Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy

Viral vectors have provided effective methods for in vivo gene delivery for therapeutic purposes. The ability of viruses to infect a wide variety of cell types in vivo has been exploited for several applications, such as liver, lung, muscle, brain, eye and many others. Immune responses directed towards the viral capsids and the transgene products have severely affected the ability of these vectors to induce long-term gene expression. This paper reviews the influence of viral vectors on antigen-presenting cells (APC), which are central to the induction of innate as well as adaptive immune responses. In this respect, we have focused on adenovirus and adeno-associated viruses because of the polar responses these vector systems induce in vivo. While adenovirus vector can induce significant inflammatory responses, adeno-associated viral vectors are characterized by their inability to consistantly induce immune responses to the transgene product. Understanding the mechanism of infection, transduction and activation of APC by viral vectors will provide strategies to develop safe vectors and prevent immune responses in gene therapies.     

Innate immune response to adenovirus.

Adenovirus is a common infectious pathogen in both children and adults. It is a significant cause of morbidity in immunocompetent people living in crowded living conditions and of mortality in immunocompromised hosts. It has more recently become a popular vehicle for gene therapy applications. The host response to wild-type infection and gene therapy vector exposure involves both the innate and adaptive immune systems. The initial innate immune response is associated with the severe acute manifestations of adenovirus infection and also plays a significant role in acute toxicity owing to adenovirus vector exposure. This review discusses the innate immune response primarily during wild-type adenovirus infection because this serves as the basis for understanding the response during both natural infection and exposure to adenovirus vectors.     

Adenoviruses as vaccine vectors.

Adenoviruses have transitioned from tools for gene replacement therapy to bona fide vaccine delivery vehicles. They are attractive vaccine vectors as they induce both innate and adaptive immune responses in mammalian hosts. Currently, adenovirus vectors are being tested as subunit vaccine systems for numerous infectious agents ranging from malaria to HIV-1. Additionally, they are being explored as vaccines against a multitude of tumor-associated antigens. In this review we describe the molecular biology of adenoviruses as well as ways the adenovirus vectors can be manipulated to enhance their efficacy as vaccine carriers. We describe methods of evaluating immune responses to transgene products expressed by adenoviral vectors and discuss data on adenoviral vaccines to a selected number of pathogens. Last, we comment on the limitations of using human adenoviral vectors and provide alternatives to circumvent these problems. This field is growing at an exciting and rapid pace, thus we have limited our scope to the use of adenoviral vectors as vaccines against viral pathogens.     

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

Recombinant vectors based on adeno-associated virus (AAV) have been shown to stably express many genes in vivo without mounting immune responses to vectors or transgenes. Thus, AAV vectors have rapidly become the reagents of choice for therapeutic gene transfer. Yet one of the first translations of AAV gene therapy into humans unexpectedly resulted in only short-term expression of the therapeutic gene accompanied by transient but significant toxicity. Immune responses to the vector capsid were held accountable for these results, confirming that a detailed understanding of the interaction of AAV vectors with the immune system is of great importance for the safety and success of gene therapy applications. Most humans display naturally acquired immunity to AAV; circumventing neutralizing antibodies and memory T-cell responses is challenging, but not impossible. This review will evaluate the strategies that have been proposed to overcome such responses and summarize recent findings about the mechanisms and circumstances that lead to the activation of innate and adaptive immune responses to AAV vector components.     

Evaluation of polyethylene glycol modification of first-generation and helper-dependent adenoviral vectors to reduce innate immune responses.

Adenoviruses are robust gene delivery vectors in vivo, but are limited by their propensity to provoke strong innate and adaptive responses. Previous work has demonstrated that polyethylene glycol (PEG) modification of adenovirus can protect the vectors from preexisting and adaptive immune responses by reducing protein-protein interactions. To test whether PEGylation can reduce innate immune responses to adenovirus by reducing their interactions with immune cells, first-generation (FG-Ad) and helper-dependent (HD-Ad) Ad5 vectors were PEGylated with SPA-PEG and tested in vitro and in vivo. We demonstrate that increasing PEGylation ablated in vitro transduction, but surprisingly had no negative effect on the level or distribution of in vivo gene delivery. This poor in vitro transduction could be rescued in part by physically forcing the PEGylated vectors onto cells, suggesting that physiological forces in vivo may enable transduction via heparin sulfate proteoglycan and integrin interactions. While transduction remained the same as for unmodified vectors, the PEGylated vectors reduced innate IL-6 responses by 70 and 50% in vivo for FG-Ad and HD-Ad. These reduced innate responses paralleled similar reductions in vector uptake by macrophages in vitro and Kupffer cells in vivo. These data suggest that PEGylation of Ad vectors can reduce innate immune responses without reducing transduction in vivo. These data also suggest that nonspecific vector uptake by macrophages and Kupffer cells may be critically involved in the initial activation of innate immune responses.     

Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia.

Adenovirus vectors are capable of high efficiency in vivo arterial gene transfer, and are currently in use as therapeutic agents in animal models of vascular disease. However, despite substantial data on the ability of viruses to cause vascular inflammation and proliferation, and the presence in current adenovirus vectors of viral open reading frames that are translated in vivo, no study has examined the effect of adenovirus vectors alone on the arterial phenotype. In a rabbit model of gene transfer into a normal artery, we examined potential vascular cell activation, inflammation, and neointimal proliferation resulting from exposure to replication-defective adenovirus. Exposure of normal arteries to adenovirus vectors resulted in: (a) pronounced infiltration of T cells throughout the artery wall; (b) upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in arterial smooth muscle cells; (c) neointimal hyperplasia. These findings were present both 10 and 30 d after gene transfer, with no evidence of a decline in severity over time. Adenovirus vectors have pleiotropic effects on the arterial wall and cause significant pathology. Interpretation of experimental protocols that use adenovirus vectors to address either biological or therapeutic issues should take these observations into account. These observations should also prompt the design of more inert gene transfer vectors.     

Use of DAF-displaying adenovirus vectors reduces induction of transgene- and vector-specific adaptive immune responses in mice

Adenovirus (Ad)-based vectors are attractive candidates for a variety of gene-transfer applications. In this study, we found that decay-accelerating factor (DAF)-displaying Ads induce significantly decreased cellular immune responses to transgenes expressed from the vectors in both Ad5-naive and Ad5-immune mice. Specifically, we found a diminished ability of splenocytes to secrete interferon-γ after recall exposure to multiple peptides derived from antigens expressed by DAF-displaying Ads. We also confirmed that DAF-displaying Ads induce decreased numbers of antigen-specific, CD8(+) effector memory and central memory CD8(+) T cells, thereby uncovering a unique role of complement in modulating the induction of robust memory T-cell responses. We also confirmed that DAF-displaying Ads generate significantly reduced titers of Ad capsid-specific neutralizing antibodies after gene transfer in vivo. In conclusion, DAF-displaying Ad5-based vectors exhibit decreased induction of complement-dependent, innate immune responses, resulting in both an improved safety profile and a decreased propensity to induce humoral and cellular adaptive immune responses to Ad capsid proteins and Ad vector-expressed transgene products. This attractive combination of features will be beneficial in a variety of clinically relevant gene-transfer applications.     

PEGylation of E1-deleted adenovirus vectors allows significant gene expression on readministration to liver

Systemic administration of adenoviral vectors leads to activation of innate and antigen-specific immunity. In an attempt to diminish T and B cell-specific immune responses to E1-deleted adenoviral vectors, capsid proteins were modified with various activated monomethoxypolyethylene glycols (MPEGs). The impact of this modification was studied in a murine model of liver-directed gene transfer in which an E1-deleted adenovirus expressing the lacZ gene was given intravenously. The efficiency of vector transduction of hepatocytes in vivo was not compromised by any of the polymer chemistries. PEGylation of the virus, however, diminished the activation of cytotoxic T lymphocytes and helper T cells of the type 1 subset (Th1 cells) against native viral antigens; neutralizing antibodies to native virus were also diminished. PEGylation prolonged transgene expression and allowed partial readministration with native virus or with a virus PEGylated with a heterologous chemical moiety. Apparently, modification of the capsid leads to a shift in antigenic epitopes because vector readministration was not possible when the immunizing vector had been modified by the same PEGylation chemistry used to modify the second vector. In light of these results, the concept of improving the performance of adenoviral vectors through modification of the capsid with PEG shows promise.     

Established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of immunity.

Preclinical arterial gene transfer studies with adenoviral vectors are typically performed in laboratory animals that lack immunity to adenovirus. However, human patients are likely to have prior exposures to adenovirus that might affect: (a) the success of arterial gene transfer; (b) the duration of recombinant gene expression; and (c) the likelihood of a destructive immune response to transduced cells. We confirmed a high prevalence (57%) in adult humans of neutralizing antibodies to adenovirus type 5. We then used a rat model to establish a central role for the immune system in determining the success as well as the duration of recombinant gene expression after adenovirus-mediated gene transfer into isolated arterial segments. Vector-mediated recombinant gene expression, which was successful in naive rats and prolonged by immunosuppression, was unsuccessful in the presence of established immunity to adenovirus. 4 d of immunosuppressive therapy permitted arterial gene transfer and expression in immune rats, but at decreased levels. Ultraviolet-irradiated adenoviral vectors, which mimic advanced-generation vectors (reduced viral gene expression and relatively preserved capsid function), were less immunogenic than were nonirradiated vectors. A primary exposure to ultraviolet-irradiated (but not nonirradiated) vectors permitted expression of a recombinant gene after redelivery of the same vector. In conclusion, arterial gene transfer with current type 5 adenoviral vectors is unlikely to result in significant levels of gene expression in the majority of humans. Both immunosuppression and further engineering of the vector genome to decrease expression of viral genes show promise in circumventing barriers to adenovirus-mediated arterial gene transfer.     

Biophysical targeting of adenovirus vectors for gene therapy

Advances in understanding the interaction of animal viruses with their cognate receptors has led to improvements in the development of cell-specific, targeted viral vectors. Research strategies to generate safe, non-inflammatory viral vectors that are capable of delivering a therapeutic gene to a specific population of cells are currently underway in many laboratories. One approach in the utilization of this cell targeting activity is to ablate the natural interaction of the virus with its native receptor, although this is not an absolute requirement. The initial development of 'viral targeting strategies' was based on the view that by modifying the viral protein/receptor interaction, it would be possible to redirect virus vectors to new host cells. As the understanding of virus/cell interactions increased it was observed, however, that many viruses can use different entry mechanisms for cell attachment and penetration. Adenovirus vectors have been used extensively for the delivery of genes to cells. The entry mechanism for adenoviruses into cells has recently been studied and is relatively well understood, however, there are many aspects of cell receptor/virus interactions, which have still to be elucidated. The single high-affinity receptor on mammalian cells for adenovirus type 5 is recognized as the coxsackie and adenovirus receptor. However, in the absence of coxsackie and adenovirus receptor other receptors are used. A thorough understanding of the biology of adenoviruses is essential in the further development of their use as vectors for cell targeting. One strategy is to modify the viral capsid, either through coating the surface using bispecific antibodies, or by chemically crosslinking the targeting ligand onto the virion surface. Another approach is to genetically modify the virus by incorporating the targeting ligand into the viral 'spike' (fiber) protein. This involves manipulating the adenovirus genome and generating a new targeting ligand on the surface of the fiber protein using recombinant DNA technology. The penton base protein has also received attention as a means of directing adenoviruses via insertion of novel targeting ligands.     

MyD88 Signaling in B Cells Regulates the Production of Th1-dependent Antibodies to AAV

The administration of recombinant adeno-associated viral vectors (rAAV) for gene transfer induces strong humoral responses through mechanisms that remain incompletely characterized. To investigate the links between innate and adaptive immune responses to the vector, rAAVs were injected intravenously into mice deficient in cell-intrinsic components of innate responses (Toll-like receptors (TLRs), type-1 interferon (IFN) or inflammasome signaling molecules) and AAV-specific antibodies were measured. Of all molecules tested, only MyD88 was critically needed to mount immunoglobulin G (IgG) responses since MyD88^−/− mice failed to develop high levels of AAV-specific IgG2 and IgG3, regardless of capsid serotype injected. None of the TLRs tested was essential here, but TLR9 ensured a Th1-biased antibody responses. Indeed, capsid-specific Th1 cells were induced upon injection of rAAV1, as directly confirmed with an epitope-tagged capsid, and the priming and development of these Th1 cells required T cell-extrinsic MyD88. Cell transfer experiments showed that autonomous MyD88 signaling in B cells, but not T cells, was sufficient to produce Th1-dependent IgGs. Therefore, rAAV triggers innate responses, at least via B cells, controlling the development of capsid-specific Th1-driven antibodies. MyD88 emerges as a critical and pivotal regulator of both T- and B-cell adaptive immunity against AAV.     

Increased virus replication in mammalian cells by blocking intracellular innate defense responses

The mammalian innate immune system senses viral infection by recognizing viral signatures and activates potent antiviral responses. Besides the interferon (IFN) response, there is accumulating evidence that RNA silencing or RNA interference (RNAi) serves as an antiviral mechanism in mammalian cells. Mammalian viruses encode IFN antagonists to counteract the IFN response in infected cells. A number of IFN antagonists are also capable of blocking RNAi in infected cells and therefore serve as RNA-silencing suppressors. Virus replication in infected cells is restricted by these innate antiviral mechanisms, which may kick in earlier than the viral antagonistic or suppressor protein can accumulate. The yield of virus vaccines and viral gene delivery vectors produced in mammalian producer cells may therefore be suboptimal. To investigate whether blocking of the innate antiviral responses in mammalian cells leads to increased viral vector production, we expressed a number of immunity suppressors derived from plant and mammalian viruses in human cells. We measured that the yield of infectious human immunodeficiency virus-1 particles produced in these cells was increased 5- to 10-fold. In addition, the production of lentiviral and adenoviral vector particles was increased 5- to 10-fold, whereas Sindbis virus particle production was increased approximately 100-fold. These results can be employed for improving the production of viral gene transfer vectors and viral vaccine strains.     

The TLR9-MyD88 pathway is critical for adaptive immune responses to adeno-associated virus gene therapy vectors in mice

Recombinant adeno-associated viruses (AAVs) have been used widely for in vivo gene therapy. However, adaptive immune responses to AAV have posed a significant hurdle in clinical application of AAV vectors. Recent advances have suggested a crucial role for innate immunity in shaping adaptive immune responses. How AAV activates innate immunity, and thereby promotes AAV-targeted adaptive immune responses, remains unknown. Here we show that AAV activates mouse plasmacytoid DCs (pDCs) via TLR9 to produce type I IFNs. In vivo, the TLR9-MyD88 pathway was crucial to the activation of CD8⁺ T cell responses to both the transgene product and the AAV capsid, leading to loss of transgene expression and the generation of transgene product–specific and AAV-neutralizing antibodies. We further demonstrate that TLR9-dependent activation of adaptive immunity targeting AAV was mediated by type I IFNs and that human pDCs could be activated in vitro to induce type I IFN production via TLR9. These results reveal an essential role for the TLR9-MyD88–type I IFN pathway in induction of adaptive immune responses to AAV and suggest that strategies that interfere with this pathway may improve the outcome of AAV-mediated gene therapy in humans.     

Antivector and antitransgene host responses in gene therapy

Current viral gene therapy vectors effectively transfer genes in vivo at the price of eliciting innate and acquired host responses against the vector and/or transgene. Antigens present in the viral vector and the expression of the transgene both cause cellular and humoral immune responses dependent on the viral vector, the route of administration, and the genotype and infection history of the host. In general, adenoviral vectors cause strong immune responses, which result in only transient expression of the therapeutic gene. Adeno-associated virus and retrovirus vectors elicit weaker immune responses and can therefore result in long-term gene transfer and expression. Methods to avoid host responses, including modification of viral vector and immunosuppression of the host, can increase the longevity and efficiency of gene transfer.     

Recognition of Virus Infection and Innate Host Responses to Viral Gene Therapy Vectors

The innate immune and inflammatory response represents one of the key stumbling blocks limiting the efficacy of viral-based therapies. Numerous human diseases could be corrected or ameliorated if viruses were harnessed to safely and effectively deliver therapeutic genes to diseased cells and tissues in vivo. Recent studies have shown that host cells recognize viruses using an elaborate network of sensor proteins localized at the plasma membrane, in endosomes, or in the cytosol. Three classes of sensors have been implicated in sensing viruses in mammalian cells—Toll-like receptors (TLRs), retinoid acid-inducible gene (RIG)-I-like receptors (RLRs), and nucleotide oligomerization domain (NOD)-like receptors (NLRs). The interaction of virus-associated nucleic acids with these sensor molecules triggers a signaling cascade that activates the principal host defense program aimed to limit or eliminate virus infection and restore tissue homeostasis. In addition, recent data strongly suggest that host cells can mount innate immune responses to viruses without prior recognition of their nucleic acids. To deliver therapeutic genes into the nuclei of diseased cells, viral gene therapy vectors must be efficient at penetrating either the plasma or endosomal membrane. The therapeutic use of high numbers of virus particles disturbs cellular homeostasis, triggering cell damage and stress pathways, or “sensing of modified self”. Accumulating data indicate that the sensing of modified self might represent a powerful framework explaining the innate immune response activation by viral gene therapy vectors.