Immunogenicity of virus-like particles: University Hospital Zürich, Switzerland

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1 Immunogenicity of virus-like particles: Use of An virus-like particles for therapeutic vaccination immunological perspective Martin Bachmann, Jenner Institute, of xford, UK against addiction and chronic diseases Regulation of anti-viral Bother celluniversity responses University Hospital Zürich, Switzerland

2 Harnessing the Immune System Clinical Use Antibodies Most Prophylactic Vaccines Self-specific Antibodies Allergies Drug-specific Antibodies T Cells Cancer Chronic Viral Infections 2

3 Three Key Features of VLPs Size Repetitiveness Toll-Like Receptor Ligands 3

4 Effector Mechanisms Induced by Vaccines The efficacy of many vaccines is based on antibodies produced by B cells What do B cells actually recognize? 4

5 What B cells recognize B cells recognize native antigen 5

6 What B cells recognize Antigens therefore should reach lymphoid organs in native form! 6

7 Dendritic cells Transport Antigen from the Periphery DCs transport antigen from the periphery But there is no evidence for skin DCs bringing antigens to lymph nodes in native form 7 Bachmann et al., ature Reviews Immunology 6, 159

8 How antigens traffic to the lymph node Cell mediated and/or free draining Injection site Cell-mediated transport Free draining Lymph node (L) 8 Modified from Randolph et al, at. Immunol. 2005

9 How antigens traffic to the lymph node Cell mediated and/or free draining Is size important for lymphoid transport? 9

10 nly particles < 200 nm freely drain to L nly particles <=200 nm drain to L in free form B cells plus anobeads Lymph node (L) 10 Bachmann & Jennings, at Rev Immunol. 10: 787 (2010) 20 nm beads 1 um beads

11 Size of Particulate Adjuvants Cell mediated and/or free draining Viral size is optimal to reach L 11 Jennings&Bachmann, Biological chemistry 389:

12 Particulate Adjuvants: Size Matters VLPs have optimal size to reach lymphoid organs in native form for interaction with B cells. 12

13 Three Key Features of VLPs Size Repetitiveness Toll-Like Receptor Ligands 13

14 Influence of Antigen Repetitiveness on B cell Responses rganization: high low absent Antibody response Induction of auto-antibodies Science 262,

15 VLPs are optimal for B cell activation Receptor Crosslinking Complement Fixation 15 Pentraxin Binding atural Ab Binding Bachmann & Jennings, at. Rev. Immunol. 10:787)

16 atural IgM recognized the VLPs (Qb) 16 Link et al., JI 188: 3724

17 Follicular Dendritic Cells and the Germinal Centre 17 ature Reviews Immunology 8, (January 2008)

18 18 atural IgM is requried for FDC-deposition

19 Activating Complement IgM 19

20 Complement-dependent FDC-Trapping Link et al., JI 188: 3724

21 FDC-Trapping: Multimers but not Dimers VLPs (180 monomers) Dimers (28 kd) 21 Qβ MMA-1 IgD Link et al., JI 188: 3724

22 How are VLPs transported? Marginal Zone???? FDC-etwork 22

23 How are VLPs transported? Transport/Deposition is Complement dependent B cells can transport immune complexes in in a complement-dependent fashion Generation of chimeric mice in which only B cells lack Complement receptors CD21/CD35 23

24 B cells transport VLPs to FDCs via C-receptor Control o C-receptor on B cells 24 Link et al., JI 188: 3724

25 nly VLPs Induce GC-Formation 25 Link et al., JI 188: 3724

26 Conclusion Repetitive VLPs fix complement B cells bind VLPs via C-receptor (Cr2) Transport to FDCs Induction of GC-response 26

27 Complement (C): Direct Effects on B cells Well described role for Complement in B cell activation C reduces threshold for B cell activation Science 256: 105 (1992) C reduces number of BCRs that need to be engaged EJI 32:3305 (2002) C enhances differentiation of long-lived plasma cells JEM 201:993 (2005) 27

28 Role of epitope densitiy and organisation Why is epitope reptitiveness so important 28 ur immune system exploits structural features for the discrimination of self and foreign. Viruses have small genomes and therefore consist of a small number of proteins. Viruses cannot help but have to express highly repetitive and highly ordered arrays of antigens on their surface Antigen rganisation is a geometric PAMP (Pathogen associated molecular pattern)

29 Three Key Features of VLPs Size Repetitiveness Toll-Like Receptor Ligands

30 ot to forget Some VLPs carry toll-like receptor ligands 30

31 RA-Phage-derived VLPs are Are highly repetitive Particulate Target dendritic cells Contain RA Activate TLR7/8 Right size to drain into lymph nodes In addition, they are very stable cheap to produce 31

32 VLPs loaded with different TLR-ligands VLP with RA Disassembled VLP Dimers (no RA) RA CpG Poly-Glutamic acid (PG) VLP-RA VLP-CpG VLP-PG 32 TLR7/8 TLR9 no TLR

33 % p33 specific CD8 T cells MyD88-dependent CTL-induction Immunize with p33-vlps Tetramer Staining for specific T cells o TLR TLR7/8 TLR9 no TLR TLR7/8 TlR9 0 VLP-PG p33 VLP-RAp33 VLP-CpGp33 VLP-PG p33 VLP-RAp33 VLP-CpGp33 C57BL/6 MyD88-/- 33 Eur. J. Immunol : 1 10

34 IgG Titer TLR-dependent IgG responses Total IgG IgG2a IgG VLP-PG VLP-RA VLP-CpG VLP-PG VLP-RA VLP-CpG VLP-PG VLP-RA VLP-CpG no TLR TLR7/8 TLR9 no TLR TLR7/8 TlR9 no TLR TLR7/8 TLR9 34 J Immunol. (2007) 178: J Immunol. (2009) 183: Immunity (2011) 34:

35 IgA Titer TLR-dependent IgA responses VLP-PG VLP-RA VLP-CpG 35 J Immunol. (2009) 183:

36 Challenges for Vaccine Design Critical Role for TLR-ligands in the design of a universal Flu Vaccine 36

37 Targeting M2 M2 Protein - Short extracellular domain (24 aa) - Can easily by made synthetically - hidden from the immune system 37

38 Variability of M2 versus HA major pandemics Representative portion of HA Complete extracellular domain of M2 38

39 Vaccine Design Antigen (M2 peptide) Linker (SMPH) Carrier (AP205) Cys Lys MSLLTEVETPIREWGCRCDSSD-GGGC 39 on-replicating Contains RA as natural TLR7/8 ligand Very stable Economic production in bacteria 2 g/l bacterial culture of GMP grade material

40 Experimental Setup Vaccination - VLP-M2 - VLP Challenge with 4x LD50 Influenza A PR8) day 0 day 14 d31 antibody response - Survival - Temperature ( Fever ) - Appearance 40

41 anti-m2 IgG Induction of M2-Specific Antibodies M2 specific titers 40'000 30'000 20'000 10'000 0 d14 d21 Strong immune responses against M2 are induced by VLP-M2 without adjuvant 41

42 Survival and Fever Post 4xLD 50 Dose Survival Fever (Strain: PR8 H11 mouse adapted) VLP-M2 vaccinated mice are protected from a lethal challenge with Influenza A 42

43 M2 does not induce neutralizing antibodies 43

44 Critical Role for Isotypes in Protection with RA TLR7/8 IgG2a VLP-M2 % survival no RA no TLR IgG1 VLP-M days after infection 44 EJI 42:863

45 Complete Protection by M2-mAB 1 Survival Ctrl Ab anti M2-mAB EJI 42:863

46 Fraction of surviving mice Active Fc-portion is critical anti M2-Fc wt anti M2-Fc mut Ctrl Ab EJI 42: Mutation in Fc constant region FcgR binding Complement binding Days after challenge 46 anti-m2 mediated protection is mediated by Fc effector mechanisms

47 Targeting Cytokines by Active Immunization: Harnessing the Immunogenicity of VLPs 47

48 Basic Concept mab therapy is highly effective, but cost-intensive many patients develop anti-antibody responses which neutralize the therapeutic potential of mabs Increasingly older populations pose a pharmacoeconomic threat to health- care systems resulting in downward pressure on drug-costs Innovative and cost-effective therapies are needed Vaccination addresses these issues 48 Drug Discov Today ov;11(21-22): at Rev Drug Discov Jan;3(1):81-8

49 The IL-1 Family Interleukin-1: Three major forms of ~ 17 kda with high structural but limited sequence similarity: IL-1, IL-1, IL-1Ra Binding to two ubiquitously expressed receptors, IL-1RI and IL-1RII IL-1 or IL-1 binding to IL-1RI leads to recruitment of IL-1RAcP and initiation of a signal transduction cascade, resulting in F- B activation and expression of proinflammatory cytokines IL-1RII lacks a cytoplasmic signaling domain and functions a decoy receptor for IL-1 or IL-1 (together with soluble IL-1RI) IL-1 /IL-1 signalling is inhibited by IL-1Ra which binds to IL-1RI with similar affinity without transducing any intracellular signal 49

50 IL-1: Disease Indications Diseases which have been shown to respond to Kineret treatment: Rheumatoid arthritis Type II diabetes Systemic onset juvenile idiopathic arthritis Muckle Wells syndrome (MWS) Adult onset Still s disease Familial mediterranean fever (FMF) Familial cold autoinflammatory syndrome (FCAS) eonatal-onset multisystem inflammatory disease (MID) Schnitzler s syndrome 50 IL-1 is responsible for many rphean Diseases

51 Genetic Background 51

52 Why Immunize Against IL-1? Life-long therapy due to life-long overproduction of IL-1 52

53 53 The Technology diameter = 30 nm SMPH chemical linker MW = IL-1b Cys on-replicating o pre-existing immunity Contains RA as natural TLR7/8 ligand Very stable Economic production in bacteria 2 g/l bacterial culture of GMP grade material

54 ELISA titer (D50%) The Response is Highly Specific mil IL-1a immunized IL-1b immunized anti-mil-1 anti-mil-1 anti-mil-1ra ELISA IL-1a IL-1b IL-1antagonist 54 Eur J Immunol. 38:877-87

55 Efficacy in Mice: Collagen-induced Arthritis 55 Eur J Immunol. 38:877-87

56 ELISA titer eutralization titer Response in Primates: Delayed euralization eutralizing Ab response is delayed: evidence for affinity maturation CYT013-IL1bQb Rhesus monkeys ELISA titer (=24) eutralization titer (=24) Days 56 Science Translational Medicine, in revision

57 A Dose Finding Study in Humans Stage I Part FU1 FU2 FU3 FU4 FU5 wk Cohort 1 =6 =2 Scr. 10 μg + Alum Placebo FLLW UP Cohort 2 =6 =2 Scr. 30 μg + Alum Placebo Cohort 3 =6 =2 Scr. 100 μg + Alum Placebo Safety, Tolerability, Antibody reversibility Cohort 4 =6 =2 Scr. 300 μg + Alum Placebo Cohort 5 =6 =2 Scr. 300 μg + Alum Placebo Cohort 6 =6 =2 Scr. 900 μg + Alum Placebo 57 Science Translational Medicine, in revision

58 Unique Dose Response 900 mg works by far the best! 58 Science Translational Medicine, in revision

59 eutralizing Capacity (expressed as ng Ilaris) Robust eutralizing Activity in 3/6 Patients Cohort 6 =6 =2 Scr. 900 μg + Alum Placebo D0260 D0401 D Weeks Robust neutralization activity detected in 3 out of 6 patients of Cohort 6 Remaining 3 patients of Cohort 6 show early sign of neutralization eutralization becomes detectable after 7 injections and was long-lived 59 Science Translational Medicine, in revision

60 Are eutralizing Ab Active in Vivo? Preliminary Evidence for in vivo Efficacy in Type II Diabetes Patients Does Presence of eutralizing Antibodies alter CRP levels or influence Glucose Levels? 60

61 % Reduction CRP Preliminary Evidence For Efficacy: CRP 100 >100 r = p = eutralization titer 61 Science Translational Medicine, in revision

62 Change in Glucose (mg/dl) from baseline Preliminary Evidence For Efficacy: Glucose Change in eutralization (%) from baseline R, 6-7x900 ug (n=3) R, 8x900 ug (n=3) 62 Science Translational Medicine, in revision

63 63 A Vaccine Against IL-17

64 IL-17: A Potent Proinflammatory Cytokine APC TH1 IL-12 TGF- IL-6 Tn CD4 + Tcell TH17 IL-23 IL-4 TH2 IF-g Cellular immunity Autoimmune responses IL-17 IL-17F IL-6 TF- IL-4 IL-5 IL-13 Humoral immunity Allergy 64

65 IL-17: A Validated Target Company Product Recently completed ngoing ovartis AI457 α-il-17a Phase II: RA Crohn s disease RR-MS Psoriasis Phase II: Ankylosing spondylitis Phase III: Psoriasis Amgen AMG 827 α-il-17r Phase II: RA Psoriasis Phase II: Asthma 1-2 doses 3-10 mg/kg i.v. Eli Lilly LY α-il-17a Phase II: Psoriasis Phase II: Psoriasis Hueber et al., Sci Transl Med ct 6;2(52):52ra72. RA 65 Hueber et al., Sci Transl Med, 2010

66 High Probability of Success Good Safety Profile: mabs and IL-17R-deficient individuals Largely only skin restricted muco-cutaneous candidiasis Good immunogenicity expected (Individuals with genetic deficiency of AIRE all develop neutralizing antibodies against IL17) Vaccinating patients instead of providing mabs would results in savings of > 1.2 Billion per year across the EU Likely implementation of a vaccine`s approach 66

67 Polyclonal sera specifically recognize IL-17A Qβ-IL-17 IL-17A VLP Linker IL-17F IL-17A 67 Eur J Immunol. 36:

68 IL-17: PoC in Mice RA and EAE Qβ-IL-17 CIA EAE VLP Linker IL-17A Röhn et al., Eur J Immunol ov;36(11): Eur J Immunol. 36:

69 Pilote Study in Primates Goals: Assess preliminary safety. Determine immunogencity and neutralizing ability of induced antibodies. Immunisation IL-17 challenge rmil-17wt i.v 1. rhil17qb 500 mg +Al(H) 3 2. rhil17qbd 500 mg +Al(H) 3 i.v IL-17 challenge rmil-17wt 200 mg/kg i.v o Alum s.c s.c s.c s.c Macacca mulatta n = 2 per group (1 male, 1 female) 69

70 ELISA titer eutralization titer ELISA titer eutralization titer Rapid Induction of eutralizing Ab ELISA titer (=4) IL17-VLP CYT017-IL17Qb Rhesus monkeys eutralization titer (=4) IL1b-VLP CYT013-IL1bQb Rhesus monkeys ELISA titer (=24) eutralization titer (=24) Study days Days 70

71 Acknowledgements University of xford Aadil El-Turabi Arturo Reyes-Sandoval Sumi Biswas University Hospital Zürich Franziska Zabel Antonia Fettelschoss Deepa Mohanan Thomas Kündig CHUV Lausanne Daniel Speiser University of Capetown Frank Brombacher 71 BRSC Riga Paul Pumpens Andris Zeltins Andris Dishlers Cytos Biotechnology, Zürich Gunther Spohn Juliana Bessa Alexander Link Susanne Keller Gary Jennings University of Basel Marc Donath Claudia Cavelti-Weder