“The Biological Basis for the Correct Interferon Usage in Clinical Practice.”

Summer School ESCMID 2011 Treviso – Italy OLL “The biological basis for the correct interferon usage in clinical practice.”

Guido AntonelliAuthor Virology Laboratory Department of Molecular Medicine ESCCMID“ Sapienza”©by University of Rome Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by OLL

Papers Reporting the Discovery of Interferon Proc. Royal Soc. London 147:258-267,Author 1957 Proc. Royal Soc. London 147:268-273, 1957 NaganoESCCMID Y and Kojima Y. Inhibition de l’infection©by vaccinale par la virus homologue Seances Soc. Biol. Fil. 152: 1627-1630, 1958. OLL

Author

ESCCMID©by Paper Reporting the Discovery of IFN-γ Wheelock, EF. Science 149:310-311, 1965 Nature. 1980 Mar 27;284(5754):316-20. Synthesis in E. coli of a polypeptide with human leukocyte interferon activity Nagata S, Taira H, Hall A, Johnsrud L, Streuli M, Ecsodi J, Boll W, Cantell K, Weissmann C. OLL

Author

ESCCMID©by Major milestones and discoveries in 50 years of interferon research (1) OLL

Author

ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by MAIN APPROVED USES OF INTERFERONS

Type of Interferon Indications Interferon alfa-2a (Roferon®) hairy cell leukemia AIDS-related Kaposi's sarcoma chronic hepatitis C Pegylated Interferon alfa-2a (PEGASYS® ) chronic hepatitis C andOLL B Interferon alfa-2b (Intron-A®) genital warts chronic hepatitis B chronic hepatitis C hairy cell leukemia AIDS-related Kaposi's sarcoma adjuvant therapy for malignant melanoma in combination with chemotherapy for non-Hodgkin's lymphoma (follicularAuthor lymphoma) Pegylated Interferon alfa-2b (PEG-Intron®) chronic hepatitis C Interferon alfa-n3 (Alferon®) condylomata acuminata Interferon alfacon-1 (Infergen®) chronic hepatitis C Interferon beta-1a ESCCMID(Avonex®, Rebif®) relapsing forms of multiple sclerosis Interferon beta-1b (Betaseron®) ©byrelapsing-remitting multiple sclerosis Interferon gamma (Actimmune®) chronic granulomatous disease malignant osteopetrosis 1 Main IFNs in clinical trials

Indication/ Trade name / product Company Clinical phase application PegIntron (pegylated rh- Schering- IFN-α2b) Plough/Enzon OLL phase III CML

malignant phase III melanoma phase I solid tumors PEG-alfacon-1 (pegylated IFN-alfacon-1 IntermuneAuthor phase I hepatitis C Hemispherx Alferon LDO (IFN-αn3) Biopharma phase I / II HIV infection Hemispherx West Nile virus Alferon (IFN-αESCCMIDn3) Biopharma phase II encephalitis ©by first trials started in Recombinant IFN-α2b Biolex Q1/2005 hepatitis C 2 Main IFNs in clinical trials Clinical Indication/ Trade name / product Company phase application Veldona (natural-oral IFN-α) Amarillo Bioscience phase III Sjogren's syndrome

fibromyalgia OLLphase II syndrome phase II pulmonary fibrosis

oral warts (HIV- phase II positive) phase II / III Behçets disease Albuferon-α (Albumin-IFN- Human Genome α2a) Science Authorphase II b CHC r-IFN-α2b XL Flamel Technologies phase I / II hepatitis B hepatitis C CHC in Asian r-IFN-β ESCCMIDAres-Serono phase III patients ©by polyneuropathy Avonex (IFN-β1a) Biogen IDEC phase II (CIDP) Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by OLL

Author

ESCCMID©by XXX X X PEG-IFN X OLL

Author

ESCCMID©by OLL Science News

Newly Discovered Interferon Response May Offer Early Control Of H5N1 Influenza Virus ScienceDaily (June 21, 2009) — Researchers from Georgia suggest that the cell-signaling protein, interferon type 1, reduced H5N1Author influenza virus replication in mice and may offer some degree of protection in the early stages of infection. ESCCMID©by OLL

Author

ESCCMID©by OLL

Author

ESCCMID©by Leukemia. 2011 Jan 28. [Epub ahead of print]

Interferon-α in acute myeloid leukemia: an old drug revisited. Anguille S, Lion E, Willemen Y, Van Tendeloo VF, Berneman ZN,OLL Smits EL. 1] Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute, Faculty of Medicine, University of Antwerp, Antwerp, Belgium [2] Division of Hematology, Antwerp University Hospital, Edegem, Belgium [3] Center for Cell Therapy and Regenerative Medicine, Antwerp University Hospital, Edegem, Belgium.

Interferon-α (IFN-α), a type I IFN, is a well-known antitumoral agent. The investigation of its clinical properties in acute myeloid leukemia (AML) has been prompted by its pleiotropic antiproliferative and immune effects. So far, integration of IFN-α in the therapeutic arsenal against AML has been modest in view of the divergent results of clinical trials. Recent insightsAuthor into the key pharmacokinetic determinants of the clinical efficacy of IFN along with advances in its pharmaceutical formulation, have sparked renewed interest in its use. This paper reviews the possible applicability of IFN-α in the treatment of AML and provides a rational basis to re-explore its efficacy in clinical trials.Leukemia advance onlineESCCMID publication, 28 January©by 2011; doi:10.1038/leu.2010.324. The Rockefeller University Press J. Exp. Med. Vol. 207 No. 10 2053-2063 www.jem.org/cgi/doi/10.1084/jem.20101664 • Type I interferon: friend or foe? • Giorgio Trinchieri OLL • Cancer and Inflammation Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health,

• Although the role of type I interferon (IFN) in the protection against viral infections has been known and studiedAuthor for decades, its role in other immunologically relevant scenarios, including bacterial infections, shock, autoimmunity, and cancer, is less well defined and potentiallyESCCMID©by much more complicated. OLL The Interferon System Author

ESCCMID©by MAIN TYPES OF IFNs (an update to 2009)

Most types of cells T lymphocyte, Most types of cells NK cells OLL

Type I - IFNs Type II - IFN Type III - IFN

α β Others: γ λ ω δ, ε, ξ, κ, τ, ?? Author ≥13 1 gene genes* 1 gene 1 gene 1 gene 3 genes - For IFN alpha the main producing cells are pDC -The IFN alpha subspeciesESCCMID do not compensate for the loss of IFN beta - Some type I IFNs may be tissue restricted©by - For sure, type III IFN are not highly expressed in hematopoietic cells and act predominantly at epithelial surfaces OLL

Author

ESCCMID©by Virus Infection

IFN gene Type III IFN expression IFNλ Type I IFN IFNβ OLL Type II IFN IFNα IFN secretion IFNγ

Binding to IFN receptor

Transcription of IFN Author Stimulated Genes (ISG) Biological activities • antiviral activity ESCCMID• inhibition of cell growth Expression©by of ISGs • blockage of protein synthesis • immune response modulation Modificata da John Hiscott • apoptosis Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by OLL

Author

ESCCMID©by OLL

Author

ESCCMID©by OLL

Author

ESCCMID©by Extracytoplasmic pathways for Cytoplasmic pathways for induction induction (RNA Helycase: retinoic acid-inducible gene I (RIG-I) and (Toll like Receptors-TLRs) Melanoma differentiation associated protein 5 (MDA-5) OLL

Author

ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Nature Reviews - December 2007 - Borden et al.

OLL

Author

ESCCMID©by interferon stimulated response element GAS interferon gamma activated sites OAS protein accumulates in the cell cytoplasm as an inactive monomer. Following activation by viral double-stranded RNA, the enzyme oligomerizes to form a tetramer that synthetizes oligoadenylates that, in turn, activate the constitutively expressed inactive ribonucleases L (RNaseL). The binding of 2’,5’ – oligoadenylates to RNaseL triggers the dimerization of enzymes monomers, through their kinase like domains, and this then enables RNaseL to cleave viral and cellular RNA. OLL

Author

ESCCMID©by OAS protein accumulates in the cell cytoplasm as an inactive monomer. Following activation by viral double-stranded RNA, the enzyme oligomerizes to form a tetramer that synthetizes oligoadenylates that, in turn, activate the constitutively expressed inactive ribonucleases L (RNaseL). The binding of 2’,5’ – oligoadenylates to RNaseL triggers the dimerization of enzymes monomers, through their kinase like domains, and this then enables RNaseL to cleave viral and cellular RNA. Picornaviridae Reoviridae OLL Togaviridae Paramixoviridae Orthomixoviridae Flaviridae RetroviridaeAuthor

ESCCMID©by PKR accumulates in the nucleus and cytoplasm as an inactive monomer. It is activated directly by viral RNAs and by several other ligands. Following activation, PKR monomers are phosphorylated and dimerize to form the active enzyme. Activated PKR regulates several cell signalling pathways through unknown mechanisms. A crucial function of PKR in viral defence is the inhibition of translation by phosphorylation of eukaryotic traslation initiation factor 2 alpha. OLL

Author

ESCCMID©by The MxA protein accumulates in the cytoplasm on intracellular membranes as oligomers. Following viral infection, MxA monomers are released and bind viral nucleocapsids or other viral components, to trap and then degrade them.

OLL

Author

ESCCMID©by FURTHER ISGs

Interferon stimulated genes IFN-induced antiviral state ISG15, ISG54, ISG56 covalently interacting withOLL many proteins (ubiquitin-like): stabilize protein against degradation facilitate the degradation of other proteins inhibit traslation of others by interacting with eIF3 Adenosine deaminase deaminate adenosines within dsRNA ADAR generating inosines and leading to mutations in viral RNAs Viperin binds and disrupteAuthor lipid rafts (CMV-induced protein) APOBEC, TRIM cytidine deamination and template mutation inhibition of reverse trascription ESCCMID©by The genome of measles virus isolates in SSPE patients show a very high rate of A-to-G mutations

Proc Natl Acad Sci U S A. 2011 Jan 4;108(1):331-6. Epub 2010 Dec 20. RNA editing enzyme adenosine deaminase is a restriction factor for controlling measles virus replication that also is required for embryogenesis. Ward SV, George CX, Welch MJ, Liou LY, Hahm B, Lewicki H, de la Torre JC, Samuel CE, Oldstone MB. OLL Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037, USA. Abstract Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to- G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN- inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150(-/-) but not WT MEF cells displayed extensive syncytiumAuthor formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150(-/-) cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150(-/-) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restrictionESCCMID factor in the replication©by of paramyxoviruses and orthomyxoviruses. Measles virus leading to SSPS are selected under selective pressure from a host defence mechanism that targets viral RNA genome – i.e. IFN-induced ADAR ABOBEC is a cytidine-deaminase that, when packaged into virions, causes extensive G-to-A hypermutation during reverse trascription compromising the stability and function of the viral cDNA

OLL

Author

ESCCMID©by

Harris and Liddament, 2004; Nature Reviews Immunology Interferon-induced microRNAs

OLL

Author

ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by IFN-repressed miRNA (miR122) is a positive regulator of HCV replication OLL HCV-specific

Author

ESCCMID©by Table 2. Baseline expression of microRNAs and MxA in healthy controls and in patients with with chronic hepatitis C (CHC).

Healthy controls* Patients with CHC* Mean ratio n = 10 n = 12 OLLCHC/healthy controls miR-1 0.30 ± 1.43 2.82 ± 5.09 9.4 (0.36) (0.10) miR-30 128.96 ± 93.99 255.57 ± 466.62 1.98 (128.61) (27.41) miR-128 1.39 ± 3.31 19.54 ± 36.15 14.05 (0.05) (0.03) miR-196 1.85 ± 3.21 0.81 ± 1.52 0.43 (0.73) (0.04) miR-296 3.39 ± 7.12 12.94 ± 40.96 3.81 (0.46) (0.31) Author MxA** 2.45 ± 1.05 9.42 ± 10.64 3.84 (0.02) (7.09)

* Data are expressed as mean ± standard deviation (median) ** MxA vs miR-1, miR-30, p < 0.05 (healthy individuals) by Wilcoxon test. ESCCMID©by Scagnolari et al. Virology Journal 2010 Virus Infection

IFN gene Type III IFN expression IFNλ Type I IFN IFNβ OLL Type II IFN IFNα IFN secretion IFNγ

Binding to IFN receptor

OLL

Author

ESCCMID©by OLL Interferon and immunity Author

ESCCMID©by OLL

Author

ESCCMID©by OLL

Author ESCCMID Apoptosis ©byApoptosis INTERFERON AND APOPTOSIS

IFN OLL

PKR OAS-RNAseL pro-caspase p53 Author ESCCMID ©byAPOPTOSIS (i.e. REMOVAL OF INFECTED CELLS) (especially when apoptosis depends on dsRNA) In addition to direct inhibition of viral replication IFNs as modulators of innate and adaptive immunity: • Upregulation of MHC I andOLL MHC II • Stimulation of the effector function and/or maturation of natural killer cells, cytotoxic T lymphocytes, dendritic cells • Induction of immunoglobulin synthesis • Proliferation of memoryAuthor T cells ESCCMID - positive regulation of many©by innate and adaptive immune responses - transition from innate to adaptive immune response In addition to direct inhibition of viral replication IFNs as modulators of innate and adaptive immunity: OLL • Overexpression of lysosomal membrane permeabilization (LPM) component of proteasome • Overexpression of vascular adhesion molecules (i.e. ICAM1) • Production of chemotactic cytokines (i.e. ISG- CXCL9 (MIG), CXCL10 (IP-10),Author CXCL11 (TAC) ESCCMID accumulation©by of immune cells at site of virus invasion Interferon production and action in vivo

IFN production mainly by circulating dendritic cells OLL

direct antiviral action immune response Author

accumulationESCCMID of leukocytes increase of specific and aspecific at site of pathogen©by invasion immune effectors mechanism Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by 2-5-OASlog 1000 *

100 Relative gene expression 2-5OAS 10 of IFN-induced 1 RSV RhinovirusOLLNo virus genes in 10000 p56 log * infants with 1000 acute 100 bronchiolites P56 10

•p<0.05 by 1 •Kruskal-Wallis Test 0.1 RSV Rhinovirus No virus AuthorPKR 1000 * 100 10 ESCCMIDPKR ©by1 0.1 Scagnolari et al., 2007 RSV Rhinovirus No virus Correlation between IFN-induced gene levels and the severity score index Scagnolari et al., 2007

1400 Disease severity score index 1200 r=-0.26, p<0.05 SCORE 0 1 2 1000 Respiratory < 45 45-60 >60 800 rateOLL (b/m) 600 Sa O2 (%) >95 95-90 <90 2-5 OAS 400 Retraction/ Nasal No Retraction nasal flaring flare 200 Respiratory Rales/ Rales/ 0 diffuse sounds Normal localized 0246810 wheezing wheezing Clinical score Feeding Normal Reduced i.v. fluids

3000 250 r=-0.29, p<0.05 r=-0.21, p<0.05 2500 Author 200 2000 150 1500 P56 PKR 100 1000 500 ESCCMID 50 0 0 0246810©by 0246810 Clinical score Clinical score IMMUNE RESPONSE AND VIRUS COUNTERMEASURES

OLL

Author

IMMUNE RESPONSE ESCCMID IFN ©by IMMUNE RESPONSE AND VIRUS COUNTERMEASURES

OLL

Author

IMMUNE RESPONSE VIRUS COUNTERMEASURES ESCCMID IFN ©byANTAGONIST OF IFN PRODUCTION AND ACTION Overview of interferon pathway and viral counteracting strategies

Inhibition of IFN induction

Inhibition of IFN binding to receptor OLL Perturbation of intracellular IFN signaling pathway

Author Downregulation of the level of expression of ISGs

Inhibition of action of IFN- induced proteins ESCCMID©by Various stages of the IFN antiviral system blocked by different virus Mechanism of action Viruses

INHIBITION IN IFN Hepatitis B virus SYNTHESIS/PRODUCTION Human papillomavirus Human herpesvirus-8 Influenza Measles virus

Adenovirus INHIBITION IN IFN SIGNALING Hepatitis B virusOLL Human papillomavirus Epstein-Barr virus Cytomegalovirus Human herpesvirus-8 Sendai virus Simian virus 5 Human respiratory syncytial virus Mumps virus Hepatitis C virus Ebola Human parainfluenza virus types 3 Human parainfluenza virus types 2 InfluenzaAuthor Adenovirus INHIBITION IN IFN-INDUCED Herpes simplex virus ANTIVIRAL Epstein-Barr virus PROTEIN FUNCTIONS Hepatitis C virus Human immunodeficiency virus-1 Vaccinia ESCCMIDPoliovirus ©byInfluenza Reovirus MECHANISM OF ACTION OF PROTEIN KINASE (PKR)

eIF2-PO4OLL (inactive form)

ATP PROTEIN IFN KINASE dsRNAAuthor

eIF2 ESCCMID©by (+GTP+Met+tRNA) MECHANISM OF ACTION OF PROTEIN KINASE (PKR) AND NS5A

eIF2-PO4OLL (inactive form)

ATP PROTEIN IFN KINASE dsRNAX HCV-NS5A AuthorX

eIF2 ESCCMID©by (+GTP+Met+tRNA) Mutations at PKR-binding domain (ISDR domain) of NS5A give a strong replicative advantage IMMUNE RESPONSE AND VIRUS COUNTERMEASURES

OLL

Author

IMMUNE RESPONSE VIRUS COUNTERMEASURES ESCCMID IFN ©byANTAGONIST OF IFN PRODUCTION AND ACTION The biological basis for a proper use of IFNs in therapy of infectious diseases

IFNs: OLL • Represent a natural defense mechanism against viral infections • Are endogenous proteins and potentially they are not “toxic” becauseAuthor they are natural proteins • HaveESCCMID a very high©by specific activity • Possess a highly pleiotropic activity Interferon and combination therapy

IFN therapy OLL

Inhibits viral replication Author

IMMUNE RESPONSE VIRUS COUNTERMEASURES ESCCMID IFN ©byANTAGONIST OF IFN PRODUCTION AND ACTION IFNs TREATMENT OF PATIENTS

IFN beta IFN alpha IFN gamma OLL

IFN-INDUCED PROTEINS (i.e. Mxs, 2’-5’ OAS, PKR, MxA, ADAR1, and other > 100 proteins) Author

IFN-induced products: ? IFN-induced biological effects (i.e.Beta2m, Neopterin, etc) ESCCMID?©by IFN therapeutic efficacy Proportion of patients with chronic hepatitis C who achieved a sustained viral response

60 OLL55% 50 44% 40 33%

30 25%

16% Percentage of SVR 20

10 6% 0 Author

Modificata da G.I.Davis and K. I. Lindsay - Lancet Infect. Dis. 2005 PRISMS-LTFU study (rIFN beta 1a). 560 pts suffering from RRMS - Kappos et al. (Neurology, 2006)

18 16 15.4 OLL 14 12 Patients relapse free after 7 years 10 8.1 8 6.5 % patients 6 Author 4 2 0 ESCCMID44 mcg©by22 mcg late treatment OLL Variability of response to the IFN therapy Author

ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by IFNs TREATMENT OF PATIENTS

IFN beta IFN alpha IFN gamma OLL

IFN-INDUCED PROTEINS (i.e. Mxs, 2’-5’ OAS, PKR, MxA, ADAR1, and other > 100 proteins) Author

IFN-induced products: ? IFN-induced biological effects (i.e.Beta2m, Neopterin, etc) ESCCMID?©by IFN therapeutic efficacy HCV-RNA in patients after a single administration of rIFN alpha2

100000000 OLL 10000000

1000000

100000 HCV-RNA HCV-RNA copies/ml 10000 Author

1000 02448 ESCCMID©by (hrs) Antonelli G. et al, Journal of Infectious Diseases (1999) GENE DISEASE COHORT IFN TYPE OUTCOME AUTHOR YEAR CD152 Hepatitis C 79R, 79 NR IFN alpha + rib yes Yee et al. 2003 HLA II MS 66R, 30NR IFN beta no effect Fernandez et al. 2005 MS 77R, 57NR IFN beta no effect Villoslada et al. 2002 IFNG Hepatitis C 40 patients IFN alpha+rib no effect Abbas et al. 2005 IL 10 Hepatitis C 24R, 29NR IFN alpha yes Edwards-smith et al. 1999 MS 38α, 25β IFN alpha IFN beta yes Wergeland et al. 2005 Hepatitis C 40 patients IFN alpha+rib yes Abbas et al. 2005 Hepatitis C 49R, 55NR IFN alpha+rib yesOLLYee et al. 2001 IRF1 Hepatitis C 18 patients IFN beta yes Saito et al. 2005 JAK1 Hepatitis B 46R, 36NR IFN alpha no effect King et al. 2002 LMP7 Hepatitis C 49R, 126NR IFN alpha yes Sugimoto et al. 2002 MS 93R, 69NR IFN beta no effect Cunningham et al. 2005 MX1 Hepatitis C 93SR, 12RR IFN alpha no effect Knapp et al. 2003 Hepatitis B 46R, 36NR IFN alpha no effect King et al. 2002 MS 93R, 69NR IFN beta yes Cunningham et al. 2005 MS 179 NR Rel IFN beta yes Weinstock-Guttman et al. 2007 OAS1 Hepatitis B 46R, 36NR IFN alpha Authorno effect King et al. 2002 OAS2 Hepatitis B 46R, 36NR IFN alpha no effect King et al. 2002 OAS3 Hepatitis B 46R, 36NR IFN alpha no effect King et al. 2002 PKR/PRKR Hepatitis C 93SR, 12RR IFN alpha yes Knapp et al. 2003 Hepatitis B 46R, 36NR IFN alpha no effect King et al. 2002 MS 93R, 69NR IFN alpha no effect Cunningham et al. 2005 RANTES/CCL5 HepatitisESCCMID C 297 patients IFN alpha yes Wasmuth et al. 2004 STAT1 Hepatitis B 46R, 36NR©by IFN alpha no effect King et al. 2002 TNF A Hepatitis C 40 patients IFN alpha+rib no effect Abbas et al. 2005 Hepatitis C 100R, 41NR IFN alpha+rib yes Dai et al. 2006 Hepatitis C 49R, 55NR IFN alpha+rib no effect Yee et al. 2001 Single nucleotide polymorphism (SNP) in IFN-induced genes and outcome of HCV infection and response to IFN-therapy (368 pts) (Knapp S. Genes and immunity 2003)

SELF-LIMITING INFECTION INTERFERON RESPONSE PERSISTENT INFECTION OLL GG TT -88 MxA -88

PERSISTENT INFECTION SELF-LIMITING INFECTION

TT AuthorCT -168 -168 PKR PERSISTENTESCCMID INFECTION SELF-LIMITING INFECTION AA ©by GG OAS-1 3’UTR 3’UTR Hepatic gene expression discriminates Responders and Nonresponders in treatment of chronic hepatitis viral infection. Chen L. et al. Gastroenterology May 2005 OLL Conclusions: -….Hepatic gene expression profiling identified consistent differences in patients who subsequently fail treatment with PEG- IFNalpha plus ribavirin ….. - …..Up-regulation of a specificAuthor set of IFN- responsive genes predicts nonresponse to exogenous therapy…………. ESCCMID©by 2’-5’OAS, Beta2m, MxA-mRNA e neopterina: variability inter-patient 2-5 OAS (pmol/dl) NEOPTERINA (nmol/l)

160 CV>100% 140 150 CV= 69% 100 CV=73% 120 100 50 80 CV= 68% 45 40 60 OLL CV=24% 35 40 30 CV=46% 25 CV=46% 20 20 15 0 CV=18% Volontari sani CM SMRR HCV 10

5 0 MxA-mRNA (Log n° copie) Volontri sani CM SMRR HCV

7,5 Beta2m (ug/ml) CV>100% CV>100% 7 16Author CV=52 % 6,5 14 CV=63% CV>100%

12 6 10 5,5 8 CV=33% CV=28% <5 6 ESCCMID4 CV=16% 4,5 ©by 2 Volontari sani SMRR HCV CM 0 Volontari sani CM SMRR HCV Change of serum levels of beta2microglobulin, neopterin, 2-5 oligoadenylate synthetase activity after the first injection of IFNs in patients with chronic diseases OLL b2mg (μg/ml) NTP (nmol/l) 2-5 OAS (pmol/dl)

RRMS CHC RRMS CHC RRMS CHC

Pre-dose (T0) Mean±SD 2.7±0.8* 2.8±0.8* 9.2±3.6* 7.2±2.6* 20.3±14.6 35.8±24.2* Median 2.6 (1.7- 2.7 (1.7-5.3) 9.6 (1.4-22) 6.6 (2.6-13) 21.5 (2.0-57) 32 (4.1-100) (range) 6.2)

Post-dose (T24) Mean±SD 3.7±0.8 3.6±0.8 21.5±9.2 20.1±11.1 29.4±19.8 91.6±74.8 Median 3.7 (2.1- 3.5 (1.8-7.6) 19.7 (6.8- 18.2 (3.4- 26.5 (3.4- 78 (15-360) (range) 6.2 Author44.5) 54.2) 90.8) Increase Mean±SD 1.3±0.3 1.3±0.2 2.5±0.9 2.9±1.8 2.2±2.3 3.9±4.7 (T24/T0) Median 1.3 (0.6- 1.3 (0.7-2.2) 2.3 (1.1-5.0) 2.9 (0.4-9.4) 1.5 (0.2-12) 2.2 (0.6- (range) 2.1) 19.25)

Pre-dose/ increase r coefficient -0.57 -0.54 -0.47 -0.44 -0.53 -0.44 correlation ESCCMID(p<0.05)©by(p<0.05) (p<0.05) (p<0.05) (p<0.05) (p<0.05) * T0 vs T24; (p<0.05) Scagnolari et al., Clin.Exp.Immunol. 2007 Magdalena Sarasin-Filipowicz et al. Interferon signaling and treatment outcome in chronic hepatitis C. Proc Natl Acad Sci U S A. 2008 May 13; 105(19): 7034–7039. OLL

Author

ESCCMID©by Patients who were subsequently identified as non responders had high baseline expression of ISGs whereas responders to IFN therapy (and those who relapsed) more closely resembled healthy controls in terms of IFN induction of ISGs OLL

Non responders have un upregulated and largely ineffective IFN response so that administration of exogenous IFN alpha adds little Author and/or ESCCMIDDownstream inhibitors, mainly viral related, are active©by rendering both endogenous and exogenous IFN ineffective Hepatitis C virus personalized therapy Genome-wide association study (GWAS) allowsOLL to associate a charcteristic of interest , usually a disease, with hundreds of thousands of single nucleotide polymorphism (SNP) that are variations in one of the 3 billion nucleotide base pairs of human genome. Identification of a variant, or polymorphism, in the human genome that is significantly associated with HCV clearance during treatment has been reported. Author - Ge D, Nature 2009 : 1137 pts North America - Suppiah V, Nature Genetics 2009 : 293 pts Australia - Tanaka Y, Nature Genetics 2009 : 142 pts Japan - Rauch AESCCMID , Gastroenterology©by 2010 : 465 pts Switzerland • - Several authors identified several SNPs near to IL-28 gene on chromosome 19 that were significantly more common in patients that responded to IFN therapy than in non-responders. • - Approximatively 80% of patients who carry two copies of the advantageous variant cleared the virus during IFN therapy and remained virus-free for a period of 24 weeksOLL post-treatment IFN lambda 3

IFN lambda 2 Author

ESCCMID©by

Hepatology. 2011 Jan;53(1):336-45 Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Possible treatment algorithms for chronic HCV genotype 1 infected patients according to IL28B alleles OLL

Author

IFN beta IFN alpha IFN gamma OLL

- Antibodies to IFN IFN-INDUCED PROTEINS (i.e. Mxs, 2’-5’ - IFN-Receptor OAS, PKR, MxA, ADAR1, and other > 100 - IFN antagonists proteins) Author

IFN-induced products: ? IFN-induced biological effects (i.e.Beta2m, Neopterin,etc)ESCCMID?©by IFN therapeutic efficacy Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by There is a potential for immunogenicity with all biopharmaceuticals……… Antonelli G. Clinical Microbiology and Infection 2008 Product Detection incidence of antibody response OKT3 (murine mAb, anti-CD3) ~80% Remicade (chimeric mAb, anti-CD20) 10-57% ReoPro (chimeric mAb, anti-GPIIb/IIIa) 7-19% Simulect (chimeric mAb, anti-IL-2 receptor) OLL≤2% Rituxan (chimeric mAb, anti-CD20) ≤1% Zenapax (humanised mAb, anti-IL-2 receptor) 8% Synagis (humanised mAb, anti-RSV) ≤1% Herceptin (humanised mAb, anti-HER2) ≤0.1% Enbrel (TNF receptor/anti-TNF IgG) 16% Peglyated-MGDF 0.5-1.6% (oncology) 80% (in platelet donors) Thrombopoietin ≤10% (oncology) Eprex, Epogen, Procrit, NeoRecormon & Aranesp (erythropoietin)AuthorVery rare Roferon (interferon α2a) 20-50% Intron (interferon α2b) 0-24% Berofor (interferon α2c) ≤1% Avonex (interferonESCCMID β1a) ~5% Betaferon (interferon β1b) ©by ~44% Proleukin (interleukin-2) 47-74% Bone morphogenic protein-7 13-38% INTRODUCTION

• Treatment with therapeutic proteins may induce an immune reaction. Occasionally, OLLthis immune response is clinically significant….. • Obviously, guidance on the investigation and assessment of immunogenicity may contribute to a more rational and systematic approach to the immunogenicity by the industry and the regulatory authorities….. Author

ESCCMID European Medicines Agency ©by London, 22 February 2006 Antibody to IFNs OLL Vallbracht A et al. Interferon neutralizing antibodies in a patient treated with human fibroblast interferon. Author Nature 1981 ESCCMID©by Inhibitors of the IFNOLL action (antibodies to IFN alpha) chronic hepatitis C and cryoglobulinemiaAuthor ESCCMID©by RESPONSE TO nIFN TREATMENT IN A PATIENT WHO REACTIVATED WHILE ON rIFN TREATMENT AFTER DEVELOPMENT OF ANTI-rIFN ANTIBODIES

OLL

Author

ESCCMID©by ( Roffi et al. Hepatology 1996) Alpha recombinant Interferon in the therapy in chronic hepatitis C (up to 2000)

PATIENTS OLL RESPONDERS NON RESPONDERS

BREAKTHROUGH (10%) Author

ESCCMIDSUSTAINED RELAPSING ©byRESPONDERS PATIENT Development of Neutralizing antibodies to IFN alpha (Santantonio et al. Journal of Hepatology 2006)

Peg-IFNα2b + RBV Peg-IFNα2a + nIFN 6MU/die+ nIFN 9 MU/die+ RBV+ AMA OLLRBV+AMA RBV 1000 miastenia HCV -RNA UI/L(x103)

800 ALT UI/L

600 400 Author 200

t 2 3 3 3 3 3 3 4 4 4 4 4 4 4 4 5 5 5 5 0 0 0 0 ESCCMID00 0 00 0 00 31-ot /2 00 /2 00 200 /2 00 /2 00 200 /2 00 /2 00 /09/2 01 /02/2 05 08/ ©by/11/2 02 04/2 06 07/ /09/2 03 05/2 09 0 18 15/ 1 24/03/20020/ 22/ 05 30/01/20026/ 02/ 03/05/20004/ 09/ 17 10/12/20021/ 04/ 06/07/20010/ Annemiek A. van der Eijk, Jan Maarten Vrolijk,OLL Bart L. Haagmans N Engl J Med 354;12 - March 23, 2006 ...... Peginterferons are widely used to treat chronic hepatitis C virus (HCV) infection, an important cause of liver cirrhosis and hepatocellular Carcinoma. We describe a patient who did not have a virologic response to peginterferon and had a virtual absence of circulating peginterferon alfa-2aAuthor but had neutralizing antibodies against interferon alfa...... ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by Viral and host factors and response to IFN therapy

OLL

Viral Countermeasures IFN Author Antagonistis of IFN action ESCCMID©by Resistance and response to IFN therapy Online Lecture Library OLL Slide withheld at request of Authorauthor ESCCMID©by