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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