Adjuvants development perspective of industry Ennio De Gregorio, Head of Immunology Italy, Novartis Vaccines and Diagnostics Workshop on Adjuvants and Innate Immunity Brussels July 2-2009
Why adjuvants are included in vaccines Modern vaccines are based on purified antigens which are often poorly immunogenic. They may require vaccine adjuvants which: Increase frequencies of effector T cells and antibody titers Induce protective responses more rapidly Enhance memory B and T cell responses Increase breadth of response heterologous activity vercome limited immune response in some populations elderly, young children, chronic diseases, immunocompromised 2 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Adjuvants included in licensed vaccines Liposomes Epaxal (HAV) Fluad (Flu) Fendrix (HBV) Cervarix (HPV) Prepadrix (pan.flu) Alum is the only adjuvant approved in USA Many vaccine adjuvants which are potent in pre-clinical models have failed in clinical trials 3 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Characteristics of optimal vaccine adjuvants Potential problems Unacceptable safety issues Significant local reactions Complex, difficult to scale up, lack of reproducibility Raw materials expensive or not available of suitable purity from reliable sources Non degradable, leaves long term residue at injection sites Difficult to formulate with diverse antigens, negative impact on antigen stability Inflexible, not easy to combine with additional components Ideal features - path to success Safe, not associated with long term effects Well tolerated Simple scale up and manufacturing, reproducible, easily characterized Made from abundant inexpensive components Biodegradable and biocompatible Compatible with many different antigens Flexible, capable of co delivery of antigen and immune potentiator 4 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Vaccine adjuvants activities: promoting antigen uptake and DC stimulation Ag uptake by DCs MF59, Alum PLG, liposomes Particulate adjuvants ANTIGEN DELIVERY SYSTEMS DC activation 1)Co-stimulation 2)Cytokine production 3)Ag presentation to T cells CpG:TLR9 R848: TLR7/8 MPL: TLR4 Microbial products (PAMPs) TLR-agonists IMMUNPTENTIATRS ptimal formulation for soluble antigen: combinations of Ag+ delivery system + immunomodulator 5 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59: a potent licensed oil in water emulsion adjuvant H H 2 2 H 2 H 2 H 2 H 2 H 2 H 2 H 2 IL H 2 H 2 H H 2 2 H 2 H 2 Composition: 0.5% Polysorbate 80 water-soluble surfactant 0.5% Sorbitan Triolate oil-soluble surfactant 4.3% Squalene oil Water for injection 10 mm Na-citrate buffer Density: 0.9963 g/ml Size: 160nm. MF59 was developed by by Chiron, now Novartis Vaccines and licensed in Europe for adjuvanted flu vaccine in 1997 MF59 is one of the most potent human vaccine adjuvants in pre-clinical and clinical studies MF59 mechanism of action is only partially understood 6 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Impact of MF59 in H5N1 vaccination The addition of MF59 to H5N1 vaccine in clinical trials resulted in: Higher frequencies of H5 CD4 T cells Higher frequencies of H5N1 memory B cells Protective antibody titers after two doses, broadly neutralizing drifted H5 clades A/VN/11194/04 MN- GMT 1000 100 * * * * * 40 Non-Adj-15 MF59-7.5 MF59-15 10 1 22 43 130 202 223 382 days 7 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Vaccination as an insurance against the risk of a pandemic influenza 6-8 years Three groups: 1. Primed with H5N3 (clade 0) with MF- 59 2. Primed with H5N3 and without MF-59 3. Unprimed subjects Boost with 2 doses of H5N1/MF59(clade 1) 8 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Pre-pandemic influenza vaccination with MF59 vaccine primes for a rapid and universal protection against H5N1 boosts GMT microneutralization antibody titer Protective titer (1:40) 10000 1000 100 10 1 With MF59 w/o MF59 By day 7 post-boost most of subjects have already protective neutralizing antibody titers against all virus strains 6-8 years Homologous H5N1 Clade 1 Heterologous H5N1 Clade 2.2 Heterologous H5N1 Clade 2.3 Heterologous H5N1 Clade 2.1 Homologous H5N1 Clade 1 No MF59 Priming with H5N3 Days Boost with H5N1 clade 1 with MF59 Months Stephesnon et al, N Engl J Med 2008; Galli et al, Proc. Natl. Acad. Sci. USA, in press 9 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Understand the MA of MF59 Two approaches: in vitro: Human PBMCs in vivo: Mouse muscle 10 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59 activates secretion of chemokines by human blood cells in vitro MF59 does not activate human DC MF59 induces secretion of CCL2 in human monocyte-derived macrophages, monocytes and granulocytes. CCL2 concentration at 48h [pg/ ml] [pg/ ml] 60000 50000 40000 30000 20000 10000 0 20000 15000 10000 5000 MoMf monocytes Mφ MF59 Alum LPS [pg/ ml] [pg/ ml] 6000 5000 4000 3000 2000 1000 0 6000 5000 4000 3000 2000 1000 total blood total blood MF59 Alum LPS MoDC [pg/ ml] 10 8 6 4 2 granulocytes myeloid DC monocytes MoDC myeloid DC* [pg/ ml] 400 350 300 250 200 150 100 50 0 granulocytes* MF59 Alum LPS MF59 alum LPS MF59 alum LPS MF59 alum LPS 0 0 MF59 Alum LPS 11 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity Seubert et al. J. Immun. 2008 MF59 Alum LPS 0 MF59 Alum LPS MF59 alum LPS MF59 alum LPS MF59 alum LPS
Classes of adjuvant responsive genes MF59 was the most potent activator of mouse transcriptome at injection site All adjuvant tested modulate a common set of 168 adjuvant core response genes 12 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59 is a strong inducer of cytokines & cytokine receptor genes at injection site Mosca et al. PNAS 2008 13 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59 is a strong inducer of genes involved in transendothelial migration in mouse muscle MF59 is the most potent inducer of Itgam/CD11b 14 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59 induces a rapid recruitmnent of CD11b+ blood cell injection site Mosca et al. PNAS 2008 Blue: Utrophin Red: PI Green: αcd11b 15 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MHCII + cells are recruited in the muscle 4 days after vaccine adjuvants injection 16 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59 induces an early recruitmemt of neutrophils followed by monocytes and eosinophils Analysis of cell recruitment by FACS analysis of muscle cell suspension 140000 Recruited recruited cells/ cells/ muscle [# cells] 120000 100000 80000 60000 40000 neutrophils infl. monocytes eosinophils macrophages mdc CD11b- DC subset pdc T B Unpublished data 20000 0 0h 1h 3h 6h 16h 24h 48h 3d 5d 11d 20d 0h 1h 3h 6h 16h 24h 48h 3d 5d 11d 20d Non-treated n.t. muscle MF59-injected muscle 17 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59 induces the expression of the early biomarker Ptx3 in muscle fibers Microarray Confocal immunofluorescence Fold change 18 16 14 12 10 8 6 4 2 0 Ptx3 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 Time (h) MF59 CpG Alum PBS Utrophin + PI Merge Ptx3 18 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
MF59: dual adjuvant function at injection site 1 Indirect DC activation MF59 2 Antigen Uptake Muscle fibers JunB, Ptx3 Macrophages/granulocytes chemokines CCL2 Ag Local immunocompetent environment TNFα, IL1β, CCLs Activation of resident DCs DC Circulating APC recruitment and activation Monocytes DC precursors 19 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity Migration to lymph node CD4 T cell activation
DC activation is a common step in adjuvanticity MF59 Muscle fibers Alum Stromal cells Uric acid Alum PLG Quil-A MSU TLR agonists: MPL, CpG, Flagellin R848, Imiquimod, Lipopeptides, PolyI:C Cytokines MF59 Alum TLR agonists Monocytes Granulocytes Macrophages Differentiation Cytokines Cytokines Direct interactions Cytokines Cytokines Mast cells NLRP3 DC Activation CD1d αgalcer itcr TLRs Dectin-1 Iscomatrix Beta glucans c48/80 inkt 20 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity
Acknowledgements MF59 MA Flaviana Mosca Elaine Tritto Anja Seubert Samuele Calabrò Andreas Wack Alessandro Muzzi Nick Valiante Gib tten Manmohan Singh Fabio Bagnoli Carlo Iavarone Barbara Baudner Elisabetta Monaci Mariagrazia Pizza Derek Hagan Rino Rappuoli Translational medicine (H5N1) Grazia Galli Duccio Medini Erica Borgogni Luisanna Zedda Carmine Malzone Monia Bardelli Sandra Nuti Flora Castellino Giuseppe Del Giudice Animal Facility Marco Tortoli Elena Amantini Stefania Torricelli 21 Adjuvants Ennio De Gregorio July 2 2009 Brussels Workshop on Adjuvants and Innate Immunity