1. STATENS SERUM INSTITUT An estimated 15 million active cases, leading to….. An estimated 9 million new infections Approx 2 million deaths Approx 2 Billion USD in direct control costs And an uncounted indirect cost in lost lives and productivity Global burden of tuberculosis(The economist’s view)

2. STATENS SERUM INSTITUT Exposure Healthy (95%) Year 1 Year 2 Year 3 thereafter TB (5%) Healthy (92%) TB (3%) Healthy (91%) TB (1%) Healthy (approx. 90%) TB (less than 0.1%/year) Development of Tuberculosis (the clinician’s view)

3. STATENS SERUM INSTITUT Early bacterial growth arrested at early time point. May (or may not) result in latent infection Initial exposure Early bacterial growth not contained. Leads to clinical illness Subsequent bacterial growth contained. Symptoms abate but latent infection established. Bacterial growth not contained. Progressive disease unless treated Reactivation of latent infection at a later point in life 33% 67% 9% 24% 2% Remain healthy but latently infected 22% These individuals do not apparently skin-test convert These individuals generally skin-test convert. They often have characteristic patterns on X-ray. Response to infection (the immunologist’s view)

4. STATENS SERUM INSTITUT Butajira Hospital Hossana Hospital Study Sites in Ethiopia

5. STATENS SERUM INSTITUT Recruitment in Ethiopia Index cases (TB, n=76) smear /culture positive X-ray confirmed Symptomatic Healthy Household Contact (HHC, n=104) smear/culture negative X-ray normal Asymptomatic Symptomatic Household Contact (XHC, n=58) smear/culture negative X-ray often, but not always, suspicious Symptomatic Community Control (CC, n=40) smear/culture negative X-ray normal Asymptomatic No known TB contact

6. STATENS SERUM INSTITUT Blood PBMCFrozen in liquid N 2 RNA Culture with specific antigens IFN-  ELISpot cDNA PCR Plasma HIV Double ELISA Agarose gel OD reading 12 bit CCD camera frozen at - 20 0 C IFN-  ELISA ELISA: sTNFR2 IL-10 Ag-specific Ab Thawed 96 hr Culture supernatant 24/48 hr cultured cells Real Time PCR Methods

7. STATENS SERUM INSTITUT TB-specific antigens M. tuberculosis Atypical mycobacteria BCG M.tuberculosis specific Antigens (100+): ESAT-6 Common mycobacterial Antigens (1000+) Ag85A/B Rv2031c Shared TB complex Antigens (4000+)

8. STATENS SERUM INSTITUT Risk of TB in ESAT+ healthy contacts from Ethiopia 35 healthy household contacts of TB patients were tested by ELISA for their response to ESAT-6 or PPD Over the next two years their clinical status was monitored Doherty et al., JCM, Feb. 2002

9. STATENS SERUM INSTITUT High ESAT-6 immune reactivity reflects high levels of M. tuberculosis replication CFU Time after infection “positivity” threshold People who fail to control bacterial replication become ESAT+ and get TB People who fail to control initial bacterial replication become ESAT+, but if they control later infection, become latently infected People who control initial bacterial replication remain ESAT-, and may or may not be latently infected “Clinical disease” threshold

10. STATENS SERUM INSTITUT Differential cytokine expression in clinical cohorts

11. STATENS SERUM INSTITUT Differential cytokine expression in healthy contacts + - IL-4 mRNA expressed as percentage  -actin

12. STATENS SERUM INSTITUT Differential cytokine expression in community controls IL-4 mRNA expressed as percentage  -actin 0 0.5 1.0 1.5 IFN-  mRNA expressed as percentage  -actin IL-4  2 mRNA expressed as percentage  -actin

13. STATENS SERUM INSTITUT Change in cytokine expression over time (TB patients) mRNA relative to  -actin

14. STATENS SERUM INSTITUT Change in cytokine expression over time (contacts) mRNA relative to  -actin

15. STATENS SERUM INSTITUT A lowered ratio of IL-4 to IFN-  and IL-4  2 reflects a shift from acute to latent TB CFU Time after infection “positivity” threshold People who fail to control bacterial replication become ESAT+ and get TB People who fail to control initial bacterial replication become ESAT+, but if they control later infection, become latently infected People who control initial bacterial replication remain ESAT-, and may or may not be latently infected “Clinical disease” threshold Th1? Th1 Th2 Th1 Th2

16. STATENS SERUM INSTITUT Early bacterial growth arrested at early time point. May (or may not) result in latent infection Initial exposure Early bacterial growth not contained. Leads to clinical illness Subsequent bacterial growth contained. Symptoms abate but latent infection established. Bacterial growth not contained. Progressive disease unless treated Reactivation of latent infection at a later point in life 33% 67% 9% 24% 2% Remain healthy but latently infected 22% These individuals do not apparently skin-test convert or become ESAT-6 positive These individuals generally skin-test convert and become ESAT-6 positive. They often have characteristic patterns on X-ray. Immunologically these individuals tend to express elevated levels of IL-4 and in advanced disease, decreased IFN-  and IL-12 Immunologically, these individuals tend to express elevated levels of IFN-  and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-4  2 is greatly increased Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals Response to infection (the immunologist’s view)

17. STATENS SERUM INSTITUT Acute infection Latent infection Expression of early phase Expression of late phase genes genes such as Ag85 such as  -crystallin and and ESAT-6 the DosR regulon CFU Acute Disease Reactivation of infection Years after exposure 1-34-50 Elimination? Latent infection Immune conversion Latency? Bacterial response to infection

18. STATENS SERUM INSTITUT 10 100 1000 10000 TBHHCLTBI p<0.001 Rv2031c response in clinical groups 10 100 1000 10000 TBHHCLTBI ESAT-6 response in clinical groups IFN-  (pg/ml) Alteration of antigen recognition as disease progresses (ET)

19. STATENS SERUM INSTITUT Alteration of antigen recognition as disease progresses (Ga and NL) Slope of linear regression no. of spots from ESAT-6 stimulation vs Rv2031c Slope of linear regression no. of spots from ESAT-6 stimulation vs Rv2031c Slope of linear regression IFN-  from ESAT-6 stimulation vs Rv2031c

20. STATENS SERUM INSTITUT ratio nHBHA-IFN-  /ESAT-6-IFN-  0.00 0.25 0.50 0.75 1.00 100 200 300 400 Active TB Latent TB Comparison of the IFN-  secretions induced by nHBHA and by ESAT-6 Latent TB Log IFN-  conc. (pg/ml) 10 100 1000 10000 100000 nHBHA Active TB p< 0.005 ESAT-6 Active TBLatent TB Figure kindly provided by Camille Locht and François Mascart

21. STATENS SERUM INSTITUT A lowered ratio of ESAT-6 immune reactivity to Rv2031c reactivity reflects a shift from acute to latent TB CFU Time after infection “positivity” threshold People who fail to control bacterial replication become ESAT+ and get TB People who fail to control initial bacterial replication become ESAT+, but if they control later infection, become latently infected People who control initial bacterial replication remain ESAT-, and may or may not be latently infected “Clinical disease” threshold ESAT-6 Rv2031c ESAT-6 Rv2031c ESAT-6 Rv2031c

22. STATENS SERUM INSTITUT Early bacterial growth arrested at early time point. May (or may not) result in latent infection Initial exposure Early bacterial growth not contained. Leads to clinical illness Subsequent bacterial growth contained. Symptoms abate but latent infection established. Bacterial growth not contained. Progressive disease unless treated Reactivation of latent infection at a later point in life 33% 67% 9% 24% 2% Remain healthy but latently infected 22% These individuals do not apparently skin-test convert or become ESAT-6 positive These individuals generally skin-test convert and become ESAT-6 positive. They often have characteristic patterns on X-ray. Immunologically these individuals tend to express elevated levels of IL-4 and in advanced disease, decreased IFN-  and IL-12. They weakly recognise Rv2031c Immunologically, these individuals tend to express elevated levels of IFN-  and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-4  2 is greatly increased. They strongly recognise Rv2031c Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals Response to infection (the immunologist’s view)

23. STATENS SERUM INSTITUT Summary  Immunity to M. tuberculosis is dependent on the generation of Th1 immunity, particularly IL-12, IFN-  and TNF-   As the bacteria persists in the face of this Th1 response, it begins to alter its proteome towards a pattern characteristic of latency, downregulating some antigens, upregulating others  At the same time, a Th2 response seems to develop  Susceptibility to infection therefore appears to correlate not so much with inability to generate a Th1 response, as with inability to maintain it long term, or perhaps inability to direct it to relevant antigens  We are starting to see evidence that M. tuberculosis-derived antigens are driving some of this Th2 response

24. STATENS SERUM INSTITUT Activation of the immune response (the molecular biologist’s view) TLR2TLR4 MYD88 +MAL IRAK TRAF6 NF  B NEMO/IKK TNF-  IL-12, IL-18, IL-6, etc Lipoproteins Peptidoglycans M.tuberculosis The activation of the inflammatory cascade is essential for protection. Defects in this pathway in either humans or animals are associated with extreme susceptibility to mycobacteria Blocking either IFN-  or TNF-  after induction of immunity also blocks control of infection However - M. tuberculosis also needs these cytokines to cause pathology so that it can spread TIRAP IRF3 IL-18,etc

25. STATENS SERUM INSTITUT PAMP binding IL-12 IL-12R IFN-  IFN-  R Uptake/ Phagocytosis Lysosome maturation and bacterial killing Jak/Stat activation TNF-  TNF-  R Presented antigen Specific T cell proliferation TCR T cellAntigen presenting cell M. tuberculosis Stat1 activation IL-18 IL-18R Mycolic acids, lipoproteins Generation of protective immunity (the cell biologist’s view)

26. STATENS SERUM INSTITUT PAMP binding IL-12 IL-12R IFN-  IFN-  R Uptake/ Phagocytosis Lysosome maturation and bacterial killing Jak/Stat activation TNF-  TNF-  R Specific T cell proliferation TCR T cellAntigen presenting cell M. tuberculosis Stat1 activation Elevated IL-10 IL-10R IL-18 IL-18R Mycolic acids, lipoproteins Blocking of phagocytic maturation Elevated IL-4/13 Presented antigen What can go wrong with protective immunity

27. STATENS SERUM INSTITUT PAMP binding IL-12 IL-12R IFN-  IFN-  R Uptake/ Phagocytosis Lysosome maturation and bacterial killing Jak/Stat activation TNF-  TNF-  R Presented antigen Specific T cell proliferation TCR T cellAntigen presenting cell M. tuberculosis Stat1 activation DC-SIGN IL-10 IL-10R IL-18 IL-18R PGL Mycolic acids, lipoproteins ESAT-6/ CFP10 Bacterial lipid- induced IL-4/13 Decoy antigens (27 kDa, PE/PPE family) 19 kDa LAM Effect of mycobacterial products on the immune response