Immune Responses to Viral Gene Therapy Vectors
Authors: Jamie L. Shirley, Ype P. de Jong, Cox Terhorst, Roland W. Herzog
Journal: Molecular Therapy (2020)

Several viral vector-based gene therapy drugs have now received marketing approval. A much larger number of additional viral vectors are in various stages of clinical trials for the treatment of genetic and acquired diseases, with many more in pre-clinical testing. Efficiency of gene transfer and ability to provide long-term therapy make these vector systems very attractive. In fact, viral vector gene therapy has been able to treat or even cure diseases for which there had been no or only suboptimal treatments. However, innate and adaptive immune responses to these vectors and their transgene products constitute substantial hurdles to clinical development and wider use in patients. This review provides an overview of the type of immune responses that have been documented in animal models and in humans who received gene transfer with one of three widely tested vector systems, namely adenoviral, lentiviral, or adeno-associated viral vectors. Particular emphasis is given to mechanisms leading to immune responses, efforts to reduce vector immunogenicity, and potential solutions to the problems. At the same time, we point out gaps in our knowledge that should to be filled and problems that need to be addressed going forward. Several viral vector-based gene therapy drugs have now received marketing approval. A much larger number of additional viral vectors are in various stages of clinical trials for the treatment of genetic and acquired diseases, with many more in pre-clinical testing. Efficiency of gene transfer and ability to provide long-term therapy make these vector systems very attractive. In fact, viral vector gene therapy has been able to treat or even cure diseases for which there had been no or only suboptimal treatments. However, innate and adaptive immune responses to these vectors and their transgene products constitute substantial hurdles to clinical development and wider use in patients. This review provides an overview of the type of immune responses that have been documented in animal models and in humans who received gene transfer with one of three widely tested vector systems, namely adenoviral, lentiviral, or adeno-associated viral vectors. Particular emphasis is given to mechanisms leading to immune responses, efforts to reduce vector immunogenicity, and potential solutions to the problems. At the same time, we point out gaps in our knowledge that should to be filled and problems that need to be addressed going forward. Gene therapy can treat a variety of both inherited and acquired diseases, and viral vectors have emerged as a preferred platform for gene delivery. Once the viral genome is replaced with a therapeutic gene cassette, stripping the virus of the replicative and pathogenic traits, such vectors are well suited as gene transfer vehicles. An ideal gene therapy vector should reliably and efficiently carry and deliver a therapeutic gene to target cells and direct long-term therapeutic expression. Viruses naturally satisfy these criteria, except that they are prone to host immune responses, as the mammalian immune system has evolved to recognize infectious agents. Past and ongoing clinical trials have utilized several different viral vectors, including adenovirus (Ad), adeno-associated virus (AAV), lentivirus (LV), murine γ-retrovirus, and herpes simplex virus (HSV). Marketing approval has been granted to two AAV-based therapies to treat a form of congenital blindness (Luxturna) and spinal muscular atrophy (Zolgensma), a γ-retrovirus-based therapy for the primary immune deficiency adenosine deaminase severe combined immunodeficiency (ADA-SCID) (Strimvelis), an LV-based therapy for CD19-directed genetically modified chimeric antigen receptor (CAR)-T cell immunotherapies for acute lymphoblastic leukemia and non-Hodgkin lymphoma (Kymriah and Yescarta), and HSV-based oncolytic virotherapy for melanoma (Imlygic).1High K.A. Roncarolo M.-G. Gene therapy.N. Engl. J. Med. 2019; 381: 455-464Crossref PubMed Scopus (27) Google Scholar The suitability of a viral vector for a given application depends on multiple factors, including target cells or tissues, tropism, use for ex vivo versus in vivo gene transfer, packaging capacity, potential for genome integration (and insertional mutagenesis), and also the propensity for immunotoxicities. While LV vectors are now preferred for ex vivo gene correction (in particular for gene transfer to hematopoietic stem cells [HSCs]), AAV has emerged as the preferred vector for in vivo gene transfer due to its favorable safety profile compared to other vectors, ability to transduce a variety of tissues, and availability of a large number of viral capsids with different tropism. Although the use of vectors derived from viruses takes advantage of their refined evolutionary fitness to transduce human cells, these advantages have co-evolved with an equally sophisticated human immune system aimed at protecting host tissues by eliminating foreign invaders perceived as dangerous. To the immune system, certain components of viral vectors are indistinguishable from their parent viruses (such as nucleic acids carried in a protein coat). Such vectors are therefore subject to similar innate and adaptive immune responses as wild-type viruses. Innate immune receptors, or pattern recognition receptors (PRRs), detect viruses by recognizing conserved molecular motifs such as unique nucleic acid conformations that trigger antiviral immunity (Figure 1). The virally derived capsid or envelope proteins constitute foreign proteins that can become the target of adaptive immune responses (Figure 2). Furthermore, a therapeutic trasn transgene product that constitutes a neo-antigen may be similarly targeted by both humoral and cellular immune responses (Figure 2). Immune-mediated rejection in viral gene therapy represents one of the most significant hurdles to human gene therapy. A comprehensive understanding of the processes underlying these deleterious responses directed against both the viral vector and transgene product is critical for developing treatment modalities that mitigate immune-mediated rejection. A body of research has interrogated these mechanisms, which are reviewed herein. We will focus on three widely utilized and studied vector systems: Ad, AAV, and LV vectors (Figure 2; Table 1).Figure 2Examples of Molecular Structures and Antigens in Viral Vectors that Are Recognized by Innate Immune Sensors or Targeted by Antigen-Specific Adaptive (B and T Cell) Immune ResponsesShow full caption(A) Adenoviral vectors. (B) AAV vectors. (C) Lentiviral vectors. Abbreviations are as follows: dsDNA, double-stranded DNA; TLR, Toll-like receptor; cGAS, cyclic guanosine monophosphate-AMP synthase; IFN, interferon; APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; dsRNA, double-stranded RNA; MDA5, melanoma differentiation-associated protein 5; pDC, plasmacytoid dendritic cell; and ssRNA, single-stranded RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Overview of Viral Vectors and Their Immune ResponsesViral VectorAdAAVLVVirion and genomecapsid; 36-kb dsDNA genomecapsid, ≤5-kb ssDNA genome (or ∼2.5-kb scDNA genome)enveloped virus containing capsid and ∼10-kb ssRNA genomeInnate immunitypotent innate response, including activation of vascular endothelial cells and platelets, inflammatory cytokine production, and macrophage cell deathcomparatively weak and transient innate response; TLR9 signaling promotes CD8+ T cell responses; complement activation and other immunotoxicities seen in some patients receiving high-dose systemic gene transferstrong IFN-α/β response limits transduction and drives adaptive responsesImmunity in human populationpre-existing immunity to human serotypespre-existing immunity varies between serotypes and geographic locationlow pre-existing immunityIn vivo transduction of antigen-presenting cellsefficientinefficientefficientUse as a vaccine carriervaccine and cancer gene therapy applicationslimitedtargeting of dendritic cells for vaccine developmentAdaptive immune responses to vectorNAB formation; CD8+ T cell responses to viral gene products (except for high-capacity vectors)NAB formation; CD8+ T cell responses to capsidNAB formation; possibly T cell responses to envelope proteinAdaptive immune responses to transgene productefficient inducer of CD8+ T cell responses; antibody formation possibleleast efficient inducer of CD8+ T cells compared to Ad and LV; risk of CD8+ T cell and antibody responses highly variable depending on vector design and dose, route of administration, and host factorsefficient inducer of B and T cell responses unless transgene expression is tightly controlled by miRNA and promoter and expression is professional APCs is eliminateddsDNA, double-stranded DNA; ssDNA, single-stranded DNA; scDNA, self-complementary DNA; TLR9, Toll-like receptor 9; IFN, interferon; NAB, neutralizing antibody; miRNA, microRNA; APC, antigen-presenting cell. Open table in a new tab (A) Adenoviral vectors. (B) AAV vectors. (C) Lentiviral vectors. Abbreviations are as follows: dsDNA, double-stranded DNA; TLR, Toll-like receptor; cGAS, cyclic guanosine monophosphate-AMP synthase; IFN, interferon; APC, antigen-presenting cell; CTL, cytotoxic T lymphocyte; dsRNA, double-stranded RNA; MDA5, melanoma differentiation-associated protein 5; pDC, plasmacytoid dendritic cell; and ssRNA, single-stranded RNA. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; scDNA, self-complementary DNA; TLR9, Toll-like receptor 9; IFN, interferon; NAB, neutralizing antibody; miRNA, microRNA; APC, antigen-presenting cell. The immune system is comprised of a complex interwoven network of multiple different cell types that collaborate to protect host tissues from further infection and mobilize a cadre of effectors tailored to specifically eliminate the invading pathogen. The cellular network can be generally broken up into innate and adaptive arms. Innate immune responses occur early, are not antigen specific, and do not result in immunological memory. Adaptive immune responses are conditioned by the inflammatory environment created by innate immune sensing, rely on activation and clonal expansion of antigen-specific (effector) B and T cell differentiation, and generate immunological memory. Viral vectors share many commonalities with natural viruses but are distinctly different in that they are non-replicative, delivered in a single high-titer bolus, and introduced at an unnatural site. So, although canonical immunological concepts can be applied, the unwanted immune response to viral vectors also has inherently unique aspects. Vector particles containing viral proteins that are identical or similar to antigens that humans are exposed to as a result of natural infection may be neutralized by antibodies upon injection into in some humans because of pre-existing immunity. Recognition of viral structures (e.g., capsids or nucleic acids) by innate immune sensors may cause tissue infiltration by innate immune cells, may trigger the production of interferon (IFN)-α/β (type 1 IFN, hereafter abbreviated as T1 IFN), thereby inducing an antiviral state in the tissue and reducing transduction, and provides an activation signal for adaptive immune responses. Activation of, and subsequent antigen presentation by, dendritic cells (DCs) is a critical step in linking innate to adaptive immunity, leading to activation/differentiation and expansion of T cells. While major histocompatibility complex (MHC) class I (MHC I)-restricted CD8+ T cells (cytotoxic T lymphocytes [CTLs]) are capable of lysing virally infected cells, MHC class II (MHC II)-restricted CD4+ T cells provide help for optimal CD8+ T cell activation and for B cell activation, leading to antibody formation. T helper (Th) cells are also critical for generation of memory responses. Ad is one of the first viruses investigated as a potential gene therapy vector and was also the subject of early failures for in vivo gene transfer that highlight how pivotal host inflammatory responses are on the durability of therapeutic gene expression and the overall safety of this type of intervention.2Crystal R.G. Adenovirus: the first effective in vivo gene delivery vector.Hum. Gene Ther. 2014; 25: 3-11Crossref PubMed Scopus (99) Google Scholar Early enthusiasm for Ad vectors was largely based on their high transduction efficiency and packaging capacity. However, robust transgene expression was met with an equally high inflammatory response that resulted in transient expression and a potential for severe immunotoxicity, resulting in the death of a patient. Because of their ability to effectively activate CD8+ T cells, subsequent efforts shifted toward their use as vaccine carriers and in cancer gene therapy. Ad vectors contain an ∼36-kb double-stranded DNA genome packaged into a viral protein capsid. Various viral genes are removed to render the virus replication defective. It is also possible to remove as viral coding sequences and produce "gutted" or "helper-dependent" adenoviral vectors. Some serotypes such as AdHu5 efficiently transduce a variety of cell types in vivo (with particularly high tropism for hepatocytes), while serotypes infecting hematopoietic cells have also been described. Upon transduction, the vector genome remains episomal. Ad vectors activate a large spectrum of innate immune pathways and have therefore been ideal tools to study innate immunity to viruses. Hepatic gene transfer is achieved by intravenous injection of adenoviral vector. However, innate responses may occur within minutes to hours, leading to blood pressure changes, thrombocytopenia, inflammation, and fever.3Seiler M.P. Cerullo V. Lee B. Immune response to helper dependent adenoviral mediated liver gene therapy: challenges and prospects.Curr. Gene Ther. 2007; 7: 297-305Crossref PubMed Scopus (57) Google Scholar Dysregulation of coagulation can spread to multiple organs and lead to DIC (disseminated intravascular coagulation). Activation of vascular endothelial cells by Ad vectors results in release of ultra-large-molecular-weight multimers of von Willebrand factor (vWF), a blood protein that is critical for platelet adhesion. Ad vectors also activate platelets and induce exposure of the adhesion molecule P-selectin and formation of platelet-leukocyte aggregates, ultimately causing thrombocytopenia and thus a risk for bleeding.4Othman M. Labelle A. Mazzetti I. Elbatarny H.S. Lillicrap D. 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Blockage of co-stimulatory pathways that are required for B and T cell activation has also been successful in pre-clinical studies.32Yang Y. Wilson J.M. CD40 ligand-dependent T cell activation: requirement of B7-CD28 signaling through CD40.Science. 1996; 273: 1862-1864Crossref PubMed Google Scholar,33Yang Y. Su Q. Grewal I.S. Schilz R. Flavell R.A. Wilson J.M. Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues.J. Virol. 1996; 70: 6370-6377Crossref PubMed Google Scholar However, the potent innate response to adenoviral vectors substantially complicates translation of such approaches, and use of adenoviral vectors for in vivo gene transfer in the treatment of genetic disease has been largely abandoned. AAV is a small non-enveloped parvovirus with a single-stranded genome of about 5 kb that is naturally non-pathogenic and replication defective. AAV vectors contain no viral coding sequences, elicit only weak inflammatory or T1 IFN responses when compared to other viruses, and their V genomes persist mostly in episomal form. These features contribute to a favorable safety profile, although immunotoxicities may still occur following systemic delivery of very high doses.34Penaud-Budloo M. François A. Clément N. Ayuso E. Pharmacology of recombinant adeno-associated virus production.Mol. Ther. Methods Clin. Dev. 2018; 8: 166-180Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar,35Colella P. Ronzitti G. Mingozzi F. Emerging issues in AAV-mediated in vivo gene therapy.Mol. Ther. Methods Clin. Dev. 2017; 8: 87-104Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar AAV vectors are being widely tested in human gene therapy trials for in vivo gene transfer to a large number of different tissues and cells types, including the CNS, liver, skeletal and cardiac muscle, eye, and lung. In addition to two US Food and Drug Administration (FDA)-approved products for treatment of Leber's congenital amaurosis (LCA) and spinal muscular atrophy (SMA), other products have reached phase III trials as in liver-directed gene therapy for hemophilia A and B.1High K.A. Roncarolo M.-G. Gene therapy.N. Engl. J. Med. 2019; 381: 455-464Crossref PubMed Scopus (27) Google Scholar,36Butterfield J.S.S. Hege K.M. Herzog R.W. Kaczmarek R. A molecular revolution in the treatment of hemophilia.Mol. Ther. 2019; (Published online November 13, 2019)https://doi.org/10.1016/j.ymthe.2019.11.006Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar Initial marketing approval was for an ocular gene transfer with limited vector doses using a subretinal route of injection. Likely facilitated by the immune privilege of the eye, vector administration to one eye could be later on followed by contralateral gene transfer to the second eye, and repeat administration to the same eye is also possible.37Bennett J. Ashtari M. Wellman J. Marshall K.A. Cyckowski L.L. Chung D.C. McCague S. Pierce E.A. Chen Y. Bennicelli J.L. et al.AAV2 gene therapy readministration in three adults with congenital blindness.Sci. Transl. Med. 2012; 4: 120ra15Crossref PubMed Scopus (259) Google Scholar,38Weed L. Ammar M.J. Zhou S. Wei Z. Serrano L.W. Sun J. Lee V. Maguire A.M. Bennett J. Aleman T.S. Safety of same-eye subretinal sequential readministration of AAV2-hRPE65v2 in non-human primates.Mol. Ther. Methods Clin. Dev. 2019; 15: 133-148Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Other routes of in vivo vector administration are more typically associated with formation of NABs that prevent re-administration of vector. Furthermore, humans develop NABs to various serotypes during childhood.35Colella P. Ronzitti G. Mingozzi F. Emerging issues in AAV-mediated in vivo gene therapy.Mol. Ther. Methods Clin. Dev. 2017; 8: 87-104Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar Seroprevalence varies geographically, and some humans have NABs against multiple serotypes, likely reflecting cross-reactivity. Some of the highest prevalences of NABs are against AAV2, the serotype that is similar to AAVs present in the human population, while the more diverse AAV5 has some of the lowest prevalences. Overall, seroprevalences appear to vary from 5% to