Developing New Vaccine Adiuvants

Transcription

1 Developing New Vaccine Adiuvants Rino Rappuoli Summer School on Influenza Siena August 3 211

2 When you run out of ideas

3 Istituto Sieroterapico e Vaccinogeno Toscano From Sclavo to Sabin exploiting innovation to solve medical needs Achille Sclavo 194 I.S.V.T. Albert Sabin

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5 The slow path to the development of vaccine adjuvants 191s192s193s194s195s196s197s198s199s2s Aluminium Salts MF59

6 The Slow Pace of Adjuvant Development Fluad (influenza Fendrix (HBV) Cervarix (HPV) Prepandrix (pandemic influenza) MPL+Alum (ASO4) Cervarix Many potent vaccine adjuvants have failed, due to safety concerns MF59 was a key innovation, first novel adjuvant in 7 years Alum and MPL (AS4 ) are the only adjuvants currently approved in US

7 Innate Response Activators and Vaccine Development TLR shown to be LPS target Rabies vaccine introduced 1996 Toll receptors linked to innate immunity 1997 Imiquimod therapy approved Typhoid vaccine introduced 1921 BCG vaccine introduced Pertussis (Pw) vaccine introduced Influenza (whole cell) vaccine introduced IPV vaccine introduced JE vaccine introduced Vaccines containing TLRagonists have been used since 1885 Hep-A vaccine introduced 25 Fendrix approved in EU. First approval of vaccine containing TLR agonist 29 Cervarix approved in U.S.A. First FDA approval of vaccine containing TLR agonist ssrna: TLR7/8 LPS, DNA: TLR 2,4,5,9 Lipoprotein, DNA: TLR 2,9 LPS, DNA: TLR 2,4,9 ssrna: TLR7/8 ssrna: TLR7/8 ssrna: TLR7/8

8 History of Emulsions Lipovaccines Water/Oil Emulsion Oil/Water Emulsion Approval of Oil/Water Emulsion Products 1916: Le Moignac, Pinoy 1919: Whitmore 1937: Freund 1952: Salk (Flu 9K doses in U.K.) 1997: Seppic Montanide 1975: Ribi 1999: Allison 1997: MF59 Fluad (Novartis) 2 : Fluarix (GSK) Prepandrix (GSK)

9 Adjuvants for influenza vaccines, an old solution Hennessy A,V, and Davenport F,M; Pub. Health Repts 76, , 1961 Polyvalent vaccines Test strain and antibody response 1 Aqueous; aqueous Swine PR8 PR31 Before second dose After second dose Adjuvant; adjuvant Before second dose After second dose 242 1, , ,818 Mineral oil adjuvant vaccine is remarkably effective for stimulating high, broad, uniform, and persistent antibody levels against prototype strains of influenza A A phenomenal economy can be affected in the requirement of antigen

10 Too many Adjuvants for preclinical studies very few for clinical studies for clinical studies

11 MF59: An established adjuvant in a European-licensed seasonal trivalent vaccine Oil-in-water emulsion adjuvant licensed for use in seasonal influenza vaccine FLUAD * since 1997 More than 1 million commercial doses distributed Adjuvanted vaccine provides heterologous responses to drifted strains >12 Clinical studies, >2, subjects No safety signals in either pharmacovigilance database or metaanalysis of clinical trial database with 6 month subject follow-up (filed with CBER) 16nm oil MF59 adjuvant emulsion Antigens SPAN 85 TWEEN 8 Pediatric studies and efficacy trial in 3, subjects *FLUAD is a registered trademark of Novartis. FLUAD is not licensed in the Unites States. FLUAD is recommended for active prophylaxis of influenza in the elderly

12 MF59 mechanism of action genes modulated by adjuvants at injection site Mosca et al. PNAS 28 MF59 was the most potent activator of mouse transcriptome at injection site All adjuvants tested modulate a common set of 168 adjuvant core response genes

13 MF59 is a strong inducer of cytokines & cytokine receptor genes at injection site (mouse muscle) Mosca et al. PNAS

14 MF59 is a potent inducer of genes involved in leukocyte transendothelial migration at injection site MF59 is the most potent and rapid inducer of Itgam/CD11b mrna Suggest a more rapid recruitment of CD11b+ blood cells into the muscle compared to CpG and Alum Mosca et al. PNAS 2

15 MF59 induces a rapid recruitmnent of CD11b+ blood cell injection site Mosca et al. PNAS 2 Blue: Utrophin Red: PI Green: acd11b

16 cells/ muscle cells/ muscle cells/ muscle cells/ muscle Early reduction of macrophage and DCs in the muscle following MF59 injection 1h 12 8 neutrophils * monocytes 4 1 n.t. MF59 n.t. MF macrophages mdc 6 * * 4 2 n.t. MF59 n.t. MF59 Which cell type participates in adjuvant/antigen uptake and transport?

17 [# cells] Kinetic of cell recruitment induced by MF59 in mouse muscle 14 Recruited cells/ muscle neutrophils infl. monocytes eosinophils macrophages mdc CD11blow DC CD11b neg DC T B 2 h 1h 3h 6h 16h 24h 48h 3d 5d 11d 2d h 1h 3h 6h 16h 24h 48h 3d 5d 11d 2d n.t. Non-treated muscle MF59-injected muscle Neutrophil depletion does not affect recruitment of other cell types and does not affect MF59 adjuvanticity

18 All CD11b+ cell types recruited in the muscle are MF59-Ag+ n.t. muscle MF59-DiO+OVA-AF647 24h CD11b- CD11b+ CD11b- CD11b+ DiO DiO OVA OVA neutrophils monocytes eosinophils macrophages mdc OVA-AF647

19 positive cells per 1E6 cells MF59 promotes OVA uptake in LNs more efficiently than alum 25 OVA-positive cells 24h OVA-AF647 OVA-AF647+Alum OVA-AF647+MF59 2 pos B cells pos mdcs Right Quadriceps 15 pos CD11b low DCs pos monocytes 1 pos neutrophils Right Inguinal LN Left Inguinal LN 5 pos pdcs pos CD8+ DCs TREATED UNTREATED OVA positive cells treated untreated treated untreated treated untreated MF59+OVA-AF647 alum+ova-af647 OVA-AF647

20 MF59 oil-in-water emulsion adjuvant progress on mechanism of action Oil: 4.3% Squalene Naturally present in human: component of cell membrane and synthetic precursor of cholesterol OIL Surfactants:.5% Tween 8 (water-soluble).5% Span 85 (oil-soluble) Commonly used in other licensed pharmaceutical products Water for injection 1 mm Na-citrate buffer - Squalene or surfactants alone no adjuvant activity - does not signal via inflammasome - does not signal via TLR - requires MyD88

21 Total IgG titers Complete MF59 emulsion is required for adjuvanticity OIL Oil: 4.3% Squalene Surfactants:.5% Polysorbate 8.5% Sorbitan Triolate 1 mm Na-citrate buffer Which MF59 component has immunostimulatory activity?

22 Number of cells Only complete MF59 emulsion induces cell migration events in mouse muscle 6 Singlets 3 Neutrophils 8 Eosinophils 6h Macrophages 8 Monocytes 1 DC OVA-A647 +OVA-A647 +OVA-A647

23 MF59 Surfac. Squalene Citrate Untreated OVA- MFI SSC Only complete MF59 emulsion increase OVA uptake by monocytes at injection site Monocytes OVA A647 MF59 Surfact. Squalene Citrate untreated mo DCs DCs 1 5 OVA-A647 6h OVA A647

24 MF59 Span+ Tween Squalene Citrate buffer MF59 Span+ Tween Squalene Citrate buffer MF59 Span+ Tween Squalene Citrate buffer MFI SSC Only complete MF59 emulsion changes morphology and differentiation state of human monocytes in vitro MF59 Surfactants Squalene Citrate untreated FSC CD14 MHC class II CD86

25 [pg/ml] [pg/ml] MF59 does not activate Nalp3 in vitro MF59 does not activate Nalp3 in vitro in LPS primed BMDC or PBMCs Mouse BMDC Adjuvant: - Alum 4 ug/ml MF59 1:1 MF59 1:1 LPS: 1/ 1/.1 ng/ml Pro-IL1b IL1b Mouse BMDC IL-1b LPS alone LPS + adjuvant Human PBMCs IL-1b LPS 1 ng/ml LPS 1 ng/ml LPS.1 ng/ml w/o alum MF59 alum MF59 w/o 4 4 1:1 1:1 1:1 wt Nalp3-/- Alum MF59

26 cells/ 1E6 cells/ 1E6 GMT MF59 adjuvanticity is Nalp3-independent WT and Nalp3 KO mice have been imunized i.p. with 3 MenB antigens alone or adjuvanted with alum, MF59 and CFA Cell recruitment and cytokine production in the peritoneum are not affected in Nalp3 KO Adjuvanticity to three different MenB Ags is Nalp3-independent (IgG and BCA titers) Nalp3 mutation affects the response to all non-adjuvanted antigens in the plain vaccine h monocytes macrophages h 24h 4h plain alum MF59 CFA wt MenB wt MenB Nalp3-/- wt MenB+alum wt MenB+alum Nalp3-/- wt MenB+MF59 wt MenB+MF59 Nalp3- /- MenB+CFA wt wt MenB+CFA Nalp3-/- Nalp3-/- Nalp3-/- Nalp3-/- Nalp3-/ IgG against MenB * ** ** * P<,5 ** P<,1 ** wt Nalp3-/- wt Nalp3-/- wt Nalp3-/- wt Nalp3-/- plain alum MF59 CFA

27 MF59 is not a TLR Agonist in Vitro

28 wt WT wt WT wt WT wt WT MyD88-/- MyD88-/- /- MyD88-/- /- MyD88-/- /- wt WT wt WT wt WT wt WT MyD88-/- MyD88-/- /- MyD88-/- /- MyD88-/- /- Log1 titers Log1 titers WT wt WT wt WT wt WT wt MyD88-/- MyD88-/- MyD88- /- MyD88-/- MyD88- /- MyD88-/- MyD88- /- MyD88-/- Log1 titers MF59-adjuvanticity depends on the adaptor molecule MyD88 A 7 6 a-nhba (total IgG) ** ** ** ** AS59 8 C57B6 WT or KO/group vaccinate (i.p.) Day: serum -- Alum MF59 CFA -- Alum MF59 CFA a-fh-bp (total IgG) ** ** ** ** a-nada (total IgG) ** ** ** * -- Alum MF59 CFA -- Alum MF59 CFA -- Alum MF59 CFA -- Alum MF59 CFA Seubert et al. PNAS 211

29 Signaling pathways required for MF59 action MF59 Release of endogenous TLR agonists? H H 2 O 2 O OIL PAMPs (pathogen-associated molecular patterns) TLR DAMPs (damage-associated molecular patterns) TACI crystals, lysosomal damage, ROS,... MyD88 Nlrp3 Asc Pro-caspase- 1 B cell NF-κB Pro-IL- 1β Caspase-1 IL-1β IL-1R IL-18R ST2/IL1R AcP Pro-IL- 18 IL-18 IL-1β IL-18 IL-33 APC, epithelial cells,... IL- 33 MyD88 MyD88 Target cells: T, B, NK, MPh,... MyD88

30 old old + MF59 young The MF59 adjuvant makes the old immune system to behave like a young one!!!!

31 Serum HI antibody titers Seum HI titers Preclinical studies show that MF59 nanoparticles are the strongest adjuvant for subunit flu vaccine nil CpG Alum CAP MF59 PLG 3 H3N2 8 H1N1 35 B post-1 post-2 post-1 post-2 post-1 post-2 FCC given at.1 micrograms/dose

32 FLUAD/Comparator Post-Immunization GMT Ratio FLUAD Immunogenicity Meta-Analysis - All Subjects Superior Immune Response Against Conventional Vaccines B A/H3N2 A/H1N st immunisati on 2st immunisati on 3st immunisati on

33 Pain Erythema Induration Malaise Headache Myalgia Fever (>=38 C) Pain Erythema Induration Malaise Headache Myalgia Fever (>=38 C) Fluad safety meta-analysis: no severe reactions 5% 4% 3% 2% 1% Control Severe Reaction (n= 1437) Control Mild/Mod. Reaction (n= 1437) FLUAD Severe Reaction (n= 2112) FLUAD Mild/Mod. Reaction (n= 2112) % Podda, Vaccine 19: , 21

34 Relative increase in % with 4-fold increase Relative increase in % with HI Titre >=16 Enhancement of adjuvant effect in elderly with low pre-immunization titer A) Proportion of subjects with 4-fold increase or seroconversion B) Proportion of subjects with HI Titre >= 16 2% Pretiter <=2 2% 15% ** ** Pretiter>4 15% ** 1% 1% ** ** 5% 5% % B A/H3N2 A/H1N1 % B A/H3N2 A/H1N1 ** Pretiter 2 vs Pretiter >4, P<.1

35 MF59-adjuvanted vaccine in children Seroprotection rates after one and two doses H3N2 H1N1 B % % % DAY 1 DAY 29 DAY 5 DAY 1 DAY 29 DAY 5 DAY 1 DAY 29 DAY 5 FLUAD Vaxigrip

36 Relative risk of adverse events following vaccination with MF59-adjuvanted or non-adjuvanted vaccines Pooled analysis of 38 randomized, controlled influenza trials, subjects 65 years of age Lower risk with MF59-adjuvanted* Higher risk with MF59-adjuvanted* Solicited local reactions Solicited systemic reactions All unsolicited adverse events Including: Cardiovascular diseases New onset of chronic diseases Serious adverse events Hospitalizations Deaths Risk ratios The risk of subjects 65 years of age developing an unsolicited reaction was Green similar indicates statistical for evidence MF59- of a decreased and risk with non-adjuvanted MF59-adjuvanted vaccine; vaccines red indicates statistical evidence of an increased risk with MF59-adjuvanted vaccine. Significance claimed if 95% CI excluded 1. * Compared to non-adjuvanted vaccine; includes or excludes study V7P35 (ClinicalTrials.gov Identifier: NCT48165). Risk ratio: risk of developing a disease after exposure to a vaccine. Pellegrini M. et al. Vaccine 29; 27:

37 14 MF59-adjuvanted vaccine is safe in children 12 (6-36 mo-old) Summary of Post-injection Reactogenicity After 1 Any Vaccination FEVER DIARRHEA VOMITING IRRITABILITY UNUSUAL CRYING SLEEPINESS CHANGE IN HABITS TENDERNESS GMTs GMTs FLUAD Vaxigrip SWELLING P=.33 INDURATION ERYTHEMA ECCHYMOSIS % 1% 2% 3% 4% 5% Local and systemic reactions were mild or moderate and transient, and comparable to those induced by the control vaccine with the exception of site injection swelling (12% vs 5% p=.33).

38 In 26 we vaccinated with clade 1 H5N1 vaccine those people that had been vaccinated with clade H5N1 in By day 7 post-boost most of subjects have already protective neutralizing antibody titers against all virus strains Homologous H5N1 Clade 1 Heterologous H5N1 Clade 2.2 Protective titer (1:4) 1 1 With MF59 Heterologous H5N1 Clade 2.3 Heterologous H5N1 Clade 2.1 w/o MF years Priming with H5N3 Days Boost with H5N1 clade 1 with MF59 Months

39 Memory T cells are induced first Memory B cells are more abundant following adjuvant priming 1 Mean T cells x nil / 15 ug MF59 / 7.5 ug MF59 / 15 ug Mean IL-2 + / IFN- - cells (x 1 6 ) Pre Post-1 Post n=17 p<.5 p<.5 n=6 n=7 day 22 unprimed plain-h5n3 primed MF59-H5N3 primed Homologous H5N1 Clade 1 1 Heterologous H5N1 Clade 2.2 Protective titer (1:4) 1 1 With MF59 w/o MF59 Heterologous H5N1 Clade 2.3 Heterologous H5N1 Clade years Priming with H5N3 Days Boost with H5N1 clade 1 with MF59 Months

40 H5-CD4 + (in 1 6 tot CD4) A/VN/11194/4 MN- GMT Vaccination with adjuvant: more CD4+ T cells, more antibodies... a different game 1 * Non-Adj-15 MF * * * MF59-15 * 1: * * * * days Galli et al, Proc Natl Acad Sci USA 29

41 Percentage of memory B cells Memory B cells are present only in those primed with adjuvanted H5N1vaccine Unprimed H5N3 primed MF59-H5N3 primed Galli et al, Proc Natl Acad Sci USA 16 (19): , 29

42 It is all about Memory Adjiuvant CD4 T cells Memory B cells Antibody titer No adjuvant Protective tit

43 MF59 works against drifetd viruses Fujan example Immunogenicity (haemagglutination inhibiting antibody titers) of 3 Chiron Vaccines inactivated influenza vaccines against H3N2 homologous vaccine strain (A/Panama) and against H3N2 heterovariant strain (A/Wyoming) Agrippal subunit vaccine (n = 29) Prevaccination GMT 95% CI Seroprotection rate No/Total (%) Postvaccination GMT 95% CI Seroprotection rate No/Total (%) A/Panama (H3N2) /29 (79.3%) /29 (96.5%) A/Wyoming@ (H3N2) /29 (65.5%) /29 (75.9%) Begrivac split vaccine (n = 3) A/Panama (H3N2) /3 (73.3%) /3 (96.7%) A/Wyoming (H3N2) /3 (36.7%) /3 (8%) Fluad adjuvanted subunit vaccine (n = 3) A/Panama (H3N2) /3 (7%) /3 (1%) Seroprotection rate: proportion of subjects with a protective HI titre A/Wyoming /3/23 is an A/Fujian/411/22-like strain. * GMR: geometrical mean ratio (post-vaccination GMT / pre-vaccination GMT) A/Wyoming (H3N2) /3 (53.3%) /3 (1%)

44 Efficacy in the elderly 1-year study, 713,872 person-seasons, average age 73 MF 59 can address low vaccine efficacy due to antigenic drift

45 Influenza Vaccines past today future

46 The race to make vaccines From June to December 29 3 H1N1 vaccines were: Developed Tested in clinical trials Licensed 18 million doses produced

47 The Infleunza Hemagglutinin

48 Structure of the antibody binding site on the stem of HA

49 F16, a truly universal antibody against influenza

50 F16 recognizes a flat surface on the stem of HA

51 5-15 mg/kg of F 16 are necessary for protection

52 MF59 induces a more broad recognition of H5N1 epitopes covering neutralizing regions of HA Data obtained through a CRADA with Hana Golding CBER Khurana et al. Sci Trans. Med. 21

53 Toll-like receptor (TLRs) recognize microbial structures TLRs on the plasma membrane recognize bacterial products TLRs in the endosome recognize microbial nucleic acids TLRs trigger different signal transduction pathways TLRs are differentially expressed in immune cells All TLRs are validated targets for vaccine adjuvants.

54 Adjuvant Discovery: High Throughput Screening for Small Molecule Immune Potentiators (SMIPs) Focused or diverse libraries TLR Screens Hit to lead optimization Structure activity assessment In vitro Hits Confirmed hits Secondary screens Novel adjuvant candidates Formulation and delivery In vivo In vivo immunogenicity evaluation Adjuvant development Lead candidates for advancement

55 percentage of coverage TLR7 agonists enhances breadth of antibody response in mice against Men B (>MF59) Alum MenB Alum MenB 1/4 OMV MF59 Alum MenB IC31 TLR7 agonist Super Alum MenB Percent coverage (15 MenB strains) for serum bactericidal titers greater than 496

56 % cytokine release relative to R848 (25ug) SBA Titers Picking the Winners Defining the optimal PK/PD profile for adjuvant safety and efficacy 1 75 Peak Cytokines IFNg IL-12t IL-6 IL-1 TNFa SBA titer level of Mice dosed IM with 3MenB antigen + SMIP 5 1 Clinical correlate for protection (titer 124) 25 5 SMIP-1 SMIP-2 SMIP-3 base SMIP-1 SMIP-2 SMIP-3 Wasted Inflammation! Systemic exposure is detrimental to efficacy

57 Toll-like receptors ( TLRs ) Adjuvant ( MF59 )

58 Acknowledgement (II) The SMIP Team Immunology Anne-Marie Pulichino Alessandro Muzzi Carla Russo Carlo Iavarone Christine Dong Lee Christine Shaw Diego Piccioli Elaine Tritto Elena Caproni Elisabetta Monaci Elisabetta Soldaini Ennio De Gregorio Flaviana Mosca Gillis Otten Igor Leykin Jason Debasitis Lamine Mbow Mary Schaefer Michaela Brazzoli Nick Valiante Rocco Cantisani Sara Valentini Susanna Aprea Ugo D Oro Valerie Pasquetto Immunology Andy Miller Ann Herman Alejandra Rocha Bill Wacknov Bishnu Nayak Christophe Filippi De Shon Hall Janice Hampton Jillian Rojek Joe Cahiwat Jonathan Deane Kelly-Anne Masterman Laura Boase Maureen Ibanez Michael Cooke Peggy Hedden Tamar Tomassian Tami Annable Genomics Loren Miraglia Raquel Vega Richard Glynne Chemistry Alex Cortez Allan Pan Chun Li (DMPK) Kathy Yue Michael Shapiro (DMPK) Porino Va Tim Hoffman Tom Wu Xiaoyue Zhang Xuebin Liao Yefen Zou Yongkai Li Protein expression Mark Knuth Sarah Cox Benjamin Bohl DMP Greg Michaud John Tallarico Joshua Schustak Qian Huang Xinming Cai Lisa Welch Rekha Panchal Sina Bavari Travis Warren Jody Puglisi Tinghe Wu Ana Avalos Hidde Ploegh 5