Immune Responses to AAV in Canine Muscle Monitored by Cellular Assays and Noninvasive Imaging
Authors: Zejing Wang, Rainer Storb, Donghoon Lee, Martin J Kushmerick, Baocheng Chu, Carolina Berger, Andrea Arnett, James Allen, Jeffrey S Chamberlain, Stanley R Riddell, Stephen J Tapscott
Journal: Molecular Therapy (2009)

We previously demonstrated that direct intramuscular injection of rAAV2 or rAAV6 in wild-type dogs resulted in robust T-cell responses to viral capsid proteins, and others have shown that cellular immunity to adeno-associated virus (AAV) capsid proteins coincided with liver toxicity and elimination of transgene expression in a human trial of hemophilia B. Here, we show that the heparin-binding ability of a given AAV serotype does not determine the induction of T-cell responses following intramuscular injection in dogs, and identify multiple epitopes in the AAV capsid protein that are recognized by T cells elicited by AAV injection. We also demonstrate that noninvasive magnetic resonance imaging (MRI) can accurately detect local inflammatory responses following intramuscular rAAV injection in dogs. These studies suggest that pseudotyping rAAV vectors to remove heparin-binding activity will not be sufficient to abrogate immunogenicity, and validate the utility of enzyme-linked immunosorbent spot (ELISpot) assay and MRI for monitoring immune and inflammatory responses following intramuscular injection of rAAV vectors in preclinical studies in dogs. These assays should be incorporated into future human clinical trials of AAV gene therapy to monitor immune responses. We previously demonstrated that direct intramuscular injection of rAAV2 or rAAV6 in wild-type dogs resulted in robust T-cell responses to viral capsid proteins, and others have shown that cellular immunity to adeno-associated virus (AAV) capsid proteins coincided with liver toxicity and elimination of transgene expression in a human trial of hemophilia B. Here, we show that the heparin-binding ability of a given AAV serotype does not determine the induction of T-cell responses following intramuscular injection in dogs, and identify multiple epitopes in the AAV capsid protein that are recognized by T cells elicited by AAV injection. We also demonstrate that noninvasive magnetic resonance imaging (MRI) can accurately detect local inflammatory responses following intramuscular rAAV injection in dogs. These studies suggest that pseudotyping rAAV vectors to remove heparin-binding activity will not be sufficient to abrogate immunogenicity, and validate the utility of enzyme-linked immunosorbent spot (ELISpot) assay and MRI for monitoring immune and inflammatory responses following intramuscular injection of rAAV vectors in preclinical studies in dogs. These assays should be incorporated into future human clinical trials of AAV gene therapy to monitor immune responses. IntroductionOne obstacle in using viral-derived vectors for in vivo gene delivery is the development of host immune responses to both viral and transgene products. Recombinant vectors based on a subset of adeno-associated virus (rAAV) serotypes have attracted increasing attention due to their high tropism for various cell types including postmitotic cells,1Athanasopoulos T Graham IR Foster H Dickson G Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD).Gene Ther. 2004; 11: S109-S121Crossref PubMed Scopus (64) Google Scholar,2Wang Z Chamberlain JS Tapscott SJ Storb R Gene therapy in large animal models of muscular dystrophy.ILAR J. 2009; 50: 187-198Crossref PubMed Scopus (45) Google Scholar,3Warrington Jr, KH Herzog RW Treatment of human disease by adeno-associated viral gene transfer.Hum Genet. 2006; 119: 571-603Crossref PubMed Scopus (116) Google Scholar absence of serious pathology associated with AAV infections in humans, and the perceived lack of strong immunogenicity of AAV capsids enabling sustained transgene expression.1Athanasopoulos T Graham IR Foster H Dickson G Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD).Gene Ther. 2004; 11: S109-S121Crossref PubMed Scopus (64) Google Scholar,3Warrington Jr, KH Herzog RW Treatment of human disease by adeno-associated viral gene transfer.Hum Genet. 2006; 119: 571-603Crossref PubMed Scopus (116) Google Scholar,4High K AAV-mediated gene transfer for hemophilia.Genet Med. 2002; 4: 56S-61SAbstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,5Gregorevic P Blankinship MJ Allen JM Crawford RW Meuse L Miller DG et al.Systemic delivery of genes to striated muscles using adeno-associated viral vectors.Nat Med. 2004; 10: 828-834Crossref PubMed Scopus (539) Google Scholar However, in a recent clinical trial of hepatic rAAV2-mediated delivery of the factor IX gene in hemophilia B patients, transient transaminitis developed after vector administration, which was accompanied by detection of AAV2 capsid-specific CD8+ T cells, and a decline in factor IX transgene expression to subtherapeutic levels by 8 weeks.6Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1536) Google ScholarWe have focused on the development of gene therapy for Duchenne muscular dystrophy using a canine model, and have found that T-cell responses to rAAV6 and rAAV2 vectors were induced after intramuscular injection.7Wang Z Allen JM Riddell SR Gregorevic P Storb R Tapscott SJ et al.Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy.Hum Gene Ther. 2007; 18: 18-26Crossref PubMed Scopus (121) Google Scholar Immune responses were elicited independent of the transgene constructs used, vector dose, or vector preparation. Furthermore, comparably robust cellular immune responses were also observed against empty AAV6 capsids. In line with the findings from the canine studies and the clinical hemophilia B trial, recent studies in various strains of mice and in nonhuman primates have demonstrated induction of CD8+ T cells specific to epitopes within AAV2 or AAV8 capsids after either direct injection of rAAV vectors or immunization with adenoviral vectors expressing AAV capsid protein.8Sabatino DE Mingozzi F Hui DJ Chen H Colosi P Ertl HC et al.Identification of mouse AAV capsid-specific CD8+ T cell epitopes.Mol Ther. 2005; 12: 1023-1033Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,9Chen J Wu Q Yang P Hsu HC Mountz JD Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy.Mol Ther. 2006; 13: 260-269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,10Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar Thus, immunity to AAV-mediated therapeutic gene delivery has emerged as a significant barrier for successful long-term gene therapy. Therefore, unless a nonimmunogenic serotype of AAV is identified, future human clinical trials will need to incorporate transient immunosuppressive therapy and validated methods of monitoring immune responses to AAV.Several different AAV serotypes with distinct patterns of tissue tropism have been isolated from human and nonhuman primates,11Gao G Vandenberghe LH Wilson JM New recombinant serotypes of AAV vectors.Curr Gene Ther. 2005; 5: 285-297Crossref PubMed Scopus (406) Google Scholar and it is conceivable that some serotypes might not induce immune responses due to serotype-specific biological characteristics. Studies in mice and nonhuman primates have suggested that AAV serotypes that cannot bind heparan sulfate proteoglycans (HSPGs) due to sequence alterations of the capsid were less able to activate T cells after intramuscular injection.10Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar Therefore, in this study, we compared the ability of rAAV serotypes containing or lacking the HSPG-binding motif to induce immune responses in the canine model. In addition to monitoring local and systemic T-cell responses using histology and interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assays, we examined the ability of magnetic resonance imaging (MRI) to serve as a noninvasive method for monitoring inflammatory responses after AAV injection into the muscle.ResultsOverall experimental designIndividual dogs from a random-bred colony received intramuscular injections of 5 × 1011 vector genomes of recombinant rAAV expressing canine factor IX (cFIX) at several distinct locations, each marked with a sterile suture. A total of four dogs were injected, two with rAAV6 and one each with rAAV1 and with the modified rAAV1 (rAAV1-E531K, described below). At 4 weeks after intramuscular injections, a time point previously shown to have the maximal immune infiltration,7Wang Z Allen JM Riddell SR Gregorevic P Storb R Tapscott SJ et al.Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy.Hum Gene Ther. 2007; 18: 18-26Crossref PubMed Scopus (121) Google Scholar the injected site(s) were imaged by MRI, and, after the MRI scan, one of the AAV-injected sites for each individual was biopsied for histology. Similarly, at 8 weeks after injection, MRI scan was performed on the injected regions prior to biopsy of a second site. Cells were collected before and 4 weeks after the intramuscular injections from a subset of the dogs for T-cell assays. In this report, we will first describe the histology, then the T-cell assays, and finally the MRI studies.Intramuscular injections of HSPG-binding and nonbinding serotypes induced cellular immune responses in dogsIt has been suggested that activation of T cells after AAV injection in mice is due to binding of the virion to HSPG, which facilitates its uptake by dendritic cells.10Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar Our prior studies showed that both AAV2 and AAV6, which contain distinct HSPG-binding motifs, were immunogenic in dogs. AAV1 shares both a high degree of sequence homology (~99.2%) and tropism for muscle with AAV6 (refs. 12Rutledge EA Halbert CL Russell DW Infectious clones and vectors derived from adeno-associated virus (AAV) serotypes other than AAV type 2.J Virol. 1998; 72: 309-319Crossref PubMed Google Scholar,13Blankinship MJ Gregorevic P Allen JM Harper SQ Harper H Halbert CL et al.Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6.Mol Ther. 2004; 10: 671-678Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar); however, AAV1 does not bind HSPG due to a single amino-acid change at position 531, with an E in AAV1 versus a K in AAV6 (ref. 14Wu Z Asokan A Grieger JC Govindasamy L Agbandje-McKenna M Samulski RJ Single amino acid changes can influence titer, heparin binding, and tissue tropism in different adeno-associated virus serotypes.J Virol. 2006; 80: 11393-11397Crossref PubMed Scopus (171) Google Scholar). Therefore, we prepared rAAV6, rAAV1, and rAAV1 modified to contain K at position 531 to confer binding to HSPG (AAV1-E531K), to determine the effects of HSPG binding on immunogenicity in dogs receiving intramuscular vector injections. All vectors expressed a canine factor IX transgene driven by a CMV promoter (CMV-cFIX). An equal amount of each vector preparation (5 × 1011 vector genomes) was injected into hindlimb muscles of different wild-type dogs (one animal for each vector preparation). Muscle biopsies obtained 4 weeks after vector administration showed uniformly similar robust infiltrations of CD8+ and CD4+ T cells (Figure 1) for each of the three vectors. Thus, the ability of AAV capsid to bind HSPG did not affect the induction of a local T-cell infiltrate after intramuscular injection.Detection of AAV capsid-specific T cells in PBMCTo determine whether the cellular responses detected by histological analysis correlated with induction of T-cell reactivity to viral capsids, we measured antigen-induced T-cell secretion of IFN-γ, as a marker of T-cell reactivity in peripheral blood mononuclear cell (PBMC), using a quantitative ELISpot assay15Schmittel A Keilholz U Scheibenbogen C Evaluation of the interferon-gamma ELISPOT-assay for quantification of peptide specific T lymphocytes from peripheral blood.J Immunol Methods. 1997; 210: 167-174Crossref PubMed Scopus (127) Google Scholar in the dog injected with rAAV6 above. The AAV virion is composed of three capsid proteins, VP1, VP2, and VP3 in a ratio of 1:1:10, which are produced from the same open reading frame through alternative splicing.1Athanasopoulos T Graham IR Foster H Dickson G Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD).Gene Ther. 2004; 11: S109-S121Crossref PubMed Scopus (64) Google Scholar,16Daly TM Overview of adeno-associated viral vectors.Methods Mol Biol. 2004; 246: 157-165PubMed Google Scholar,17Geoffroy MC Salvetti A Helper functions required for wild type and recombinant adeno-associated virus growth.Curr Gene Ther. 2005; 5: 265-271Crossref PubMed Scopus (58) Google Scholar VP1 is the full-length protein; therefore, a peptide library spanning the entire capsid protein VP1 and consisting of 15 mer peptides each overlapping by 11 amino acids with adjacent peptides was generated (Figure 2a). The peptides were organized into 27 pools and arranged in a grid such that pools 1–14 each consisted of peptides from its corresponding column, and pools 15–27 each consisted of peptides from its corresponding row. Therefore, each peptide was present in two individual pools (Figure 2b,c). PBMC collected before and after intramuscular injection of rAAV6 vectors (from the dog used above) were stimulated with each individual pool and IFN-γ secretion was measured. AAV6 peptide-specific IFN-γ secretion was not detected before vector injection (Figure 2b), but was detected 4 weeks after injection (Figure 2c). Positive results after stimulation with pools 4, 6, 8, 10, 22, 25, and 27 predicted overlapping peptides: 102, 104, 106, 108, 144, 146, 148, 150, 172, 174, 176, and 178 as potentially immunogenic. To confirm this result, PBMC obtained before and after vector injections were expanded by incubation for 10 days with each of the six pools containing the potential immunogens (Supplementary Figure S1). The cells were then subjected to subsequent ELISpot assays with each individual peptide from the pools. A T-cell response to peptide 174 (sequence: KRWNPEVQYTSNYAK) was confirmed by expansion with pool 6, and responses to peptides 144 (sequence: ATERFGTVAFNLQSS), 174 (sequence: KRWNPEVQYTSNYAK), and 178 (sequence: ANVDFTVDNNGLYTE) were confirmed by expansion with pools 25 and 27, respectively.Figure 2Enzyme-linked immunosorbent spot assay with rAAV6 peptide pools. The number of positive IFN-γ spots measured in 1 × 106 cells is shown. Medium and PHA were used as negative and positive controls, respectively. (a) A total of 182 peptides was synthesized, each of which is 15 amino acids long and overlapping by 11 amino acids with adjacent peptides. Twenty-seven peptide pools are shown in yellow. (b) Preinjection PBMCs were stimulated with individual peptide pools. (c) Postinjection PBMCs at week 4 were stimulated with individual pools. DVI, direct viral injection; IFN-γ, interferon-γ; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to validate the assay, we used the same strategy to identify immunogenic peptides in a second wild-type dog following intramuscular injection with rAAV6. In the second dog, five immunogenic peptides were identified, including one common peptide (peptide 144, ATERFGTVAFNLQSS) that was recognized by both dogs. The sequences of the immunogenic peptides in both dogs are listed in Table 1, as well as whether the individual peptides are present in other commonly used AAV serotypes (Table 2). Therefore, the results showed that AAV6 capsid proteins were recognized by T-cell responses induced after intramuscular injection of AAV in dogs, and confirmed the utility of ELISpot assays for detecting such cellular responses in peripheral blood.Table 1Summary of immunogenic AAV6 epitopes identified from two dogsDogAmino-acid positionPeptide sequence1573-587ATERFGTVAFNLQSS693-707KRWNPEVQYTSNYAK709-723ANVDFTVDNNGLYTE2309-323PKRLNFKLFNIQVKE359-373YQLPYVLGSAHQGCL405-419RTGNNFTFSYTFEDV573-587ATERFGTVAFNLQSS669-683SFITQYSTGQVSVEI Open table in a new tab Table 2Individual peptides conserved in commonly used AAV serotypesPeptide sequencesAAV6AAV1AAV2AAV8PKRLNFKLFNIQVKExxxYQLPYVLGSAHQGCLxxxxRTGNNFTFSYTFEDVxxATERFGTVAFNLQSSxSFITQYSTGQVSVEIxxxxKRWNPEVQYTSNYAKxxANVDFTVDNNGLYTExx Open table in a new tab Noninvasive MRI can detect AAV-induced inflammation in canine muscleAAV vectors are currently being used in human trials for intramuscular delivery of transgenes, so it will be important to develop noninvasive methods of monitoring inflammatory responses to minimize the need for repeated muscle biopsies.18Wang Z Kuhr CS Allen JM Blankinship M Gregorevic P Chamberlain JS et al.Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression.Mol Ther. 2007; 15: 1160-1166Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar MRI provides anatomic information and identifies inflammatory processes and progressive fibrosis by contrast derived from differences in local water proton relaxation time of transverse (T2) magnetization, as changes in T2 values and in intensity of T2 signals on T2-weighted images.19Ruiz-Cabello J Carrero-González B Avilés P Santisteban C Méndez RJ Ferreirós J et al.Magnetic resonance imaging in the evaluation of inflammatory lesions in muscular and soft tissues: an experimental infection model induced by Candida albicans.Magn Reson Imaging. 1999; 17: 1327-1334Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Therefore, we tested whether MRI could be used as a noninvasive method to detect inflammatory reactions resulting from intramuscular injection of rAAV vectors by performing MRI studies on the dogs injected with different vectors (rAAV6, rAAV1, and rAAV1-E531K) described above.Our prior studies demonstrated that the maximum lymphocytic infiltration was consistently present 4 weeks after vector administration but was reduced by week 8 and negligible by week 12. In order to correlate MRI findings with histopathology, each dog received intramuscular rAAV injections at two different muscle sites, and MRI examinations were performed before muscle biopsies of one site at week 4 and the second site at week 8. Contralateral hindlimbs were either injected with buffer or left untreated to serve as controls. We analyzed T2 values 4 weeks after vector administration in the four wild-type dogs, given either rAAV6 (two dogs, one of which used in HSPG study and see Figure 1 for histology), rAAV1 or rAAV1-E531K (see Figure 1 for histology), all carrying the same CMV-cFIX construct. The mean T2 value was significantly higher (P < 0.0003) at the sites of AAV injection (60.1 ± 5.4) than that from the uninjected or buffer-injected control muscles (33.6 ± 0.5) in the contralateral or the same limbs (Figures 3 and 4). In Figure 4, a and b showed T2 map and T2 values from the dogs injected with rAAV6 and rAAV1-E351K, respectively, in the left biceps, and buffer or no injection in the right biceps 4 weeks after injection. Figure 4c showed T2 map and T2 values from the dog injected with rAAV1 and buffer in the same muscle. Hyperintense regions were observed on T2 maps of rAAV-injected biceps (Figure 4 a1, b1, and c1), but not buffer-injected ones (Figure 4 a1, 3 and 4; and 1 in 4c1); and T2 values were increased in rAAV-injected biceps compared to controls (Figure 4). The hyperintense regions were manually contoured to define regions of interest for the T2 measurements and compared to similar-sized regions away from the injection sites. The injection sites had been marked by taping a vitamin E capsule and agarose (1%) sample to the incision scar (labeled as 5 and 6, respectively, in Figure 4 a1). Histological analysis (Figure 1) following MRI at the 4-week time point showed a significant inflammatory response in the regions demonstrating a hyperintense T2 signal, but not in the buffer-injected control sites (Figure 1 b4, c4, and d4).Figure 3Magnetic resonance imaging T2 values at week 4. Median T2 values in rAAV-injected and uninjected muscles of contralateral limb from four wild-type dogs (P < 0.0003).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Magnetic resonance imaging and corresponding muscle biopsy analysis at week 4 in dogs receiving different AAV vectors. (a) rAAV6-CMV-cFIX. a1, T2 map; a2, T2 values. 1 and 2: rAAV6-injected sites; 3 and 4: buffer-injected sites; 5 and 6: vitamin E capsule and agarose (1%) sample, respectively, as markers. BF, biceps femoris; ST, semitendinosus. (b) rAAV1-E351K. b1, T2 map; b2, T2 values. (c) rAAV1. c1, T2 map; c2, T2 values (see Figure 1 for immunohistological analysis on muscle biopsy injected with corresponding vectors). ROI, regions of interest.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Eight weeks after the intramuscular injection, MRI showed reduced T2 intensity at the site of AAV injection, compared to the signal at week 4 (Figure 5a). This observation correlated with reduced inflammation and reduced cFIX expression by histological analysis (Figure 5b,c) of corresponding muscle tissues. These results demonstrated that MRI was a sensitive and potentially useful noninvasive modality for monitoring AAV-induced inflammatory responses.Figure 5Progression of inflammatory responses to rAAV6 from week 4 to week 8 after intramuscular injection in dog. (a) T2-weighted images at 4 weeks (a1) and 8 weeks (a2) after vector injection. Arrows point at the two rAAV-induced inflammatory sites. (b) Muscle biopsy analysis at week 4 (b1-H&E, and b2-CD8 in green), and week 8 (b3-H&E, and b4-CD8, DAPI-stained nuclei (blue). (c) cFIX expression at week 4 (c1-green) and week 8 (c2-green).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionVectors derived from AAV are in general less immunogenic than adenoviral and other viral vector systems, and have shown promise as delivery vehicles for human gene therapy.1Athanasopoulos T Graham IR Foster H Dickson G Recombinant adeno-associated viral (rAAV) vectors as therapeutic tools for Duchenne muscular dystrophy (DMD).Gene Ther. 2004; 11: S109-S121Crossref PubMed Scopus (64) Google Scholar,2Wang Z Chamberlain JS Tapscott SJ Storb R Gene therapy in large animal models of muscular dystrophy.ILAR J. 2009; 50: 187-198Crossref PubMed Scopus (45) Google Scholar,3Warrington Jr, KH Herzog RW Treatment of human disease by adeno-associated viral gene transfer.Hum Genet. 2006; 119: 571-603Crossref PubMed Scopus (116) Google Scholar,4High K AAV-mediated gene transfer for hemophilia.Genet Med. 2002; 4: 56S-61SAbstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,20Halbert CL Standaert TA Aitken ML Alexander IE Russell DW Miller AD Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.J Virol. 1997; 71: 5932-5941Crossref PubMed Google Scholar,21High KA Adeno-associated virus-mediated gene transfer for hemophilia B.Int J Hematol. 2002; 76: 310-318Crossref PubMed Scopus (13) Google Scholar However, recent studies in dogs, nonhuman primates, and humans have shown development of T-cell immunity against different AAV serotypes after either intramuscular or intravenous administration.6Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1536) Google Scholar,8Sabatino DE Mingozzi F Hui DJ Chen H Colosi P Ertl HC et al.Identification of mouse AAV capsid-specific CD8+ T cell epitopes.Mol Ther. 2005; 12: 1023-1033Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,9Chen J Wu Q Yang P Hsu HC Mountz JD Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy.Mol Ther. 2006; 13: 260-269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,10Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar,22Mingozzi F Meulenberg JJ Hui DJ Basner-Tschakarjan E Hasbrouck NC Edmonson SA et al.AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.Blood. 2009; 114: 2077-2086Crossref PubMed Scopus (217) Google Scholar,23Mendell JR Rodino-Klapac LR Rosales-Quintero X Kota J Coley BD Galloway G et al.Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.Ann Neurol. 2009; 66: 290-297Crossref PubMed Scopus (163) Google Scholar In line with these reports, our previous studies in dogs demonstrated robust T-cell-mediated immune responses to AAV capsid following intramuscular injection.7Wang Z Allen JM Riddell SR Gregorevic P Storb R Tapscott SJ et al.Immunity to adeno-associated virus-mediated gene transfer in a random-bred canine model of Duchenne muscular dystrophy.Hum Gene Ther. 2007; 18: 18-26Crossref PubMed Scopus (121) Google ScholarDifferences in capsid sequences among various AAV serotypes can influence the biodistribution of vectors, vector interaction with antigen-presenting cells, vector uncoating, and intracellular vector trafficking and processing. These differences might account for the lower immunogenicity reported for some AAV serotype capsids compared to others. It has been suggested that rAAV2 is more immunogenic than rAAV8 because rAAV2 capsids contain a heparin-binding motif that facilitates rAAV2 binding to HSPG on dendritic cells with resultant AAV2 antigen uptake, processing, and major histocompatibility complex class I presentation. One study both in mice and nonhuman primates showed that high-frequency, capsid-specific T cells were only elicited against natural or recombinant AAV vectors that contained a heparin-binding motif.10Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar On the other side, recent studies from the High and Mendell groups showed immunogenicity of AAV1 capsids following intramuscular injection in humans, another nonheparin-binding serotype;22Mingozzi F Meulenberg JJ Hui DJ Basner-Tschakarjan E Hasbrouck NC Edmonson SA et al.AAV-1-mediated gene transfer to skeletal muscle in humans results in dose-dependent activation of capsid-specific T cells.Blood. 2009; 114: 2077-2086Crossref PubMed Scopus (217) Google Scholar however, in the Mendell study,23Mendell JR Rodino-Klapac LR Rosales-Quintero X Kota J Coley BD Galloway G et al.Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.Ann Neurol. 2009; 66: 290-297Crossref PubMed Scopus (163) Google Scholar the immune response apparently did not diminish transgene expression. In another report from the High group,24Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8(+) T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (516) Google Scholar humans were shown to have pre-existing CD8+ memory T cells specific for AAV capsids. The cells were at a frequency too low for detection by a direct ex vivo assay. However, these T cells could be expanded by culturing in the presence of rAAV vectors and were broadly cross-reactive with different serotypes, including those that lacked HSPG binding.24Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8(+) T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (516) Google Scholar These results extended previous observation of immune responses to additional AAV serotype, AAV1, and suggested that HSPG binding might not be critical in activation of memory T cells, and that immune responses to AAV would be a barrier for gene therapy using rAAV in patients with pre-existing immunity, and for readministration of therapeutic vectors. In the current study, we compared the immunogenicity of capsids from AAV1, a serotype without a heparin-binding domain; AAV6, with a natural heparin-binding motif; an