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).

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.

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.

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.

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).

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.