A BCG kill switch strain protects against Mycobacterium tuberculosis in mice and non-human primates with improved safety and immunogenicity

Abstract

Improved vaccination strategies for tuberculosis are needed. Intravenous (i.v.) delivery of live attenuated Mycobacterium bovis BCG provides protection against Mycobacterium tuberculosis (Mtb) in macaques but poses safety challenges. Here we genetically engineered two strains, BCG-TetON-DL and BCG-TetOFF-DL, to either induce or inhibit expression of two phage lysin operons, respectively, upon tetracycline exposure. We show that lysin expression kills BCG in vitro, in infected macrophages, and following infection of immunocompetent (C57BL/6) and immunocompromised (SCID) mice. Modified BCG elicited similar immune responses and provided similar protection against Mtb challenge as wild-type BCG in mice. In macaques, cessation of tetracycline treatment reduced i.v.-administered BCG-TetOFF-DL numbers. Intravenous BCG-TetOFF-DL increased pulmonary CD4 T-cell responses compared with wild-type BCG-induced responses and provided robust protection against Mtb challenge. Sterilizing immunity occurred in 6 of 8 macaques compared with 2 of 8 wild-type BCG-immunized macaques. Thus, a 'kill-switch' BCG strain provides additional safety and robust protection against Mtb infection.

Extended Data Fig. 1 |. Single and dual lysin kill switches.

(a) TetON single lysin kill switch schematic. Lysin expression is repressed by tetracycline repressor (TetR) and induced by anhydrotetracycline (atc) or doxycycline (doxy) which bind TetR and prevent it from binding DNA. (b) Impact of atc on growth of BCG-TetON single lysin strains. The paper disc contains 1 μg of atc. (c) TetOFF dual lysin kill switch schematic. Reverse TetR (Rev TetR) binds to DNA in complex with atc or doxy to repress D29L. TetR represses PipR and in the presence of atc/doxy PipR is expressed and represses L5L. (d) Impact of atc on growth of BCG-TetOFF-DL. The paper disc contains 1 μg of atc.

Extended Data Fig. 2 |. In vitro characterization of BCG kill switch strains.

(a) Growth of BCG TetON single and dual lysin kill switch strains with and without atc. OD₅₈₀ data are means from duplicate cultures. Data are representative of two independent experiments. (b) Western blot analysis of culture filtrates from BCG-TetON-DL and BCG-TetOFF-DL strains grown in the absence of detergent. A whole cell lysate of WT BCG serves as control. Eno and PrcB were enriched in culture filtrate of BCG-TetON-DL after 6 and 9 days of growth in the presence of atc and in culture filtrate of BCG-TetOFF-DL after 6 and 9 days of growth in the absence of atc indicating cell lysis. Data are representative of two independent experiments. (c) Expression of two lysins reduces the fraction of escape mutants compared to expression of single lysins. Cultures were grown in the presence of atc. Symbols represent data from 3–6 individual cultures and means ± SD are depicted. Significance was determined by one-way analysis of variance (ANOVA) with two-sided Tukey’s adjusted p-values shown. (d) Growth of BCG TetOFF single and dual lysin kill switch strains with and without atc. Data are means ± SD from triplicate cultures. Error bars are too small to be seen. Data are representative of two independent experiments. (e) Expression of two lysins reduces the fraction of escape mutants compared to expression of single lysins. Cultures were grown in the absence of atc. Symbols represent data from 6 individual cultures and means ± SD are depicted. Statistical significance was assessed by one-way ANOVA with two-sided Tukey’s adjusted p-values shown.

Extended Data Fig. 3 |. Survival of BCG, BCG-TetOFF-DL and BCG-TetON-DL following intravenous vaccination.

(a) CFU from lungs and spleens of mice infected with BCG and BCG-TetOFF-DL not receiving doxy. Data are means ± SD from 4–5 mice per group and time point. Multiple unpaired two-sided t-tests run on log₁₀-transformed data at each time point with Holm-Šídák adjusted p-values shown. (b) CFU quantification from lungs and spleens of mice infected with BCG and BCG-TetON-DL treated with doxy. Data are means ± SD from 4–5 mice per group and time point. Multiple unpaired two-sided t-tests run on log₁₀transformed data at each time point with Holm-Šídák adjusted p-values shown. (c) CFU quantification from lungs and spleens of BCG-TetOFF-DL infected SCID mice treated or not with doxy for the indicated times. Data are means ± SD from 4 mice per group and time point. Multiple unpaired two-sided t-tests run on log₁₀-transformed data at each time point with Holm-Šídák adjusted p-values shown. (d, e) Lung sections stained with hematoxylin and eosin from SCID mice infected with BCG-TetOFF-DL for 84 days. (d) Mice were treated with doxy from day 1–14. (e) Mice were treated with doxy from day 1–84. Each section is from an individual mouse and is representative of each lung. Scale bar represents 2 mm. This analysis was done in one experiment.

Extended Data Fig. 4 |. BCG-TetON-DL and wt BCG provide similar protection against Mtb infection in mice.

Mice were i.v. vaccinated with BCG or BCG-TetON-DL or received PBS. (a, b) Quantification of effector memory CD4 and CD8 T cells in mouse lungs from mice vaccinated with BCG and BCG-TetON-DL. Symbols are data from 4–5 individual mice in each group with lines indicating mean ± SD. Two-way ANOVA was performed with two-sided Tukey’s adjusted p-values shown for each time point. (c) Quantification of cytokine producing antigen specific CD4 T cells from mice vaccinated with BCG and BCG-TetON-DL on day 30 post vaccination. Lung cells were restimulated ex vivo with PPD prior to intracellular cytokine staining. Data are means ± SEM from 5 mice per group. Two-way ANOVA with two-sided Tukey’s adjusted p-values shown for each cytokine producing population. For most of the cytokine producing cells, there were no statistically significant differences among the treatment groups. (d) Bacterial burden in lungs and spleens of vaccinated and PBS treated mice. Mice were infected with Mtb H37Rv by aerosol 90 days post vaccination. Symbols represent data from 5–8 individual mice with lines indicating mean ± SEM. Two-way ANOVA performed on log₁₀-transformed data with two-sided Tukey’s adjusted p-values shown at each time point.

Extended Data Fig. 5 |. Histologic analysis indicates microgranulomas in spleen, liver and lymph nodes 8 weeks post- BCG-TetOFF-DL.

H&E staining of fixed spleen (a), lymph node (b) and liver (c) tissue. (a-c) Scale bar in lower right corner indicates 100 micrometer scale. (d) Spleen size at necropsy for macaques in persistence study and macaques vaccinated and challenged with Mtb. Dashed lines represent normal spleen size range of adult male MCMs. (d, e) Lymphocyte composition of lung tissue (e, n = 4) and thoracic lymph nodes (f, n = 3) from NHP in persistence study at necropsy.

Extended Data Fig. 6 |. T cell responses in airways are similar between BCG-TetOFF-DL and WT BCG after vaccination.

Total number of cells, CD4+ , CD8+ , effector memory CD4+ and CD8+ cells during the vaccination phase with BCG-TetOFF-DL, WT BCG or unvaccinated macaques. BAL samples were obtained pre-vaccination and 4, 12, 20 weeks post vaccination and stimulated with Mtb WCL. Flow cytometry was performed with intracellular staining for effector molecules IFN-γ, TNF, IL-17, IL-2, GzmB, GzmK, granulysin or perforin (vaccinated groups n = 8, unvaccinated n = 2). Multiple unpaired two-sided t-tests were used to compare groups at each time with Holm-Šídák adjusted p-values (# 0.05 < p < 0.10). Median and IQR shown.

Extended Data Fig. 7 |. Individual effector molecules produced by T cells in lungs of vaccinated and challenged NHP.

Total number of CD4 + , CD8 + , effector memory CD4+ and CD8αβ+ cells in the lung at necropsy (vaccinated groups n = 8, unvaccinated n = 2). Cells producing either cytokines or cytotoxic molecules (IFN-γ, TNF, IL-17, IL-2, GzmB, GzmK, granulysin or perforin) were analyzed. Two-sided Mann-Whitney p-values reported. Median shown.

Extended Data Fig. 8 |. Splenomegaly reduces over time post IV-BCG vaccination.

Spleen size at necropsy for macaques in persistence study and macaques vaccinated and challenged with Mtb. Dashed lines represent normal spleen size range of adult male MCMs.

Extended Data Fig. 9 |. Gating strategy for flow cytometry.

Representative plots of WCL stimulated, homogenized lung tissue.

Fig. 1 |. Induction of phage lysins results in cell lysis with low frequency of escape and promotes macrophage proinflammatory cytokine production.

a, Growth of BCG-TetON single and dual lysin kill switch strains with and without atc. Individual c.f.u. data points from 2 replicate cultures are depicted. b, Impact of BCG-TetON dual lysin kill switch induction at different times of growth. c, Growth of BCG-TetOFF single and dual lysin kill switch strains with and without atc. Data are means ± s.d. of triplicate cultures. Error bars are frequently too small to be seen. d, Fraction of resistant mutants per culture and mutation rate per cell division of BCG-TetOFF-DL. The resistance rate in 20 individual cultures was determined in a fluctuation assay. The number of mutations per cell division was calculated using the bz-rates web tool. e, C.f.u. quantification of WT BCG and BCG-TetON-DL (plus or minus atc or rifampin) during infection of murine BMDMs. Data are means ± s.d. of triplicate cultures. Multiple unpaired two-sided t-tests were performed on log₁₀-transformed data comparing BCG-TetON-DL treated with rif to BCG-TetON-DL treated with atc at each time point, with Holm–Šídák adjusted P values shown. f–h, Quantification of TNF (f), IL-12 p40 (g) and IL-6 (h) production by macrophages infected with BCG or BCG-TetON-DL and treated with rifampin or atc for 20 h. Data are means ± s.d. of triplicate cultures. Significance was determined using one-way ANOVA with Tukey’s adjusted P values shown. ND, not detected. All data are representative of 2 or 3 independent experiments.

Fig. 2 |. Phage lysin induction kills BCG in immune competent and immune deficient mice.

a,b, C.f.u. quantification from lungs (a) and spleens (b) of BCG and BCG-TetON-DL-infected C57BL/6 mice treated or not with doxycycline (doxy) starting on day 7 post infection. Data are means ± s.d. of c.f.u.s from 3–5 mice per group and time point. c,d, C.f.u. quantification from lungs (c) and spleens (d) of BCG-TetOFF-DL-infected C57BL/6 and SCID mice treated or not with doxy for the indicated times. Data are means ± s.d. of c.f.u.s from 4–5 mice per group and time point. Multiple two-sided unpaired t-tests run on log₁₀-transformed data at each time point, with Holm–Šídák adjusted P values shown comparing BCG with BCG + doxy (black P values), BCG-TetON-DL with BCG-TetON-DL + doxy (purple P values), BCG-TetOFF-DL with BCG-TetOFF-DL + doxy in C57BL/6 (blue P values) and in SCID (red P values) mice.

Fig. 3 |. BCG-TetOFF-DL and WT BCG provide similar protection against Mtb infection in mice.

Mice were i.v. vaccinated with BCG or BCG-TetOFF-DL or received PBS. a–e, Quantification of T-cell subsets in mouse lungs from mice vaccinated with BCG or BCG-TetOFF-DL: (a) effector memory CD4 T cells; (b) effector memory CD8 T cells; (c) CD153 expressing CD4 T cells; (d) lung-resident memory CD4 T cells; (e) cytokine (TNF, IFNγ, IL-2, IL-17A)-expressing lung cells following ex vivo restimulation with PPD before intracellular cytokine staining and Boolean OR gating. Two-way ANOVA was performed and Tukey’s adjusted P values are shown for each time point. Symbols are data from 4–5 individual mice in each group, with lines indicating means ± s.d. f, Bacterial burden in lungs and spleens of vaccinated and PBS-treated mice. Mice were infected with Mtb H37Rv by aerosol at 90 days post vaccination. C.f.u.s on day 1 post infection were 90 ± 17. Symbols represent data from 12 individual mice, with lines indicating means ± s.e.m.; n = 12 lung and spleen samples used in each treatment group and in each time point. Two-way ANOVA was performed on log₁₀-transformed data and Tukey’s adjusted P values are shown for each time point.

Fig. 4 |. Discontinuation of doxy limits BCG-TetOFF-DL persistence in NHP.

a, Persistence study design. Created with BioRender.com (/u75q436). b, Memory CD4⁺ pre-vaccination, memory effector CD4⁺ 4 weeks post vaccination, memory CD8⁺ pre-vaccination and memory CD8⁺ 4 weeks post vaccination (n = 3 per group) total cell numbers isolated from the BAL, stimulated with H37Rv WCL. Two-way ANOVA was performed. There were no treatment effects, but total cells increased over time in all cell types. Error bars represent median ± range. c, Gross pathology scores at necropsy (Nx). d–g, Bacterial burden of BCG-TetOFF-DL c.f.u.s recovered from total body of animal (d), thoracic cavity (e), lungs (f) and thoracic lymph nodes (LN) (g) (n = 3 per group, mean and s.d. shown). h,i, Number of effector CD4⁺ cells (h) and CD8⁺ cells (i) per gram of lung tissue, stimulated with media or H37Rv WCL (average of 4 lung tissues per animal, n = 3 animals per group). Memory is defined as CD45RA⁺CD28⁻, CD45RA⁻CD28⁺ or CD45RA⁻CD28⁻.

Fig. 5 |. BCG-TetOFF-DL provides robust protection against Mtb infection in macaques.

a, Efficacy study design. Created with BioRender.com (/e14h603). b,c, Number of lung granulomas over time (b) and at necropsy (c). d,e, Total lung FDG activity by PET imaging over time (d) and at necropsy (e). f, Gross pathology scores at necropsy. g, Total thoracic bacterial burden at necropsy. h, Fisher’s exact test showing trend of higher levels of sterility in animals vaccinated with BCG-TetOFF-DL versus WT BCG Pasteur; two-sided Fisher’s exact P value reported. i,j, Total bacterial burden in lungs (i) and thoracic lymph nodes (j). WT BCG (n = 8), BCG-TetOFF-DL (n = 8) and unvaccinated animals (n = 10). Stars represent historical controls. Line represents the median. For c, e, f, g, i and j, Kruskal–Wallis tests were performed and Dunn’s P values (adjusted for multiple comparisons) are reported.

Fig. 6 |. BCG-TetOFF-DL induces more CD4 T cells producing cytokines in lung tissue compared with WT BCG.

a, Frequency of CD4⁺ and CD8⁺ T cells as a percentage of CD3⁺ cells isolated from homogenized lung tissue at necropsy (WT BCG, BCG-TetOFF-DL n = 8, unvaccinated n = 2). b,c, Frequency of effector CD4⁺ and CD8⁺ T cells in the lung (b) and thoracic lymph nodes (c) (WT BCG n = 8, BCG-TetOFF-DL n = 7, unvaccinated n = 1) producing cytokines or cytotoxic molecules. Mean ± s.d. shown. Two-sided Mann–Whitney P values reported, comparing BCG-TetOFF-DL and WT BCG.