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

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original article

A Preclinical Animal Model to Assess the Effect of Pre-existing Immunity on AAV-mediated Gene Transfer Hua Li1, Shih-Wen Lin1,2, Wynetta Giles-Davis1, Yan Li1, Dongming Zhou1, Zhi Quan Xiang1, Katherine A High3 and Hildegund CJ Ertl1 Immunology Program, Wistar Institute, Philadelphia, Pennsylvania, USA; 2Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania, USA; 3Department of Pediatrics, The Children’s Hospital of ­Philadelphia and Howard Hughes Medical Institute, Philadelphia, Pennsylvania, USA 1

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. Received 5 December 2008; accepted 24 March 2009; published online 14 April 2009. doi:10.1038/mt.2009.79

Introduction In a clinical trial for correction of hemophilia B, hepatic transfer of a therapeutic dose of an adeno-associated virus (AAV)2 vector encoding factor (F).IX resulted in a transient transaminitis, which was accompanied by loss of F.IX expression in a patient.1 Overall the clinical course was suggestive of T cell–mediated destruction of AAV2-F.IX-transduced hepatocytes. In a second patient treated with a lower dose, T cells to AAV capsid and F.IX were monitored before and after AAV2-F.IX vector transfer. Neither AAV capsid nor hF.IX specific T cells circulated in blood before treatment. After AAV2-F.IX infusion, interferonγ-producing CD8+ T cells to AAV2 capsid antigens became detectable 2 weeks later and then declined to pretreatment levels by week 12 (refs. 1,2). These results were in contrast to those obtained in mice3,4 or hemophilic dogs5 in which hepatic AAV2-F.IX gene transfer resulted in sustained expression of F.IX. We hypothesized that

humans, unlike mice or dogs, have memory T and B cells to AAV due to natural exposures during childhood, which are reactivated upon AAV gene transfer. Reactivated immune mechanisms such as CD8+ T cells in turn could then cause rejection of the AAV2transduced liver cells. Subsequent studies indeed showed that ~60% of human children or adults carry AAV capsid–specific CD8+ memory T cells.2 Initial attempts to recapitulate the clinical finding in mice failed. In four independent studies,6–9 AAV capsid–specific CD8+ T cells did not succeed in eliminating AAV-transduced hepatocytes in vivo. In human subjects, AAV capsid–specific CD8+ T  cells were shown to belong to the memory subset.2 All of the above cited studies tested AAV capsid–specific effector CD8+ T cells. CD8+ T cells undergo finely tuned differentiation steps after their exposure to antigen8 and one could argue that CD8+ T cells may need to mature and differentiate into memory cells before they are able to expand upon AAV gene transfer to levels that suffice to efficiently lyse AAV-transduced cells. We therefore readdressed whether AAV capsid–specific immune responses may eliminate AAV-transduced hepatocytes using a mouse model, in which animals had AAV-specific memory T and B cells. Our results ­conclusively show a reduction of human (h)F.IX expression in mice that receive a hepatic transfer of AAV2-hF.IX several months after having been immunized against a heterologous clade of AAV. This reduction was primarily caused by AAV-binding antibodies and required neither AAV-neutralizing antibodies, CD8+ T cells, natural killer (NK) cells, or natural killer T (NKT) cells.

Results Properties of AAV capsid–specific effector and memory CD8+ T cells induced by vaccination with an adenovirus vector We and others reported previously that AAV capsid–specific effector CD8+ T cells do not affect hepatic AAV gene transfer in mice.6–9 We repeated these studies in mice that carried memory CD8+ T cells, which may differ phenotypically and functionally from ­effector CD8+ T cells. To show such differences, groups of three mice were immunized with adenovirus (Ad) vector expressing AAV8 capsid (Ad-AAV8cap). Ad vectors were given intramuscularly at 1011 virus particles per mouse for all of the

Correspondence: Hildegund CJ Ertl, Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected] Molecular Therapy vol. 17 no. 7, 1215–1224 july 2009

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experiments described in this article. Mice were killed 9 days or 3 months after immunization and splenocytes were tested for production of cytokines (interferon-γ, tumor necrosis ­factor (TNF)-α, or interleukin-2) in response to the immunodominant epitope of AAV capsid. Figure  1a shows the distributions of effector and memory CD8+ T cells producing one, two, or all three of the cytokines. CD8+ T cells that produced interferon-γ and TNF-α or TNF-α only were more frequent in the memory CD8+ T-cell population, while CD8+ T cells producing all three cytokines or interleukin-2 only were more frequent in the effector cell population. Additional differences were revealed upon staining of the CD8+ T cells with an AAV capsid epitope–specific major ­histocompatibility complex (MHC) class I tetramer and antibodies to differentiation/activation markers (Figure 1b). Memory CD8+ T cells expressed lower levels of CD44, PD-1, Ki67, and granzyme

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Figure 1  Functional properties of effector and memory CD8+ T cells specific for AAV capsid. Groups of three BALB/c mice were immunized i.m. with 1 × 1011 vp/mouse of Ad-AAV8cap. Nine days (effector cells) or 3 months (memory cells) after immunization, mice were killed and splenocytes were isolated. (a) Splenocytes were tested for secretion of IFN-γ, TNF-α, or IL-2 by intracellular cytokine staining of CD8+ T cells that had been stimulated with the peptide of AAV capsid for 5 hours in presence of Brefeldin. The pie charts show the relative distributions of CD8+ T cells that secreted one, two or three cytokines. (b) Splenocytes were stained for CD8 and the T-cell receptor with a tetramer. In addition, they were stained with antibodies carrying different fluorochromes to a number of differentiation markers. Cells were analyzed by multicolor flow cytometry. Data were analyzed by FlowJo and the graph shows the MGFI for the different markers. Both sets of graphs show the mean ± SD in b for splenocytes analyzed from three individual mice per group. AAV, adeno-associated virus; Ad, adenovirus; IFN, interferon; IL, interleukin; i.m., intramuscularly; MGFI, mean geometric fluorescent intensity; TNF, tumor necrosis factor; vp, virus particle.

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B. They expressed higher levels of CD127 although this did not reach statistical significance (P = 0.051 by t-test). Levels of CD62L were comparable. CD62L expression is in general increased once CD8+ T cells transit to central memory. The persistence of Ad vector delays upregulation of CD62L as we reported previously.10 Results obtained with CD8+ T cells from mice immunized with an Ad-AAV2cap vector were comparable (data not shown).

Pre-existing AAV capsid–specific immunity affects AAV2-mediated transgene expression To assess the role of memory immune responses on AAV-mediated gene transfer, mice were immunized with an Ad-AAV8cap vector to induce AAV-specific immunity, or as a control with an Ad vector expressing green fluorescent protein (Ad-GFP). Other groups were injected with an Ad vector expressing AAV2 capsid (Ad-AAV2cap) or the Ad-GFP control vector. After 3 months, after Ad vector– induced immune responses had differentiated into memory, Ad-AAV8cap immunized mice were infused with 2 × 1011 vector genomes/mouse of an AAV2 vector expressing hF.IX (AAV2-hF. IX) while Ad-AAV2cap immunized mice received the same dose of an AAV8 vector expressing hF.IX (AAV8-hF.­IX). In both AAV constructs, hF.IX expression was controlled by a hepatocyte-specific promoter. As we reported previously,7,11 CD8+ T-cell epitopes are conserved between the capsid antigens of AAV2 and AAV8 while binding sites for neutralizing antibodies differ, thus preventing antibody-mediated neutralizing of the gene transfer vehicle in mice immunized to the capsid antigens of a heterologous clade of AAV, while allowing for crossreactive T-cell activity. To ensure that antibodies to AAV induced by the Ad-AAV8cap or Ad-AAV2cap vectors given several months earlier still failed to neutralize the heterologous AAV vector upon additional affinity maturation, we conducted a series of neutralizing studies, which all conclusively showed that sera from mice immunized 3–8 months earlier with Ad-AAV2cap neutralized AAV2 but not AAV8, while sera from mice immunized with Ad-AAV8cap neutralized AAV8 but not AAV2 (data are shown in legend to Figure 2). We also tested mice shortly before gene transfer for frequencies of AAVcap-specific CD8+ T cells and such CD8+ T cells could be detected in blood at frequencies of ~1%. After AAV2-hF.IX or AAV8-hF.IX transfer, mice were monitored for hF.IX expression. Plasma levels of hF.IX upon ­AAV2-hF.­IX transfer into AAV8 capsid–immune mice were significantly lower at all time points tested than those achieved in Ad-GFP immunized control mice (Figure 2a). In contrast, pre-existing immunity to AAV2 did not consistently affect levels of hF.IX expression upon transfer of the AAV8-hF.IX vector (Figure 2b), showing a significant difference only at some time points. Furthermore, this difference was less pronounced than that seen in AAV8-immune mice, which received the AAV2-hF.IX vector. This experiment was conducted repeatedly with >14 mice per group and with similar results. Levels of AAV2-mediated hF.IX expression in AAV8-immune mice were partially rescued by increasing the dose of the AAV2-hF.X vector to 1 × 1012 vector genomes/mouse (Figure 2c). Notwithstanding, even at this high dose, hF.IX levels in Ad-AAV8cap immunized mice were still lower than in Ad-GFP immunized control mice. www.moleculartherapy.org vol. 17 no. 7 july 2009

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Figure 2  Pre-existing immunity to AAV capsid affects transgene product expression upon AAV2- but not AAV8-mediated gene transfer. (a) BALB/c mice were i.m. immunized with 1 × 1011 vp/mouse of Ad-GFP (dash lines) or Ad-AAV8cap (solid lines). After 3 months, AAV-specific CD8+ T cells were tested from blood of Ad-AAV8cap immunized mice and the average frequencies were 1.46% (AAV-specific CD8+ T cells over all CD8+ T cells). Shortly thereafter, all mice received 2 × 1011 vg of AAV2-hF.IX by tail-vein injection and circulating hF.IX levels were monitored. Each line represents the hF.IX level in one individual mouse. hF.IX levels were significantly different between the two groups at all the timepoints. (b) Similar experiments were performed as in a. Mice were preimmunized with 1 × 1011 vp/mouse of Ad-GFP or Ad-AAV2cap. After 4 months, frequencies of circulating AAV2 capsid–specific CD8+ T cells were 1.39%. Mice received 2 × 1011 vg of AAV8-hF.IX given i.v. Circulating hF.IX levels were analyzed. For panels a and b, one out of three independent experiments (total 14 mice for each group) with similar results is shown here. (c) Mice were immunized with 1 × 1011 vp/mouse of Ad-GFP or Ad-AAV8cap. After 4 months, half of the mice received 1 × 1012 vg of AAV2-hF.IX and the other half received 2 × 1011 vg of AAV2-hF.IX. Circulating hF.IX levels are shown at different time points after the gene transfer. Error bars represent SEM for five mice per group. (a,b) Mice were tested for neutralizing antibodies. Plasma from mice immunized with Ad-AAV2capsid neutralized AAV2 at ­dilutions of up to 1:640. They did not neutralize AAV8 even when the assay was conducted with undiluted plasma. Similarly plasma from Ad-AAV8cap immunized mice neutralized AAV8 at dilutions of >1:640 but failed to neutralize AAV2 even when the assay was conducted with undiluted plasma. AAV, adeno-associated virus; Ad, adenovirus; GFP, green fluorescent protein; hF.IX, human factor IX; i.m., intramuscularly; i.v., intravenously; vg, vector genomes; vp, virus particle.

Pre-existing immunity to AAV capsid affects levels of transgene To ensure that the differences in hF.IX expression observed in AAV-immune mice related to loss of vector rather than other mechanisms, such as downregulation of the promoter driving expression of the transgene or differential targeting of vector to cells other than hepatocytes, we analyzed relative hF.IX gene copy numbers as a correlate for AAV vector presence in the liver of AAV capsid–immune and control mice at different times after injection of AAV2-hF.IX or AAV8-hF.IX vectors. At 24 hours and 3 days after transfer of AAV2-hF.IX or AAV8-hF.IX vector, levels of hF.IX gene copies were comparable in livers of AAV-immune and control mice (Figure 3a,b). By day 7, hF.IX gene copy numbers started to show a decline in AAV8-immune mice which received AAV2-hF.IX. In these mice, hF.IX gene copy numbers continued to decline gradually and reached baseline levels by day 60 after gene transfer (Figure 3a). AAV2 immune mice that received AAV8-hF.IX showed a slight but statistically significant Molecular Therapy vol. 17 no. 7 july 2009

reduction of hF.IX gene copy numbers by day 7. This reduction was transient and could no longer be detected at later time points (Figure 3b). Neutralizing antibodies to AAV capsid are known to reduce gene transfer.12 To determine whether the kinetics of the ­reduction of hF.IX gene copy numbers in mice preimmune to a heterologous clade of AAV contrasted to that caused by neutralizing antibodies in mice preimmune to the homologous clade, we immunized mice with Ad-AAV2cap, Ad-AAV8cap, or Ad-GFP vectors before transfer of the homologous vector. Upon injection of the homologous AAV-hF.IX vector, genome copies of hF.IX in liver were already reduced by day 1 after AAV gene transfer and became undetectable by day 7 (Figure 3c,d). The initial reduction on day 1 was more pronounced in ­recipients of the AAV8-hF.IX vectors, which may relate to ­differences in ­pre-AAV transfer–neutralizing antibody titers or antibody affinities. Overall these data show a clear ­difference in the ­kinetics of AAV vector decline caused by clade-specific 1217

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Figure 3  Pre-existing immunity to AAV capsid affects levels of transgene. (a,b) Mice were intramuscularly immunized with Ad-GFP, Ad-AAV8cap or Ad-AAV2cap at 1 × 1011 vp/mouse. After 3–4 months, AAV8-immune and control mice received AAV2-hF.IX gene transfer at 2 × 1011 vg/mouse and AAV2 and control mice received the same dose of AAV8-hF.IX. Mice were killed at different time points after the gene transfer and livers were isolated. hF.IX genome copy numbers were determined. Error bars represent SEM for five mice per group. (c,d) Mice were intramuscularly injected with Ad-AAV2cap or Ad-AAV8cap at 1 × 1011 vp/mouse to induce specific neutralizing antibodies against AAV2 or AAV8 capsid. After 24 days, mice received the homologous clade of AAV expressing hF.IX at 2 × 1011 vg/mouse. Control mice were preimmunized with 1 × 1011 vp/mouse of Ad-GFP before gene transfer. Liver hF.IX genome copy numbers were analyzed 1, 7, and 14 days after the gene transfer. Error bars show SEM. AAV, adenoassociated virus; Ad, adenovirus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; hF.IX, human factor IX; vg, vector genomes; vp, virus particle.

neutralizing antibodies as compared to ­pre-existing immunity to a ­heterologous clade.

containing AAV-specific CD8+ T cells were given 24 hours after AAV2-hF.IX gene ­transfer (Figure 4d–f).

The effect of adoptive transfer of plasma or CD8+ T cells from AAV8-immune mice on AAV2-mediated hF.IX gene expression We next determined by adoptive transfer whether CD8+ T cells were causative for a loss of AAV2-mediated hF.IX gene copy numbers in Ad-AAV8cap immune mice. To this end, we transferred 1  × 107 purified CD8+ T cells from mice that had been immunized 3 months earlier with Ad-AAV8cap or Ad-GFP vectors into recipients that received AAV2-hF.IX 24 hours later (Figure  4a). Transfer of purified CD8+ T cells resulted in a slight (~25%) decrease in hF.IX expression in plasma (Figure 4c) that was well below the reduction seen in immune mice. To further determine whether immune mechanisms other than CD8+ T cells contributed to loss of hF.IX gene copies, we injected recipient mice with plasma from donor mice that had been immunized 3–4 months ago with Ad-AAV8cap; control mice received plasma from Ad-GFP-immune mice. One day after the passive immunization, mice were injected with AAV2-hF.IX and serum hF.IX levels were monitored (Figure 4a). Mice that received AAV8-immune plasma 24 hours before AAV2-hF.IX gene transfer showed a strong reduction in hF.IX expression compared to control mice that received plasma from Ad-GFP-immune mice (Figure 4b). Such a reduction was not observed if AAV8-immune plasma or splenocytes

Antibodies to AAV8 capsid bind to AAV2 The Ad-AAV8cap-induced plasma preparations that reduced hF.IX expression upon AAV2-hF.IX transfer did not have detectable levels of neutralizing antibodies to AAV2 even when tested undiluted, and vice versa, plasma from mice immunized with Ad-AAV2cap lacked neutralizing antibodies to AAV8. To test whether the plasma preparations had antibodies that could bind the heterologous AAV clade, we conducted enzyme-linked immunosorbent assays on plates coated with AAV vectors. Plasma from Ad-AAV8cap immune mice showed low but detectable binding to AAV2 (Figure 5a), while plasma from mice immunized with AAV2 lacked antibodies that bound to AAV8 (Figure  5b). The presence of non-neutralizing binding antibodies thus mirrored the pattern of reduction of AAV2-mediated hF.IX expression in AAV8-immune mice. Both plasmas from Ad-AAV8cap and Ad-AAV2cap-immune mice showed high titers of antibodies that bound to the homologous AAV vectors (Figure 5c,d). The antibody isotypes were determined by enzyme-linked immunosorbent assay on AAV-coated plates. Ad-AAVcapspecific antibodies that bound to the homologous virus were mainly of IgG isotypes with IgG2a predominating (Figure 5e,f). Ad-AAV8cap-induced antibodies that bound to AAV2 were of the IgG2a isotype.

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Figure 4 The effect of adoptive transfer of plasma or CD8+ T cells from AAV8-immune mice on AAV2-mediated hF.IX gene expression. (a–c) Blood, liver, and spleens were harvested from BALB/c mice immunized for 3.5 months with Ad-GFP or Ad-AAV8cap. CD8+ T cells were purified from pooled lymphocytes of spleen, blood, and liver. Plasma was preserved from blood. Mice were i.p. injected with 1 ml of plasma or i.v. with 1 × 107 CD8+ T cells and then received the AAV2-hF.IX at 2 × 1011 vg/mouse by tain vein injection 24 hours later. Circulating hF.IX levels were monitored. (d–f) Mice first received the AAV2-hF.IX gene transfer at 2 × 1011 vg/mouse. After 24 hours, mice were injected i.p. with 1-ml plasma or i.v. with 8 × 107 splenocytes from mice immunized for 3.5 months with Ad-GFP or Ad-AAV8cap. Circulating hF.IX levels were tested. (a–f) Each line represents the hF.IX level in one individual mouse. AAV, adeno-associated virus; Ad, adenovirus; GFP, green fluorescent protein; hF.IX, human factor IX; i.p., ­intraperitoneally; i.v., intravenously; vg, vector genomes.

Pre-existing immunity to AAV8 capsid does not affect AAV2-mediated hF.IX expression in B-cell deficient mice To further ensure that the effect of AAV8-immune plasma on AAV2 gene transfer was mediated by antibodies, B-cell deficient mice were immunized with Ad-AAV8cap or Ad-GFP 3 months before AAV2-hF.IX gene transfer. To ensure that B-cell deficient mice developed T-cell responses to the Ad-AAV8cap vector, mice were bled shortly before the AAV2-hF.IX gene transfer and frequencies of AAVcap-specific CD8+ T cells were determined and found to be comparable to those in wild-type mice. Circulating hF.IX levels and hF.IX gene copy numbers were comparable in AAV-immune and control B-cell deficient mice (Figure  6a,b) confirming that the effect observed in normal AAV-immune mice depended on antibodies. It should be pointed out here that B-cell deficient mice showed very poor transduction by AAV2, suggesting that immunoglobulins may promote AAV-mediated gene transfer through mechanisms that remain to be elucidated.

requires NK cells. To further analyze the mechanisms of the antibody-mediated clearance of AAV vectors, we depleted NK cells from mice immunized for 3 months with Ad-AAV8cap or Ad-GFP, before AAV2-hF.IX gene transfer by injection of an anti-asialo GM1 antibody. Depletion of NK cells did not rescue the expression of AAV2-mediated hF.IX and the liver hF.IX gene copy number in Ad-AAV8cap immune mice (Figure 7a,b). The NK depleting antibodies do not affect NKT cells, which are common in liver.13 We therefore conducted additional experiments in RAG−/− mice, which lack B, T, and NKT cells. RAG−/− mice were transfused with plasma from Ad-AAV8 or Ad-GFP immune mice and then 24 hours later were injected with AAV2-hF.IX. RAG−/− recipients of AAV-immune plasma developed reduced levels of circulating hF.IX (Figure 6c), indicating (together with data obtained in NK-depleted mice) that antibody-dependent cell-mediated cytotoxicity by NK cells or NKT cells does not contribute to loss of hF.IX gene copy numbers.

The effect of AAV8cap-specific immunity does not require NK or NKT cells Antibodies can reduce viral loads not only by neutralization, but also by antibody-dependent cell-mediated cytotoxicity, which

AAV-mediated gene transfer to liver has brought about long-lasting gene expression and phenotypic cures in small and large animal models for a wide range of disorders,14–18 but successful translation to the clinic has been stymied by an apparent immune response in

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Discussion

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Figure 5  Antibodies to AAV8 capsid bind to AAV2. Plasma from mice intramuscularly immunized with 1 × 1011 vp Ad-GFP, Ad-AAV2cap or Ad-AAV8cap 3–5 months ago were harvested and tested for (a,c) binding to AAV2 or (b,d) binding to AAV8 by an ELISA. Each circle or square ­represents the plasma from one individual mouse. (e,f) Antibody isotypes were also determined by ELISA. AAV, adeno-associated virus; ELISA, enzymelinked immunosorbent assay, GFP, green fluorescent protein; OD, optical density; vp, virus particle.

humans, who generally encounter AAV during childhood. Clinical studies showed that humans carry AAV capsid–specific CD8+ T cells,2 which expand upon AAV gene transfer to liver or muscle. It was thus assumed that these CD8+ T cells present in humans but not other species interfere with sustained transgene expression upon AAV-mediated gene transfer to peripheral sites. Although this hypothesis was compatible with the clinical data, which showed a slow decline of F.IX expression accompanied by a transaminitis1 and a rise in circulating AAV capsid–specific CD8+ T cells,2 skepticism on its validity lingered for the following reasons:

1. In patients, decline of hF.IX expression occurred with a delay of several weeks, and it was argued that by that time epitopes from the capsid of the AAV vectors should have declined to levels that no longer permitted recognition of transduced hepatocytes by CD8+ T cells. 2. Nonhuman primates commonly have pre-existing AAV capsid–specific CD8+ T cells (H. Li and H.C.J. Ertl, unpublished results); nevertheless in this species hepatic AAV gene transfer resulted in sustained transgene expression. 1220

3. Initial attempts to recapitulate loss of transgene expression in mice with pre-existing CD8+ T cells to AAV ­capsid failed. Validation or rebuttal of the hypothesis that AAV-specific CD8+ T cells interfere with AAV-mediated gene transfer continues to be of utmost importance, as it affects the design of future AAV gene replacement trials, some of which have now been designed to combine gene transfer with a short regimen of immunosuppression that aims to prevent expansion of T cells. Data presented here confirm that immunological memory to AAV capsid can impact the efficacy of transgene product expression upon hepatic transfer of an AAV2 vector into mice. The gradual loss of AAV vector appears to be caused primarily by binding antibodies that partially crossreact between different serotypes. In our experimental design, antibodies induced against AAV8 capsid bound AAV2 and affected AAV2–mediated gene transfer, while antibodies to AAV2 capsid failed to bind AAV8 and accordingly failed to affect AAV8-mediated gene transfer. One could envision several pathways by which non-neutralizing antibodies could www.moleculartherapy.org vol. 17 no. 7 july 2009

© The American Society of Gene Therapy

a

Ad-GFP + AAV2-hF.IX

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Relative copies of hF.IX in liver/ 100 GAPDH copies

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15 10 5 0

.IX -hF

-GF

Ad

.IX -hF

2 AV

2 AV

A P+

+A V8 -AA

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Figure 6  Pre-existing immunity to AAV8 capsid does not affect AAV2-mediated hF.IX expression in B-cell deficient mice. B-cell ­deficient BALB/c mice were first i.m. immunized with Ad-GFP or Ad-AAV8cap at 1 × 1011 vp/mouse. After 3 months, mice received 2 × 1011 vg of AAV2‑hF.­IX by tail-vein injection. (a) Circulating hF.IX levels were ­monitored. Each line represents the hF.IX level in one individual mouse. AAV-specific CD8+ T cells were tested from blood of Ad-AAV8cap immunized B cell deficient mice by intracellular cytokine staining and the ­frequency was 1.2% (AAV-specific CD8+ T cells over all CD8+ T  cells), which is comparable to frequencies in wild-type mice which typically range from 1 to 2% at 3 months after immunization. (b) Mice were killed 82 days after the gene transfer and hF.IX copy numbers in mice liver were tested. AAV,  adeno-associated virus; Ad, adenovirus; GFP, green fluorescent ­protein; hF.IX, human factor IX; i.m., intramuscularly; vg, vector genomes; vp, virus particle.

impede gene transfer. It is feasible that in vitro neutralization assays do not accurately mimic neutralization in vivo and thus the observed reduction of AAV2-mediated hF.IX expression reflected neutralization by crossreactive antibodies. Our finding that passive transfer of AAV8 immune plasma only affected AAV2-hF.­IX gene transfer if mice were passively immunized before gene transfer, while passive immunization after gene transfer was ineffective supports this assumption. The observed reduction of hF.IX expression in AAV8-immune mice required neither NK cells nor NKT cells, which could have acted in concert with AAV-binding antibodies, again supporting the notion that the antibodies primarily may have prevented AAV uptake rather than affecting lysis of already transduced cells. Other results argue against antibodymediated neutralization as the culprit for loss of hF.IX gene copy numbers in AAV8-immune mice that received AAV2-hF.IX vector. Most notably, kinetics of loss of hF.IX gene copy numbers in liver of mice that had been immunized with a heterologous AAV differed from those of mice that, due to immunization against the homologous AAV capsid, carried AAV-neutralizing antibodies. Molecular Therapy vol. 17 no. 7 july 2009

In mice with neutralizing antibodies to AAV capsid, AAV2, or AAV8-hF.IX, gene copies were reduced in liver as of day 1 after gene transfer, suggesting that the neutralizing antibodies had affected retargeting of the vector. By day 7 after gene transfer, AAV-neutralizing antibodies caused a near complete clearance of the homologous AAV vector. In contrast, in AAV8-immune mice, levels of AAV2 vector in liver did not show a significant decline till day 14 after gene transfer and then declined further by month 2, arguing against direct extracellular neutralization. It is feasible that transduced hepatocytes were eliminated by antibody-mediated complement-dependent cytolysis. This would require presence of B-cell epitopes on the surface of the transduced cells. AAV vectors are taken up by endocytosis and one would thus not expect that capsid antigens would remain for a prolonged period of time on the cell surface. Synthesis of AAV capsid antigens through AAV vectors that inadvertently packaged the capsid genome could also result in expression of AAV capsid antigens on the cell surface. We do not favor this explanation, and in fact previous studies showed that our method of vector preparation does not generate AAV vectors that encapsidate the cap encoding genome. One could make a case that binding antibodies retargeted the AAV vector to cells within the liver other than hepatocytes.19,20 This would not have been detected in our molecular assays, as gene copy numbers were tested from whole liver rather than from cell subsets. Nevertheless, hF.IX levels upon AAV2-hF.IX gene transfer were comparable on day 7 between AAV8-immune mice and control mice. As hF.IX expression in our vectors is controlled by a hepatocyte-specific promoter, retargeting of vector to cells other than hepatocytes should have resulted in an immediate rather than a delayed ­reduction in hF.IX expression. We failed to observe a reduction of hF.IX expression in our previous publication, which described a series of results in mice that were immunized to AAV capsid with an Ad vector shortly before hepatic transfer of an AAV-hF.IX vector.7 In these experiments, mice were injected with AAV-hF.IX vector at the peak of the Ad-induced CD8+ T-cell response. Antibody responses to the transgene product of an Ad vector do not reach maximal titers till later.21,22 Furthermore, B cells continue after the initial ­effector phase to differentiate causing isotype switching and affinity maturation of antibodies.23 It is thus not surprising that the initial studies failed to observe an effect of capsid-specific antibodies on AAV-mediated gene transfer. Our results do not conclusively rule out that CD8+ T cells contributed to loss of hF.IX gene copies in the clinical trial. In our studies, adoptive transfer of AAV capsid–specific CD8+ T cells resulted in a slight decrease of hF.IX expression that was by no means as severe as that seen in the clinical trial, where circulating levels of F.IX eventually declined to zero.1 Humans develop CD8+ T cell responses to natural infection with AAV and a helper virus such as an Ad. Infections most likely occur through mucosal routes. AAV capsid–specific CD8+ T cells in humans are presumably repeatedly restimulated by recurrent infections with different serotypes of AAV or by reactivation of persisting virus. Functionality of CD8+ T cells is dependent on route of induction and on immunization history, i.e., CD8+ T cells that encounter their antigen repeatedly develop distinct phenotypes and cytokine secretion patterns compared to those that only encounter their 1221

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Immunity to AAV Capsid

b

28 days after gene transfer 2,000

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Relative copies of hF.IX in liver/ 100 GAPDH copies

Circulating hF.IX level (ng/ml)

a

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1,500 1,000 500 0

1) 1) S) S) GM GM NR (NR (a(aX( X I X I . X I . I F . F. -h -hF -hF V2 2-h V2 V2 AA AV AA AA +A 8+ P+ 8+ V P F V A F -G -A -G -AA Ad Ad Ad Ad

c

28 days after gene transfer 50

P < 0.01

40 30 20 10 0

1) 1) S) S) GM GM (NR (NR (a(a.IX .IX .IX .IX F F -hF -hF h h 2 2 V V 2V2 AA AA AV AA +A 8+ P+ 8+ AV FP AV -GF A G d A d A A Ad Ad

Ad-GFP plasma + AAV2-hF.IX

P < 0.05

Ad-AAV8 plasma + AAV2-hF.IX

Circulating hF.IX level (ng/ml)

2,000

P < 0.01

*

* *

1,500

1,000

500

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20 40 60 80 Days after AAV2-hF.IX gene transfer

100

Figure 7 The effect of AAV8cap-specific immunity does not require NK or NKT cells. Mice were i.m. immunized with 1 × 1011 vg of Ad-GFP or Ad-AAV8cap. After 3 months, mice were injected i.p. with anti-asialo GM1 (a-GM1) or normal rabbit serum (NRS) every 4 days. AAV2-hF.IX gene transfer (2 × 1011 vg/mouse) was performed 2 days after the first dose of antibody by tail-vein injection. (a) Circulating hF.IX level and (b) liver hF.IX copy numbers were tested 28 days after the gene transfer. (c) RAG−/− mice were injected i.p. with 1-ml plasma harvested from mice immunized for 9  months with Ad-GFP or Ad-AAV8cap. Mice were infused with 2 × 1011 vg/mouse of AAV2-hF.IX 24 hours later and circulating hF.IX levels were tested. Each line represents the hF.IX level in one individual mouse. AAV, adeno-associated virus; Ad, adenovirus; GFP, green fluorescent protein; hF.IX, human factor IX; i.p., intraperitoneally; NK, natural killer cells; NKT, natural killer T cells; vg, vector genomes.

antigen once.24,25 CD8+ T cells induced in this study by a single systemic immunization of mice with an Ad vector may thus have distinct functional properties from those in humans, which may affect their ability to recognize and eliminate AAV-transduced hepatocytes. Our results point to additional immune mechanisms that need to be considered in AAV gene transfer trials. Such trials typically assess pretransfer neutralizing antibody titers to the AAV vector used for treatment, as such antibodies are known to impact AAV transduction rates. Here, we show that antibodies that can bind AAV without causing neutralization can result in loss of AAV vectors upon hepatic gene transfer. This loss occurs with a delay, which is shorter than that seen in the clinical trial but nevertheless precludes that the antibodies operate by preventing transduction of hepatocytes. We assume that the antibodies act intracellularly by interfering with the processing of AAV particles. Clinical trials for AAV-mediated gene transfer now routinely measure pre- and post-treatment CD8+ T-cell frequencies to AAV capsid26 and some trials transiently immunosuppress AAV gene transfer recipients to prevent a reactivation and expansion of such CD8+ T cells.27 Results presented here suggest that AAV gene transfer recipients may also need to be monitored for AAV-binding antibodies, which would not be affected by immunosuppression 1222

but may require alternative interventions such as plasmapheresis before injection of the AAV vector.

Materials And Methods Animals. Six- to eight-week-old male BALB/c mice were purchased from

ACE Animals (Boyertown, PA). BALB/c RAG–/– mice were purchased from The Jackson Laboratory (Bar Harbor, ME). The B-cell deficient BALB/c mice, which have a deletion of the endogenous murine J segments of the Ig heavy-chain locus and have no detectable IgM or IgG in their sera, were purchased from Taconic (Hudson, NY). All mice were housed at the Wistar Institute Animal Facility. Animals were treated according to the institutional rules for animal welfare.

Ad and AAV vectors. E1- and E3-deleted Ad human serotype 5 vectors

expressing capsid antigens of AAV2 or AAV8 or enhanced GFP under a cytomegalovirus promoter were grown on HEK 293 cells, purified by CsCl density–gradient centrifugation followed by column purification (Bio-Gel P-6DG; Bio-Rad, Hercules, CA). Vectors were diluted in phosphate buffered saline (PBS) supplemented with 10% glycerol and stored at −80 °C. Content of virus particles was determined by spectrophotometry at 260 and 280 nm. Viral titer (virus particles) was determined using the formula: OD260 × ­dilution × 1.1 × 1012. Vectors were quality controlled for contamination with endotoxin and replication competent Ad. Vectors used in these studies contained neither. AAV vectors were produced by the triple transfection method into HEK 293 cells and purified by CsCl density–gradient purification. AAV2 and AAV8 vectors expressing hF.IX have been described previously.7 www.moleculartherapy.org vol. 17 no. 7 july 2009

© The American Society of Gene Therapy

Animal procedures. Mice were injected intramuscularly into the upper leg muscles with 1011 virus particles of Ad vectors in 100 µl of PBS. Each leg received 50 µl. AAV vectors diluted in 200 µl of PBS were given at 2 × 1011 vector genomes per mouse by tail-vein injection unless stated otherwise. Mice were bled by retro-orbital puncture using heparinized capillaries. Intracellular cytokine staining. Lymphocytes (1 × 106) were stimulated with 5 μg/ml of AAV2 (VPQYGYLTL) or AAV8 (IPQYGYLTL) peptides or nonspecific peptides in complete Dulbecco’s modified Eagle’s medium with 50 U/ml mouse recombinant interleukin-2 (Roche, Mannheim, Germany) and 1 μl/ml Golgi plug (BD Pharmingen, San Diego, CA) in 96-well plates. After 5 hours of incubation at 37 °C/10% CO2, the cells were washed and stained with a PercP-Cy5.5-labeled antibody to CD8. Cells were then fixed and permeabilized using the Cytofix/Cytoperm (BD Pharmingen), and intracellular staining was performed using fluorescein ­isothiocyanate-labeled anti-mouse interferon-γ antibody, allophycocyaninlabeled interleukin-2 antibody, and phycoerythrin-Cy7-labeled TNF-α antibody. Phycoerythrin-Cy7-labeled TNF-α antibody was purchased from eBioscience (San Diego, CA) and all other antibodies were purchased from BD Pharmingen. The cells were washed and resuspended in PBS and analyzed by flow cytometry. Data were analyzed by FlowJo software (Tree Star, Ashland, OR). Tetramer and phenotypic marker analyses. Cells were stained with an allophycocyanin-conjugated AAV2 VPQYGYLTL peptide H-2Ld tetramer (NIH Tetramer Facility, Atlanta, GA) and antibodies to the surface markers CD8, CD44, CD127, CD62L, and PD-1. The antibodies were purchased from BD Pharmingen. For staining of intracellular markers, cells were first permealized with Cytofix/Cytoperm. Antibodies for the intracellular markers granzyme B and Ki67 were purchased from eBioscience and BD Pharmingen, respectively. Before analysis, cells were fixed with 2% paraformaldehyde in PBS and analyzed by flow cytometry. Preparation and adoptive transfer of plasma. Mouse blood was spun down for 10 minutes at 8,000 rpm at 4 °C and plasma was collected. For the adoptive transfer of plasma, each mouse was injected intraperitoneally with 1 ml of plasma pooled from several mice. Enzyme-linked immunosorbent assay for hF.IX. Nunc 96-well plates were

coated with monoclonal antihuman F.IX antibody (Sigma, St Louis, MO) diluted 1:600 in 0.1 mol/l carbonate coating buffer (pH 9.6). Plates were washed with 0.05% Tween-20/PBS, blocked with 1% bovine serum albumin, 0.05% Tween-20 in PBS, incubated with diluted mouse plasma samples. Plates were then washed and treated with a 1:500 dilution of horseradish peroxidase–conjugated goat antihuman F.IX antibody (Enzyme Research Laboratories, South Bend, IN). Levels of human F.IX were determined in an enzyme-linked immunosorbent assay reader at OD450 after adding 100 µl tetramethylbenzidine substrate and quantified against the linear standard curve generated with serially diluted normal human plasma. Antibody binding assay and subisotype analyses. Nunc 96-well plates were coated with 100 µl of AAV2-empty or AAV8-empty vectors diluted to 5 × 1011 vector genomes/ml in PBS. Plates were washed with 0.05% surfactant/PBS and blocked with serum. Mouse plasma was serially diluted and 50 µl of the diluted plasma was added into the plates. After 1-hour incubation, plates were washed. Then 100-µl alkaline phosphatase–­ conjugated goat affinity-purified antibody to mouse immunoglobulins (ICN Pharmaceuticals/Cappel, Aurora, OH) diluted to 1:200 was added to the plates. Plates were incubated 1 hour at room temperature and washed again. Substrate was added to the plates, which were then read at a wavelength of 405 nm. To analyze antibody isotypes, mouse plasma was diluted to 1:200 with saline and tested with the Mouse Hybridoma Subisotyping Kit purchased from Calbiochem (cat. no. 386445; La Jolla, CA). Molecular Therapy vol. 17 no. 7 july 2009

Immunity to AAV Capsid

Quantification of human F.IX in liver by real-time PCR. Genomic DNA

of mice liver tissue was extracted with the DNeasy Tissue kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions and amplified using primers for glyceraldehyde 3-phosphate dehydrogenase in a single real-time PCR. Then genomic DNA containing 1 × 106 molecules of glyceraldehyde 3-phosphate dehydrogenase was amplified again using primers specific for hF.IX to quantify the relative copy number of hF.IX DNA in liver tissues. The primers used for amplifying hF.IX were 5′-ACC AGC AGT GCC ATT TCC A-3′ and 5′-GAA TTG ACC TGG TTT GGC ATC T-3′. This experiment was controlled with liver tissues from naive mice. Data are expressed as hF.IX DNA copy numbers per 100 glyceraldehyde 3-phosphate dehydrogenase DNA copy numbers. CD8+ T cell purification by negative selection. Splenocytes were ­isolated from mice and treated with ACK Lysing Buffer (Invitrogen, Carlsbad, CA) to lyse red blood cells. Then cells were resuspended at a concentration of 2 × 108 cells/ml in PBS + 1% fetal bovine serum and incubated with the following anti-rat antibodies: anti-MHCII, MHC-B220, MHC-CD4, MHCTER119, MHC-CD19, and MHC-GR1/Ly69 (BD Biosciences, Franklin Lakes, NJ) on ice for 30 ­minutes. Cells were washed twice with PBS + 1% fetal bovine serum and then incubated with goat anti-rat IgG microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) for 15 ­minutes on ice. Cells were washed twice as above. Cells were then resuspended in PBS + 0.5% bovine serum albumin + 2 mmol/l ethylenediaminetetraacetic acid and run over ­magnetic-activated cell sorting magnetic separation ­columns (Miltenyi Biotec) following the manufacturer’s protocol. The effluent containing an enriched CD8+ T-cell population of ~90% purity was collected. NK cell depletion. Mice were injected intraperitoneally with 50 µl each of

an anti-asialo GM1 (rabbit) antibody (Wako Chemicals, Richmond, VA) diluted in 150 µl PBS. The control mice were injected the same amount of normal rabbit serum (Calbiochem) diluted in 150 µl PBS. Antibodies were injected eight times every 4 days. NK cells were analyzed from spleens of control mice after the antibody injection using the PE rat anti-mouse CD49b (BD Pharmingen). The antibody treatment resulted in depletion of ~90% of NK cells.

Statistical analyses. Significance was determined by one-tailed Student’s t-tests comparing results obtained with individual mice from one group to those obtained with individual mice from the other group. Significance was set at P = 0.05.

Acknowledgments This work was funded by the National Institute of Health grant P01HL078810, and the Wistar Cancer Center Support Grant (NCI— P30 CA 010815). KA.H. is supported by the Howard Hughes Medical Institute. We thank Christina Cole and Colin Barth for help in preparation of the manuscript. H.L, S.-W.L, W.G.-D., Y.L., D.Z., Z.Q.X, and H.C.J.E have no financial interest in any of the work presented. K.A.H. holds patents on AAV-Factor IX gene transfer, licensed to Genzyme. She has waived any financial interest in these patents.

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