How to make better vaccines in the future?

1. How to make better vaccines in the future?

2. Vaccines interfere at more than one point Vaccine Adjuvant + Antigen “Inflam-mation” APC T cells B cells Ab Inflammation: Cytokines Cell recruitment Neutrophils Monocytes Macrophages Granulocytes Antigen Presenting Cells: Monocytes Macrophages Dendritic cells Protection adaptive innate

3. How to enhance efficiency of vaccines? Find better antigens Adjuvants Delivery systems Mucosal immunisation

4. Requirements for a good vaccine Safety Protection : most vaccinees against most substrains Long-lasting Neutralising Abs: important against bacterial toxins Mucosal immunity: presence of Abs on mucosae T cells Stability, ease of administration etc.

5. How do you test the quality of your vaccine? Animal model Correlate of protection in humans (what aspect of the immune response is crucial for protection) Efficacy trials (high numbers)

6. Adjuvants Necessary to induce, enhance and direct an Ag specific immune response Why and how?

7. The immune response in one slide

8. The immune response in one slide

9. Necessity of adjuvants I In a real infection, pathogens FIRST activate innate immunity (macrophages, neutrophils, DCs, NK cells etc.) The activated innate immune system then TRIGGERS and STEERS adaptive immunity: Priming of naive T cells only by activated DCs Th1 or Th2 type of T cell response depends on cytokines present during priming

10. Necessity of adjuvants II Immunologist’s dirty secret: provide the signals to activate innate immunity and generate inflammation Many whole-cell vaccines contain ligands for TLRs Subunit vaccines made from purified proteins don’t The only adjuvants allowed for use in humans are alum (1940) and MF59 (1995) Both tend to induce Th2 responses More efficient Th1 inducing adjuvants are needed

11. The immune response at a glance Pathogen “Inflam-mation” APC T cells B cells Ab Inflammation: Cytokines Cell recruitment Neutrophils Monocytes Macrophages Granulocytes Antigen Presenting Cells: Monocytes Macrophages Dendritic cells Protection

12. Vaccines interfere at more than one point Vaccine Adjuvant + Antigen “Inflam-mation” APC T cells B cells Ab Inflammation: Cytokines Cell recruitment Neutrophils Monocytes Macrophages Granulocytes Antigen Presenting Cells: Monocytes Macrophages Dendritic cells Protection adaptive innate

13. Future vaccine challenges Vaccines not available/ inadequate: HIV, HCV, tuberculosis, malaria, Group A and B streptococci, ... emerging or re-emerging pathogens: West Nile, SARS, Ebola, Hanta, Dengue, pandemic influenza therapeutic vaccines: against established infections e.g. antibiotic resistent bacteria, chronic infections cancer vaccines

14. Different Vaccines require different Adjuvants Safety Gradient Pediatric vaccines Th2 vs. Th1 Ab vs. CTL Traveler vaccines allergy vaccines Vaccine Bioterrorism military therapeutic vaccines Cancer vaccines

15. 1910s 1920s 1930s 1940s 1950s 1960s 1970s 1980s 1990s 2000s Alum MPL (TLR4) Insoluble Aluminium Salts MF59 o/w The Slow Pace of Adjuvant Development • Thousands of adjuvants have been described in pre clinical studies. • Three are included in approved vaccine products. • Many have failed, including a licensed product (Merck w/o emulsion for influenza,1960’s): • Many have failed due to safety (Merck emulsion; Chiron with MTP-PE in Phase I) • Difficult to manufacture, raw materials unavailable • Impossible to scale up • Lack of reproducibility • Too complex or expensive • There will continue to be failures (Adjuvant development not for the easily discouraged)

16. Adjuvants = Immune potentiators + Delivery systems • Delivery systems: • Ag in particulate form is taken up better • by DCs and Macrophages than Ag in soluble form • This allows more efficient Ag presentation to T cells • Immune potentiators: • Substances with direct stimulatory effect on APCs, • often with a specific receptor • Not always clear distinction

17. Key Components of Effective Vaccines Delivery Systems Immune- potentiators Antigens Potent and Durable Immune Responses

18. Comparison pathogen - vaccine Synthetic Vaccine Bacterium Protein antigens LPS Flagellae DNA Immune Potentiators Delivery system ~1m ~1m

19. A classification system for vaccine adjuvants • Antigen delivery systems • Alum • Calcium phosphate • Tyrosine • Liposomes • Virosomes • Emulsions • Microparticles • Iscoms • Virus-like particles • Immunopotentiators • MPL and derivatives • MDP • CpG oligos • Alternative PAMPs – flagellin etc. • Lipopeptides • Saponins • dsRNA • Small molecules e.g. Resiquimod “the others” Mechanism of action??? TLR-agonists

20. Types of immune potentiators and delivery systems Adjuvant category Representative examples Brief description Mineral salts Aluminum and calcium salts Licensed for human use Many bacterial and viral antigens have been adsorbed onto alum and Ca salts Emulsions and surfactant- MF59, AS02, montanide ISA-51 Micro-fluidized detergent-stabilized based formulations and ISA-720, QS21 emulsions Surfactants derived from natural sources Particulate delivery vehicles Microparticles, immuno- Antigens and adjuvants can be stimulatory complexes; trapped inside or coated onto the liposomes, virosomes, virus- surface of particles like particles Microbial derivatives Monophosphoryl lipid A, CpG Bacterial products or synthetic mimics oligonucleotides, cholera toxin are potent stimulators of the innate and heat labile toxin from immune system. Most of Escherichia coli, lipoproteins these agents signal through TLRs Cells and cytokines Dendritic cells; IL-12 and Cytokines stimulate cells of the GM-CSF immune system Autologous dendritic cells pulsed with tumor-derived peptides efficiently present antigenic epitopes

21. Types of immune potentiators and delivery systems Adjuvant category Representative examples Brief description Mineral salts Aluminum and calcium salts Licensed for human use Many bacterial and viral antigens have been adsorbed onto alum and Ca salts Emulsions and surfactant- MF59, AS02, montanide ISA-51 Micro-fluidized detergent-stabilized based formulations and ISA-720, QS21 emulsions Surfactants derived from natural sources Particulate delivery vehicles Microparticles, immuno- Antigens and adjuvants can be stimulatory complexes; trapped inside or coated onto the liposomes, virosomes, virus- surface of particles like particles Microbial derivatives Monophosphoryl lipid A, CpG Bacterial products or synthetic mimics oligonucleotides, cholera toxin are potent stimulators of the innate and heat labile toxin from immune system. Most of Escherichia coli, lipoproteins these agents signal through TLRs Cells and cytokines Dendritic cells; IL-12 and Cytokines stimulate cells of the GM-CSF immune system Autologous dendritic cells pulsed with tumor-derived peptides efficiently present antigenic epitopes

23. Possible mechanisms of delivery systems • presentation of Ag in an ideal conformation • render Ag polymeric • render Ag particulate [PLG] • protect Ag from degradation • retention of Ag at injection site: depot-effect [alum] • target Ag to antigen presenting cells [mAbs] • immune cell activation [PAMPS, cytokines, mAbs] • recruitment of additional immune cells[MF59]

24. Examples for delivery systems

25. Cationic PLG/CTAB microparticle for DNA adsorption - - - - Polylactide-co- glycolide DNA DNA - - - - + + CTAB + + - - + + + - + + - 1um + DNA + - DNA PLG Microparticle - - + + + - - + - - + + - + + + - + + - - DNA DNA - - Delivers DNA to APC Protects DNA from degradation Safe, Stable, Scalable - - DNA

26. – – – – – – – – – – – – – – – – – – – – – – – – – – – – Ag Ag 1m Ag Ag PLG microparticle – – – – – – Ag Ag – – Ag – – PLG microparticles for vaccine delivery Ag Ag IP IP IP IP Ag Ag IP IP IP IP – – IP – – – – Ag Ag Ag PLG/Immune Potentiator/Antigen PLG/Antigen The antigen is adsorbed after preparation of the microparticle Immune potentiators can be entrapped in the microparticle during preparation

27. Adjuvant performance with different Ag

28. H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O OIL OIL H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O H2O MF59 - o/w emulsion adjuvant Appearance:milky white oil in water (o/w emulsion) Composition: 0.5% Polysorbate 80 water-soluble surfactant 0.5% Sorbitan Triolate oil-soluble surfactant 4.3% Squalene oil Water for injection 10 nM Na-citrate buffer Density:0.9963 g/ml Viscosity:close to water, easy to inject. Size: 160nm.

29. H2O H2O H2O H2O H2O H2O H2O OIL H2O H2O H2O H2O H2O H2O H2O H2O MF59 - Overview MF59… • has a good safety profile • increases immunogenicity of a wide range of vaccine Ags tested in preclinical and clinical trials • induces immune responses in immune compromised individuals • induces predominantly Th0/Th2 responses • allows reduction of Ag dose • increases the breadth of cross reactivity of Ab responses

30. Adjuvant performance with different Ag

31. Addition of MF59 allows to reduce Ag dose Plain + MF59 mg/dose 3.75 7.5 15 30 GMT Pandemic H9N2 clinical trial: HAI Geometric Mean titers

32. MF59 - Mechanism of Action MF59 is not only a delivery system, but also an immunopotentiator MF59 has three human target cell types: monocytes, macrophages and granulocytes MF59 induces chemoattractants for cell recruitment MF59 enhances and accelerates development of dendritic cells The signature of MF59 (and alum) greatly differs from that of LPS: “adjuvant” signature vs. “danger” signature? Consistency with mouse serum cytokine and cell recruitment data

33. MF59 effects in the context of vaccination

34. Why is that good? • increased recruitment of immune cells to the injection site: • more cells to start the immune response • increased endocytosis by MC: • Ag is taken up to be presented to the immune system • differentiation of MC towards immature DCs: • these are the best cells to activate T cells • up-regulation of chemokine receptor CCR7 on DCs: • helps DCs to migrate to draining lymph nodes where T cells are

35. How does MF59 increase the immune response to vaccine antigens?

36. Examples for immune potentiators

37. The immune response in one slide

38. Naïve T cells are primed by antigen presented on activated dendritic cells

39. Naïve T cells are primed by antigen presented on activated dendritic cells PAMP= Pathogen Associated Molecular Pattern PRR= Pattern Recognition Receptor

40. Many types of PRR

44. Structure of bacterial LPS LPS: Endotoxin from outer membrane of Gram-negative bacteria stimulates innate immune response via TLR4

45. Eritoran (E5564) (TLR4 Antagonist) E. Coli lipid A (TLR4 Agonist) Westphal, et al, Angew. Chem.1954, 66, 407. E6020 (ER-804057) (TLR4 Agonist) (R,R,R,R) Rossignol, et al.; In: “Endotoxin in Health and Disease,” Brade, Opal, Vogel and Morrison, eds., Marcel Dekker, Inc., 1999, 699. E6020 is a completely synthetic TLR4 agonist Fully synthetic molecules

46. The search for new immune potentiators

47. Small Molecules SMIPs Human PBMCs Cytokine ELISA IFNs IL-1 IL-12 IL-6 TNF GM-CSF Screens for Immune Potentiators A. Functional HTS to Identify SMIPs

48. Screen for immune potentiators Use small molecule libraries, do high throughput screen for Th1 related cytokine production of total PBMCs Modify positive scorers to improve them Check for cell type specificity on purified populations Test in vivo in mice in combination with known Ags