, S, Schlesinger, PH, Russell, DG. Cytokine activation leads to acidification and increases maturation of Mycobacterium avium-containing phagosomes in murine macrophages. J. Immunol. 1998. 160:1290-1296.
    View this article via: PubMed Google Scholar
17. Kaufmann, SHE. Immunity to intracellular bacteria. Annu. Rev. Immunol. 1993. 11:129-163.
    View this article via: PubMed Google Scholar
18. Cirman, T, et al. Selective disruption of lysosomes in HeLa cells triggers apoptosis mediated by cleavage of bid by multiple papain-like lysosomal cathepsins. J. Biol. Chem. 2004. 279:3578-3587.
    View this article via: PubMed Google Scholar
19. Rogers, HW, Callery, MP, Deck, B, Unanue, ER. Listeria monocytogenes induces apoptosis of infected hepatocytes. J. Immunol. 1996. 156:679-684.
    View this article via: PubMed Google Scholar
20. Guzman, CA, et al. Apoptosis of mouse dendritic cells is triggered by listeriolysin, the major virulence determinant of Listeria monocytogenes. Mol. Microbiol. 1996. 20:119-126.
    View this article via: PubMed Google Scholar
21. Clemens, DL, Lee, BY, Horwitz, MA. Purification, characterization, and genetic-analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction. J. Bacteriol. 1995. 177:5644-5652.
    View this article via: PubMed Google Scholar
22. Sendide, K, Deghmane, AE, Reyrat, JM, Talal, A, Hmama, Z. Mycobacterium bovis BCG urease attenuates major histocompatibility complex class II trafficking to the macrophage cell surface. Infect. Immun. 2004. 72:4200-4209.
    View this article via: PubMed Google Scholar
23. Bifani, PJ, Mathema, B, Kurepina, NE, Kreiswirth, BN. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol. 2002. 10:45-52.
    View this article via: PubMed Google Scholar
24. Filliol, I, et al. Global distribution of Mycobacterium tuberculosis spoligotypes. Emerg. Infect. Dis. 2002. 8:1347-1349.
    View this article via: PubMed Google Scholar
25. van Soolingen, D. Molecular epidemiology of tuberculosis and other mycobacterial infections: main methodologies and achievements [review]. J. Intern. Med. 2001. 249:1-26.
    View this article via: PubMed Google Scholar
26. Rad, ME, et al. Mutations in putative mutator genes of Mycobacterium tuberculosis strains of the W-Beijing family. Emerg. Infect. Dis. 2003. 9:838-845.
    View this article via: PubMed Google Scholar
27. Toungoussova, OS, Mariandyshev, A, Bjune, G, Sandven, P, Caugant, DA. Molecular epidemiology and drug resistance of Mycobacterium tuberculosis isolates in the archangel prison in Russia: predominance of the W-Beijing clone family. Clin. Infect. Dis. 2003. 37:665-672.
    View this article via: PubMed Google Scholar
28. van Soolingen, D, et al. Predominance of a single genotype of Mycobacterium tuberculosis in countries of east Asia. J. Clin. Microbiol. 1995. 33:3234-3238.
    View this article via: PubMed Google Scholar
29. Lopez, B, et al. A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin. Exp. Immunol. 2003. 133:30-37.
    View this article via: PubMed Google Scholar
30. Fayyazi, A, et al. Apoptosis of macrophages and T cells in tuberculosis associated caseous necrosis. J. Pathol. 2000. 191:417-425.
    View this article via: PubMed Google Scholar
31. Keane, J, Remold, HG, Kornfeld, H. Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages. J. Immunol. 2000. 164:2016-2020.
    View this article via: PubMed Google Scholar
32. Lee, KD, Oh, YK, Portnoy, DA, Swanson, JA. Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes. J. Biol. Chem. 1996. 271:7249-7252.
    View this article via: PubMed Google Scholar
33. Hess, J, et al. Mycobacterium bovis bacille Calmette-Guerin strains secreting listeriolysin of Listeria monocytogenes. Proc. Natl. Acad. Sci. U. S. A. 1998. 95:5299-5304.
    View this article via: PubMed Google Scholar
34. Rossi, L, et al. Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages. J. Antimicrob. Chemother. 2004. 53:863-866.
    View this article via: PubMed Google Scholar
35. Hess, J, et al. Mycobacterium bovis bacille Calmette-Guerin strains secreting listeriolysin of Listeria monocytogenes. Proc. Natl. Acad. Sci. U. S. A. 1998. 95:5299-5304.
    View this article via: PubMed Google Scholar
36. Reyrat, JM, Lopez-Ramirez, G, Ofredo, C, Gicquel, B, Winter, N. Urease activity does not contribute dramatically to persistence of Mycobacterium bovis Bacillus Calmette-Guerin. Infect. Immun. 1996. 64:3934-3936.
    View this article via: PubMed Google Scholar
37. Horwitz, MA, Harth, G, Dillon, BJ, 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. U. S. A. 2000. 97:13853-13858.
    View this article via: PubMed Google Scholar
38. McShane, H, et al. Recombinant modified vaccinia virus Ankara expressing antigen 85A boosts BCG-primed and naturally acquired antimycobacterial immunity in humans. Nat. Med. 2004. 10:1240-1244.
    View this article via: PubMed Google Scholar
39. Brooks, JV, Frank, AA, Keen, MA, Bellisle, JT, Orme, IM. Boosting vaccine for tuberculosis. Infect. Immun. 2001. 69:2714-2717.
    View this article via: PubMed Google Scholar
40. Vordermeier, HM, et al. Cellular immune responses induced in cattle by heterologous prime-boost vaccination using recombinant viruses and bacille Calmette-Guerin. Immunology. 2004. 112:461-470.
    View this article via: PubMed Google Scholar
41. Goonetilleke, NP, et al. Enhanced immunogenicity and protective efficacy against Mycobacterium tuberculosis of bacille Calmette-Guerin vaccine using mucosal administration and boosting with a recombinant modified vaccinia virus Ankara. J. Immunol. 2003. 171:1602-1609.
    View this article via: PubMed Google Scholar
42. Mollenkopf, HJ, et al. Application of mycobacterial proteomics to vaccine design: improved protection by Mycobacterium bovis BCG prime-Rv3407 DNA boost vaccination against tuberculosis. Infect. Immun. 2004. 72:6471-6479.
    View this article via: PubMed Google Scholar
43. Nicoletti, I, Migliorati, G, Pagliacci, MC, Grignani, F, Riccardi, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow-cytometry. J. Immunol. Methods. 1991. 139:271-279.
    View this article via: PubMed Google Scholar
44. Palendira, U, Bean, AGD, Feng, CG, Britton, WJ. Lymphocyte recruitment and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior Mycobacterium bovis BCG immunization. Infect. Immun. 2002. 70:1410-1416.
    View this article via: PubMed Google Scholar