A Preclinical Animal Model to Assess the Effect of Pre-existing Immunity on AAV-mediated Gene Transfer

Hepatic adeno-associated virus (AAV)-serotype 2–mediated gene transfer results in sustained transgene expression in experimental animals but not in human subjects. We hypothesized that loss of transgene expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer. Here, we tested the effect of immunological memory to AAV capsid on AAV-mediated gene transfer in a mouse model. Upon hepatic transfer of an AAV2 vector expressing human factor IX (hF.IX), mice immunized with adenovirus (Ad) vectors expressing AAV8 capsid before AAV2 transfer developed less circulating hF.IX and showed a gradual loss of hF.IX gene copies in liver cells as compared to control animals. This was not observed in mice immunized with an Ad vectors expressing AAV2 capsid before transfer of rAAV8-hF.IX vectors. The lower hF.IX expression was primarily linked to AAV-binding antibodies that lacked AAV-neutralizing activity in vitro rather than to AAV capsid–specific CD8⁺ T cells.     

Cross-presentation of adeno-associated virus serotype 2 capsids activates cytotoxic T cells but does not render hepatocytes effective cytolytic targets

Liver toxicity observed in a clinical trial of adeno-associated virus serotype 2 (AAV2) delivered systemically to patients with hemophilia was ascribed to killing of vector-transduced hepatocytes by capsid-specific T cells. This study evaluated the biology of T cell activation in response to AAV capsids in murine models. CD8(+) T cell epitopes were mapped to capsids from AAV2, AAV7, and AAV8. A tetramer generated in response to a dominant capsid epitope in BALB/c mice was shared between these AAV serotypes. Administration of AAV2 vector resulted in the activation of capsid-specific CD8(+) T cells as evidenced by binding to tetramer and production of capsid-induced interferon-gamma expression this was not observed with the AAV7 and AAV8 vectors. CD8(+) T cells specific to AAV2 capsids demonstrate functional cytolytic activity in vivo to peptide-loaded target cells. The frequency of capsid-specific T cells was much higher in liver than in blood or spleen. The performance of liver-directed AAV-mediated gene transfer was not diminished in animals with high levels of pre-existing capsid-specific T cells. We conclude that cross-presentation of AAV capsids does result in activation of cytotoxic T lymphocytes (CTLs) in a serotype-specific manner; however, there is no evidence that vector-transduced hepatocytes are targets for CTL effector activity.     

Identification of mouse AAV capsid-specific CD8+ T cell epitopes.

Adeno-associated virus has been developed for use as a gene transfer vector. To understand the impact of AAV capsid-specific CD8(+) T cells on AAV-mediated gene transfer, we identified CD8(+) T cell epitopes for AAV-2 and AAV-8 capsid in C57BL/6 (H-2(b) MHC haplotype) and BALB/c (H-2(d) MHC haplotype) mice. Mice of both the H-2(b) and the H-2(d) haplotypes recognized epitopes on AAV-2 and AAV-8 capsid. T cells from H-2(b) mice recognized an epitope that was conserved between AAV-2 and AAV-8 capsid. Cross-reactivity of AAV-specific CD8(+) T cells induced by different AAV serotypes may have important implications for gene transfer. Identification of these epitopes will facilitate studies of immune response to AAV capsid in mouse models.     

Proteasome Inhibitors Decrease AAV2 Capsid derived Peptide Epitope Presentation on MHC Class I Following Transduction

Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize that in the first clinical trial using AAV to treat hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8⁺ T cells and consequent loss of therapeutic transgene expression. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.     

CD8+ T Cell Recognition of Epitopes Within the Capsid of Adeno-associated Virus 8–based Gene Transfer Vectors Depends on Vectors' Genome

Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8⁺ T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8⁺ T cells. Our results clearly show that depending on the vectors'' genome, CD8⁺ T cells can detect such epitopes on AAV8''s capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.     

Capsid-specific T-cell Responses to Natural Infections With Adeno-associated Viruses in Humans Differ From Those of Nonhuman Primates

Hepatic adeno-associated virus serotype 2 (AAV2)-mediated gene transfer failed to achieve sustained transgene product expression in human subjects. We formulated the hypothesis that rejection of AAV-transduced hepatocytes is caused by AAV capsid-specific CD8⁺ T cells that become reactivated upon gene transfer. Although this hypothesis was compatible with clinical data, which showed a rise in circulating AAV capsid-specific T cells following injection of AAV vectors, it did not explain that AAV vectors achieved long-term transgene expression in rhesus macaques, which are naturally infected with AAV serotypes closely related to those of humans. To address this apparent contradiction, we tested human and rhesus macaque samples for AAV capsid-specific T cells by intracellular cytokine staining combined with staining for T-cell subset and differentiation markers. This highly sensitive method, which could provide a tool to monitor adverse T-cell responses in gene transfer trials, showed that AAV capsid-specific CD8⁺ and CD4⁺ T cells can be detected in blood of naturally infected humans and rhesus macaques. They are present at higher frequencies in rhesus macaques. Furthermore, T cells from humans and rhesus macaques exhibit striking differences in their differentiation status and in their functions, which may explain the disparate duration of AAV-mediated gene transfer in these two species.     

Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors

Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid–derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.     

Pharmacological Modulation of Humoral Immunity in a Nonhuman Primate Model of AAV Gene Transfer for Hemophilia B

Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.     

Muscle as a target for supplementary factor IX gene transfer

Immune responses to the factor IX (F.IX) transgene product are a concern in gene therapy for the X-linked bleeding disorder hemophilia B. The risk for such responses is determined by several factors, including the vector, target tissue, and others. Previously, we have demonstrated that hepatic gene transfer with adeno-associated viral (AAV) vectors can induce F.IX-specific immune tolerance. Muscle-derived F.IX expression, however, is limited by a local immune response. Here, skeletal muscle was investigated as a target for supplemental gene transfer. Given the low invasiveness of intramuscular injections, this route would be ideal for secondary gene transfer, thereby boosting levels of transgene expression. However, this is feasible only if immune tolerance established by compartmentalization of expression to the liver extends to other sites. Immune tolerance to human F.IX established by prior hepatic AAV-2 gene transfer was maintained after subsequent injection of AAV-1 or adenoviral vector into skeletal muscle, and tolerized mice failed to form antibodies or an interferon (IFN)-gamma(+) T cell response to human F.IX. A sustained increase in systemic transgene expression was obtained for AAV-1, whereas an increase after adenoviral gene transfer was transient. A CD8(+) T cell response specifically against adenovirus-transduced fibers was observed, suggesting that cytotoxic T cell responses against viral antigens were sufficient to eliminate expression in muscle. In summary, the data demonstrate that supplemental F.IX gene transfer to skeletal muscle does not break tolerance achieved by liver-derived expression. The approach is efficacious, if the vector for muscle gene transfer does not express immunogenic viral proteins.     

Characterization of the relationship of AAV capsid domain swapping to liver transduction efficiency.

Recombinant adeno-associated virus (AAV) vectors show promise for use in gene therapy. For liver-targeted gene transfer in animals, AAV vectors pseudotyped with the AAV serotype 8 (AAV8) capsid have definite advantages over the widely used but less efficient serotype AAV2, even though the capsid amino acid sequences are 82% conserved. To demonstrate the mechanism behind the higher liver transduction efficiency associated with AAV8 capsids, we adopted a domain-swapping strategy that would generate 27 chimeric capsid genes containing exchanged domains between AAV2 and AAV8. The resulting chimeric capsids were then used to package AAV genomes with a liver-specific human coagulation factor IX (hFIX) expression cassette. By comparing the transduction efficiencies between vectors pseudotyped with chimeric, AAV2 and AAV8 capsids, we found that the more efficient liver transduction achieved by AAV8 was closely related to the components of its interstrand Loop IV domain, particularly the subloops 1 and 4. These subloops are exposed on opposite sides of a threefold proximal peak on the virion surface, which may function as a critical structural determinant for AAV transduction. Because a single specific peptide component could not explain all the observed differences in the transduction parameters, we suggest that important subloop regions require interaction with other portions of the capsid for their functioning.     

Tolerance induction by viral in vivo gene transfer

Treatment of genetic disease by protein or gene replacement therapy is hampered by immune responses to the therapeutic protein. An excellent example is formation of inhibitory antibodies to coagulation factors in treatment of the X-linked bleeding disorder hemophilia. Experiments in murine and canine models of hemophilia B (deficiency in factor IX) have demonstrated sustained therapeutic levels of factor IX transgene expression following hepatic adeno-associated viral gene transfer in animals with deletion and nonsense mutations in the factor IX gene. This article reviews experimental evidence for induction of immune tolerance to the factor IX transgene product by hepatic adeno-associated viral gene transfer, which has been shown to limit T helper cell responses and to substantially reduce the risk of antibody responses. Tolerance induction is associated with activation of regulatory CD4(+) T cells capable of suppressing antibody formation to factor IX protein. Hepatic administration of adeno-associated viral vector expressing ovalbumin in mice transgenic for a T cell receptor specific for this antigen provided direct evidence for induction of CD4(+) T cell tolerance, including T cell anergy and clonal deletion. Taken together, these data indicate the potential for viral in vivo gene transfer not only to provide sustained systemic expression, but moreover to induce immunological hypo-responsiveness to the therapeutic gene product.     

The Pleiotropic Effects of Natural AAV Infections on Liver-directed Gene Transfer in Macaques

Adeno-associated viral (AAV) vectors hold great potential for liver-directed gene therapy. Stable and high levels of transgene expression have been achieved in many murine models. Systemic delivery of AAV vectors in nonhuman primates (NHPs) that are natural hosts of AAVs appear to be challenging due to the high prevalence of pre-existing neutralizing antibodies (NAbs). This study evaluates the performance of AAV8, hu.37, and rh.8 vectors expressing green fluorescent protein (GFP) from a liver-specific promoter in rhesus macaques. Two of the animals that received AAV8 showed transduction of 24 and 40% of hepatocytes 7 days after systemic vector delivery. Importantly, expression was detected in several animals after 35 days despite the elevation of liver enzymes and development of transgene-specific T cells in liver. Pre-existing low levels of NAbs profoundly impacted the outcome of gene transfer and redirected vector DNA to spleen. We developed a sensitive in vivo passive transfer assay to detect low levels of NAbs to these novel AAV serotypes. Other strategies need to be developed to reduce immune response to the transgene in order to maintain long-term gene expression.     

Role of Vector in Activation of T Cell Subsets in Immune Responses against the Secreted Transgene Product Factor IX

Defining immune responses against the secreted transgene product in a gene therapy setting is critical for treatment of genetic diseases such as hemophilia B (coagulation factor IX deficiency). We have previously shown that intramuscular administration of an adeno-associated viral (AAV) vector results in stable expression of therapeutic levels of factor IX (F.IX) and may be associated with humoral immune responses against F.IX. This study demonstrates that intramuscular injection of an AAV vector expressing F.IX fails to activate F.IX-specific cytotoxic T lymphocytes (CTLs) in hemostatically normal or in hemophilia B mice, so that there is an absence of cellular immune responses against F.IX. However, transgene-derived F.IX can cause B cell responses characterized by production of T helper cell-dependent antibodies (predominantly IgG1, but also IgG2 subclasses) resulting from activation of CD4⁺ T helper cells primarily of the Th2 subset. In contrast, administration of an adenoviral vector efficiently activated F.IX-specific CTLs and T helper cells of both Th1 and Th2 subsets, leading to inflammation and destruction of transduced muscle tissue and activation of B cells as well. Therefore, vector sequences fundamentally influence T cell responses against transgene-encoded F.IX. In conclusion, activation of the immune system in AAV-mediated gene transfer is restricted to pathways mediated by F.IX antigen presentation through MHC class II determinants resulting in T and B cell responses that are more comparable to responses in the setting of protein infusion rather than of viral infection/gene transfer.     

Evaluation of adeno-associated viral vectors for liver-directed gene transfer in dogs

This study evaluated six adeno-associated viral (AAV) vectors expressing green fluorescent protein (GFP) from the liver-specific thyroid hormone-binding globulin (TBG) promoter made with novel capsids in canine liver-directed gene transfer. Studies in 1.5-month-old dogs, which were administered vector through a peripheral vein, showed that AAV8 capsid vectors had the most favorable performance profiles. Interestingly, the absolute levels of hepatocyte transduction achieved with AAV8 were lower in dogs compared with what had been achieved in mice and nonhuman primates. Additional studies were performed with AAV8 delivered into the hepatic artery in adult dogs, with higher doses of vector used to assess potential dose-limiting toxicities. These studies showed good transduction on day 7 in one dog that apparently was lost by day 28 in another dog through the generation of GFP-specific T cells. Each adult dog was carefully monitored for any hemodynamic changes associated with vector infusion. Both animals demonstrated mild to moderate hypotension and bradycardia, which appeared to be anesthesia-related, making it difficult to evaluate contributions of the vector.     

Enhancing transduction of the liver by adeno-associated viral vectors

A number of distinct factors acting at different stages of the adeno-associated virus vector (AAV)-mediated gene transfer process were found to influence murine hepatocyte transduction. Foremost among these was the viral capsid protein. Self-complementary (sc) AAV pseudotyped with capsid from serotype 8 or rh.10 mediated fourfold greater hepatocyte transduction for a given vector dose when compared with vector packaged with AAV7 capsid. An almost linear relationship between vector dose and transgene expression was noted for all serotypes with vector doses as low as 1 x 10(7) vg per mouse (4 x 10(8) vg kg(-1)) mediating therapeutic levels of human FIX (hFIX) expression. Gender significantly influenced scAAV-mediated transgene expression, with twofold higher levels of expression observed in male compared with female mice. Pretreatment of mice with the proteasome inhibitor bortezomib increased scAAV-mediated hFIX expression from 4+/-0.6 to 9+/-2 microg ml(-1) in female mice, although the effect of this agent was less profound in males. Exposure of mice to adenovirus 10-20 weeks after gene transfer with AAV vectors augmented AAV transgene expression twofold by increasing the level of proviral mRNA. Hence, optimization of individual steps in the AAV gene transfer process can further enhance the potency of AAV-mediated transgene expression, thus increasing the probability of successful gene therapy.     

Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes

Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8⁺ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4⁺CD25⁺FoxP3⁺ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8⁺ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4⁺CD25⁺FoxP3⁺ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8⁺ T cell reactivity to an antigen using regulatory T cell epitopes is possible.     

Recombinant adeno-associated virus vectors induce functionally impaired transgene product-specific CD8+ T cells in mice

Recombinant adeno-associated virus (rAAV) vectors were used in human trials as carriers of vaccines for HIV-1 after encouraging preclinical results. However, the clinical trials yielded disappointing results. Here we demonstrated that in mice, rAAV vectors expressing the gene encoding HIV-1 gag stimulated gag-specific CD8(+) T cells, but these T cells failed to expand after a booster immunization with a replication-defective adenoviral (Ad) vector also expressing gag. We tested rAAV vectors of different serotypes expressing HIV-1 gag for induction of transgene product-specific CD8(+) T cells and found that the immunoinhibitory effect of rAAV priming observed with different AAV serotypes was transgene product specific, was independent of the interval between prime and boost, and extended to boosts with vaccine modalities other than Ad vectors. rAAV vector-induced CD8(+) T cells proliferated poorly, produced low levels of IFN-gamma in response to gag stimulation, and upregulated immunoinhibitory molecules. These T cells did not protect efficiently against challenge with a surrogate pathogen. Finally, we showed that the impaired proliferative capacity of the T cells was caused by persistence of the antigen-encoding rAAV vectors and could be reversed by placing the CD8(+) T cells in an antigen-free environment. Our data suggest that rAAV vectors induce functionally impaired T cells and could dampen the immune response to a natural infection.     

Muscle‐directed gene therapy for hemophilia B with more efficient and less immunogenic AAV vectors

Summary. Background: Adeno‐associated viral vector (AAV)‐mediated and muscle‐directed gene therapy is a safe and non‐invasive approach to treatment of hemophilia B and other genetic diseases. However, low efficiency of transduction, inhibitor formation and high prevalence of pre‐existing immunity to the AAV capsid in humans remain as main challenges for AAV2‐based vectors using this strategy. Vectors packaged with AAV7, 8 and 9 serotypes have improved gene transfer efficiencies and may provide potential alternatives to overcome these problems. Objective: To compare the long‐term expression of canine factor IX (cFIX) levels and anti‐cFIX antibody responses following intramuscular injection of vectors packaged with AAV1, 2, 5, 7, 8 and 9 capsid in immunocompetent hemophilia B mice. Results: Highest expression was detected in mice injected with AAV2/8 vector (28% of normal), followed by AAV2/9 (15%) and AAV2/7 (10%). cFIX expression by AAV2/1 only ranged from 0 to 5% of normal levels. High incidences of anti‐cFIX inhibitor (IgG) were detected in mice injected with AAV2 and 2/5 vectors, followed by AAV2/1. None of the mice treated with AAV2/7, 2/8 and 2/9 developed inhibitors or capsid T cells. Conclusions: AAV7, 8 and 9 are more efficient and safer vectors for muscle‐directed gene therapy with high levels of transgene expression and absence of inhibitor formation. The absence of antibody response to transgene by AAV7, 8 and 9 is independent of vector dose but may be due to the fact that these three serotypes are associated with high level distribution to, and transduction of, hepatocytes following i.m. injection.     

Efficacy and safety of adeno-associated viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene therapy

BACKGROUND: Adeno-associated viral (AAV) and lentiviral vectors are promising vectors for gene therapy for hemophilia because they are devoid of viral genes and have the potential for long-term gene expression. OBJECTIVES: To compare the performance of different AAV serotypes (AAV8 and AAV9) vs. lentiviral vectors expressing factor (F) IX. METHODS AND RESULTS: AAV-based and lentiviral vectors were generated that express FIX from the same hepatocyte-specific expression cassette. AAV9 transduced the liver as efficiently as AAV8 and resulted in supra-physiological FIX levels (3000-6000% of normal) stably correcting the bleeding diathesis. Surprisingly, AAV9 resulted in unprecedented and widespread cardiac gene transfer, which was more efficient than with AAV8. AAV8 and AAV9 were not associated with any proinflammatory cytokine induction, in accordance with their minimal interactions with innate immune effectors. In contrast, lentiviral transduction resulted in modest and stable FIX levels near the therapeutic threshold (1%) and triggered a rapid self-limiting proinflammatory response (interleukin-6), which probably reflected their ability to efficiently interact with the innate immune system. CONCLUSIONS: AAV8 and 9 result in significantly higher FIX expression levels and have a reduced proinflammatory risk in comparison with lentiviral vectors. The unexpected cardiotropic properties of AAV9 have implications for gene therapy for heart disease.     

Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy.

The application of AAV2 or AAV8 vectors for delivery of human coagulation factor IX (hF.IX) is a promising gene therapy for hemophilia B. One major limitation of this therapy is the development of antibodies and a cytotoxic T lymphocyte (CTL) response against both the vector capsid and the transgene. We determined the class I and class II MHC peptide epitopes for AAV2, AAV8, and hF.IX after administration of AAV-2-hF.IX or AAV8-hF.IX in H2(b) (C57BL/6), H2(d) (BALB/c), or H2(k) (C3H) strains of mice. The results indicate that the AAV2 capsid peptide AA(373-381), the AAV8 capsid peptide AA(50-58), and the hF.IX transgene peptide AA(311-319) can elicit a CTL response as indicated by an IFN-gamma ELISPOT assay and an in vivo CTL assay. Furthermore, a strong H2(k) MHC II-restricted Th1 response can be elicited in C3H mice by the AAV8 capsid peptide AA(126-140) and the hF.IX peptide AA(108-122), whereas a strong Th2 response can be elicited by the AAV2 peptide AA(475-489). These results show that specific CTL responses are generated to both AAV capsid epitopes and hF.IX epitopes after injection of AAV-hF.IX, and MHC class II epitopes derived from AAV-hF.IX promote development of either Th1 or Th2 cells.