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Gene Therapy (2003) 10, 935–940
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REVIEW
Molecular basis of the inflammatory response to
adenovirus vectors
Q Liu and DA Muruve
Libin Gene Therapy Unit, Department of Medicine, University of Calgary, Calgary, AB, Canada
Adenovirus vectors are extensively studied in experimental
and clinical models as agents for gene therapy. Recent
generations of helper-dependent adenovirus vectors have
the majority of viral genes removed and result in vectors with
a large carrying capacity, reduced host adaptive immune
responses and improved gene transfer efficiency. Adenovirus vectors, however, activate innate immune responses
shortly after administration in vivo. Unlike the adaptive
response, the innate response to adenovirus vectors is
transcription independent and is caused by the viral particle
or capsid. This response results in inflammation of transduced tissues and substantial loss of vector genomes in the
first 24 h. The adenovirus capsid activates a number of
signaling pathways following cell entry including p38 mitogenactivated protein kinase and extracellular signal-regulated
kinase (ERK) that ultimately lead to expression of proinflammatory genes. Various cytokines, chemokines and
leukocyte adhesion molecules are induced by the adenovirus
particle in a wide range of cell types providing a molecular
basis for the inflammatory properties of these vectors. An
understanding of the innate response to adenovirus vectors
is essential to overcome the last remaining hurdle to improve
the safety and effectiveness of these agents.
Gene Therapy (2003) 10, 935–940. doi:10.1038/sj.gt.3302036
Keywords: adenovirus vectors; innate immunity; inflammation; signal transduction; cytokines; chemokines
Introduction
Gene therapy is a promising advance in science that will
lead to the treatment of many genetic and nongenetic
diseases. The success of gene therapy relies largely on the
availability of gene delivery vectors that confer therapeutic gene expression in desired organs with regulated
kinetics and minimal adverse effects on the host. To
achieve efficient gene transfer, viral vectors will be
usually administered to humans in titers many logs
greater than the number of viral particles required to
cause a wild-type infection. Therefore, to improve the
safety and effectiveness of these vectors for human gene
therapy, a need exists to understand the interaction of
viral vectors with host immune systems in the context of
their proposed use.
Adenoviridae are nonenveloped viruses with a 30–
40 kb linear double-stranded DNA genome. There are
approximately 50 serotypes of adenoviridae with the
group C viruses (serotypes 2 and 5) most extensively
studied and developed for gene therapy applications. In
addition, the application of replication-competent adenoviridae in gene therapy and cancer therapy is being
extensively evaluated and beyond the scope of this
review. Replication-deficient adenovirus vectors have
several advantages, including the ability to package large
quantities of DNA, ease to produce and broad cell
tropism.1 First-generation adenovirus vectors are derived
from E1-deleted wild-type adenoviridae. The nonessential E3 region is also removed in first-generation vectors
Correspondence: Dr DA Muruve, University of Calgary, 3330 Hospital
Dr NW, Calgary, AB Canada T2N 4N1
to increase capacity. Newer generations of adenovirus
vectors have been engineered to increase DNA-carrying
capacity and to alleviate host adaptive immune responses. These include adenovirus vectors deleted of
various early viral genes such as the E2 and E4 regions
(second-generation) or the entire coding region (helperdependent gutted Ad vectors).2 The development of
helper-dependent adenovirus vectors has minimized the
host adaptive response to these agents and improved the
efficacy and duration of gene transfer in vivo.3,4 Adenovirus vectors however activate the innate immune
system. The innate immune response to adenovirus
vectors is dose dependent and induced by the viral
particle or capsid independent of viral gene transcription.5,6 At high titers, adenovirus vectors can trigger
significant inflammation in transduced tissues with rapid
loss of vector and transgene.7 The transcription-independent innate response therefore remains a significant
problem for all generations of adenovirus vectors. In this
article, we will review our current understanding of the
molecular basis underlying the early host inflammatory
response to adenovirus vectors.
Innate immunity and the early host response
to adenovirus vectors
The primary function of the host immune response to a
virus is to detect rapidly, limit and ultimately eradicate
an infection. The innate immune system plays a key role
as the first line of defense in this process. An infecting
virus can trigger a variety of responses in a target cell or
resident macrophage that will lead to the production of
Biology of adenovirus vector inflammation
Q Liu and DA Muruve
936
cytokines and chemokines and the recruitment of
effector cells to the site of infection.8 These effector cells,
which include neutrophils, monocytes/macrophages
and natural killer cells in turn, limit the infection directly
by killing infected cells or indirectly by secreting
antiviral cytokines and chemokines. In addition, the
recruitment and activation of antigen-presenting cells to
the site of infection is essential for the development of an
optimal adaptive immune response.8 The ability of cells
to detect an invading virus is essential for triggering a
cascade of events that ultimately leads to eradication of
infection. The MAP kinases play a central role in this
process, and include the extracellular signal-regulated
kinases (ERK), the p38 kinases (p38) and the c-Jun NH2terminal kinases (JNK). In addition, signal transduction
via molecules containing Toll-like receptor/IL-1 receptor
(TIR) domains is essential in mediating specific aspects
of the innate immune response.9 Cellular activation by
one or more of these pathways is an essential component
of the early host response to infection.
Adenovirus vectors are well known to induce host
adaptive immunity.10 The Th1 dominant antiviral immune response occurs 5–7 days following transduction
and is directed against the residual expression of viral
genes that are still present in first-generation recombinant Ad-vectors.10,11 The development of newer generations of Ad-vectors deleted of the viral genome has
reduced the cell-mediated immune response and improved the duration of gene expression in vivo.3,4 Advectors, in addition to inducing adaptive antiviral
immunity, activate the innate arm of the immune
system.6,12–15 The acute inflammation triggered by Advectors impacts gene transfer efficiency and causes
significant morbidity in transduced hosts.14,16 Our
studies and others have characterized the innate
response to Ad-vectors in vivo. In contrast to the adaptive
response, the innate response is dose-dependent, occurs
within 24 h of transduction and is independent of viral or
transgene transcription.5,6 Histologically, Ad-vectortransduced tissues contain inflammatory infiltrates that
consist of CD11b+ neutrophils, natural killer cells and
macrophages.6,12 In the liver, resident macrophages
(Kupffer cells) efficiently take up vectors and release
proinflammatory cytokines and chemokines such as
tumor necrosis factor-alpha (TNF-a), IP-10 and
RANTES.6,14 The expression of proinflammatory cytokines and chemokines is associated with leukocyte
recruitment mediated by P-selectin, E-selectin and a4integrin.17 The prototypical activation of the innate
immune response by Ad-vectors is associated with rapid
clearance of the delivered vector dose (480%) in the first
24 h.7 A thorough understanding of the mechanism by
which Ad-vectors activate the innate arm of the immune
system is required to overcome the last remaining hurdle
limiting the efficiency and safety of adenovirus-mediated
gene therapy in humans.
Biology of adenovirus cell entry
Adenovirus attachment and internalization into the cell
is the first step of viral transduction and also the
triggering event for host responses to viral infection.
Therefore, understanding viral–cell interaction and the
biology of cell entry is essential to dissect the molecular
Gene Therapy
mechanisms underlying the inflammatory response to
adenovirus vectors. Group C adenoviridae (includes
serotypes 2 and 5) first attach to the cell through an
interaction between the carboxy terminus of the adenovirus fiber knob protein and a high-affinity receptor, the
coxsackievirus–adenovirus receptor (CAR).18 Heparan
sulfate glycosaminoglycans can also mediate initial cell
binding of group C adenoviridae.19 Following the highaffinity binding, a lower affinity interaction between av
integrins (avb3, avb5, avb1) and an arginine–glycine–
aspartic acid (RGD) motif on the adenovirus penton base
capsid protein mediates virus internalization by receptor-mediated endocytosis through clathrin-coated vesicles.20,21 At 10 min following internalization, pHdependent penetration of the endosome occurs.22 Adenovirus penetration into the cytoplasm is believed to
involve a pH-dependent conformational change in the
adenovirus penton base and an interaction with avb5
integrins.23,24 Following endosomal disruption, the partially uncoated virions traffic through the cytoplasm
along microtubules and reach the nuclear pore complex
by 30–40 min.22,25,26 In macrophages, group C adenovirus
vectors utilize different cell surface molecules for
binding. In the monocytic cell line, THP-1, RGDdependent interactions with the aM subunit of the b2
integrin, CD11b (aMb2) proved to be essential in mediating adenovirus high-affinity binding.27 In addition,
adenovirus has also been shown to bind aLb2 integrin
(LFA-1), suggesting a significant role for b2-integrins in
mediating adenovirus binding to macrophages.27
Adenovirus vectors and inflammatory gene
expression
The activation of innate responses by adenovirus vectors
in vivo has been followed by numerous studies detailing
the direct activation of inflammatory gene expression by
adenovirus vectors in vitro. Adenovirus vectors induce
the expression of various cytokines and inflammationassociated genes in innate cells such as macrophages and
noninnate targets such as epithelial and endothelial cells.
Similar to the response in vivo, the adenovirus vector
induction of inflammatory gene expression in vitro is
dose-dependent. As vector titers increase, a saturation
effect occurs where further increases in vector titers are
associated with minimal or no further increases in
inflammatory gene expression.28,29 These results are
consistent with the studies by Hidaka et al,30 who
demonstrated a saturation effect related to adenovirus
vector titer and binding in CAR-expressing A549 cells.
Cell type and high-affinity receptor density are likely
factors that determine at which titer vector saturation
occurs. These observations are relevant for in vivo gene
therapy. First, escalating titers of adenovirus vectors may
not translate into improved gene transfer efficiency and
may worsen the inflammatory response to these agents.
Second, many of the adverse inflammatory effects related
to the adenovirus particle may be avoided by simply
using lower titers.31
Activation of innate cells such as monocytes and
resident macrophages is an essential component of the
innate response to viral infection. In the lung, Zsengeller
et al15 showed the rapid accumulation of Ad5LacZ
vectors in alveolar macrophages 10 min after vector
Biology of adenovirus vector inflammation
Q Liu and DA Muruve
administration. This was associated with the upregulation of inflammatory cytokines and chemokines IL-6,
TNF-a, MIP-2, and MIP-1a. In situ hybridization studies
confirmed that alveolar macrophages were the source of
the expressed inflammatory genes. In vitro, transduction
of the macrophage cell line RAW264.7 with adenovirus
vectors resulted in the rapid stimulation of TNF-a
expression as early as 2 h post-transduction.15 In these
cells, adenovirus vector-induced TNF-a expression required vector internalization since chemical inhibition of
endosome acidification and/or lysis attenuated TNF-a
expression.15 Combining this observation with the lack of
transgene expression in macrophages would suggest that
the triggering event might reside after endosomal escape
but prior to nuclear localization of adenovirus vectors.
Consistent with these findings, adenovirus vectors also
stimulate the expression of cytokines and chemokines in
human peripheral blood mononuclear cells (PBMC) in
vitro.28 At a dose of 1000 PFU per cell, serotype 5
adenovirus (Ad5) vectors caused a minimal release of
TNF-a and IL-1b, a significant upregulation of IL-6 and
RANTES, and a steady increase of GM-CSF, MIP-1a, Groa, and IL-8 over a 96-h window. The use of UV-psoraleninactivated vector particles or empty capsids did not
diminish the inflammatory response confirming the
importance of the viral particle or capsid in this
process.28
Adenovirus vectors also induce the expression of
inflammatory genes in noninnate cells. In primary
kidney epithelial cells, adenovirus vectors and UVpsoralen-inactivated adenovirus particles induced the
expression of the chemokines RANTES, IP-10 and MIP-2
within 6 h of transduction.6 A variety of chemokines
including RANTES, IP-10 and IL-8 have been induced
early by adenovirus vectors in HeLa cells, the respiratory
epithelial cell line A549 and the mouse insulinoma cell
line TGP61.6,32–35 The activation of RANTES and IP-10 in
HeLa cells and the epithelial-derived cell line, REC,
occurred in the absence of a second messenger or
cytokine such as TNF-a, IL-1b or interferon-g suggesting
that the adenovirus particle is capable of directly
activating nonmacrophage target cells to express inflammatory mediators.34,35 In addition to inducing inflammatory cytokines, adenovirus vectors can induce the
expression of other genes involved in the inflammatory
process. In epithelial A549 cells and in endothelial cells
(human umbilical vein and bone marrow microvascular
endothelial cells), adenovirus vectors induced the expression of various leukocyte adhesion molecules such
as ICAM-1 and VCAM-1.36,37 The expression of leukocyte
adhesion molecules facilitates leukocyte recruitment to
adenovirus vector-transduced tissues.17 Thus, adenovirus vectors induce a broad array of inflammatory
genes in innate and noninnate cells that underlie the
inflammatory response to these agents in vivo.
aV integrins. While signal transduction related to CAR
binding has yet to be demonstrated, integrins are
involved in a wide variety of signaling events regulating
protein kinases, growth factor receptors and organization
of actin cytoskeleton.38,39 The adenovirus penton base
triggers a number of integrin-linked signaling pathways
in noninnate target cells.39 Nemerow and colleagues
have shown the activation of various components of the
integrin-associated focal adhesion complex by serotype 2
adenovirus (Ad2) infection in the colon adenocarcinoma
cell line, SW480. Adenovirus infection resulted in a fiveto seven-fold increase in tyrosine phosphorylation of
p125FAK (focal adhesion kinase) and p130CAS (Crkassociated substrate) and a 14- to 15-fold increase of
phosphotyrosine-associated
p85/phosphoinositide-3OH kinase (PI3 K).40,41 The activation of PI3 K was
essential for adenovirus internalization and downstream
of p130CAS.40 Interestingly, although p125FAK was activated, signaling via ERK or c-Jun NH2-terminal kinase
(JNK) was not induced by adenovirus entry in these
cells.41,42 Purified penton base proteins, but not fiber
proteins, have a similar effect, indicating that the
interaction of adenovirus penton base with aV integrin
is the triggering event for p125FAK, p130CAS and PI3K
activation.40,41 In a follow-up study, the same group
found that the Rho GTPases Cdc42 and Rac1 were
activated downstream of PI3K following infection with
Ad2 and required for actin cytoskeleton reorganization.41,42 The activation of small G proteins by adenovirus-mediated endocytosis was selective since H-Ras
GTPase did not play a role in adenovirus internalization.42 Recently, Hautala and co-workers reported that
the activation of rab5 GTPase also occurred during Ad2
internalization. Activation of p125FAK or p130CAS was not
detected; however, a different cell type (HeLa) and lower
titers were used for these experiments.
Cell signaling pathways are required not only for
adenovirus vector cell entry, but are also important for
subsequent intracellular trafficking. Greber and co-workers have shown in HeLa cells that Ad2 infection results
in a 3.5-fold upregulation of protein kinase A (PKA) at
15–30 min.43 PKA activation was required for sufficient
adenovirus nuclear targeting since the application of
PKA inhibitor PKI-myr reduced the nuclear localization
of adenovirus particles. Signaling via p38/MAPK (p38)
was also activated shortly following Ad2 infection in
HeLa cells. Similar to PKA, p38 enhanced nuclear
targeting of adenovirus particles that was dependent
on the downstream kinase MAPKAP kinase 2 (MK2).
Unlike the activation of integrin signaling, adenovirus
activation of p38 occurred independent of RGD-aV
integrin interactions.43 As seen with cytokine gene
expression, adenovirus induction of PKA and p38 was
also dose-dependent.
Adenovirus vectors and signal transduction
The in vivo and in vitro studies demonstrating early
activation of host innate immune responses independent
of viral transcription point to a significant role for the
adenovirus particle or capsid in triggering inflammation
and subsequent antiviral immunity. This is not surprising given the signaling events linked to adenovirus cell
entry. In epithelial cells, adenovirus vectors attach to and
enter cells through interactions with CAR receptor and
Adenovirus vector induced signaling and host
inflammatory responses
A significant understanding exists for the signaling
pathways utilized by group C adenoviridae internalize
and infect cells (Figure 1). The impact of signal activation
during cell entry on host inflammation and antiviral
responses are not well known. Bruder and Kovesdi33
have linked adenovirus vector induction of ERK signaling to the expression of the chemokine IL-8 in HeLa cells.
937
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Biology of adenovirus vector inflammation
Q Liu and DA Muruve
938
Ad
CAR α β
v 1,3,5
?
FAK
Ad
pI30
Ad
RAS
αvβ1,3,5
PI3K
Ad
Raf-1
CAS
Cdc42/Rac1
?
MKK6
p38
ERK1/2
?
NFκB
cytokine/chemokine expression
Figure 1 General scheme of adenovirus-induced signaling and inflammatory gene expression.
Raf-1, the downstream effector of the Ras GTP binding
protein, was activated as early as 5 min after Ad5LacZ
transduction in HeLa cells. This was followed by p42/
MAPK phosphorylation 10 min after Ad5LacZ transduction. The activation of ERK signaling was required for IL8 expression in these cells.
Studies from our laboratory have demonstrated the
activation of p38, ERK, but not JNK within minutes of
adenovirus vector cell entry in a mouse kidney-derived
epithelial cell line (REC cells).29 ERK and p38 were
activated as early as 10 min and persisted up to 3 h
following transduction with Ad5 vectors. The activation
of p38 and ERK were directly linked to the expression of
the chemokine IP-10 since chemical inhibition of these
pathways decreased adenovirus vector induction of this
chemokine. Furthermore, we found that signaling via
p38 played a larger role in the induction of IP-10
compared to ERK. Blockade of both pathways however
proved to be synergistic.29 We have also shown a
significant role for NF-kB in adenovirus vector induction
of chemokine genes.34,35 In HeLa and REC cells,
adenovirus vectors induced the nuclear translocation of
NF-kB within 2 h. The activation of NF-kB was directly
involved in the transcription of the chemokines IP-10 and
RANTES. NF-kB was the minimal promoter element
required for the transcriptional activation RANTES and
IP-10 genes. The inhibition of NF-kB activity by overexpressing the natural inhibitor Ik-Ba effectively blocked
adenovirus vector induction of both these chemokines.34,35
The early activation of signaling and subsequent
chemokine gene expression confirm that the viral cell
entry process is an early event in the host inflammatory
Gene Therapy
response to adenovirus vectors. Several studies have
evaluated the role of adenovirus cell surface receptors in
the induction of inflammatory gene expression. The
high-affinity receptor CAR does not appear to be
specifically required for the adenovirus vector signal
activation.29,34,41 In REC cells, the CAR-ablated fiber knob
mutant AdL.F44 induced similar levels of IP-10 gene
expression compared to the wild-type capsid vector
AdLuc when corrected for differences in transduction
efficiency.29 Furthermore, using group B adenovirus
particles that do not use CAR as a high-affinity
receptor,45 we have demonstrated equal activation of
both IP-10 and RANTES in epithelial cell lines.29,34 These
studies suggest that adenovirus vector-induced inflammatory gene expression can occur independent of CAR.
Adenovirus-induced signaling occurs via aV integrins as
stated above; however, RGD-dependent interactions
with integrins do not appear to be essential for the
activation of inflammatory pathways in epithelial
cells.29,34,43 RGD peptides did not affect p38 activation
following Ad2 infection in HeLa cells.43 Similarly, we
demonstrated a reduction in adenovirus vector induction
of RANTES following competition with RGD peptides;
however, a parallel reduction in transduction was also
seen. Studies in CAR-deficient cells confirmed that vector
interaction with aV-integrins in the absence of internalization was insufficient to induce the expression of
RANTES.34 This observation suggests that vector internalization rather than RGD-dependent integrin interaction is the critical step in the activation of a transduced
cell. Studies with the RGD-deleted vector AdL.PB46
confirmed that the induction of proinflammatory signals
was mainly RGD independent. At equal levels of
transduction, AdL.PB activated similar levels of p38
and IP-10 gene expression as the wild-type vector
AdLuc.29 On the other hand, ERK activation by AdL.PB
was not as pronounced suggesting that RGD-dependent
and -independent mechanisms of activation exist for this
signaling pathway.29 Nevertheless, the evidence supports
the notion that efficient activation of inflammatory
signals and gene expression requires vector internalization.
The importance of internalized particles to induce an
inflammatory response has been suggested in several
studies. Trapnell and co-workers attenuated adenovirus
vector-induced TNF-a gene expression in alveolar
macrophages using several inhibitors of adenovirus
endocytosis and endosomal penetration.15 In a similar
vein, studies in our laboratory have shown that impairing endosomal escape with bafilomycin A1 or ammonium chloride significantly reduced p38 and ERK
activation by adenovirus vectors. A significant reduction
in IP-10 gene expression was also seen.29 Furthermore, a
temperature-sensitive adenovirus mutant that has a
defect in endosomal penetration was unable to activate
p38 in HeLa cells.43 These data suggest that the activation
of proinflammatory signals by adenovirus vectors occurs
mainly in a postinternalization step, likely following
endosomal penetration.
The exact upstream mediators of ERK and p38
signaling are not known. The MAPK kinase MKK6 is
activated upstream of p38 in HeLa cells following
infection with Ad2.43 Further upstream, Rac1, Cdc42
and PAK1 have been implicated in signaling to p38.47,48
Although PAK1 was not activated following Ad2
Biology of adenovirus vector inflammation
Q Liu and DA Muruve
infection in SW480 cells,42 it remains to be seen whether
Rac1 and Cdc42 are upstream of p38 in adenovirusinduced signaling. The mechanism of ERK activation is
also not clear. ERK is linked to integrin signaling via
p125FAK in the focal adhesion complex; however, this
relation in adenovirus-mediated signaling can only be
assumed. Future studies will be required to unravel the
complexity of adenovirus-induced signaling and its
impact on viral and host biology.
Future perspectives
Adenovirus vectors have several advantages including
ease to produce and the ability to package large
quantities of DNA. Newer generations of gutted adenovirus vectors have greatly diminished the adaptive
immune response to these vectors and improved the
efficiency and duration of gene transfer. The activation of
innate immune responses by adenovirus vectors can be
of benefit in certain applications such as cancer gene
therapy and vaccine development, where adjuvant
responses can enhance the therapeutic effect. On the
other hand, the innate immune response to adenovirus
vectors can also result in inflammation of nontarget
tissues and reduced gene transfer efficiency. It is therefore essential to understand the innate response to
adenovirus vectors to improve the safety profile of these
agents when an inflammatory response is not desired or
to enhance the response when an adjuvant effect is
desired. Studies in vitro have begun to examine the
mechanism by which adenovirus vectors trigger inflammatory signaling and gene expression following cell
transduction. The adenovirus capsid triggers a series of
events during viral cell entry that results in inflammation
and ultimately antiviral immunity. Future studies will
focus on the upstream and downstream mediators
activated in the p38 and ERK signaling pathways that
ultimately control expression of antiviral and inflammatory genes. This will include studies that aim to identify
the viral determinants that trigger inflammatory signaling. Finally, the involvement of other innate signaling
pathways has yet to be explored. The demonstration that
respiratory syncitial virus envelope proteins recognize
and activate Toll-like receptor-4 raises the possibility that
a different spectrum of innate signaling may be involved
in the inflammatory response to adenovirus vectors.49
Ultimately, research in this area will result in strategies to
modify the vector capsid or the host and improve the
application of adenovirus vectors in humans.
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