A multistage tuberculosis vaccine that confers efficient protection before and after exposure

Journal name:
Nature Medicine
Year published:
Published online


All tuberculosis vaccines currently in clinical trials are designed as prophylactic vaccines based on early expressed antigens. We have developed a multistage vaccination strategy in which the early antigens Ag85B and 6-kDa early secretory antigenic target (ESAT-6) are combined with the latency-associated protein Rv2660c (H56 vaccine). In CB6F1 mice we show that Rv2660c is stably expressed in late stages of infection despite an overall reduced transcription. The H56 vaccine promotes a T cell response against all protein components that is characterized by a high proportion of polyfunctional CD4+ T cells. In three different preexposure mouse models, H56 confers protective immunity characterized by a more efficient containment of late-stage infection than the Ag85B-ESAT6 vaccine (H1) and BCG. In two mouse models of latent tuberculosis, we show that H56 vaccination after exposure is able to control reactivation and significantly lower the bacterial load compared to adjuvant control mice.

At a glance


  1. Immunogenicity and protective efficacy of H56 and its components.
    Figure 1: Immunogenicity and protective efficacy of H56 and its components.

    (a) Purified Ag85B, ESAT-6, Rv2660c and H56 were visualized in Coomassie-stained gels. Ag85B, lane 1; ESAT-6, lane 2; Rv2660c, lane 3 and H56, lane 4. MW, molecular weight. (b) Protective efficacies in H56/CAF01 vaccinated CB6F1 mice challenged with Mtb (Erdman). One BCG vaccinated group is included as positive control. The differences between vaccinated and nonvaccinated mice 6 weeks after challenge are shown from one of two experiments. (c) Antigen-specific IFN-γ released from splenocytes purified from Ag85B-, ESAT-6–, Rv2660c- or H56-immunized CB6F1 mice (n = 3 per group) after in vitro stimulation with Ag85B, ESAT-6 or Rv2660c. Results are shown from one of three experiments. (d) Bacterial numbers in the lung of individual mice (CB6F1) 6 weeks after challenge (n = 6 per group). Data from one of three experiments are shown. (e) The development of the infection in mice (C57BL/6) immunized with BCG, H56 or saline and challenged with Mtb (H37Rv) was followed by enumerating the bacilli in the lung of individual mice (n = 6) over a period of 24 weeks. In d and e, one-way analysis of variance (ANOVA) was used for group comparisons. *P < 0.05; **P < 0.01; ***P < 0.001 compared to the adjuvant control group or BCG (}). Data are means ± s.e.m.

  2. Immune responses and vaccine efficacy of H56 compared to H1.
    Figure 2: Immune responses and vaccine efficacy of H56 compared to H1.

    (a) Antigen-specific IFN-γ release from in vitro–stimulated splenocytes isolated from mice (CB6F1) vaccinated with H1 or H56. Representative results from one of three experiments are shown. *P < 0.05, one-way ANOVA. (b) IFN-γ released from splenocytes isolated after challenge with Mtb (Erdman) from groups of CB6F1 mice vaccinated with CAF01 adjuvant, BCG, H1 or H56 and stimulated in vitro with the indicated antigens. Results from one of two independent experiments are shown. (c) The frequency of Ag85B-specific CD4+ T cells (CD44high) producing IFN-γ, TNF-α or IL-2 measured in cells isolated from perfused lungs from mice immunized with CAF01, BCG, H1 or H56. The cytokine profile in individual cells was measured by multicolor flow cytometry by gating for lymphocytes and CD4+ T cells. All possible combinations of cytokine expression were tabulated, and, after subtracting the background (nonstimulated samples), the results for the seven combinations expressing at least one of the cytokines are shown. Each of the panels ac show results from the same experiment. Two independent experiments were performed. (d) The bacterial load (CFUs) in the lungs of individual mice. The results are pooled values from two experiments, and each time point represents results from 10–12 mice per group. *P < 0.05; **P < 0.01 using one-way ANOVA. Data are means ± s.e.m.

  3. Evaluation of H56 as a BCG booster.
    Figure 3: Evaluation of H56 as a BCG booster.

    (a,b) Bacterial numbers (CFUs) 6 (a) and 24 (b) weeks after challenge with Mtb (Erdman) in the lung (n = 6 per group) of mice (CB6F1) vaccinated with BCG and boosted twice with either H1 or H56. Representative data from one of two experiments are shown as log10 CFU. ***P < 0.001; **P < 0.01; *P < 0.05, one-way ANOVA with Tukey's post test. Data are means ± s.e.m.

  4. Vaccination with H56 after exposure.
    Figure 4: Vaccination with H56 after exposure.

    (a) The model used for evaluation of the H56 vaccine after exposure. Mice infected with Mtb (Erdman) were treated with antibiotic for 6 weeks (shaded area). After treatment, mice were killed and bacteria were enumerated in the lungs at the indicated time points (n = 6). The arrows indicate the vaccination time points used in b, c and d. Log10 CFUs are given as mean values ± s.e.m. (b) IFN-γ released from PBMCs isolated 35 weeks after infection with Mtb (Erdman) from mice vaccinated at weeks 10, 13 and 16 and from nonvaccinated mice (n = 16 per group, pooled PBMCs). PBMCs were stimulated in vitro with Ag85B, ESAT-6 or Rv2660c. (c) At the same time point, cytokine profiles of antigen-specific CD4+ T cells were measured in Ag85B-stimulated splenocytes by flow cytometry as described in Figure 2c. (d) The protective efficacy of H56 was measured in two different laboratories. Experiments 1–3 were done in CB6F1 mice at Statens Serum Institut and experiments 4–6 were done in C57BL/6 mice at Unitat de Tuberculosi Experimental. In experiments 1–3, bacteria were enumerated in the lungs of individual mice 35 weeks (exp. 1 and 3) and 43 weeks (exp. 2) after challenge. In experiments 1 and 2, mice received two vaccinations, and in experiment 3, three. In experiments 4–6, mice received two vaccinations, and the bacterial load was measured 23 weeks after infection. The CFU values are shown as scattered plots with the median indicated (n = 12–16 per group in each experiment). We used the Mann-Whitney U test for comparison among groups. *P < 0.05; **P < 0.01; ***P < 0.001. Data are means ± s.e.m.


  1. Fine, P.E. Variation in protection by BCG: implications of and for heterologous immunity. Lancet 346, 13391345 (1995).
  2. Andersen, P. Tuberculosis vaccines—an update. Nat. Rev. Microbiol. 5, 484487 (2007).
  3. Doherty, T.M., Dietrich, J. & Billeskov, R. Tuberculosis subunit vaccines: from basic science to clinical testing. Expert Opin. Biol. Ther. 7, 15391549 (2007).
  4. Olsen, A.W., van Pinxteren, L.A.H., Okkels, L.M., Rasmussen, P.B. & Andersen, P. Protection of mice with a tuberculosis subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Infect. Immun. 69, 27732778 (2001).
  5. Skeiky, Y.A. et al. Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein. J. Immunol. 172, 76187628 (2004).
  6. Corbett, E.L. et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163, 10091021 (2003).
  7. Wilkinson, D. & Davies, G.R. The increasing burden of tuberculosis in rural South Africa—impact of the HIV epidemic. S. Afr. Med. J. 87, 447450 (1997).
  8. Horwitz, M.A., Lee, B.W., Dillon, B.J. & Harth, G. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis . Proc. Natl. Acad. Sci. USA 92, 15301534 (1995).
  9. Baldwin, S.L. et al. Evaluation of new vaccines in the mouse and guinea pig model of tuberculosis. Infect. Immun. 66, 29512959 (1998).
  10. Brandt, L., Elhay, M., Rosenkrands, I., Lindblad, E.B. & Andersen, P. ESAT-6 subunit vaccination against Mycobacterium tuberculosis . Infect. Immun. 68, 791795 (2000).
  11. Olsen, A.W., Williams, A., Okkels, L.M., Hatch, G. & Andersen, P. Protective effect of a tuberculosis subunit vaccine based on a fusion of antigen 85B and ESAT-6 in the aerosol guinea pig model. Infect. Immun. 72, 61486150 (2004).
  12. Dietrich, J., Billeskov, R., Doherty, T.M. & Andersen, P. Synergistic effect of bacillus Calmette Guerin and a tuberculosis subunit vaccine in cationic liposomes: increased immunogenicity and protection. J. Immunol. 178, 37213730 (2007).
  13. Langermans, J.A. et al. Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT-6. Vaccine 23, 27402750 (2005).
  14. Agger, E.M. et al. Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements. PLoS ONE 3, e3116 (2008).
  15. Dye, C. Global epidemiology of tuberculosis. Lancet 367, 938940 (2006).
  16. Finlay, B.B. & Falkow, S. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61, 136169 (1997).
  17. Voskuil, M.I., Visconti, K.C. & Schoolnik, G.K. Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy. Tuberculosis (Edinb.) 84, 218227 (2004).
  18. Rustad, T.R., Harrell, M.I., Liao, R. & Sherman, D.R. The enduring hypoxic response of Mycobacterium tuberculosis . PLoS ONE 3, e1502 (2008).
  19. Betts, J.C., Lukey, P.T., Robb, L.C., McAdam, R.A. & Duncan, K. Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol. Microbiol. 43, 717731 (2002).
  20. Muttucumaru, D.G., Roberts, G., Hinds, J., Stabler, R.A. & Parish, T. Gene expression profile of Mycobacterium tuberculosis in a non-replicating state. Tuberculosis (Edinb.) 84, 239246 (2004).
  21. Schnappinger, D. et al. Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J. Exp. Med. 198, 693704 (2003).
  22. Butcher, P.D., Mangan, J.A. & Monahan, I.M. Intracellular gene expression. Analysis of RNA from mycobacteria in macrophages using RT-PCR. Methods Mol. Biol. 101, 285306 (1998).
  23. Dolganov, G.M. et al. A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na+-K+-Cl cotransporter (NKCC1) in asthmatic subjects. Genome Res. 11, 14731483 (2001).
  24. Brosch, R. et al. Genome plasticity of BCG and impact on vaccine efficacy. Proc. Natl. Acad. Sci. USA 104, 55965601 (2007).
  25. Lin, M.Y. et al. Lack of immune responses to Mycobacterium tuberculosis DosR regulon proteins following Mycobacterium bovis BCG vaccination. Infect. Immun. 75, 35233530 (2007).
  26. McCune, R.M. Jr., McDermott, W. & Tompsett, R. The fate of Mycobacterium tuberculosis in mouse tissues as determined by the microbial enumeration technique. II. The conversion of tuberculous infection to the latent state by the administration of pyrazinamide and a companion drug. J. Exp. Med. 104, 763802 (1956).
  27. Manganelli, R., Voskuil, M.I., Schoolnik, G.K. & Smith, I. The Mycobacterium tuberculosis ECF σ factor σE: role in global gene expression and survival in macrophages. Mol. Microbiol. 41, 423437 (2001).
  28. Fontán, P.A. et al. Mycobacterium tuberculosis σ factor E regulon modulates the host inflammatory response. J. Infect. Dis. 198, 877885 (2008).
  29. Voskuil, M.I. et al. Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J. Exp. Med. 198, 705713 (2003).
  30. Govender, L. et al. Higher human CD4 T cell response to novel Mycobacterium tuberculosis latency associated antigens Rv2660 and Rv2659 in latent infection compared with tuberculosis disease. Vaccine 29, 5157 (2010).
  31. Dietrich, J. et al. Exchanging ESAT6 with TB10.4 in an Ag85B fusion molecule-based tuberculosis subunit vaccine: efficient protection and ESAT6-based sensitive monitoring of vaccine efficacy. J. Immunol. 174, 63326339 (2005).
  32. Palendira, U., Spratt, J.M., Britton, W.J. & Triccas, J.A. Expanding the antigenic repertoire of BCG improves protective efficacy against aerosol Mycobacterium tuberculosis infection. Vaccine 23, 16801685 (2005).
  33. Darrah, P.A. et al. Multifunctional TH1 cells define a correlate of vaccine-mediated protection against Leishmania major . Nat. Med. 13, 843850 (2007).
  34. Wille-Reece, U. et al. Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates. J. Exp. Med. 203, 12491258 (2006).
  35. Beveridge, N.E. et al. Immunisation with BCG and recombinant MVA85A induces long-lasting, polyfunctional Mycobacterium tuberculosis–specific CD4+ memory T lymphocyte populations. Eur. J. Immunol. 37, 30893100 (2007).
  36. Kannanganat, S., Ibegbu, C., Chennareddi, L., Robinson, H.L. & Amara, R.R. Multiple-cytokine–producing antiviral CD4 T cells are functionally superior to single-cytokine–producing cells. J. Virol. 81, 84688476 (2007).
  37. Kannanganat, S. et al. Human immunodeficiency virus type 1 controllers but not noncontrollers maintain CD4 T cells coexpressing three cytokines. J. Virol. 81, 1207112076 (2007).
  38. Heeney, J.L. & Plotkin, S.A. Immunological correlates of protection from HIV infection and disease. Nat. Immunol. 7, 12811284 (2006).
  39. Lindenstrøm, T. et al. Tuberculosis subunit vaccination provides long-term protective immunity characterized by multifunctional CD4 memory T cells. J. Immunol. 182, 80478055 (2009).

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Author information

  1. These authors contributed equally to this work.

    • Claus Aagaard &
    • Truc Hoang


  1. Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark.

    • Claus Aagaard,
    • Truc Hoang,
    • Jes Dietrich,
    • Rolf Billeskov &
    • Peter Andersen
  2. Unitat de Tuberculosi Experimental, Institut per a la Investigació en Ciències de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Catalonia, Spain.

    • Pere-Joan Cardona
  3. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA.

    • Angelo Izzo
  4. Department of Microbiology and Immunology, Stanford University School of Medicine, California, USA.

    • Gregory Dolganov &
    • Gary K Schoolnik
  5. Veterinary Sciences Centre, School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland.

    • Joseph P Cassidy


C.A. conceived of the study, produced H56, conducted preexposure vaccine studies and prepared the manuscript. T.H. developed the CB6F1 latency model and conducted latency studies in this model. J.D. conducted the BCG boost studies. P.-J.C. developed the C57BL/6 latency model and conducted latency studies in this model. A.I. conducted preexposure vaccine studies. G.D. designed and performed gene expression analyses. G.K.S. designed and performed gene expression analyses. J.P.C. performed histological evaluation of lung specimens. R.B. contributed to the latency vaccine studies. P.A. conceived of the study and prepared the manuscript. All authors discussed the results and commented on the manuscript at all stages.

Competing financial interests

C.A. and P.A. are co-inventors on a patent application to the Danish patent office covering the use of H56 as a vaccine. All rights have been assigned to Statens Serum Institut, a Danish not-for-profit governmental institute.

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