1. STATENS SERUM INSTITUT Advances in TB vaccinology There have been several major changes in the way we look at vaccine in development in the last few years: 1.Advances in bioinformatics/genomics at the bacterial level a)Identification of targets for vaccines has expanded enormously b)We are beginning to understand how M. tuberculosis reacts to the host’s immune response 2.Advances in understanding immunology and disease processes in patients and animal models a)Choosing targets and designing interventions has become much more sophisticated b)Our understanding of what constitutes a desirable immune response has broadened 3.The first clinical trials have started

2. STATENS SERUM INSTITUT Antigen discovery in the pre-genomic era 2244 3765 The Ag85 family (Ag85A,B and C) The ESAT6 family (20 members organized pairwise) + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ++ ++ + + + + + + 0288 3875 3648c 0287 1038c/1197/1792 3914 2031c 2878c 1926c 0652 3874 3418c 2140c 0009 1984c 1932 1827 2534c + 2882c 3803c 2109c 1980c 0733 3036c 0577 1886c 0129c 3804c 0363c 0798c 2716 1626 0036c 0934 3045 2780 0884c 0462 1098c 1860 2220 1077 3842c 0350 Definition of secreted antigens Size fractionation Antigen recognition

3. STATENS SERUM INSTITUT Human recognition of antigens (ranked by mean value of responses)

4. STATENS SERUM INSTITUT Finding Vaccine Targets Of the hundreds of antigens screened, the vast majority are not strongly immunogenic. However, of the the antigens that are immunogenic, most come from a relatively small number of gene families. Thus, looking at genomic organization has proven to be a very efficient route of finding antigens to screen. Many of these antigens have also proven to be virulence factors, suggesting that functional analysis might also be a useful way to identify targets

5. STATENS SERUM INSTITUT Lifetime risk of Tuberculosis (the clinician’s view) Exposure Healthy (97%) Year 1 Year 2 Year 3 thereafter TB (3%) Healthy (95%) TB (2%) Healthy (94%) TB (1%) Healthy (approx. 90%) TB (less than 0.1%/year)

6. STATENS SERUM INSTITUT Acute infection Latent infection Expression of early phase Expression of late phase genes genes such as Ag85 such as a-crystallin and and ESAT-6 the DosR regulon Immune response initiated Immune response alters Progress of infection (the microbiologist’s view) CFU Acute Disease Reactivation of infection Years after exposure 1-34-50 Elimination? Latent infection Immune conversion Latency?

7. STATENS SERUM INSTITUT Alteration of antigen recognition as disease progresses TBHHC LTBI 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Clinical status Linear regression of ratio

8. STATENS SERUM INSTITUT Response to infection (the immunologist’s view) Early bacterial growth arrested at early time point. May (or may not) result in latent infection Initial infection Early bacterial growth not contained. Leads to clinical illness Subsequent bacterial growth contained. Symptoms abate but latent infection established. Bacterial growth not contained. Progressive and eventually fatal disease unless treated Reactivation of latent infection at a later point in life 33% 67% 8% 25% 2% Remain healthy but latently infected 23% 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

9. STATENS SERUM INSTITUT The balance of Th1 and Th2 cytokines - not the absolute level - correlates with disease status Ratio IL-4/IL-4  2 Ratio IL-4/IFN-  TBHHCLTBI 0.0 2.5 5.0 Clinical status TBHHCLTBI 0 5 10 15 p<0.001 p<0.01 p<0.001 p<0.01 mRNA expression in unstimulated PBMC

10. STATENS SERUM INSTITUT The infection process (the cell biologist’s point of view) 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 MHC II T cellAntigen presenting cell M. tuberculosis Stat1 activation IL-18 IL-18R Mycolic acids, lipoproteins

11. STATENS SERUM INSTITUT The infection process (the cell biologist’s point of view) 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 MHC II T cellAntigen presenting cell M. tuberculosis Stat1 activation DC-SIGN IL-10 IL-10R IL-18 IL-18R PGL Mycolic acids, lipoproteins LAM Multiple factors ESAT-6/ CFP10 Bacterial lipid- induced IL-4/13 Decoy antigens (27 kDa, PE/PPE family) 19 kDa LAM

12. STATENS SERUM INSTITUT What does this mean from a vaccine designer’s perspective? The fact that most exposed individuals develop a protective immune response, shows that immunity is possible The fact that even taking this into account, BCG can reduce mortality shows that boosting that immune response is possible The fact that BCG has not performed well against adult pulmonary TB shows the need for a new vaccine The fact that M. tuberculosis can survive for extended periods in people with strong antigen-specific immune responses, shows that a strong IFN-  response is not, by itself, enough

13. STATENS SERUM INSTITUT Why can’t BCG be used against adult pulmonary TB? BCG Systemic protection BCG Eliminated No protection Proliferation and dissemination Naive Recipient Sensitized Recipient Pre-existing immunity

14. STATENS SERUM INSTITUT BCG - boost or replace? Booster vaccine BCG-induced level of immunity 0-1415-2425-3435-4445-5455-6465+ Age (years) Improved priming vaccine BCG-induced level of immunity 0-1415-2425-3435-4445-5455-6465+ Age (years) After Hart, 1977 and Sterne, 1998 TB incidence per 100,000 0-1415-2425-3435-4445-5455-6465+ Age (years) 0 250 500 750 1000

15. STATENS SERUM INSTITUT Vaccines in, or on their way to, clinical trials Priming vaccines Boosting vaccines

16. STATENS SERUM INSTITUT Priming vaccines (BCG-derived) rBCG30 Recombinant BCG Tice which over-expresses Ag85b Protects guinea pigs better than BCG Completed phase I trials - vaccine is immunogenic and apparently safe - currently being reworked for further testing rBCG::  ureC-llo+ Recombinant BCG which expresses Lysteriolysin O to cause leakage from the endosome, and and urease C to alter vacuole pH. The idea is to improve CD8 response via “cross-priming” Protects mice better than BCG, but is less virulent than BCG Clinical trials planned for 2006/7

17. STATENS SERUM INSTITUT Priming vaccines (M. tuberculosis-derived) PanD-/Leu- auxotroph Recombinant M. tuberculosis lacking both the PanD and Leu genes Protects guinea pigs and is much less virulent than BCG: but it also grows less well in the host, so is slightly less protective  phoP/R Recombinant M. tuberculosis in which the phoP virulence factor has been knocked out by the insertion of an antibiotic gene Protects guinea pigs better than BCG and is less virulent Will probably need further manipulation before it could be used in human trials

18. STATENS SERUM INSTITUT Priming vaccines (viral vectors) MVA85A Recombinant, replication deficient vaccinia virus, expressing the strongly immunogenic antigen 85A from M. tuberculosis Protects mice and guinea pigs as well as BCG, can boost BCG effect Has completed early clinical trials: is apparently safe and immunogenic Other viruses Adenovirus - a variety of different constructs have shown efficacy in animal models Fowlpox - has also shown efficacy in animal models

19. STATENS SERUM INSTITUT Priming vaccines (recombinant proteins) ESAT-6-Ag85B Recombinant fusion protein, composed of the strongly immunogenic antigens ESAT-6 and Antigen 85B from M. tuberculosis Two forms of the vaccine using different adjuvants IC31, for intramuscular administration LTK63 for nasal administration Protects mice and guinea pigs as well as BCG, can boost BCG effect Will enter clinical trials in September 2005 72f Recombinant fusion protein, composed of the strongly immunogenic antigens Rv1196 and Rv0125 from M. tuberculosis Administered in AS2 adjuvant Protects mice and guinea pigs as well as BCG Has completed early clinical trials: is apparently safe

20. STATENS SERUM INSTITUT Adjuvants for human use Recombinant vaccines are now routinely used in humans, but only for limited categories of disease - there are few adjuvants that combine low toxicity with the ability to stimulate good CMI responses However, our improving understanding of the interaction of bacteria and the immune system - primarily through APCs - has led to the development of new adjuvant systems that mimic bacterial infection and which look promising for new vaccines.

21. STATENS SERUM INSTITUT Adjuvants for human use Already approved for human use Alum (AlOH) MF59 Virosomes The first human adjuvant, alum promotes a strong humoral response and is widely used in viral vaccines. However it generates strongly Th2- polarised responses and is not suitable for use as a TB vaccine. An oil-in-water emulsion composed of 5% v/v squalene, 0.5% v/v Tween 80 and 0.5% v/v Span 85. Like alum, it generates primarily humoral immunity and is currently used mostly in influenza vaccines Similar in structure to liposomes, virosomes are differentiated by containing viral proteins embedded in their membrane, which are delivered into host cells by membrane fusion. Currently used in vaccines against viral targets such as influenza and hepatitis A, where humoral immunity is most important.

22. STATENS SERUM INSTITUT Adjuvants for human use Adjuvants tested in clinical trials CAP (calcium phosphate) nanoparticles. Currently in early clinical trials, CAP have been used to generate humoral responses, but the lower levels of IgE induced suggest it may not be as polarized towards the Th2 pole of the immune response as alum. LTK63. A modified and detoxified heat labile toxin from Escherichia coli tested in human volunteers as an influenza vaccine. Generates strongly Th1-polarised responses and therefore being considered for vaccines against M. tuberculosis and HIV. AS2. An oil-in-water emulsion containing 3-deacylated-monophosphoryl lipid A(a detoxified form of lipid A from Salmonella minnesota), and a purified fraction of Quillaria saponaria, known as Quil A. Currently in early clinical trials as a TB vaccine. A synthetic analogue of monophosphoryl lipid A called RC-529) is in clinical trials in an HIV vaccine. Generates strongly Th1-polarised responses.

23. STATENS SERUM INSTITUT Adjuvants for human use Adjuvants in, or approaching, clinical trials IC31. A mixture of oligodeoxynucleotides and polycationic amino acids. Generates strong Th1 responses and planned to enter phase I clinical trials in 2005 as part of a TB vaccine. Montanide. A water/oil emulsion, two variants exist, based on mineral and non-mineral oil. Tested initially as a cancer immunotherapeutic agent, Montanide has now been through a variety of clinical trials through to phase III. It generates a mixed cell-mediated and humoral response, which may render it less attractive for a TB vaccine. ISCOM. A formulation of Quillaja saponins, cholesterol, phospholipids, and protein, typically self-assembling into small icosahedral cage-like particles. Used initially for veterinary vaccines, ISCOMS have recently shown promise in late phase human clinical trials for viral vaccines. Their potential for M. tuberculosis vaccines remains unknown, as they generate a mixed humoral and cell-mediated response. OM-174. A modified and detoxified lipid A from Escherichia coli. Synthetic analogues also exist. Currently in early clinical trials for cancer immunotherapy and suggested for TB vaccine use. Generates strongly Th1-polarised responses.

24. STATENS SERUM INSTITUT Shared characteristics for ”good” TB adjuvants Cationic vehicle – interacts with cell membranes and accelerates antigen uptake Immunomodulator – activates APC/DC Cationic Liposomes (CAT-1/2) DDA MPL-A (Monophosphoryl lipid) or TDB (synthetic cord factor ) IC31 Poly leucine/lycine peptide (KLKLLLLLKLK) Poly-IC analogue (TLR 3/9)

25. STATENS SERUM INSTITUT Shared characteristics for ”good” TB adjuvants Vehicle – forms a depot, for longer release Immunomodulator – activates APC/DC LTK63 a modified, heat-labile enterotoxin from E. coli AS2 A fraction of Quillaria saponaria, known as Quil A. 3-deacylated-monophosphoryl lipid A

26. STATENS SERUM INSTITUT New TB Vaccines Today There are 5 novel vaccines in the early clinical pathway There are at least as many vaccines in the preclinical phases We have novel delivery systems that can be used for TB vaccines However…. None of these vaccines have yet shown proof of efficacy in humans It is unknown if these vaccines will be effective in people who are already infected Research on improving TB vaccination is therefore still very much ongoing

27. STATENS SERUM INSTITUT TB research at the Dept. of Infectious Disease Immunology, SSI: Immunology Anja Olsen Else Marie Agger Søren Hoff Thomas Bennekov Karen Korsholm Mark Doherty Jes Dietrich Carina V. Lundberg Claire Andersen Jesper Davidsen Protein chem. Ida Rosenkrands Karin Weldingh Molecular biology Claus Aagaard Head. Peter Andersen