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Host Viral Vector Interactions: Overview Roland W. Herzog, PhD Division of Cellular and Molecular Therapy University of Florida Activation Signals for the Immune System Danger TLR - Examples of Pattern Recognition Receptors The Adaptive Immune Response - Antigen Presentation to T and B Cell Receptors “Immunological Synapse” B7-1 = CD80 B7-2 = CD86 CD3 MHC I or II CD4 or CD8 CD4+ T Helper Cell Subsets APC IL-6 +TGF- IL-17, IL-23 IL-4 IL-10 TGF- Th17 IL-12 IL-4 Th1 IL-2 IFN- Inflammation (neutrophils) Th2 CD8+ CTL B cell (Ig2a ) Tr1 IL-10, No IL-4 IL-4 IL-10 TGF- IFN- IL-10 IL-4 B cell (IgG1, IgE) Th3 TGF-, No/low IL-4 TGF- IL-10 Suppression TGF- B cell (IgA) Suppression An Adaptive Immune Response Generates Memory Cells Characteristics Memory Cells: • Long-lived • One MHC:peptide complex maybe enough to reactivate • Less co-stimulation required Adenovirus: Capsid, Genome, Gene Products Innate immunity (cytokine, chemokine, coagulation, blood pressure) TLR9 NAB TLR2 Complement CTL aby Interactions with Kupffer Cells • Rise in systemic cytokines (TNF, IL-6, IL-12) • Destruction of Kupffer cells • Activation of endothelial cells leading to hemodynamic changes • Recruitment of neutrophils leading to hepatic injury Interactions with Endothelial and Dendritic Cells • Transduction and direct activation of endothelial cells • Transduction and maturation of DCs Platelet Events CAR 1) Activation 2) Degranulation Thrombin l GPIb/IX/V + AD Resting Platelet CAR CAR + 3) Aggregation l GPIb/IX/V GPIb/IX/V l PSGL-1 P-selectin Othman et al. Blood 2007 Activated Platelet Endothelium Transduction of APCs by Adenoviral Vector - Implications for CTL and Aby Responses to the Transgene Product • Eliminate viral genes • Use hepatocyte-specific promoter for transgene expression Schnell et al. Mol Ther. 2001 Immune Response to Adenovirus - Summary Seiler et al. CGT 2007 Adenovirus Problem: Potential solution: • Strong innate immunity • Limit (systemic) vector dose • Capsid • Chemical modification of • Genome (TLR9, capsid inflammasome) • Transduction of APCs (DCs) • Tissue -specific promoters • Adaptive immune responses • High capacity vectors to viral gene products • Pre-existing immunity to • Alternate serotypes (non- human serotypes (NAB, human) memory T cells) • Chemical modification of capsid Does Pre-existing Immunity to Adenoviral Vectors Increase the Risk of HIV Infection? Adeno-associated Virus (AAV) Vectors The positive: Inefficient transduction of APCs Low innate immunity No viral gene products Immune tolerance to transgene products after hepatic gene transfer Tolerance Induction by Hepatic AAV Gene Transfer F.IX -Galactosidase Destruction of hepatocytes -Glucosidase Ovalbumin APC CD4+ Th1 ApoAI Epo hAAT OTC CD8+ CTL CD4+ Th2 Neutralization of systemic expression IgG B Immune Regulation by CD4+CD25+FoxP3+ Treg IL-2 = Suppression CD25 Antibody Treg MHC II CTLA4-CD28 APC MHC II MHC I TGF- T help T help Cao et al. Current Gene Ther. 2007 Tolerance Induction by Hepatic Gene Transfer IL-10 LoDuca et al. Curr Gene Ther. 2009 Immune Privilege in the Eye Injection of Viral Vector Expressing Ovalbumin into Mouse Muscle AA VF .I X Day 5 B C CFSE KJ1-26 CD4 TCR Day 10 CFSE A Wang et al. Blood 2005 20 18 16 14 12 10 8 6 4 2 0 1200 1000 F.IX (%) 800 11.8 600 10.7 10.2 400 6.1 1.4 0.6 0 1 2 3 4 5 6 7 200 ALT 0.9 AST 0.6 2.7 8 9 10 11 Liver enzymes (I.U.) F.IX activity (%) Hepatic F.IX Gene Transfer Trial: Subject E 0 12 13 14 Weeks post vector infusion Manno et al., Nat. Med. 2006 CTL Responses to AAV Capsid in Humans Mingozzi et al. Nat Med. 2007 Proposed Model - Humans vs. Animals? Transduction Cross-presentation by hepatocytes? of liver AAV-F.IX particles Destruction of transduced hepatocytes Uptake and processing APC CTL Activation of memory cells? Capsidspecific CD8+ T cell Herzog, Mol Ther 2007 Ubiquitination of AAV Capsid in Transduced Target Cells Li et al. Mol Ther. 2007 CTL Responses to AAV Capsid - Summary Humans harbor memory CD8+ T cells to AAV capsid, and human hepatocytes present input capsid on their surface Multiple CD8+ capsid epitopes have been mapped in humans Several epitopes are highly conserved between serotypes AAV vectors can also activate a primary T cell response to capsid AAV capsids that do not contain HSPG binding domain are less efficient in activation of CD8+ T cells but are, nonetheless, cross-reactive with AAV capsid antigen-experienced CD8+ T cells Still lack of animal model that shows destruction of hepatocytes by AAVspecific CTLs Potential solutions to the problem: Transient immune suppression More efficient vectors that work at lower capsid antigen doses or that are less efficiently presented by MHC I Immune Responses to AAV - Summary Problems: • Inhibitor formation more Potential solutions: • Transient immune suppression frequent for some target organs • Pre-existing immunity to human serotypes: • NAB • Alternate serotypes (?) • memory CTL • Capsid engineering (molecular evolution, specific aa changes in B cell epitopes) • Transient immune suppression • More efficient vectors Lentiviral Vectors The positive: No preexisting antiviral immunity in HIV-neg subjects Choice of envelope protein to avoid pre-existing NAB No viral gene products Lentiviral Vectors TLR7 pcDC IFN/ Adaptive immune responses? Adaptive immune responses to transgene product Type I Interferon Response to Lentiviral Vectors • Activation of pcDC via TLR7 • Type I interferon response • Restriction of hepatocyte transduction • Activation signal for subsequent adaptive immune response to the transgene product Brown et al. Blood 109: 2797, 2007 Transduction of APCs by Lentiviral Vector - Tissue-specific Promoter not Enough? Liver Spleen VandenDriessche et al. Blood 2002 De-targeting from APCs - a Major Advance toward Avoiding Immune Responses to the Transgene Product Liver Liver Spleen Brown et al. Nat. Med. 2006 Sustained Expression of Factor IX after Hepatic Lentiviral Gene Transfer to Hemophilia B Mice Brown et al. Blood 110: 4144, 2007 Lentivirus Problems: • TLR7 innate immunity to ssRNA genome Potential solutions: • Block IFN/ expression • Transduction of hepatocytes limited by type I IFN response • Transduction of APCs • Strong adaptive responses to transgenes • Tissue-specific promoter and/or miRNA In Vivo vs. Ex Vivo Gene Transfer HSC Immune Tolerance Induction by Ex Vivo Gene Transfer T Cells B Cells Immune Suppression/Modulation Reagent Mode of action Gene transfer protocol Target of Suppression Cyclophosphamide DNA alkylating agent, dividing cells AAV-F.IX IM Aby to F.IX CTLA4-Ig + anti-CD40L Co-stimulation Hepatic plasmid-F.VIII; Hepatic adenovirus Aby to F.VIII; CTL to adeno Anti-ICOS Co-stimulation Hepatic plasmid-F.VIII Aby to F.VIII Rapamycin + MMF Lymphocyte proliferation Hepatic AAV-F.IX CTL to AAV capsid Immune Suppression/Modulation Reagent Mode of action Gene transfer protocol Target of Suppression Cyclophosphamide DNA alkylating agent, dividing cells AAV-F.IX IM Aby to F.IX CTLA4-Ig + anti-CD40L Co-stimulation Hepatic plasmid-F.VIII; Hepatic adenovirus Aby to F.VIII; CTL to adeno Anti-ICOS Co-stimulation Hepatic plasmid-F.VIII Aby to F.VIII Rapamycin + MMF Lymphocyte proliferation Hepatic AAV-F.IX CTL to AAV capsid Important to preserve CD4+CD25+ Treg!!! Immunology of Gene Therapy (Wiley Publishing, 2008) Chapter Corresponding Author Institution 1. The immunology of gene transfer: an overview David Lillicrap Queen’s University, Canada 2. Innate immune responses to nucleic acids Ishii and Akira Osaka University, Japan 3. The anti-viral Innate immune response Daniel Muruve U. Calgary, Canada 4. Immune responses to adenoviral vectors Dmitry M. Shayakhmetov U. Washington, Seattle 5. Immune responses to lentiviral vectors Lung-Ji Chang U. Florida 6. Immune responses to adeno-associated virus vectors Hildegund Ertl Wistar Institute and U. Pennsylvania 7. Immunology after in utero and neonatal gene transfer Simon Waddington Royal Free and University College Medical School, London, UK 8. Immunology of hepatic gene transfer Roland Herzog U. Florida 9. Immune responses to adenoviral, AAV, and lentiviral vector used for gene therapy of brain diseases Pedro Lowenstein Cedars-Sinai Medical Center 10. Immunology of cutaneous gene transfer Soosan Ghazizadeh Stony Brook University 11. Immune responses to pulmonary gene therapy Terence Flotte U. Mass. 12. Induction of immune tolerance through gene transfer to hematopoietic stem cells John Iacomini Harvard University 13. Reconstitution of the immune system by gene therapy Adrian Thasher Institute of Child Health, London, UK 14. Genetics of the immune response to proteins expressed after gene transfer Jay Lozier National Institutes of Health 15. Immune modulation Valder Arruda Children’s Hospital of Philadelphia and U. Pennsylvania 16. Gene therapy for autoimmune diseases Nicholas Giannoukakis U. Pittsburgh 17. Engineering of viral capsids to evade the host immune system Hildegard Büning U. Cologne, Germany