PERSPECTIVE
Engineering Viral Vectors to Subvert the Airway Defense Response

Dwight C. Look and Steven L. Brody

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri

The potential treatment of lung disease by delivery of exogenous DNA to cells for expression of therapeutic proteins is an exciting benefit of the recent revolution in molecular engineering (1). Adenovirus-based vectors have received considerable attention for DNA delivery to lung cells for several reasons: adenoviruses can infect nondividing cells in the airway epithelium; they are stable; they can be produced in high titer; and they are easily modified to be replication-deficient and express exogenous genes. Adenoviral vectors have already demonstrated the capacity for gene transfer in cell culture systems (4, 5), animal models (4, 6, 7), and human trials (8). However, as development of this therapeutic technology progressed to in vivo systems, biologic obstacles to gene therapy have emerged that limit high-level and prolonged transgene expression (1). The antiviral defense response of the host is one obstacle that has particularly tempered enthusiasm for early-generation adenoviral vectors, although recent modifications of vectors or host immunity have had some success at inhibiting this response. Accordingly, the purpose of this perspective is to review briefly the host response to adenoviruses and to discuss potential strategies to overcome this obstacle to adenovirus-based gene therapy to the lung.

Administration of adenoviral vectors into the lung generates a complex, multicomponent host response that involves innate and adaptive immunity, resulting in specific inhibitory effects on gene therapy efficacy (2). Components of the pulmonary response to adenovirus include: (1) an antigen-nonspecific, cytokine-dependent response resulting in acute inflammation, which may have a neurogenic component and can cause systemic toxicity (7, 9); (2) a major histocompatibility complex (MHC) class I- restricted, cytotoxic (CD8⁺) T lymphocyte-dependent response directed at cells expressing viral or transgene proteins, resulting in chronic inflammation and a lack of persistent transgene expression (7, 11, 13); and (3) a helper (CD4⁺) T lymphocyte-dependent response directed at adenoviral capsid proteins present during vector delivery, resulting in the production of neutralizing antibodies that limit repeated vector administration (if required to sustain a therapeutic effect) (10, 13, 14). It should not be surprising that the response to viral pathogens occurs through multiple, interrelated mechanisms because a multicomponent system can probably adapt for host-defense against a variety of pathogens. However, it is apparent that all components of the antiviral response in the lung will need to be identified and controlled in order to improve gene therapy efficacy with adenoviral vectors.

In this issue, Thorne and colleagues (15) present a murine model in which high-dose exposure to inactivated adenoviral particles elicits an acute airway inflammatory response marked by interleukin (IL)-6 and tumor necrosis factor- release and consequent neutrophil recruitment into the lung. This model appears relevant to gene therapy in humans because IL-6 release was observed after adenoviral vector administration to the lungs of patients with cystic fibrosis (CF) (9). The early pulmonary response to the adenovirus studied by Thorne and colleagues does not appear to be additive with the response to endotoxin, suggesting that both stimuli may activate inflammation by overlapping pathways. This finding has implications for adenovirus-based gene therapy in patients with preexisting inflammation from endotoxin, as this is part of the airway milieu in patients with lung diseases such as CF, characterized by chronic infection with gram-negative bacilli. This study also confirms that a portion of the host response to adenovirus is independent of vector gene expression, but may be modulated pharmacologically (16). Because this acute response appears to be independent of viral gene expression, future work will need to be directed toward identification of the initiating elements in adenoviral particles with the hope that these elements might be altered to limit the host response.

As host responses to adenoviral vectors that limit the utility of adenovirus-based gene transfer have been identified, two major strategies to blunt these responses have been tested. The first strategy is formed on the basis of identifying specific viral molecules that initiate or amplify the pulmonary defense response with subsequent vector reengineering to delete their expression. Work in this direction spans from removal of specific viral gene products (e.g., early region 2A) to total removal of all genes in the vectors that express viral proteins (17, 18). A second strategy is to pharmacologically manipulate the host response using drugs (e.g., corticosteroids, cyclophosphamide), mediators (e.g., IL-12), or other blocking strategies (e.g., anticell receptor proteins, oral tolerization) (19). Both strategies have produced some success in improving the persistence of transgene expression in animal studies. However, because the immune response to vectors varies with the genetic program of the host (23), the benefit of using these strategies in humans must also be evaluated.

Another path to modulation of the pulmonary response to adenoviral vectors takes a cue from the viruses themselves. Evolution of viruses (particularly DNA viruses) has generated several viral strategies to subvert antiviral mechanisms in host cells, allowing viruses to escape the immune response (24). Mimicry of these viral mechanisms by reinsertion of specific portions of adenoviral genomes that modulate host-cell defense responses may be another strategy for improving gene therapy. In fact, strategies that use this concept are already being explored. For example, a 19-kD glycoprotein encoded in the adenoviral early region 3 (E3) reduces the cytotoxic T-cell response to virus-infected host cells by inhibiting MHC class I transport to the cell surface (25). E3 coding sequences have been left in some adenoviral vectors, but it is unclear if enough expression of E3 occurs in these vectors to affect the immune response (26). An adenoviral vector in which high-level E3 expression was insured by promoter selection has been demonstrated to improve transgene persistence in an animal model (27). The use of this strategy with gene products that affect the antiviral immune response and that are encoded in other viruses might also be useful (2). Further work directed at isolating structural determinants in viral molecules that subvert the immune response might allow for selective insertion of sequences encoding only biologically active domains, thereby minimizing limitation of space for transgene insertion and viral DNA packaging in vectors.

In that context, we have been interested in understanding the biochemical basis for another mechanism for subversion of host cell defense by adenoviruses: resistance to antiviral effects of interferon (IFN)-. IFN--dependent expression of genes in airway epithelial cells is controlled by a specific signal transduction pathway that relies on the signal transducer and activator of transcription-1 (Stat1) transcription factor to first be phosphorylated at the IFN- receptor-Janus family kinase (Jak) complex and then to bind to specific recognition sequences in the promoter region of IFN--responsive genes (28) (Figure 1). At the promoter, Stat1 interacts with other transcription factors and specific coactivator molecules, including members of the cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-binding protein (CBP)/ p300 family to induce gene transcription (32). In addition, activated Stat1 participates in regulation of antiviral genes through its induction of other transcription factors, such as IFN regulatory factor-1 (IRF-1), and its role as a constituent of the IFN-/-activated transcription factor IFN-stimulated gene factor-3 (33, 34). Through these pathways, Stat1 is positioned as the trigger for an entire set of immune-response genes oriented toward antiviral defense, including MHC class I and transporter associated with antigen processing-1 (TAP1) for antigen recognition, intercellular adhesion molecule-1 (ICAM-1) for T-cell costimulation and immune cell recruitment, and inducible nitric oxide synthase (iNOS) and IFN-/ for antiviral toxicity (35). The central nature of Stat1 signaling in mucosal antiviral defense to viruses (36) is probably reflected in the fact that adenoviral evolution has developed multiple potent strategies to block Stat1 actions. In what appears to be a critical virus-specific event, the adenoviral early region 1A (E1A) gene expresses within the first hours of infection a protein that inhibits IFN--dependent activation of immune response genes in airway epithelial cells through direct interaction with Stat1 and its CBP/p300 coactivator proteins (35, 39). In addition, during the late phase of adenoviral infection the virus inhibits Stat1 phosphorylation, and through this effect also blocks IFN--dependent gene expression (35). The ability of wild-type adenoviruses to inhibit IFN--inducible genes by multiple distinct mechanisms is presumably designed to insure viral subversion of the airway immune response and the establishment of a productive viral infection.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Adenoviral effects on the IFN--driven Jak-Stat pathway in airway epithelial cells. Activation of this pathway is triggered by IFN--dependent oligomerization of the IFN--receptor and consequent phosphorylation of the -chain-associated Jak1- and -chain- associated Jak2-tyrosine kinases followed by phosphorylation of the receptor -chain (48). Receptor phosphorylation enables -chain recruitment of Stat1 and subsequent Stat1 phosphorylation and release from the receptor as a homodimer (48). Stat1 then translocates to the nucleus and binds inverted-repeat sequences, where it works in concert with adjacent transcription factors (e.g., specificity protein 1 or Sp1) and coactivators (e.g., CBP/p300) to facilitate enhanceosome formation (29). Activation of IFN--responsive gene transcription through this Stat1-dependent mechanism results in expression of several antiviral genes, including ICAM-1, TAP1, and IRF-1. The IRF-1 gene product is itself a transcription factor that induces other antiviral genes, including iNOS, IFN-/, and MHC class I (35). Adenovirus can affect this pathway at multiple locations, including: (1) inhibition of IFN--receptor complex activation; (2) direct interaction between E1A and Stat1; (3) interaction between E1A and CBP/p300; and (4) E3-dependent inhibition of MHC class I transport to the cell surface (25, 35).

Understanding E1A effects on Stat1 might be relevant to improving the persistence of transgene expression after adenoviral gene therapy. The original reason E1A was removed from adenoviral vectors was to ensure replication deficiency, but this also abrogates potential effects of E1A on host-cell proteins like Stat1. Because IFN--dependent expression of antiviral genes through activated Stat1 is particularly important for immune system recognition and cytotoxic T-cell attack of virus-infected cells (40), the inability of E1A-deficient adenoviral vectors to block Stat1-dependent gene expression may promote the host response during gene therapy with adenoviral vectors. Could this E1A-dependent mechanism for inhibition of IFN--dependent gene expression be replaced in adenoviral vectors and thereby be exploited for gene therapy? Like E3 reinsertion, this mechanism could downregulate MHC class I presentation of antigens, but in addition would inhibit expression of other IFN--dependent antiviral genes. A potential roadblock to this strategy is that E1A is a complex molecule that interacts with many host-cell proteins. Several E1A interactions (through specific domains) have detrimental effects on host-cell functions (see Figure 2), including: (1) E1A N-terminus and conserved region (CR) 1 interaction with members of the CBP/p300 family, thereby affecting cell-cycle regulation and gene activation requiring CBP/p300 transcriptional coactivator function (44, 45); (2) E1A CR1 and CR2 interaction with a family of antioncoproteins typified by retinoblastoma protein (Rb), thereby affecting cell-cycle regulation requiring Rb transcriptional corepressor function (45, 46); and (3) E1A CR3 interaction with specific viral and cellular promoters, thereby affecting gene expression through CR3 transactivation function (45, 47). Our studies, however, suggest that determinants in E1A (located in the N-terminus) for interaction with Stat1 are distinct and can be separated from those for interaction with other host-cell proteins (35). Understanding specific molecular determinants of E1A-Stat1 interaction may allow for development of selective strategies that exploit the specificity of E1A-Stat1 interaction to downregulate only IFN-dependent gene expression, but not affect other cellular functions.

View larger version (11K): [in this window] [in a new window]   Figure 2.   Model for major interaction sites in the adenoviral E1A protein. These sites in E1A include a CBP/p300 coactivator family binding site, which uses residues near the N-terminus and in the C-terminal portion of CR1; Rb antioncoprotein family binding site, which uses residues in CR2 and the N-terminal portion of CR1; and transcriptional activation site (TA), which uses residues in CR3 (45). E1A binds Stat1 using residues near the N-terminus that appear distinct from other binding sites (35).

Modulation of the IFN--dependent immune response through specific Stat1 inhibition might also be exploited using other strategies, such as treatment with selective kinase inhibitors or coexpression of Stat1 dominant-negative or other viral anti-Stat1 molecules (2, 31, 35). However, engineering viruses that eliminate host-cell immune detection may bring up theoretical concerns. Cells that lack responsiveness to IFN- and/or present antigens poorly might be more easily infected and destroyed by other viruses. Immune system detection of malignant transformation of cells with this defect might also be impaired. In addition, this strategy would probably not prevent the early host response to adenoviral particles or the production of neutralizing antibodies to adenoviral capsid proteins. In any event, this evolution of adenoviral vector engineering illustrates the importance of experimental work directed toward understanding viral protein expression and function during both wild-type and mutant viral infection. The utility of these approaches to gene therapy can only be fully understood by further study; first in the laboratory, and then in the clinic.

	Footnotes

Abbreviations: cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-binding protein, CBP; cystic fibrosis, CF; conserved region, CR; early region 1A, E1A; early region 3, E3; inducible nitric oxide synthase, iNOS; intercellular adhesion molecule-1, ICAM-1; interferon, IFN; interferon regulatory factor-1, IRF-1; interleukin, IL; Janus family kinase, Jak; major histocompatibility complex, MHC; retinoblastoma protein, Rb; signal transducer and activator of transcription, Stat; transporter associated with antigen processing-1, TAP1.

(Received in original form April 2, 1999).

Acknowledgments: Our ongoing research is supported by grants from the National Institutes of Health, Cystic Fibrosis Foundation, American Lung Association, and March of Dimes.

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