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Advances in TB vaccinology

Advances in TB vaccinology. There have been several major changes in the way we look at vaccine in development in the last few years: Advances in bioinformatics/genomics at the bacterial level Identification of targets for vaccines has expanded enormously

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Advances in TB vaccinology

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

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

  3. 400 300 200 100 100 %response of PPD 75 50 25 0 Ag85B TB42.9 CFP 10 ESAT 6 TB 10.4 TB 9.56 TB 37.6 TB 9.58 TB 10.3 TB 9.81 TB 12.9 TB 7.7 pep. TB 12.3 pep. TB 27.4 pep. Human recognition of antigens(ranked by mean value of responses)

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

  6. Progress of infection (the microbiologist’s view) Acute infectionLatent 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 Acute Disease Reactivation of infection CFU Immune conversion Latent infection Latency? Elimination? 1-3 4-50 Years after exposure

  7. 10000 1000 100 10 TB HHC LTBI ESAT-6 response in clinical groups IFN-g (pg/ml) p<0.001 p<0.001 10000 1000 100 10 TB HHC LTBI Rv2031c response in clinical groups Alteration of antigen recognition as disease progresses 3.5 3.0 2.5 Linear regression of ratio 2.0 1.5 1.0 0.5 0.0 TB HHC LTBI Clinical status

  8. Response to infection (the immunologist’s view) These individuals do not apparently skin-test convert or become ESAT-6 positive Early bacterial growth arrested at early time point. May (or may not) result in latent infection Immunologically, little is known about these individuals as they cannot be distinguished from uninfected individuals 67% Initial infection 33% 23% 25% Early bacterial growth not contained. Leads to clinical illness Subsequent bacterial growth contained. Symptoms abate but latent infection established. Remain healthy but latently infected 8% 2% Bacterial growth not contained. Progressive and eventually fatal disease unless treated Reactivation of latent infection at a later point in life 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-g and IL-12 Immunologically, these individuals tend to express elevated levels of IFN-g and IL-12, and while IL-4 often remains slightly increased, its antagonist IL-4d2 is greatly increased

  9. The balance of Th1 and Th2 cytokines - not the absolute level - correlates with disease status 15 p<0.001 10 p<0.01 Ratio IL-4/IL-4d2 5 0 TB HHC LTBI mRNA expression in unstimulated PBMC 5.0 p<0.001 2.5 Ratio IL-4/IFN-g p<0.01 0.0 TB HHC LTBI Clinical status

  10. The infection process (the cell biologist’s point of view) Mycolic acids, lipoproteins M. tuberculosis PAMP binding IL-18 IL-18R IL-12 Jak/Stat activation Uptake/ Phagocytosis IL-12R Specific T cell proliferation Lysosome maturation and bacterial killing Presented antigen MHC II Stat1 activation IFN-g IFN-gR TNF-a-R TNF-a T cell Antigen presenting cell

  11. The infection process (the cell biologist’s point of view) PGL Mycolic acids, lipoproteins M. tuberculosis ESAT-6/ CFP10 LAM PAMP binding IL-18 IL-18R IL-12 DC-SIGN Jak/Stat activation IL-10 Uptake/ Phagocytosis Decoy antigens (27 kDa, PE/PPE family) IL-12R Specific T cell proliferation LAM 19 kDa Lysosome maturation and bacterial killing IL-10R Presented antigen MHC II Stat1 activation IFN-g IFN-gR IL-10R TNF-a-R Bacterial lipid-induced IL-4/13 Multiple factors TNF-a T cell Antigen presenting cell

  12. 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-g response is not, by itself, enough

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

  14. BCG - boost or replace? 1000 After Hart, 1977 and Sterne, 1998 750 TB incidence per 100,000 500 250 0 65+ 0-14 15-24 25-34 35-44 45-54 55-64 Improved priming vaccine Booster vaccine Age (years) BCG-induced level of immunity BCG-induced level of immunity 65+ 65+ 0-14 0-14 15-24 25-34 35-44 45-54 55-64 15-24 25-34 35-44 45-54 55-64 Age (years) Age (years)

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

  16. 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. 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. 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. 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. 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. 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. Adjuvants for human use

  22. 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. Adjuvants for human use

  23. 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 . Adjuvants for human use

  24. Shared characteristics for ”good” TB adjuvants Immunomodulator – activates APC/DC Cationic vehicle – interacts with cell membranes and accelerates antigen uptake • 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. Shared characteristics for ”good” TB adjuvants Immunomodulator – activates APC/DC Vehicle – forms a depot, for longer release • LTK63 • a modified, heat-labile enterotoxin from E. coli • AS2 • A fraction of Quillaria saponaria, known as Quil A. • 3-deacylated-monophosphoryllipid A

  26. 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. TB research at the Dept. of Infectious Disease Immunology, SSI: Head. Peter Andersen 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

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