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Molecular basis of the inflammatory response to adenovirus vectors

Abstract

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 mitogen-activated 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.

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 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 (helper-dependent 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 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 NH2-terminal 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 Ad-vectors, in addition to inducing adaptive antiviral immunity, activate the innate arm of the immune system.6,12,13,14,15 The acute inflammation triggered by Ad-vectors 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-vector-transduced 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-α), 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 α4-integrin.17 The prototypical activation of the innate immune response by Ad-vectors is associated with rapid clearance of the delivered vector dose (>80%) 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 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 high-affinity binding, a lower affinity interaction between αv integrins (αvβ3, αvβ5, αvβ1) 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, pH-dependent 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 αvβ5 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, RGD-dependent interactions with the αM subunit of the β2 integrin, CD11b (αMβ2) proved to be essential in mediating adenovirus high-affinity binding.27 In addition, adenovirus has also been shown to bind αLβ2 integrin (LFA-1), suggesting a significant role for β2-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 inflammation-associated 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 administration. This was associated with the upregulation of inflammatory cytokines and chemokines IL-6, TNF-α, MIP-2, and MIP-1α. 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-α expression as early as 2 h post-transduction.15 In these cells, adenovirus vector-induced TNF-α expression required vector internalization since chemical inhibition of endosome acidification and/or lysis attenuated TNF-α 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-α and IL-1β, a significant upregulation of IL-6 and RANTES, and a steady increase of GM-CSF, MIP-1α, Gro-α, and IL-8 over a 96-h window. The use of UV-psoralen-inactivated 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 UV-psoralen-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 TGP61.6,32,33,34,35 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-α, IL-1β or interferon-γ 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.

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 αV 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 five- to seven-fold increase in tyrosine phosphorylation of p125FAK (focal adhesion kinase) and p130CAS (Crk-associated substrate) and a 14- to 15-fold increase of phosphotyrosine-associated p85/phosphoinositide-3-OH 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 αV 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-αV integrin interactions.43 As seen with cytokine gene expression, adenovirus induction of PKA and p38 was also dose-dependent.

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. 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 IL-8 expression in these cells.

Figure 1
figure1

General scheme of adenovirus-induced signaling and inflammatory gene expression.

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-κB in adenovirus vector induction of chemokine genes.34,35 In HeLa and REC cells, adenovirus vectors induced the nuclear translocation of NF-κB within 2 h. The activation of NF-κB was directly involved in the transcription of the chemokines IP-10 and RANTES. NF-κB was the minimal promoter element required for the transcriptional activation RANTES and IP-10 genes. The inhibition of NF-κB activity by overexpressing the natural inhibitor Iκ-Bα 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 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 αV 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 αV-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-α 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 infection in SW480 cells,42 it remains to be seen whether Rac1 and Cdc42 are upstream of p38 in adenovirus-induced 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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Correspondence to D A Muruve.

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Liu, Q., Muruve, D. Molecular basis of the inflammatory response to adenovirus vectors. Gene Ther 10, 935–940 (2003). https://doi.org/10.1038/sj.gt.3302036

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Keywords

  • adenovirus vectors
  • innate immunity
  • inflammation
  • signal transduction
  • cytokines
  • chemokines

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