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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Gene Ther. Author manuscript; available in PMC 2011 February 24.
Published in final edited form as:
Published online 2009 November 12. doi:  10.1038/gt.2009.148
PMCID: PMC3044498

Progress and Prospects: Immune Responses to Viral Vectors


Viral vectors are potent gene delivery platforms for treatment of genetic and acquired diseases. However, just as viruses have evolved to infect cells efficiently, the immune system has evolved to fight off what it perceives as invading pathogens. Therefore, innate immunity and antigen-specific adaptive immune responses against vector-derived antigens reduce the efficacy and stability of in vivo gene transfer. In addition, a number of vectors are derived from parent viruses that humans encounter through natural infection, resulting in pre-existing antibodies and possibly memory responses against vector antigens. Similarly, antibody and T cell responses may be directed against the therapeutic gene products which often differs from the endogenous non-functional or absent protein that is being replaced. As details and mechanisms of such immune reactions are uncovered, novel strategies are being developed, and vectors are being specifically engineered to avoid, suppress, or manipulate the response, ideally resulting in sustained expression and immune tolerance to the transgene product. This review provides a summary of our current knowledge of the interactions between the immune system adeno-associated virus, adenoviral and lentiviral vectors, as well as their transgene products.


Viral vectors are optimal vehicles for gene transfer because of their ability to efficiently infect host cells. The removal of the replicative and pathogenic ability of viruses, combined with their capacity to carry the therapeutic transgene and an ability to efficiently infect a variety of mammalian cell types makes them amenable for use in gene therapy (Figure 1). However, the immune system has evolved to fight off invading pathogens, which makes viral vectors subject to immune responses that have to be blocked or avoided to achieve therapeutic transgene expression. Administration of viral vectors can lead to the initiation of innate and adaptive immune responses against the viral particles and gene products, leading to decreased efficiency of gene transfer or elimination of the transduced cells over time (Table 1). Recent research has concentrated on various immune modulatory regimens utilizing immune suppressive drugs in combination with gene therapy, modification of viral capsids or choice of viral envelope. Immunogenicity of viral gene transfer can also provoke an immune response against the therapeutic transgene product, which may represent a neo-antigen owing to the type of gene mutation present, rendering patients with e.g. null mutations, susceptible to recognizing the transgene product as a foreign antigen. While there are similarities in immunity to different viruses, each vector contains its own set of activation signals, which are further modified by the environment of a specific tissue.1

Figure 1
Overview of immune responses to viral vectors. Targeting specific organs, engineering viral envelopes, switching serotypes, modifying the transgene cassette, utilizing tissue-specific promoters, or immune modulation regimens can result in immune avoidance ...
Table 1
Summary of immune responses in viral gene transfer.

Overview of immune responses in Adeno-Associated Virus mediated gene therapy

Adeno-associated virus (AAV) vectors are derived from a non-pathogenic replication deficient parvovirus. The AAV vector genome typically is a ≤5-kb single-stranded DNA Numerous serotypes, mostly isolated from humans or non-human primates, have now been characterized to improve transduction of specific organs and circumvent immune responses., The popularity of AAV as a vector stems from a broad host range, non-pathogenic nature, ability to transduce dividing and non-dividing cells, low innate immunity, and low efficiency of transduction of professional antigen presenting cells (APCs) such as dendritic cells (DC) or macrophages, possibly due to a post entry block which limits its immunogenicity.2 Preclinical trials in animal models of human disease have shown long-term correction of genetic disease using AAV vectors, and clinical trials have begun in a number of areas.

Innate immune responses to Adeno-Associated Virus; TLR-9 and Complement

AAV is a weak innate immunogen; microarray studies have shown that AAV does not elicit the robust type I IFN response as is seen for adenoviral vectors.3 Similarly, cytokine and chemokine responses in the transduced tissue are limited and highly transient. AAV triggers Toll-like receptor signaling (e.g. TLR-9, which senses DNA).4 AAV has also been shown to interact with complement.5 The complement cascade, an important component of the innate immune system, leads to opsonization of foreign bodies and lysis of target cells. The three complement pathways include the classical, alternative, and lectin binding pathways, all of which involve C3 convertases. Recent data show that the AAV2 capsid binds to the C3 complement proteins C3, C3b, iC3b and complement regulatory factor H, hence increasing the uptake of AAV into macrophages and enhancing their activation (Figure 2).5 C3-AAV capsid interactions are direct and can occur independently of anti-AAV antibodies. However, complement activation by AAV is primarily antibody dependent (classical pathway). Complement-dependent activation of macrophages is not restricted to the AAV2 serotype. For example, AAV1 and AAV8 have been found to induce inflammatory gene expression in macrophages. Deficiency of C3 or complement receptor 1/2 results in the impairment of the humoral response to AAV.5 C3 and CR 1/2 are essential for humoral but not innate immune responses to AAV in vivo.

Figure 2
Activation of complement by viral vector particles. AAV capsids bind to the C3 complement proteins C3, C3b, iC3b and complement regulatory factor H, hence increasing the uptake of virus into macrophages and enhancing their activation. C3-AAV capsid interactions ...

Adaptive immune responses to AAV vectors and their transgene products

Humoral immune responses against AAV capsid or the transgene product can occur following exposure to AAV vectors. Such responses differ depending on the target organ, location within the target organ (in the case of eye and brain), route of administration, serotype, transgene and expression cassette, and dosing schedule of inject6 ion (Figure 1).2 Humans are a natural host to AAV. A recent study concerning the prevalence of neutralizing antibody titers (NAB) to the various AAV serotypes spanning humans in 4 continents has shown that the most prevalent NABs are to AAV2 followed by AAV1, while AAV8 and AAV7 have the least prevalent responses.7 Interestingly, the structurally modified AAVrh32.33 serotype was rarely neutralized by human sera.7 However different studies in mice and rhesus monkeys in mice showed robust T cell responses to AAVrh32.33 capsid and transgene.6, 8

IgG1 is the predominant antibody subclass response against AAV capsid antigen in humans.9 Pre-existing NAB may not necessarily block in vivo gene transfer to some organs such as skeletal muscle following intramuscular injection. However injections into blood vessels e.g. portal vein injections and direct injection into liver parenchyma resulted in reduced transduction due to the presence of pre-existing NAB. Local delivery of the vector outside blood vessels may reduce exposure to NAB,. In addition to isolation of novel serotypes, shuffling of capsid sequences between serotypes and molecular evolution techniques are being employed to create AAV particles that are more resistant to neutralization by human sera. Although it is unlikely that such vectors can be re-administered, these may improve initial gene transfer in humans.

While long-term expression in skeletal muscle and a lack of inflammatory responses were observed in a clinical trial in hemophilia B patients using an AAV-2 vector, in a subsequent trial, initial therapeutic expression of the factor IX (F.IX) transgene declined starting 6 weeks after hepatic gene transfer. This decline of F.IX expression in a patient enrolled in the highest dose cohort was accompanied by transient elevations of liver enzyme levels, suggesting destruction of hepatocytes.10 Another subject, who had similar low titer of pre-existing NAB to AAV-2, was subsequently treated with a somewhat lower vector dose and showed a lower, but measurable, increase in liver enzyme levels, which correlated with emergence of AAV2 capsid-specific CD8+ T cells in peripheral blood, indicating T cell-mediated immunity.10 Capsid specific CD8+ T cells may have been re-activated by the infused vector and eliminated vector-transduced hepatocytes.11 About 2.5 years after initial vector infusion, capsid-specific functional CD8+ T cells were still present and cross-reacted with a common epitope of AAV serotypes 1, 6, 7 and 8, suggesting that secondary infusions with different naturally occurring serotypes may not circumvent the T cell response.10 AAV capsid-specific CD8+ memory T cells are present in humans at very low frequency but may become reactivated upon AAV gene transfer. Hepatic AAV2 infusion over a range of doses in mice transgenic for human HLA-B*0702 MHC locus failed to elicit capsid-specific CD8+ T cell responses.10 It is likely that natural infection with AAV in the presence of a helper virus causes T cell responses in humans, which would not be the case in animals that are not natural hosts for AAV. However, although mice immunized with AAV capsid or adenoviral vectors expressing AAV capsid developed CD8+ T cells against capsid epitopes, these failed to eliminate AAV transduced hepatocytes in several studies.1215 This lack of an animal model that reproduces the observations in humans has hampered preclinical studies on immune responses to AAV transduced liver. Considering that AAV vectors do not express capsid, input capsid derived from vector particles would have to be efficiently cross-presented by the transduced cell to CD8+ T cells via MHC I molecules. AAV capsid is ubiquitinated and degraded by proteosomes, which may occur over a period of time.16 These observations support a model of CTL-mediated destruction of transduced cells, but do not explain why capsid-specific CD8+ T cells failed to attack AAV-transduced liver in experimental animals.

Recently, several strategies have been suggested to avoid CTL responses to AAV capsid antigen. For example, alternate serotypes that do not contain a heparin binding site are processed differently by dendritic cells and activate CD8+ T cells less efficiently.17 Elimination of surface exposed tyrosine residues on the capsid, enhances gene transfer to the nucleus and substantially reduces accumulation of ubiquitinated capsids in the cytoplasm.18 AAV capsids with more rapid uncoating/degradation kinetics may also prove advantageous. Mycophenolate mofetil (MMF) and cyclosporine A blocked T cell responses at least at lower vector doses.19, 20 AAV1 mediated muscle gene transfer in lipoprotein lipase (LPL) deficiency patients resulted in capsid specific and dose dependent activation of CD4+ and CD8+ T cells 21

Disorders of the central nervous system (such as Parkinson’s disease) are promising targets for AAV-based gene therapies, and despite the fact that the brain is an immune privileged site, immune responses to gene transfer vectors have been observed. AAV vectors may cause transient innate immune responses at high vector doses in naïve mouse brain parenchyma, and additional injections to the opposite hemisphere induce a significantly greater response and reduced transgene expression.22 It has been speculated that the antigen that sparks a brain immune response to rAAV is the capsid protein when injected into the brain striata. Consistent with this hypothesis, delayed re-administration of vector, or switching serotype for a second gene transfer to the striata, resulted in no transgene loss or striatal inflammation, hence overcoming the danger of pre-existing immunity. The authors of these studies also concluded that intracellular processing of AAV capsid generates the immunogenic antigen, and that capsid serotypes that are processed more quickly than rAAV2/2 are less immunogenic.23

Preventing immune responses in AAV mediated gene transfer

Immune responses against AAV-encoded transgene products vary substantially and are influenced by the target organ, route of delivery and dosing schedule. For example, in Hemophilia B mice with a F.IX gene deletion, intramuscular (IM) vector administration caused a local immune response characterized by activation of CD4+ T and B cells to F.IX, which eliminated systemic expression. B cell activation has also been a complication upon over-expression of erythropoietin (epo) in skeletal muscle, which induced an autoantibody against epo resulting in autoimmune anemia. Although CD8+ T cell responses occur and are of particular concern in inflamed muscle (which is typical for some forms of muscular dystrophy), these T cells are often not fully functional in healthy muscle.20, 24, 25 Recent studies found that IM injection of AAV vectors often induces transgene product-specific CD8+ T cell that express markers of T cell functional exhaustion and T cell suppression, and ultimately undergo programmed cell death in skeletal muscle.26, 27 Several immune suppression protocols have been successfully used in different animal models to block humoral and cellular immune responses to transgene products expressed in skeletal muscle.20 For example, the prophylactic use of immunosuppressant drug rapamycin in combination with IL-10 in presence of Factor IX (F.IX) dominant epitopes induces Treg and long term tolerance to the F.IX transgene product expressed in muscles of hemophilia B mice..28

As opposed to muscle gene transfer, hepatic gene transfer with AAV vectors, has been shown to induce immune tolerance to a number of transgene products via induction of Treg and other mechanisms.2932 In addition to direct tolerization of transgene product-specific CD4+ T cells by induction of non-responsiveness (anergy) or deletion, B and T cell responses (including CTL responses) are actively suppressed by induction of regulatory T cells (Treg) with an immune suppressive phenotype.30 These Treg are phenotypically similar to naturally occurring Treg and express, among others, the surface markers CD4 and CD25, and transcription factor FoxP3, the master switch for Treg development.30 Antibody-mediated depletion studies suggest that Treg are critically required for tolerance to the transgene product following hepatic gene transfer.30, 33 IL-10 expression by Treg and Kupffer cells may directly suppress immune responses in the liver. Once tolerance is established, the transgene product can safely be expressed in sites that would otherwise predispose to immune reponses 34, 35

In some cases, one may simply avoid immunity by taking advantage of immune privileged sites. The ocular disease Leber’s Congenital Amaurosis (LCA), caused by an autosomal recessive mutation of RPE65, is being successfully treated in several ongoing clinical trials. Single eye injections of rAAV2-CBSB-hRPE65 resulted in an increase of visual sensitivity in several patients.3639 Due to the immune privileged nature of the eye, one should be able to improve treatment by re-injection in the previously injected eye or the partner eye, as has been suggested by animal studies in subretinal vector administration. This route causes a deviant immune response, resulting in a lack of NAB formation. However, intravitreal administration of AAV2 vector in mice resulted in a humoral response against the capsid that blocked transgene expression on re-administration of vector in a partner eye.40

Overview of immune responses in Adenoviral vector mediated gene therapy

Adenoviruses (Ad) are used in gene delivery and vaccine applications, transduce various cell types, incorporate large transgenes (~35 kb) with a high level of expression, and are easy to manufacture. Immune responses against adenovirus may be directed against the capsid, double-stranded DNA genome, viral proteins expressed from the vector backbone, or incorporated transgenes, and severely limit in vivo gene therapy. Systemic delivery of Ad vectors results in rapid physiological responses that include activation of innate immunity, induction of cytokines, inflammation, transient liver toxicity, and thrombocytopenia.41 Dose-dependent activation of innate and adaptive immune responses has been observed. Adenovirus vectors are able to transduce peripheral blood mononuclear cells as well as dendritic cells.

Innate immune responses to adenoviral vectors; TLRs, inflammatory cytokines, inflammasome and complement

Adenoviral vector particles tend to elicit strong innate immune responses, and 90% of vector DNA is cleared from the tissue within 24 hours of intravenous vector administration. Adenoviruses activate the innate immunity through Toll-like receptor–dependent (TLR-dependent) as well as TLR-independent pathways, causing up-regulation of type I IFNs and inflammatory cytokines.42 TLR-9 has been identified as a pattern recognition receptor (PRR) for DNA containing unmethylated CpG motifs.43, 44 TLR-2 and TLR-9 have been implicated in the innate response to Ad.45 TLR-2 is found on the cell surface and TLR-9 is an endosomal receptor. The adeoviral-ligand to TLR-2 is yet to be identified, in other viruses, glycoprotein’s can trigger this pathway. TLR-9 can recognize DNA. Signaling through these receptors typically leads to Th1 immunity which may drive cellular and humoral responses to the vector and transgene. Sensing of Ad particles and genomes via these receptors results in induction of inflammatory mediators and IFN-α,β. These Type I IFNs activate NK cells and regulate the innate immune response against the vector.46 Induction of cytokine IL-1, TNF and chemo-attractant MIP-2 cytokines also occurs, promoting leukocyte migration and infiltration. Uptake by NK cells results in further release of cytokines and priming for an adaptive immune response. Ad vectors induce innate immune responses through (MyD88)/TLR-dependent and/or-independent pathways, depending on the cell type.47 GM-CSF stimulated DCs and conventional DCs use both MyD88 and TLR-9 for Ad vector-induced IL-6 and IL-12 production. However, neither MyD88 nor TLR-9 was crucial for Ad vector-induced IL-6 production in peritoneal macrophages. Ad vector-infected DCs can also mature through a MyD88-independent pathway. The spleen is also a major contributor to Ad vector-triggered production of various cytokines and chemokines. Conventional DCs, (in contrast to plasmacytoid DCs, pDCs) in the spleen play an important role in the induction of IL-6 and IL-12 after systemic administration of Ad vectors.42

Internalized adenoviral DNA induces maturation of pro-IL-1β in macrophages, which is dependent on the innate cytosolic molecular complex called the inflammasome.48 The inflammasome consists of NALP3/ASC adaptor proteins, which recruit the inflammatory caspase-1 into a molecular complex. This pro-inflammatory pathway functions independently of TLRs and interferon regulatory factors, and leads to cell death of macrophages.48 The complement system also plays a role in vector opsonization and clearance as a part of the innate immune system. Adenovirus has been shown to bind C3-derived fragments directly or activate complement via antibodies in individuals having preexisting immunity (Figure 2).49 Ad interactions with the mammalian complement system are significant and likely initiate inflammatory responses. Thrombocytopenia is caused by interactions between adenoviral particles and the coagulation system, resulting in platelet activation, binding to endothelial cell surfaces, and formation of platelet-leukocyte aggregates.50 Finally, Ad vectors directly and indirectly activate endothelial cells.

Adaptive immune responses against adenoviral vectors

Rapid increases in IL-6, IFN-α, IFN-β, RANTES, IL-12 (p40), IL-5, G-CSF, and GM-CSF are observed. Furthermore, a complex set of interactions between the innate and the adaptive immune system results in activation of CD4+ and CD8+ T cells, and B cells.41 Type I IFN signaling is important for T help-dependent antibody formation by B cells. IFNs also induce DC maturation by up-regulating co-stimulatory molecules like CD80, CD86, and CD40. Neutralizing antibodies against IFN-α and IFN-β have been found to be effective in blocking both innate as well as adaptive immune responses to the viral vector.51

Humoral immune responses preclude re-administration of the vector by reducing efficiency of transduction in vivo due to NAB formation. Antiviral antibodies enhance the interaction and internalization of adenovirus with leukocytes via the Fc receptor and complement receptor. Over 97% of humans have pre-existing antibodies against group C adenovirus, while about 50% have antibodies against the commonly used adenovirus serotype 2. Higher seroprevalence of neutralizing antibodies against Ad 5 is observed in Asian and African populations. Subgroup C infection is endemic in the human population; the majority of individuals seroconvert within the first 5 years of life as the result of natural infections. Pre-existing immunity to Adhu5 virus strongly impairs the B- and T-cell responses to the transgene product of vaccines based on E1-deleted Ad of the same serotype.52 CD4+ and CD8+ cross-reactive T cells against different Ad serotypes were found in human peripheral blood mononuclear cells (PBMCs). Adenovirus-specific CD4+ T cells recognize conserved epitopes among different serotypes, with the majority of people developing long-lived CD4+ T cell responses to adenovirus, perhaps suggesting that alternate serotype vectors should be devoid of these epitopes. Adenovirus-specific secretion of IFN-γ from PBMCs was found within 12 hours of incubation, suggesting prior activation of Ad-specific CTLs. Most adults retain adenovirus-specific cellular memory after childhood exposure. Transduction of APCs by Ad vectors contributes to CTL responses, which are directed against viral gene products and transgene products and are dependent on T help.

Overcoming immune responses in adenoviral gene transfer

Ad vectors with modified capsid sequences such as Ad5/f45 appear to have significantly reduced seroprevalence. Other serotypes, for example Ad-35, may have less NAB and T cell cross-reactivity with Adhu5. Preexisting immunity to Adhu5completely inhibited induction of transgene product-specific antibodies by an Adhu5 vector, but did not affect antibody responses to chimpanzee-, derived vectors Ad vectors.53 Mastrangeli et al showed that alternating serotypes Ad5 (Subgroup C), Ad4 (subgroup E) and Ad30 (subgroup D) for repeated respiratory administration showed some success in circumventing humoral responses in a cystic fibrosis model. The high innate immunogenicity of Ad vectors has a negative impact on their use in gene therapy, but made these vectors more attractive as vaccine carriers. For example, Ad vectors are being developed for use as cancer vaccines, and vaccine prototypes for human immunodeficiency virus type 1 based on E1-deleted Ad recombinants are in Phase II clinical trials.53 A rAd5 based HIV-1 vaccine in a clinical trial resulted in increased HIV infection by 2.3 fold in Ad seropositive individuals. One initial hypothesis was that the vaccination of seropositive individuals caused the expansion of CD4+ Ad specific T cells, which served as targets for HIV. However, activated Ad5 specific lymphocytes were determined not to be the cause of increased susceptibility, hence the area remains under investigation.54 This surprising development in HIV vaccine biology shows the importance of studies of the immune systems’ interactions with vectors in disease models.54, 55

The removal of all viral coding sequences from Ad vectors and their replacement with human non-coding intron (stuffer) sequences has resulted in helper-dependent, high capacity (gutted) vectors, which show reduced CTL responses and therefore increase the potential for stable transgene expression, gutted Ad vectors still leave much to be desired for re-administration. Use of organ-specific promoters (e.g. the hepatocyte-specific human α1-antitrypsin promoter when targeting the liver) reduces expression of the transgene in professional APCs, thereby further reducing the risk of CTL responses. Utilization of strong promoters for improved transgene expression, in combination with limited vector doses, may also prove to be helpful in curtailing Ad vector-directed innate immune responses and inflammation. Innovative delivery techniques, such as hydrodynamic injections and other approaches toward local delivery of low vector doses directly into the liver, diminish systemic vector dissemination and reduce inflammatory responses and immunotoxicity.56 Other strategies to subdue immune responses associated with Ad viruses include the use of immune suppression, pretreatment with gulucocortocoid, use of lipid bilayer envelopes, and preventing binding to the CAR receptor by masking the adenoviral fiber knob 5759.

Overview of Immune responses to Lentiviral vector mediated gene transfer

Lentiviral vectors can transduce non-dividing cells and are able to integrate into the host cell genome, hence being advantageous for long-term expression of the therapeutic transgene (but posing a risk for insertional mutagenesis).60 Duration of transgene expression and potency of immune responses to LV encoded transgene products have varied substantially for different studies on in-vivo transfer. LV delivery in immunocompetent mice results in efficient transduction and transgene expression in several cell types like retina, muscle and hematopoetic cells, while transduction of hepatocytes is relatively inefficient. Potent immune responses directed against the transgene product however have been shown to clear the transduced liver cells within 4 weeks after the injection. Efficient LV-mediated transduction of APCs is responsible for the induction of potent CTL and antibody responses against the transgene product. Pseudotyped LV containing glycoproteins from Ebola Zaire Virus, Lymphocytic choriomenengitis virus, mokola virus and vesicular somatitis virus G (VSV) delivered to mouse lung resulted in activation of transgene specific T cells against the GFP transgene as well as the vector itself. VSV pseudotyped LV showed large reduction in transgene expression within 90 days.61 In contrast, a Baculovirus GP64 pseudotyped feline immunodeficiency vector (AcGP64-FIV) showed persistant (50 weeks) expression of luciferase in nasal epithelia when delivered to the nostril, similarly LacZ was expressed for 90 days, as observed by Sinn et al. Studies by Kremer et al. however showed that in a different strain of mice, and using a HIV-derived LacZ vector, pseudotyped with GP64, transgene expression declined to undetectable levels by 6 months.62 The difference in results may lie in the different vector designs, routes of administration and dosage of injection etc. Different routes of administration (nasal vs. intratracheal) may affect longevity of gene expression due to differential activation of the immune system. Nasal application of peptides is known to produce Treg to the applied antigen and may indicate a mode of tolerization in nasally applied LV vectors. One would also expect the nature of the transgene and the underlying mutation to have an effect. While CTL responses to envelope proteins are also a potential concern, data in mice thus far suggest that CTL-mediated elimination of transduced cells is: i) caused by T cells specific to the transgene product ii) a result of transgene expression in APCs even if a tissue-specific promoter is used and iii) the envelope plays a role in the infection of APCs.

Preventing immune responses in Lentiviral gene transfer

Hepatic LV administration in mice has been observed to induce a rapid and transient IFN-α,β response, which is dependent on infectious vector particles.63 In vitro challenge of APCs suggested that plasmacytoid dendritic cells (pDCs) drive this response. Despite the delivery of replication-defective vectors, type I IFN responses effectively interfere with transduction in a cell type-specific manner and promote functional CTL responses. Consequently, high level of stable LV GFP gene transfer to hepatocytes was observed in mice deficient in IFN-α,β signaling, while wild-type mice showed only transient (<25 Days) transduction in a small proportion of hepatocytes, which were rapidly targeted by CTLs.63 These responses were seen despite the fact that LV elicit weaker IFNα responses from pDC compared to the parent HIV-I virus. Systemically injected LV is preferentially sequestered by liver macrophages. Chimeric GP64/Sendai envelope proteins pseudotyped LV vectors have been shown to reduce macrophage transduction, thereby possibly helping to evade the immune system.64 Stimulation of murine pDC by lentivirus particles is weak compared to other single-stranded RNA viruses such as VSV, influenza, or Sendai virus, which activate murine pDC by ligation of TLR-7, the PRR that sense single-stranded RNA molecules in endosomes.65 LV induces low levels of cytokine secretion by pDC, possibly via a TLR7-dependent pathway.63 VSV-G protein-pseudotyped LV may also contain tubulovesicular structures with DNA fragments that can stimulate TLR9. 65 Blocking of TLR-7 or TLR9 pathways, however, has been observed to be insufficient to prevent LV from inducing IFN-α responses.60

Similar to adenoviral vectors, use of a hepatocyte-restricted promoter reduces the risk for immune responses to the transgene product in liver-directed gene transfer. However, depending on the mouse strain tested, this effect is not robust enough, possibly because of leaky expression and the high transduction efficiency of APCs with LV. For example, LV efficiently infect sinusoidal cells in the liver, including resident macrophages (Kupffer cells) and also splenic macrophages and DCs. Brown and Naldini developed an innovative strategy to solve this dilemma. Incorporation of several repeats of a target sequence for a hematopoietic cells lineage-specific micro-RNA into the 3′ end of the transcript is highly effective in blocking transgene expression in professional (i.e. bone marrow-derived) APCs. The therapeutic value of this approach has been documented in hemophilia B mice. Following LV transfer of a F.IX gene to the liver, sustained correction of coagulation was achieved using the miRNA regulated expressing cassette, and formation of inhibitory antibodies against F.IX were avoided (ultimately results in tolerance induction).66, 67


Each vector system faces its own set of immunological hurdles for gene therapy, some more related to innate immunity, others to adaptive immunity (including memory and pre-existing immunity). Nonetheless, similar to transplantation biologists, gene therapists are learning to circumvent, manipulate, or suppress unwanted immune responses. Advances in vector engineering (such as capsid engineering, miRNA-regulated expression cassettes, etc) and delivery techniques, administration to immune privileged sites, taking advantage of organ-specific immune responses, immune suppression and modulation regimens represent promising strategies to overcome immunological hurdles. While translation of knowledge gained from preclinical studies in animal models to human therapeutics in gene therapy is not always straightforward, immune modulation protocols are nonetheless being refined and tailored towards specific vector/target tissue/disease combination.

With reference to AAV, more clinical results are expected to emerge in the near future from ongoing or yet to be initiated clinical trials. These will help us understand the effect of target organ (e.g. brain, central nervous system, eye, muscle, liver) on T cell responses to capsid and its consequences for long term expression. Interestingly, a recent paper describes a patient that received IM AAV1-mediated gene transfer and continued to express the transgene despite a detectable CD8 response to capsid.68 However a different study suggested transient expression as a result of capsid-specific response in muscle.21 More human data are required to resolve these issues.

In the case of AAV and Ad, continued engineering of capsids should establish whether pre-existing NAB/cross-reactive antibodies to the vector can be avoided without loss of gene transfer in humans.69 Ad vectors have been an excellent model for obtaining a detailed understanding of innate immune responses to DNA viruses. A fairly complete picture of the interactions between Ad and the innate immune system is emerging. These vectors remain attractive vaccine carriers and new vaccines based on human and primate serotypes are being developed. With regard to using Ad vector for treatment of genetic disease, limited doses and local delivery of gutted vectors will likely be required to avoid immunotoxicity.

Results from LV based treatment of genetic disease in large animal models are expected to be produced in the near future. It will be of interest to compare efficacy and immune responses using optimized envelope and miRNA regulated expression cassettes to current data from rodent models. Effective de-targeting of professional APCs will decrease immune responses to LV gene transfer. In general, additional clinical experience with viral vectors combined with advances in the laboratory should generate a more complete assessment of immune responses to these gene transfer vectors and their transgene products.


Conflict of interest: RWH has been receiving royalty payments from Genzyme Corp. for license of AAV-FIX technology.


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