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Pre-existing AAV Capsid-specific CD8+ T Cells are Unable to Eliminate AAV-transduced Hepatocytes
original article
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Pre-existing AAV Capsid-specific CD8 þ T Cells are Unable to Eliminate AAV-transduced Hepatocytes Hua Li1,4, Samuel L Murphy2,4, Wynetta Giles-Davis1, Shyrie Edmonson2,3, Zhiquan Xiang1, Yan Li1, Marcio O Lasaro1, Katherine A High2,3 and Hildegund CJ Ertl1 1 Wistar Institute, Philadelphia, Pennsylvania, USA; 2The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; 3Howard Hughes Medical Institute, Philadelphia, Pennsylvania, USA
The goal of these studies was to test whether adenoassociated virus (AAV) capsid-specific CD8 þ T cells cause loss of hepatic AAV-mediated gene expression in experimental animals. Mice immunized with adenoviral vectors expressing AAV capsid or with AAV vectors developed CD8 þ T cells in blood, lymphatic tissues, and liver to epitopes shared between AAV2 and AAV8, and serotype-specific neutralizing antibodies. At the height of the T cells’ effector phase, mice were infused with a heterologous AAV vector expressing human factor IX under a hepatocyte-specific promoter. Despite the presence of lytic CD8 þ T cells in the liver, hepatic Factor IX expression was sustained and comparable in AAV-preimmune and naı¨ve animals. These results suggest that, in mice, pre-existing CD8 þ T cells to AAV capsid do not affect the longevity of AAV-mediated hepatic gene transfer. These results are in contrast to the outcome of a recent gene therapy trial of hemophilia B patients who were treated by hepatic gene transfer of AAV2 vectors expressing Factor IX. The loss of Factor IX expression, accompanied by a rise in liver enzymes and detectable frequencies of circulating AAV capsid-specific T cells, suggested T-cell-mediated destruction of transduced hepatocytes following reactivation of AAV-specific T cells upon AAV transfer. Received 3 November 2006; accepted 29 November 2006; advance online publication, 23 January 2007. doi:10.1038/mt.sj.6300090
INTRODUCTION In a recent gene transfer trial, human subjects with severe hemophilia B were infused with recombinant adeno-associated virus (AAV) vectors derived from the human serotype 2 (AAV2) expressing factor IX (AAV2-F.IX) under the control of a hepatocyte-specific promoter for intrahepatic expression, a procedure that resulted in sustained F.IX expression in preclinical animal models of hemophilia B. One subject in the highest dose group developed therapeutic levels of F.IX by week 2, but 4 weeks after vector infusion, the levels of F.IX started to decrease and, within a few weeks, returned to pre-gene therapy
levels.1 Concomitantly, the subject developed transaminitis, which resolved after F.IX had decreased to baseline levels. The subject’s clinical course was highly suggestive of immunemediated destruction of AAV-transduced hepatocytes. The trial was continued with a reduced dose of vector and again the next subject presented with transaminitis after gene transfer with a time course relative to therapy identical to that seen in the other subject. In the second patient, T-cell responses to AAV-2 capsid were monitored before and after gene transfer. The subject had no detectable AAV2 capsid-specific T cells in his peripheral blood before gene transfer. Such a response developed after gene transfer and then eventually subsided, suggesting that indeed T-cell-mediated destruction of AAV transduced hepatocytes may have contributed to the failure of persistent gene expression. T cells to the transgene product could not be detected at any time point, indicating that the response was directed against the capsid antigens of AAV. T-cell-mediated clearance of AAV-transduced hepatocytes had not been encountered during the extensive preclinical testing of AAV2 vectors in experimental animals including rodents, canines, and nonhuman primates.2–4 Humans may have reacted differently because most are naturally exposed to AAV2 during childhood. Infections occur concomitantly with a helper virus such as an adenovirus (Ad). Humans thus have immunological memory to AAV2, unlike experimental animals. Memory T cells can be triggered more readily than naı¨ve T cells, which was not taken into account by the preclinical animal experiments conducted thus far. Here we tested whether pre-existing CD8 þ effector T cells to AAV capsid affect the duration of transgene expression following hepatic AAV-mediated human F.IX gene transfer in mice. To this end, mice were immunized against AAV capsid antigen using either E1-deleted Ad vectors expressing capsid antigens of AAV2 or AAV8 or AAV vectors expressing an immunogenic transgene product. Mice were then infused with a distinct serotype of AAV vector expressing F.IX, i.e., AAV2 capsid-immune mice received AAV8-F.IX or vice versa. As we showed previously, BALB/c mice immunized with the adenoviral vector expressing capsid developed T cells that crossreacted between epitopes from AAV2 and AAV8 (VPQYGYLTL or IPQYGYLTL).5 As they lack
Correspondence: Hildegund CJ Ertl, The Wistar Institute, 3601 Spruce St, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected] 4 These authors contributed equally to this work.
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conserved binding sites for virus neutralizing antibodies, this protocol allows us to assess the effect of pre-existing T cells on the longevity of F.IX gene transfer in animals in which transduction efficacy is not impaired by pre-existing AAV neutralizing antibodies. The results show conclusively that, in mice, pre-existing lytic CD8 þ T cells to AAV capsid fail to eliminate AAV transduced hepatocytes.
RESULTS Vectors for induction of AAV capsid-specific CD8 þ T cells Immunogens were constructed to induce immune responses to AAV capsid antigens in mice. The first set of immunogens was composed of E1 deleted Ad vectors encoding the entire sequence of AAV2 or AAV8 capsid. A second set of immunogens was composed of AAV2 and AAV8 vectors encoding a fusion protein of glycoprotein D (gD) of herpes simplex virus 1 and a truncated gag of human immunodeficiency virus 1. These constructs were used to ensure that results obtained with the Ad vectors could also be obtained with AAV vectors. The AAV vectors expressed an immunogenic viral antigen to stimulate an innate immune response which, in turn, is required for induction of adaptive immunity. All of the vectors expressed the transgene product, i.e., the AAV capsid or the herpes simplex virus 1 gD-gag fusion protein (not shown). AAV capsid-specific CD8 þ T-cell responses in mice Vectors were tested for induction of AAV capsid-specific CD8 þ T cells in groups of BALB/c mice (n ¼ 4–5). Mice were injected intramuscularly into the quadriceps with 1 1011 vector particles (VP) or vector genomes (VG) of Ad-AAV2capsid, AdAAV8capsid, AAV2-gDgag37, or AAV8-gDgag37 vectors. AAVspecific acute and memory CD8 þ T-cell responses were tested from spleens of individual mice and from pooled lymphocytes of blood, lymph nodes and livers. Frequencies of AAV capsidspecific CD8 þ T cells were measured by intracellular cytokine staining for interferon (IFN)-g (Figure 1). Immunization of mice with the Ad-AAV2capsid (Figure 1a) and Ad-AAV8capsid (Figure 1b) vectors resulted in comparable frequencies of CD8 þ T cells that, in spleens, ranged from 8 to 10% of all CD8 þ T cells at the height of the acute response and then declined to 3–5% within 8 weeks after immunization. Frequencies were higher in blood than in spleens early after immunization. Frequencies in lymph nodes were modest at both time points. The highest frequencies were observed in livers. Immunization of mice with AAV2 vectors elicited AAV capsid-specific CD8 þ T cells (Figure 1c). Frequencies were markedly lower than those induced by the Ad vectors expressing AAV2 capsid. This is not surprising, as Ad vectors have been described previously as inducing exceptionally high frequencies of transgene product-specific CD8 þ T cells when compared to other types of vectors.6 Immunization of mice with the AAV8-gDgag37 vector elicited only a marginal CD8 þ T-cell response to AAV capsid, which was detectable in spleen and liver but not in blood during the acute response (Figure 1d). To ensure that both AAV2 and AAV8 vectors were immunogenic, CD8 þ T-cell frequencies to gag were also measured (Figure 1e). Both types of vectors induced CD8 þ T Molecular Therapy vol. 15 no. 4, april 2007
AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
cells to the immunodominant epitope of gag detectable in spleens, and at higher frequencies in blood and liver by day 14 after immunization.
Crossreactivity of AAV capsid-specific CD8 þ T cells The sequences of different AAV serotypes are highly conserved, and it has been reported that, in mice of the H-2d haplotype, CD8 þ T cells to AAV2 crossreact with AAV8 capsid.5 This is to be expected as one of the H-2d epitopes of AAV2 differs only by one amino acid from the corresponding sequence of AAV8. Upon immunization with either Ad-AAV2 or Ad-AAV8 vectors, mice developed titers of neutralizing antibodies 41:100 to the homologous AAV vector, whereas no such antibodies could be detected against the heterologous vector, indicating lack of detectable cross-reactivity of AAV neutralizing antibodies (Figure 2). In contrast, CD8 þ T cells induced against AAV2 capsid showed crossreactivity with the AAV8 epitope and vice versa. The effect of pre-existing AAV-specific CD8 þ T cells on AAV-mediated gene transfer Lack of serological crossreactivity and complete cellular crossreactivity between MHC class I binding epitopes of the capsid antigen of AAV2 and AAV8 allowed us to assess the role of preexisting CD8 þ T cells in mice vaccinated to AAV2 capsid using AAV8 vectors for gene transfer and vice versa. AAV-mediated gene transfer is reduced by pre-existing AAV neutralizing antibodies, necessitating the use of different serotypes for preexposure and gene transfer. In the first set of experiments, we immunized BALB/c mice intramuscularly with Ad-AAV2 vectors or a control vector. They received AAV8-hF.IX, given intravenously (i.v.) 9 days later (Figure 3a).7 In some of the experiments, mice were bled before gene transfer, and frequencies of AAV capsid-specific CD8 þ T cells were measured from blood mononuclear cells and found to be similar to those shown in Figure 1 (not shown). Circulating human Factor IX (F.IX) levels, evaluated by enzyme-linked immunosorbent assay (ELISA) at 2, 4, and 6 weeks after AAV8 transfer, showed no significant difference in AAV pre-immunized or control mice (Figure 3b). We tested liver transaminases to determine potential liver damage caused by immune-mediated destruction of AAV transduced cells, and no increase in liver transaminase was seen in comparison to different groups (data not shown). These data indicate that, even in the presence of circulating and liver-homing AAV capsid-specific effector CD8 þ T cells, hepatic AAV-mediated gene transfer results in stable expression of the transgene product. Similar experiments were conducted by immunizing mice i.v. with Ad-AAV2 followed by AAV8-F.IX gene transfer or by immunizing mice intramuscularly with Ad-AAV8 followed by AAV2-F.IX gene transfer (Figure 3e). Under both experimental conditions, F.IX levels were sustained (in part shown in Figure 3f), and transaminase levels remained at physiological levels (not shown). Transfer of F.IX by the AAV8 vectors resulted in higher circulating levels of F.IX than transfer by AAV2 vectors. To examine whether AAV capsid-specific CD8 þ T cells were still detectable after gene transfer or whether gene transfer had 793
AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
% Of IFN-+CD8+ cells / CD8+ cells
Ad-GFP Ad-AAV2Capsid (acute)
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Ad-AAV2Capsid (memory)
20 10 0 Spleen
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AAV2 capsid-specific T-cell response 40
PBMC
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AAV8 capsid-specific T-cell response 40
Ad-GFP Ad-AAV8Capsid (acute)
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20 10 0 Spleen
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AAV2 capsid-specific T-cell response 2.5
% Of IFN-+CD8+ cells / CD8+ cells
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Liver
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AAV8 capsid-specific T-cell response 0.5
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gag-specific T-cell response 6 5 4
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3 2 1 0 Spleen
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Figure 1 AAV capsid and gag specific CD8 T-cell responses in BALB/c mice intramuscularly immunized with different experimental vectors. (a) Ad-AAV2capsid and Ad-GFP control, (b) Ad-AAV8capsid and Ad-GFP control, (c) AAV2-gDgag37, (d) AAV8-gDgag37, and (e) AAV2-gDgag37 and AAV8-gDgag37. The dose of all vectors was 1 1011 vp (vg)/mouse. Frequencies of AAV-specific and gag-specific CD8 þ T cells were determined by intracellular cytokine staining (ICS) from spleens of individual mice and pooled peripheral blood mononuclear cell (PBMC), lymph nodes and livers. The acute response induced by Ad-AAV2 and Ad-AAV8 was tested 9 days after injection. The acute response induced by AAV2-gDgag37 and AAV8gDgag37 was tested 2 weeks after injection. All memory responses were examined 2 months after immunization. The responses induced by Ad-GFP control were analyzed 9 days postinjection.
boosted the capsid-specific CD8 þ T-cell response, mice were euthanized after gene transfer and tested for CD8 þ T cells to the AAV epitopes. Robust AAV2- and AAV8-specific T-cell responses were detected in mice injected with Ad-AAV vectors followed by AAV gene transfer (Figure 3c–d, g–h). Responses were not increased in mice primed with Ad-AAV and treated with AAV gene transfer when compared to mice injected with Ad-AAV, indicating that Ad-AAV-induced CD8 þ T cells were not boosted by the AAV vector used for gene transfer. The argument has been made that the epitopes that are recognized on AAV capsid as expressed by an Ad vector, are not efficiently recognized on capsid antigen expressed by AAV particles.8 We therefore repeated the gene transfer experiments in mice immunized with AAV2-gDgag37 (Figure 3i). Again, induction of AAV-specific CD8 þ T cells was confirmed and, upon testing for AAV neutralizing antibodies, mice were shown 794
to have such antibodies to AAV2 but not to AAV8 (not shown). Mice were then injected with AAV8-F.IX vector given i.v, and F.IX expression levels were tested by ELISA at 2, 4, 6, and 8 weeks after gene transfer (Figure 3j). Persistent and comparable levels of F.IX were detected in both AAV pre-exposed and control mice.
CD4 þ CD25 þ regulatory T-cell depletion does not change AAV-mediated F.IX expression level in mice with pre-existing T-cell immunity to AAV Lack of elimination of AAV-transduced hepatocytes by AAV capsid-specific CD8 þ T cells may reflect that T cells are impaired because of the activity of negative immunoregulatory mechanisms. CD4 þ CD25 þ Foxp3 þ regulatory T cells (Tregs) have been shown to play a role in AAV-mediated gene transfer.9,10 To determine if Tregs contribute to sustained AAV-mediated gene transfer in mice with pre-existing CD8 þ T cells to AAV capsid, www.moleculartherapy.org vol. 15 no. 4, april 2007
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AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
(Figure 5a). F.IX levels were lower in SCID mice than in BALB/c mice used in the other experiments, which presumably relates to genetic differences between the mouse strains, which can affect the efficiency of hepatic AAV8-mediated transgene expression.11 SCID mice killed 1 month after cell transfer were tested for AAVspecific CD8 þ T cells in blood, spleens, and livers (Figure 5b). AAV capsid-specific CD8 þ T cells could be detected, albeit only at low levels in all compartments in SCID mice that received AdAAV-immune splenocytes. Again, these data indicate that the inability of AAV capsid-specific CD8 þ T cells to eliminate AAVtransduced hepatocytes was not related to suppression mediated by a CD8 cell subset.
Figure 2 Neutralizing antibodies to AAV2 capsid or AAV8 capsid do not crossreact with AAV8 or AAV2, respectively. BALB/c mice were immunized with Ad-AAV2 or Ad-AAV8 using 1 1011vp/mouse. Three weeks later, sera from these mice and naı¨ve BALB/c mice were collected, and titers of neutralizing antibodies to AAV2 and AAV8 were tested.
we immunized BALB/c mice with AAV2-gDgag37 or phosphatebuffered saline (PBS), respectively, and then depleted Tregs by treatment with an antibody to CD25. Analysis of splenocytes showed that the antibody to CD25 reduced the frequencies of CD4 þ CD25 þ T cells from 12% in the control mice to 3% in the anti-CD25 antibody-treated mice. In addition, mice were tested for frequencies of AAV capsid-specific CD8 þ T cells in spleen, blood, and liver (Figure 4a). Mice treated with the antiCD25 antibody showed slightly increased frequencies of specific CD8 þ T cells in spleens and liver. Mice were then infused with AAV8-F.IX, and circulating levels of F.IX were monitored. As shown in Figure 4b, levels of F.IX were comparable and sustained in the control groups that had not been pretreated with AAV2 and in the experimental groups that had been preimmunized with AAV capsid, regardless of treatment with the control antibody or the anti-CD25 antibody. The results suggest that Tregs do not impair the function of AAV capsid-specific CD8 þ T cells. The anti-CD25 antibody treatment did not result in complete depletion of CD25 þ cells, and one could argue for an effect of the remaining Tregs. Furthermore, cell types other than CD4 þ CD25 þ Tregs can downregulate adaptive immune responses. To circumvent the potential activity of suppressive cells, we injected CB17 SCID mice i.v. with AAV8-F.IX vector and transfused them i.v. 24 h later with purified CD8 þ T cells from Ad-AAV2 or Ad-GFP immunized BALB/c mice (Figure 5). Successful transfer was monitored by screening CB17 SCID mice for CD8 þ T cells, and such cells could readily be detected in spleens (Figure 5c). F.IX levels were monitored over time and found to be sustained and comparable in SCID mice transfused with control CD8 þ T cells or AAV capsid-immune CD8 þ T cells Molecular Therapy vol. 15 no. 4, april 2007
AAV-specific CD8 þ T cells lyse AAV capsid epitope-loaded target cells in vivo Elimination of AAV-transduced hepatocytes would most likely require direct cell lysis by CD8 þ T cells, as was indicated by the rise of transaminase levels in the gene therapy subjects. To test whether the AAV capsid-specific CD8 þ T cells were able to lyse target cells, we performed in vivo cytotoxicity assays in BALB/c mice immunized with Ad-AAV2. Nine days after Ad-AAV2 immunization, mice were injected with AAV8-F.IX or left untreated. Control mice were immunized with Ad-GFP. All mice then received AAV8 peptide-pulsed naı¨ve splenocytes given i.v. Transferred splenocytes had been labeled with an intermediate amount of carboxyfluorescein diacetate succinimidyl ester (CFSE) to allow for their tracking. AAV peptide-pulsed splenocytes were mixed with splenocytes that were incubated with a control peptide and labeled with a high dose of CFSE. Twenty-four hours later, lymphocytes were isolated from spleens, blood, and livers, and analyzed by flow cytometry for CFSE expression. In sham-vaccinated mice, two peaks of cells with distinct fluorescent intensity could readily be detected, with the low-intensity peak reflecting the AAV peptide-pulsed cells and the high-intensity peak reflecting the control cells. In Ad-AAV2 immune mice that had not received further treatment and in AdAAV2 immune mice that had received AAV8-F.IX, the number of cells of the low-intensity peak was reduced in all three compartments (i.e., spleen, blood, and liver) by around 80% compared to those from control mice, while numbers of cells of the high-intensity peak were comparable to those in control mice (Figure 6). Similar results were obtained upon transfer of AAV2 peptide-pulsed target cells (data not shown). These results demonstrate that Ad-AAV-induced CD8 þ T cells can lyse AAV2 or AAV8 epitope-expressing target cells in vivo, although they are unable to eliminate AAV-hF.IX-transduced cells. The results also show that transfer of AAV8-F.IX neither increases nor decreases the lytic potential of AAV capsid-specific pre-existing CD8 þ T cells.
DISCUSSION While the high immunogenicity of Ad vectors limits their use for gene therapy,12–14 AAV vectors have yielded promising results3,15–20 indicating that they are comparatively non-immunogenic.21 Nevertheless, in a clinical trial with AAV2 vectors expressing human F.IX, human subjects showed evidence of immune-mediated rejection of AAV-transduced hepatocytes.1 It 795
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Figure 3 AAV-mediated human F.IX expression is sustained in BALB/c mice with pre-existing immunity to AAV capsid. (a–d) Groups of BALB/c mice were immunized with Ad-GFP or Ad-AAV2, respectively. After 9 days, some of the mice were injected with AAV8-F.IX. (b) Circulating human F.IX level was tested by ELISA at 2, 4, and 6 weeks after AAV8-F.IX injection. (c) AAV2- and (d) AAV8-specific CD8 þ T-cell responses were measured by intracellular cytokine staining (ICS) 7 days after AAV8-F.IX injection. (e–h) Similar experiments were performed as in a in mice immunized with AdAAV8 before AAV2-hF.IX gene transfer. In (f) F.IX expression was tested 2 and 4 weeks after AAV2-F.IX injection. (g) AAV2- and (h) AAV8-specific CD8 þ T cells were measured 4 weeks after AAV2-F.IX injection. (i, j) BALB/c mice were immunized with PBS or AAV2-gDgag37 at 1 1011vg/mouse. Fourteen days later, mice were injected with AAV8-F.IX. (j) F.IX expression was tested at 2, 4, 6, and 8 weeks after AAV8-F.IX injection.
was assumed that rejection was caused by AAV capsid-specific memory CD8 þ T cells that were present in the subjects owing to natural infections, and that became re-activated and expanded upon AAV-mediated gene transfer. Pre-clinical testing of AAVmediated gene replacement therapy had not taken pre-existing immunity to AAV into account and was conducted in AAV naı¨ve animals. Here, we tested whether pre-existing CD8 þ T cells to AAV capsid affects the longevity of AAV-mediated hepatic gene transfer of human F.IX in mice. We took two approaches to induce AAV-specific CD8 þ T cells to AAV capsid epitopes. In one approach, AAV capsid antigens were expressed by E1-deleted Ad vectors and immunization of mice resulted in robust frequencies of AAV capsidspecific CD8 þ T cells in lymphatic tissues, blood, and liver. In a second approach, CD8 þ T cells were induced with AAV2 or 796
AAV8 vectors expressing a fusion protein of herpes simplex virus 1 gD and human immunodeficiency virus 1 gag. The HSV-gD has immunomodulatory activity and presumably promotes an inflammatory reaction.22,23 The gag part of the fusion protein contains an immunodominant MHC class I epitope for CD8 þ T cells of the H-2d haplotype, and thus allows for tracking of transgene product-specific CD8 þ T cells. AAV vectors induced markedly lower frequencies of capsid-specific CD8 þ T cells compared to Ad vectors. This is to be expected as capsid proteins synthesized in Ad vector transduced cells more readily enter the MHC class I processing and presentation pathway as compared with AAV capsid proteins expressed on viral particles.24 The latter requires cross-presentation of the antigen through an alternative processing pathway. AAV8 vectors induced lower but nevertheless statistically significant frequencies of capsid-specific www.moleculartherapy.org vol. 15 no. 4, april 2007
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Figure 4 CD4 þ CD25 þ regulatory T-cell depletion does not change AAV-mediated F.IX expression level in BALB/c mice with preexisting T cells to AAV. BALB/c mice were injected with PBS or AAV2-gDgag37. Anti-CD25 antibody was injected intraperitoneally into half of the mice in each group at 7 and 10 days postimmunization. After 14 days, part of the mice in each group was sacrificed. (a) The AAV2 capsid-specific CD8 þ T-cell responses were analyzed by intracellular cytokine staining (ICS). (b) One more day later, AAV8-F.IX vector was injected into all the remaining mice, and F.IX expression levels were monitored by ELISA at 1, 3, and 5 weeks after AAV8-F.IX gene transfer.
CD8 þ T cells than AAV2 vectors. This is in contrast to a recent report that AAVs such as AAV2, which binds to heparan sulfate proteoglycan, a receptor that is expressed on dendritic cells, can induce capsid-specific CD8 þ T cells, whereas those such as AAV8 or AAV1, which lack binding to heparan sulfate proteoglycan, fail to induce this T-cell subset.25 Both AAV2 and AAV8 vectors stimulated transgene product-specific CD8 þ T-cell responses, which were higher in AAV8-gDgag injected mice, again arguing against the assumption that direct transduction of dendritic cells through heparan sulfate proteoglycan is required to elicit vector or transgene product-specific CD8 þ T cells. Unexpectedly, mice with circulating AAV capsidspecific CD8 þ T cells showed long-term expression of F.IX derived from AAV-transduced hepatocytes. It should be noted here that, in the AAV-F.IX vector, the transgene was under the control of a hepatocyte specific promoter, and although other cell types presumably became transduced, circulating F.IX was derived from hepatocytes. Lack of lysis of AAV transduced hepatocytes could reflect a number of mechanisms. Regulatory T cells may have affected the Molecular Therapy vol. 15 no. 4, april 2007
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activity of CD8 þ T cells. This is unlikely as depletion of Tregs or transfer of purified CD8 þ T cells from AAV capsid-immune mice into AAV-F.IX-treated SCID mice failed to affect the longevity of F.IX expression. AAV capsid-specific CD8 þ T cells may have been impaired and incapable of cell lysis. This seems unlikely as in vivo killing assays showed that the number of AAV epitope-presenting cells was substantially diminished in all compartments tested, including the liver. Hepatic AAV gene transfer may have caused a decrease in MHC class I expression on hepatocytes. This pathway is also unlikely as staining of hepatocytes from AAV-F.IX- or AAV-GFP-treated mice that had or had not been preimmunized to AAV capsid showed comparable levels of MHC class I expression to those of naı¨ve mice (data not shown). It is feasible that processing of AAV capsid is inefficient in mouse hepatocytes, which thus fail to present a target for AAV capsid-specific CD8 þ effector T cells. In summary, it is clear that studies in mice, and in other species as well, failed to predict the outcome of AAV vector infusion into the liver in human subjects. The data in humans document several weeks of expression of a transgene at therapeutic levels, followed by development of a T-cell response to capsid, decline in transgene expression, and evidence of liver injury in the form of elevated transaminases. In this study, we used a reductionist approach to determine why AAV transduction of mouse liver failed to predict this result. We showed that we can clearly induce robust levels of capsid-specific CD8 þ T cells in mice by two different approaches, but this fails to suffice for destruction of transduced hepatocytes in an animal subsequently injected with an AAV vector. It is possible that in the human patients, immune mechanisms other than CD8 þ T cells resulted in loss of AAV-transduced hepatocytes. Such immune mechanisms could include lysis by natural killer cells or AAV capsid-specific antibodies. One could also argue that AAV capsid-specific CD8 þ T cells, induced in humans upon a natural infection, are fundamentally different from those induced in mice by Ad vectors or AAV particles. Humans are expected to carry low frequencies of memory CD8 þ T cells to AAV capsid antigens, whereas our studies were conducted in recently immunized mice that had high frequencies of AAV capsidspecific effector CD8 þ T cells. Finally, as noted above, the finding may reflect differences in antigen processing and presentation in murine versus human hepatocytes. Future studies should be directed at identifying the factors responsible for the difference in outcome in the two species, as this would provide the basis for sustained AAV-mediated gene transfer in humans.
MATERIALS AND METHODS Ad and AAV vectors. The E1- and E3-deleted, replication-deficient,
AAV2capsid and AAV8capsid-encoding Ad vectors were propagated on HEK 293 cells, purified by cesium chloride gradient centrifugation followed by column purification (Bio-Gel P-6DG). Vectors were diluted in PBS supplemented with 10% glycerol and stored at 801C. Content of virus particles (vps) was determined by spectrophotometry at 260 nm and 280 nm. Viral titers (vp) were determined using the formula: OD260 dilution 1.1 1012. AAV construct encoding gDgag37 was made by inserting the transgene into SnaBI sites of pSub201 vector and then packaged in capsids from AAV2 or AAV8. The plasmid pRE4 expressing gD was provided by Dr G Cohen and Dr R Risenbag in the
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Figure 5 The AAV8-mediated F.IX gene expression level in SCID mice is not affected by adoptive transfer of AAV2-specific CD8 þ T cells. SCID mice (n ¼ 10) were injected with AAV8-F.IX. After 24 h, mice were separated into two groups. They received CD8 þ splenocytes from BALB/c mice immunized with Ad-GFP or Ad-AAV2capsid. (a) Circulating human F.IX level in SCID mice after AAV8-F.IX infusion. (b) After 36 days of AAV8-F.IX injection, SCID mice were euthanized, and the frequency of capsid-specific CD8 þ T cells was determined. (c) SCID mice that did or did not receive CD8 þ T cells were tested for the presence of the transferred cells by flow cytometry.
University of Pennsylvania. The gDgag37 was made by first PCR amplification of gag37 fragment followed by insertion into the NarI site inside the gD fragment. AAV vectors were produced by the triple transfection method into 293 cells and purified using an enhanced CsCl density gradient purification. AAV2 and AAV8 vector expressing human factor IX (AAV2-F.IX, AAV8-F.IX) have been described previously.26 Mice. Male BALB/c mice were purchased from ACE Laboratory. SCID mice were bred at the Wistar Institute. Animals were used at 6–10 weeks of age. Animals were treated according to the institutional rules for animal welfare. Intracellular cytokine staining. Lymphocytes were isolated from
spleens, blood, inguinal and popliteal lymph nodes, and livers of different groups of mice. Spleens, lymph nodes and livers were harvested into Liebowitz’s-15 (L-15) medium and homogenized, then filtered through a 70-mm cell strainer. For spleen and lymph node, cells were centrifuged for 5 min at 1500 r.p.m. at room temperature and incubated with ACK lysing buffer (Invitrogen, Carlsbad, CA) for 5 min, then washed two times with L-15 medium and resuspended in 2-MLC medium (Dulbecco’s modified Eagle’s mediuim, 2% heat-inactivated fetal bovine serum, 1% Pen-Strep, 10 mM N-2-hydroxyethylpiperazineN0 -2-ethanesulfonic acid , 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 106 M 2-mercaptoethanol). For liver, cells were first centrifuged 10 min at 300 r.p.m.. Supernatant were harvested and centrifuged 10 min at 1500 r.p.m. Then cell pellets were resuspended in 8 mL 40% percoll (Amersham Bioscience, Piscataway, NJ), and
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applied on the top of 3 mL 70% percoll in 15 mL tube. After centrifugation at 2200 r.p.m. for 20 min at room temperature, the interface of 40% and 70% percoll were harvested and lysed with ACK lysis buffer and resuspended in 2-MLC medium. For peripheral blood mononuclear cell isolation, blood was harvested by heart punctuation and red blood cells were lysed by ACK lysing buffer. Cells were then washed twice with L-15 medium and resuspended in 2-MLC medium. 1 106 lymphocytes were stimulated with 5 mg/ml of AAV peptides or nonspecific peptides in complete medium with 50 U/ml mouse rIL-2 (Roche) and 1 ml/ml Golgi Plug (BD Pharmingen, San Diego, CA) in 96well plates. After 5 h of incubation at 371C/10% CO2, the cells were washed and stained for surface markers. They were then fixed and permeabilized using the Cytofix/Cytoperm (BD Pharmingen), and intracellular staining was performed using PE labeled mouse IFN-g antibody. The cells were washed and resuspended in PBS and analyzed by flow cytometry. Antibodies purchased from BD PharMingen were fluorescein isothiocyanate anti-CD8a, PE anti-IFN-g. AAV neutralization antibody assay. Test mouse serum was serially
diluted in normal mouse serum, then mixed with AAV2-GFP or AAV8GFP vector (2 105 vg/cell), and incubated at 371C for 1 h. Then the serum-vector mixture was added to HEK293 cells stably transfected with an inducible Ad E4 gene (ATCC). Addition of Ponasterone A (Invitrogen) to the media (1 ug/mL) was used to induce E4 expression 18 h before the addition of AAV. Cells were then incubated at 371C for 1 h before addition of 100 ml of complete medium and further incubated at 371C for 24 h. GFP-positive cells were counted and compared to those
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AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
Figure 6 AAV2-specific CD8 þ T cells lyse AAV8 peptide-pulsed target cells. Groups of BALB/c mice were first immunized with Ad-GFP or Ad-AAV2capsid. After 9 days, half of the Ad-AAV2capsid immunized groups were treated with AAV8-F.IX. In vivo CTL assay were performed 13 days later. CFSE level of unpulsed (CFSEhigh) and AAV8-peptide pulsed (CFSElow) target cells in spleen, blood, and liver of these mice are shown.
of AAV2/8-GFP mixed with normal mouse serum to calculate the percentage of inhibition of AAV2/8-GFP transduction. The neutralization titer was determined as the highest serum dilution at which X50% inhibition occurred.
purchased from BD Pharmingen. The positive magnetic separation was performed with LS column (Miltenyi Biotec, Auburn, CA) according to the suggested protocol. The purity of the CD8 þ cells was about 75%. Then 1.5 107 CD8 þ T cells were transferred into each of the SCID mice, which were injected AAV8-F.IX for 24 h.
ELISA for human F.IX in mouse plasma. Nunc 96-well plates were
coated with monoclonal anti-human F.IX antibody (Sigma) diluted 1:600 in 0.1 M carbonate coating buffer (pH 9.6). Plates were washed three times with 0.05% Tween-20/PBS, blocked 2 h in room temperature with 1% bovine serum albumin, 0.05% Tween-20 in PBS, then incubated with diluted mouse plasma samples at 41C overnight, and detected by 1:500 dilution of horseradish peroxidase-conjugated goat anti-human F.IX antibody (Enzyme Research Laboratories, South Bend, IN). Levels of human F.IX were determined by OD450 and quantified against the linear standard curve generated with serially diluted normal human plasma. CD4 þ CD25 þ regulatory T-cell depletion in BALB/c mice. BALB/c
mice were treated twice with 0.5 mg of anti-CD25 antibody (clone PC61, BioExpress, West Lebanon, NH) or its isotype control antibody (rat IgG1, BioExpress, West Lebanon, NH) by intraperitoneal injection. The interval between two treatments was 3 days. The kinetics of depleted mice were studied 14 days after the second injection.
In vivo cytotoxicity assay. Splenocytes were isolated from naı¨ve BALB/
c mice and lysed with ACK lysing buffer (Invitrogen) to eliminate red blood cells. Half of the cells were pulsed with 5 mg/ml AAV2 peptide (VPQYGYLTL) or AAV8 peptide (IPQYGYLTL) for 1 h at 371C, and the remaining cells were treated with control peptide. Then all cells were washed twice with PBS. The pulsed cells were labeled with 0.2 mM CFSE (CFSElow), whereas the unpulsed cells were labeled with 2 mM CFSE (CFSEhigh) for 10 min. CFSE was obtained from Molecular Probes (Engene, OR). The cells were washed extensively and counted, and approximately equal numbers of the two different populations were mixed together and injected i.v. into the BALB/c mice, which had immunized with different vectors. Approximately 10 106 cells from each of the target groups were injected per mouse. The mice were euthanized 20 h later, and the various organs were harvested to test CFSE stained cells. The cell populations of CFSElow and CFSEhigh in different tissues of control mice (AdHu5-GFP immunized mice) were treated as 100%. The cell populations in other groups were normalized against the populations in control group correspondingly.
CD8 þ T-cell purification and adoptive transfer in SCID mice. Spleno-
cytes were isolated from BALB/c mice immunized with AdHu5-GFP or AdHu5-AAV2capsid 9 days ago. CD8 þ T cells were purified by incubating the splenocytes with rat anti-I-A/I-E MHC II, anti-CD45R/ B220, anti-CD4, anti-Ter-119/Ly-76, anti-CD19, anti-Ly-6G antibodies in PBS/1% fetal bovine serum for 30 min at 41C. All the antibodies were
Molecular Therapy vol. 15 no. 4, april 2007
ACKNOWLEDGMENTS This work was supported by a program project grant administered through the National Institutes of Health (P01HL078810) and a training grant T32-DK-007748 (SLM).
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Schulick, AH, Vassalli, G, Dunn, PF, Dong, G, Rade, JJ and Zamarron, C et al. (1997). Established immunity precludes Ad-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of immunity. J Clin Invest 99: 209–219. Takahashi, H, Hirai, Y, Migita, M, Seino, Y, Fukuda, Y and Sakuraba, H et al. (2002). Long-term systemic therapy of Fabry disease in a knockout mouse by adenoassociated virus-mediated muscle-directed gene transfer. Proc Natl Acad Sci USA 99: 13777–13782. Walsh, CE and Chao, H (2002). Parvovirus-mediated gene transfer for the haemophilias. Haemophilia 8 (Suppl 2): 60–67. Arruda, VR, Schuettrumpf, J, Herzog, RW, Nichols, TC, Robinson, N and Lotfi, Y et al. (2004). Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1. Blood 103: 85–92. Greelish, JP, Su, LT, Lankford, EB, Burkman, JM, Chen, H and Konig, SK et al. (1999). Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector. Nat Med 5: 439–443. Snyder, RO, Spratt, SK, Laqarde, C, Bohl, D, Kaspar, B and Sloan, B et al. (1997). Efficient and stable adeno-associated virus-mediated transduction in the skeletal muscle of adult immunocompetent mice. Hum Gene Ther 8: 1891–1900. Fields, PA, Kowalczyk, DW, Arruda, VR, Armstrong, E, McCleland, ML and Hagstrom, JN et al. (2000). Role of vector in activation of T cell subsets in immune responses against the secreted transgene product factor IX. Mol Ther 1: 225–235. Jooss, K and Chirmule, N (2003). Immunity to adenovirus and adeno-associated viral vectors: implications for gene therapy. Gene Ther 10: 955–963. La, S, Kim, J, Kwon, BS and Kwon, B (2002). Herpes simplex virus type 1 glycoprotein D inhibits T-cell proliferation. Mol Cell 14: 398–403. Quenelle, DC, Collins, DJ, Marciani, DJ and Kern, ER (2006). Effect of immunization with herpes simplex virus type-1 (HSV-1) glycoprotein D (gD) plus the immune enhancer GPI-0100 on infection with HSV-1 or HSV-2. Vaccine 24: 1515–1522. Chen, W, Masterman, KA, Basta, S, Haeryfar, SM, Dimopoulos, N and Knowles, B et al. (2004). Cross-priming of CD8+ T cells by viral and tumor antigens is a robust phenomenon. Eur J Immunol 34: 194–199. Vandenberghe, LH, Wang, L, Somanathan, S, Zhi, Y, Figueredo, J and Calcedo, R et al. (2006). Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid. Nat Med 12: 967–971. Grimm, D, Zhou, S, Nakai, H, Thomas, CE, Storm, TA and Fuess, S et al. (2003). Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy. Blood 102: 2412–2419.
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Pre-existing AAV Capsid-specific CD8 þ T Cells are Unable to Eliminate AAV-transduced Hepatocytes Hua Li1,4, Samuel L Murphy2,4, Wynetta Giles-Davis1, Shyrie Edmonson2,3, Zhiquan Xiang1, Yan Li1, Marcio O Lasaro1, Katherine A High2,3 and Hildegund CJ Ertl1 1 Wistar Institute, Philadelphia, Pennsylvania, USA; 2The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; 3Howard Hughes Medical Institute, Philadelphia, Pennsylvania, USA
The goal of these studies was to test whether adenoassociated virus (AAV) capsid-specific CD8 þ T cells cause loss of hepatic AAV-mediated gene expression in experimental animals. Mice immunized with adenoviral vectors expressing AAV capsid or with AAV vectors developed CD8 þ T cells in blood, lymphatic tissues, and liver to epitopes shared between AAV2 and AAV8, and serotype-specific neutralizing antibodies. At the height of the T cells’ effector phase, mice were infused with a heterologous AAV vector expressing human factor IX under a hepatocyte-specific promoter. Despite the presence of lytic CD8 þ T cells in the liver, hepatic Factor IX expression was sustained and comparable in AAV-preimmune and naı¨ve animals. These results suggest that, in mice, pre-existing CD8 þ T cells to AAV capsid do not affect the longevity of AAV-mediated hepatic gene transfer. These results are in contrast to the outcome of a recent gene therapy trial of hemophilia B patients who were treated by hepatic gene transfer of AAV2 vectors expressing Factor IX. The loss of Factor IX expression, accompanied by a rise in liver enzymes and detectable frequencies of circulating AAV capsid-specific T cells, suggested T-cell-mediated destruction of transduced hepatocytes following reactivation of AAV-specific T cells upon AAV transfer. Received 3 November 2006; accepted 29 November 2006; advance online publication, 23 January 2007. doi:10.1038/mt.sj.6300090
INTRODUCTION In a recent gene transfer trial, human subjects with severe hemophilia B were infused with recombinant adeno-associated virus (AAV) vectors derived from the human serotype 2 (AAV2) expressing factor IX (AAV2-F.IX) under the control of a hepatocyte-specific promoter for intrahepatic expression, a procedure that resulted in sustained F.IX expression in preclinical animal models of hemophilia B. One subject in the highest dose group developed therapeutic levels of F.IX by week 2, but 4 weeks after vector infusion, the levels of F.IX started to decrease and, within a few weeks, returned to pre-gene therapy
levels.1 Concomitantly, the subject developed transaminitis, which resolved after F.IX had decreased to baseline levels. The subject’s clinical course was highly suggestive of immunemediated destruction of AAV-transduced hepatocytes. The trial was continued with a reduced dose of vector and again the next subject presented with transaminitis after gene transfer with a time course relative to therapy identical to that seen in the other subject. In the second patient, T-cell responses to AAV-2 capsid were monitored before and after gene transfer. The subject had no detectable AAV2 capsid-specific T cells in his peripheral blood before gene transfer. Such a response developed after gene transfer and then eventually subsided, suggesting that indeed T-cell-mediated destruction of AAV transduced hepatocytes may have contributed to the failure of persistent gene expression. T cells to the transgene product could not be detected at any time point, indicating that the response was directed against the capsid antigens of AAV. T-cell-mediated clearance of AAV-transduced hepatocytes had not been encountered during the extensive preclinical testing of AAV2 vectors in experimental animals including rodents, canines, and nonhuman primates.2–4 Humans may have reacted differently because most are naturally exposed to AAV2 during childhood. Infections occur concomitantly with a helper virus such as an adenovirus (Ad). Humans thus have immunological memory to AAV2, unlike experimental animals. Memory T cells can be triggered more readily than naı¨ve T cells, which was not taken into account by the preclinical animal experiments conducted thus far. Here we tested whether pre-existing CD8 þ effector T cells to AAV capsid affect the duration of transgene expression following hepatic AAV-mediated human F.IX gene transfer in mice. To this end, mice were immunized against AAV capsid antigen using either E1-deleted Ad vectors expressing capsid antigens of AAV2 or AAV8 or AAV vectors expressing an immunogenic transgene product. Mice were then infused with a distinct serotype of AAV vector expressing F.IX, i.e., AAV2 capsid-immune mice received AAV8-F.IX or vice versa. As we showed previously, BALB/c mice immunized with the adenoviral vector expressing capsid developed T cells that crossreacted between epitopes from AAV2 and AAV8 (VPQYGYLTL or IPQYGYLTL).5 As they lack
Correspondence: Hildegund CJ Ertl, The Wistar Institute, 3601 Spruce St, Philadelphia, Pennsylvania 19104, USA. E-mail: [email protected] 4 These authors contributed equally to this work.
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conserved binding sites for virus neutralizing antibodies, this protocol allows us to assess the effect of pre-existing T cells on the longevity of F.IX gene transfer in animals in which transduction efficacy is not impaired by pre-existing AAV neutralizing antibodies. The results show conclusively that, in mice, pre-existing lytic CD8 þ T cells to AAV capsid fail to eliminate AAV transduced hepatocytes.
RESULTS Vectors for induction of AAV capsid-specific CD8 þ T cells Immunogens were constructed to induce immune responses to AAV capsid antigens in mice. The first set of immunogens was composed of E1 deleted Ad vectors encoding the entire sequence of AAV2 or AAV8 capsid. A second set of immunogens was composed of AAV2 and AAV8 vectors encoding a fusion protein of glycoprotein D (gD) of herpes simplex virus 1 and a truncated gag of human immunodeficiency virus 1. These constructs were used to ensure that results obtained with the Ad vectors could also be obtained with AAV vectors. The AAV vectors expressed an immunogenic viral antigen to stimulate an innate immune response which, in turn, is required for induction of adaptive immunity. All of the vectors expressed the transgene product, i.e., the AAV capsid or the herpes simplex virus 1 gD-gag fusion protein (not shown). AAV capsid-specific CD8 þ T-cell responses in mice Vectors were tested for induction of AAV capsid-specific CD8 þ T cells in groups of BALB/c mice (n ¼ 4–5). Mice were injected intramuscularly into the quadriceps with 1 1011 vector particles (VP) or vector genomes (VG) of Ad-AAV2capsid, AdAAV8capsid, AAV2-gDgag37, or AAV8-gDgag37 vectors. AAVspecific acute and memory CD8 þ T-cell responses were tested from spleens of individual mice and from pooled lymphocytes of blood, lymph nodes and livers. Frequencies of AAV capsidspecific CD8 þ T cells were measured by intracellular cytokine staining for interferon (IFN)-g (Figure 1). Immunization of mice with the Ad-AAV2capsid (Figure 1a) and Ad-AAV8capsid (Figure 1b) vectors resulted in comparable frequencies of CD8 þ T cells that, in spleens, ranged from 8 to 10% of all CD8 þ T cells at the height of the acute response and then declined to 3–5% within 8 weeks after immunization. Frequencies were higher in blood than in spleens early after immunization. Frequencies in lymph nodes were modest at both time points. The highest frequencies were observed in livers. Immunization of mice with AAV2 vectors elicited AAV capsid-specific CD8 þ T cells (Figure 1c). Frequencies were markedly lower than those induced by the Ad vectors expressing AAV2 capsid. This is not surprising, as Ad vectors have been described previously as inducing exceptionally high frequencies of transgene product-specific CD8 þ T cells when compared to other types of vectors.6 Immunization of mice with the AAV8-gDgag37 vector elicited only a marginal CD8 þ T-cell response to AAV capsid, which was detectable in spleen and liver but not in blood during the acute response (Figure 1d). To ensure that both AAV2 and AAV8 vectors were immunogenic, CD8 þ T-cell frequencies to gag were also measured (Figure 1e). Both types of vectors induced CD8 þ T Molecular Therapy vol. 15 no. 4, april 2007
AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
cells to the immunodominant epitope of gag detectable in spleens, and at higher frequencies in blood and liver by day 14 after immunization.
Crossreactivity of AAV capsid-specific CD8 þ T cells The sequences of different AAV serotypes are highly conserved, and it has been reported that, in mice of the H-2d haplotype, CD8 þ T cells to AAV2 crossreact with AAV8 capsid.5 This is to be expected as one of the H-2d epitopes of AAV2 differs only by one amino acid from the corresponding sequence of AAV8. Upon immunization with either Ad-AAV2 or Ad-AAV8 vectors, mice developed titers of neutralizing antibodies 41:100 to the homologous AAV vector, whereas no such antibodies could be detected against the heterologous vector, indicating lack of detectable cross-reactivity of AAV neutralizing antibodies (Figure 2). In contrast, CD8 þ T cells induced against AAV2 capsid showed crossreactivity with the AAV8 epitope and vice versa. The effect of pre-existing AAV-specific CD8 þ T cells on AAV-mediated gene transfer Lack of serological crossreactivity and complete cellular crossreactivity between MHC class I binding epitopes of the capsid antigen of AAV2 and AAV8 allowed us to assess the role of preexisting CD8 þ T cells in mice vaccinated to AAV2 capsid using AAV8 vectors for gene transfer and vice versa. AAV-mediated gene transfer is reduced by pre-existing AAV neutralizing antibodies, necessitating the use of different serotypes for preexposure and gene transfer. In the first set of experiments, we immunized BALB/c mice intramuscularly with Ad-AAV2 vectors or a control vector. They received AAV8-hF.IX, given intravenously (i.v.) 9 days later (Figure 3a).7 In some of the experiments, mice were bled before gene transfer, and frequencies of AAV capsid-specific CD8 þ T cells were measured from blood mononuclear cells and found to be similar to those shown in Figure 1 (not shown). Circulating human Factor IX (F.IX) levels, evaluated by enzyme-linked immunosorbent assay (ELISA) at 2, 4, and 6 weeks after AAV8 transfer, showed no significant difference in AAV pre-immunized or control mice (Figure 3b). We tested liver transaminases to determine potential liver damage caused by immune-mediated destruction of AAV transduced cells, and no increase in liver transaminase was seen in comparison to different groups (data not shown). These data indicate that, even in the presence of circulating and liver-homing AAV capsid-specific effector CD8 þ T cells, hepatic AAV-mediated gene transfer results in stable expression of the transgene product. Similar experiments were conducted by immunizing mice i.v. with Ad-AAV2 followed by AAV8-F.IX gene transfer or by immunizing mice intramuscularly with Ad-AAV8 followed by AAV2-F.IX gene transfer (Figure 3e). Under both experimental conditions, F.IX levels were sustained (in part shown in Figure 3f), and transaminase levels remained at physiological levels (not shown). Transfer of F.IX by the AAV8 vectors resulted in higher circulating levels of F.IX than transfer by AAV2 vectors. To examine whether AAV capsid-specific CD8 þ T cells were still detectable after gene transfer or whether gene transfer had 793
AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
% Of IFN-+CD8+ cells / CD8+ cells
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3 2 1 0 Spleen
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Figure 1 AAV capsid and gag specific CD8 T-cell responses in BALB/c mice intramuscularly immunized with different experimental vectors. (a) Ad-AAV2capsid and Ad-GFP control, (b) Ad-AAV8capsid and Ad-GFP control, (c) AAV2-gDgag37, (d) AAV8-gDgag37, and (e) AAV2-gDgag37 and AAV8-gDgag37. The dose of all vectors was 1 1011 vp (vg)/mouse. Frequencies of AAV-specific and gag-specific CD8 þ T cells were determined by intracellular cytokine staining (ICS) from spleens of individual mice and pooled peripheral blood mononuclear cell (PBMC), lymph nodes and livers. The acute response induced by Ad-AAV2 and Ad-AAV8 was tested 9 days after injection. The acute response induced by AAV2-gDgag37 and AAV8gDgag37 was tested 2 weeks after injection. All memory responses were examined 2 months after immunization. The responses induced by Ad-GFP control were analyzed 9 days postinjection.
boosted the capsid-specific CD8 þ T-cell response, mice were euthanized after gene transfer and tested for CD8 þ T cells to the AAV epitopes. Robust AAV2- and AAV8-specific T-cell responses were detected in mice injected with Ad-AAV vectors followed by AAV gene transfer (Figure 3c–d, g–h). Responses were not increased in mice primed with Ad-AAV and treated with AAV gene transfer when compared to mice injected with Ad-AAV, indicating that Ad-AAV-induced CD8 þ T cells were not boosted by the AAV vector used for gene transfer. The argument has been made that the epitopes that are recognized on AAV capsid as expressed by an Ad vector, are not efficiently recognized on capsid antigen expressed by AAV particles.8 We therefore repeated the gene transfer experiments in mice immunized with AAV2-gDgag37 (Figure 3i). Again, induction of AAV-specific CD8 þ T cells was confirmed and, upon testing for AAV neutralizing antibodies, mice were shown 794
to have such antibodies to AAV2 but not to AAV8 (not shown). Mice were then injected with AAV8-F.IX vector given i.v, and F.IX expression levels were tested by ELISA at 2, 4, 6, and 8 weeks after gene transfer (Figure 3j). Persistent and comparable levels of F.IX were detected in both AAV pre-exposed and control mice.
CD4 þ CD25 þ regulatory T-cell depletion does not change AAV-mediated F.IX expression level in mice with pre-existing T-cell immunity to AAV Lack of elimination of AAV-transduced hepatocytes by AAV capsid-specific CD8 þ T cells may reflect that T cells are impaired because of the activity of negative immunoregulatory mechanisms. CD4 þ CD25 þ Foxp3 þ regulatory T cells (Tregs) have been shown to play a role in AAV-mediated gene transfer.9,10 To determine if Tregs contribute to sustained AAV-mediated gene transfer in mice with pre-existing CD8 þ T cells to AAV capsid, www.moleculartherapy.org vol. 15 no. 4, april 2007
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AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
(Figure 5a). F.IX levels were lower in SCID mice than in BALB/c mice used in the other experiments, which presumably relates to genetic differences between the mouse strains, which can affect the efficiency of hepatic AAV8-mediated transgene expression.11 SCID mice killed 1 month after cell transfer were tested for AAVspecific CD8 þ T cells in blood, spleens, and livers (Figure 5b). AAV capsid-specific CD8 þ T cells could be detected, albeit only at low levels in all compartments in SCID mice that received AdAAV-immune splenocytes. Again, these data indicate that the inability of AAV capsid-specific CD8 þ T cells to eliminate AAVtransduced hepatocytes was not related to suppression mediated by a CD8 cell subset.
Figure 2 Neutralizing antibodies to AAV2 capsid or AAV8 capsid do not crossreact with AAV8 or AAV2, respectively. BALB/c mice were immunized with Ad-AAV2 or Ad-AAV8 using 1 1011vp/mouse. Three weeks later, sera from these mice and naı¨ve BALB/c mice were collected, and titers of neutralizing antibodies to AAV2 and AAV8 were tested.
we immunized BALB/c mice with AAV2-gDgag37 or phosphatebuffered saline (PBS), respectively, and then depleted Tregs by treatment with an antibody to CD25. Analysis of splenocytes showed that the antibody to CD25 reduced the frequencies of CD4 þ CD25 þ T cells from 12% in the control mice to 3% in the anti-CD25 antibody-treated mice. In addition, mice were tested for frequencies of AAV capsid-specific CD8 þ T cells in spleen, blood, and liver (Figure 4a). Mice treated with the antiCD25 antibody showed slightly increased frequencies of specific CD8 þ T cells in spleens and liver. Mice were then infused with AAV8-F.IX, and circulating levels of F.IX were monitored. As shown in Figure 4b, levels of F.IX were comparable and sustained in the control groups that had not been pretreated with AAV2 and in the experimental groups that had been preimmunized with AAV capsid, regardless of treatment with the control antibody or the anti-CD25 antibody. The results suggest that Tregs do not impair the function of AAV capsid-specific CD8 þ T cells. The anti-CD25 antibody treatment did not result in complete depletion of CD25 þ cells, and one could argue for an effect of the remaining Tregs. Furthermore, cell types other than CD4 þ CD25 þ Tregs can downregulate adaptive immune responses. To circumvent the potential activity of suppressive cells, we injected CB17 SCID mice i.v. with AAV8-F.IX vector and transfused them i.v. 24 h later with purified CD8 þ T cells from Ad-AAV2 or Ad-GFP immunized BALB/c mice (Figure 5). Successful transfer was monitored by screening CB17 SCID mice for CD8 þ T cells, and such cells could readily be detected in spleens (Figure 5c). F.IX levels were monitored over time and found to be sustained and comparable in SCID mice transfused with control CD8 þ T cells or AAV capsid-immune CD8 þ T cells Molecular Therapy vol. 15 no. 4, april 2007
AAV-specific CD8 þ T cells lyse AAV capsid epitope-loaded target cells in vivo Elimination of AAV-transduced hepatocytes would most likely require direct cell lysis by CD8 þ T cells, as was indicated by the rise of transaminase levels in the gene therapy subjects. To test whether the AAV capsid-specific CD8 þ T cells were able to lyse target cells, we performed in vivo cytotoxicity assays in BALB/c mice immunized with Ad-AAV2. Nine days after Ad-AAV2 immunization, mice were injected with AAV8-F.IX or left untreated. Control mice were immunized with Ad-GFP. All mice then received AAV8 peptide-pulsed naı¨ve splenocytes given i.v. Transferred splenocytes had been labeled with an intermediate amount of carboxyfluorescein diacetate succinimidyl ester (CFSE) to allow for their tracking. AAV peptide-pulsed splenocytes were mixed with splenocytes that were incubated with a control peptide and labeled with a high dose of CFSE. Twenty-four hours later, lymphocytes were isolated from spleens, blood, and livers, and analyzed by flow cytometry for CFSE expression. In sham-vaccinated mice, two peaks of cells with distinct fluorescent intensity could readily be detected, with the low-intensity peak reflecting the AAV peptide-pulsed cells and the high-intensity peak reflecting the control cells. In Ad-AAV2 immune mice that had not received further treatment and in AdAAV2 immune mice that had received AAV8-F.IX, the number of cells of the low-intensity peak was reduced in all three compartments (i.e., spleen, blood, and liver) by around 80% compared to those from control mice, while numbers of cells of the high-intensity peak were comparable to those in control mice (Figure 6). Similar results were obtained upon transfer of AAV2 peptide-pulsed target cells (data not shown). These results demonstrate that Ad-AAV-induced CD8 þ T cells can lyse AAV2 or AAV8 epitope-expressing target cells in vivo, although they are unable to eliminate AAV-hF.IX-transduced cells. The results also show that transfer of AAV8-F.IX neither increases nor decreases the lytic potential of AAV capsid-specific pre-existing CD8 þ T cells.
DISCUSSION While the high immunogenicity of Ad vectors limits their use for gene therapy,12–14 AAV vectors have yielded promising results3,15–20 indicating that they are comparatively non-immunogenic.21 Nevertheless, in a clinical trial with AAV2 vectors expressing human F.IX, human subjects showed evidence of immune-mediated rejection of AAV-transduced hepatocytes.1 It 795
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Figure 3 AAV-mediated human F.IX expression is sustained in BALB/c mice with pre-existing immunity to AAV capsid. (a–d) Groups of BALB/c mice were immunized with Ad-GFP or Ad-AAV2, respectively. After 9 days, some of the mice were injected with AAV8-F.IX. (b) Circulating human F.IX level was tested by ELISA at 2, 4, and 6 weeks after AAV8-F.IX injection. (c) AAV2- and (d) AAV8-specific CD8 þ T-cell responses were measured by intracellular cytokine staining (ICS) 7 days after AAV8-F.IX injection. (e–h) Similar experiments were performed as in a in mice immunized with AdAAV8 before AAV2-hF.IX gene transfer. In (f) F.IX expression was tested 2 and 4 weeks after AAV2-F.IX injection. (g) AAV2- and (h) AAV8-specific CD8 þ T cells were measured 4 weeks after AAV2-F.IX injection. (i, j) BALB/c mice were immunized with PBS or AAV2-gDgag37 at 1 1011vg/mouse. Fourteen days later, mice were injected with AAV8-F.IX. (j) F.IX expression was tested at 2, 4, 6, and 8 weeks after AAV8-F.IX injection.
was assumed that rejection was caused by AAV capsid-specific memory CD8 þ T cells that were present in the subjects owing to natural infections, and that became re-activated and expanded upon AAV-mediated gene transfer. Pre-clinical testing of AAVmediated gene replacement therapy had not taken pre-existing immunity to AAV into account and was conducted in AAV naı¨ve animals. Here, we tested whether pre-existing CD8 þ T cells to AAV capsid affects the longevity of AAV-mediated hepatic gene transfer of human F.IX in mice. We took two approaches to induce AAV-specific CD8 þ T cells to AAV capsid epitopes. In one approach, AAV capsid antigens were expressed by E1-deleted Ad vectors and immunization of mice resulted in robust frequencies of AAV capsidspecific CD8 þ T cells in lymphatic tissues, blood, and liver. In a second approach, CD8 þ T cells were induced with AAV2 or 796
AAV8 vectors expressing a fusion protein of herpes simplex virus 1 gD and human immunodeficiency virus 1 gag. The HSV-gD has immunomodulatory activity and presumably promotes an inflammatory reaction.22,23 The gag part of the fusion protein contains an immunodominant MHC class I epitope for CD8 þ T cells of the H-2d haplotype, and thus allows for tracking of transgene product-specific CD8 þ T cells. AAV vectors induced markedly lower frequencies of capsid-specific CD8 þ T cells compared to Ad vectors. This is to be expected as capsid proteins synthesized in Ad vector transduced cells more readily enter the MHC class I processing and presentation pathway as compared with AAV capsid proteins expressed on viral particles.24 The latter requires cross-presentation of the antigen through an alternative processing pathway. AAV8 vectors induced lower but nevertheless statistically significant frequencies of capsid-specific www.moleculartherapy.org vol. 15 no. 4, april 2007
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Figure 4 CD4 þ CD25 þ regulatory T-cell depletion does not change AAV-mediated F.IX expression level in BALB/c mice with preexisting T cells to AAV. BALB/c mice were injected with PBS or AAV2-gDgag37. Anti-CD25 antibody was injected intraperitoneally into half of the mice in each group at 7 and 10 days postimmunization. After 14 days, part of the mice in each group was sacrificed. (a) The AAV2 capsid-specific CD8 þ T-cell responses were analyzed by intracellular cytokine staining (ICS). (b) One more day later, AAV8-F.IX vector was injected into all the remaining mice, and F.IX expression levels were monitored by ELISA at 1, 3, and 5 weeks after AAV8-F.IX gene transfer.
CD8 þ T cells than AAV2 vectors. This is in contrast to a recent report that AAVs such as AAV2, which binds to heparan sulfate proteoglycan, a receptor that is expressed on dendritic cells, can induce capsid-specific CD8 þ T cells, whereas those such as AAV8 or AAV1, which lack binding to heparan sulfate proteoglycan, fail to induce this T-cell subset.25 Both AAV2 and AAV8 vectors stimulated transgene product-specific CD8 þ T-cell responses, which were higher in AAV8-gDgag injected mice, again arguing against the assumption that direct transduction of dendritic cells through heparan sulfate proteoglycan is required to elicit vector or transgene product-specific CD8 þ T cells. Unexpectedly, mice with circulating AAV capsidspecific CD8 þ T cells showed long-term expression of F.IX derived from AAV-transduced hepatocytes. It should be noted here that, in the AAV-F.IX vector, the transgene was under the control of a hepatocyte specific promoter, and although other cell types presumably became transduced, circulating F.IX was derived from hepatocytes. Lack of lysis of AAV transduced hepatocytes could reflect a number of mechanisms. Regulatory T cells may have affected the Molecular Therapy vol. 15 no. 4, april 2007
AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
activity of CD8 þ T cells. This is unlikely as depletion of Tregs or transfer of purified CD8 þ T cells from AAV capsid-immune mice into AAV-F.IX-treated SCID mice failed to affect the longevity of F.IX expression. AAV capsid-specific CD8 þ T cells may have been impaired and incapable of cell lysis. This seems unlikely as in vivo killing assays showed that the number of AAV epitope-presenting cells was substantially diminished in all compartments tested, including the liver. Hepatic AAV gene transfer may have caused a decrease in MHC class I expression on hepatocytes. This pathway is also unlikely as staining of hepatocytes from AAV-F.IX- or AAV-GFP-treated mice that had or had not been preimmunized to AAV capsid showed comparable levels of MHC class I expression to those of naı¨ve mice (data not shown). It is feasible that processing of AAV capsid is inefficient in mouse hepatocytes, which thus fail to present a target for AAV capsid-specific CD8 þ effector T cells. In summary, it is clear that studies in mice, and in other species as well, failed to predict the outcome of AAV vector infusion into the liver in human subjects. The data in humans document several weeks of expression of a transgene at therapeutic levels, followed by development of a T-cell response to capsid, decline in transgene expression, and evidence of liver injury in the form of elevated transaminases. In this study, we used a reductionist approach to determine why AAV transduction of mouse liver failed to predict this result. We showed that we can clearly induce robust levels of capsid-specific CD8 þ T cells in mice by two different approaches, but this fails to suffice for destruction of transduced hepatocytes in an animal subsequently injected with an AAV vector. It is possible that in the human patients, immune mechanisms other than CD8 þ T cells resulted in loss of AAV-transduced hepatocytes. Such immune mechanisms could include lysis by natural killer cells or AAV capsid-specific antibodies. One could also argue that AAV capsid-specific CD8 þ T cells, induced in humans upon a natural infection, are fundamentally different from those induced in mice by Ad vectors or AAV particles. Humans are expected to carry low frequencies of memory CD8 þ T cells to AAV capsid antigens, whereas our studies were conducted in recently immunized mice that had high frequencies of AAV capsidspecific effector CD8 þ T cells. Finally, as noted above, the finding may reflect differences in antigen processing and presentation in murine versus human hepatocytes. Future studies should be directed at identifying the factors responsible for the difference in outcome in the two species, as this would provide the basis for sustained AAV-mediated gene transfer in humans.
MATERIALS AND METHODS Ad and AAV vectors. The E1- and E3-deleted, replication-deficient,
AAV2capsid and AAV8capsid-encoding Ad vectors were propagated on HEK 293 cells, purified by cesium chloride gradient centrifugation followed by column purification (Bio-Gel P-6DG). Vectors were diluted in PBS supplemented with 10% glycerol and stored at 801C. Content of virus particles (vps) was determined by spectrophotometry at 260 nm and 280 nm. Viral titers (vp) were determined using the formula: OD260 dilution 1.1 1012. AAV construct encoding gDgag37 was made by inserting the transgene into SnaBI sites of pSub201 vector and then packaged in capsids from AAV2 or AAV8. The plasmid pRE4 expressing gD was provided by Dr G Cohen and Dr R Risenbag in the
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Figure 5 The AAV8-mediated F.IX gene expression level in SCID mice is not affected by adoptive transfer of AAV2-specific CD8 þ T cells. SCID mice (n ¼ 10) were injected with AAV8-F.IX. After 24 h, mice were separated into two groups. They received CD8 þ splenocytes from BALB/c mice immunized with Ad-GFP or Ad-AAV2capsid. (a) Circulating human F.IX level in SCID mice after AAV8-F.IX infusion. (b) After 36 days of AAV8-F.IX injection, SCID mice were euthanized, and the frequency of capsid-specific CD8 þ T cells was determined. (c) SCID mice that did or did not receive CD8 þ T cells were tested for the presence of the transferred cells by flow cytometry.
University of Pennsylvania. The gDgag37 was made by first PCR amplification of gag37 fragment followed by insertion into the NarI site inside the gD fragment. AAV vectors were produced by the triple transfection method into 293 cells and purified using an enhanced CsCl density gradient purification. AAV2 and AAV8 vector expressing human factor IX (AAV2-F.IX, AAV8-F.IX) have been described previously.26 Mice. Male BALB/c mice were purchased from ACE Laboratory. SCID mice were bred at the Wistar Institute. Animals were used at 6–10 weeks of age. Animals were treated according to the institutional rules for animal welfare. Intracellular cytokine staining. Lymphocytes were isolated from
spleens, blood, inguinal and popliteal lymph nodes, and livers of different groups of mice. Spleens, lymph nodes and livers were harvested into Liebowitz’s-15 (L-15) medium and homogenized, then filtered through a 70-mm cell strainer. For spleen and lymph node, cells were centrifuged for 5 min at 1500 r.p.m. at room temperature and incubated with ACK lysing buffer (Invitrogen, Carlsbad, CA) for 5 min, then washed two times with L-15 medium and resuspended in 2-MLC medium (Dulbecco’s modified Eagle’s mediuim, 2% heat-inactivated fetal bovine serum, 1% Pen-Strep, 10 mM N-2-hydroxyethylpiperazineN0 -2-ethanesulfonic acid , 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 106 M 2-mercaptoethanol). For liver, cells were first centrifuged 10 min at 300 r.p.m.. Supernatant were harvested and centrifuged 10 min at 1500 r.p.m. Then cell pellets were resuspended in 8 mL 40% percoll (Amersham Bioscience, Piscataway, NJ), and
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applied on the top of 3 mL 70% percoll in 15 mL tube. After centrifugation at 2200 r.p.m. for 20 min at room temperature, the interface of 40% and 70% percoll were harvested and lysed with ACK lysis buffer and resuspended in 2-MLC medium. For peripheral blood mononuclear cell isolation, blood was harvested by heart punctuation and red blood cells were lysed by ACK lysing buffer. Cells were then washed twice with L-15 medium and resuspended in 2-MLC medium. 1 106 lymphocytes were stimulated with 5 mg/ml of AAV peptides or nonspecific peptides in complete medium with 50 U/ml mouse rIL-2 (Roche) and 1 ml/ml Golgi Plug (BD Pharmingen, San Diego, CA) in 96well plates. After 5 h of incubation at 371C/10% CO2, the cells were washed and stained for surface markers. They were then fixed and permeabilized using the Cytofix/Cytoperm (BD Pharmingen), and intracellular staining was performed using PE labeled mouse IFN-g antibody. The cells were washed and resuspended in PBS and analyzed by flow cytometry. Antibodies purchased from BD PharMingen were fluorescein isothiocyanate anti-CD8a, PE anti-IFN-g. AAV neutralization antibody assay. Test mouse serum was serially
diluted in normal mouse serum, then mixed with AAV2-GFP or AAV8GFP vector (2 105 vg/cell), and incubated at 371C for 1 h. Then the serum-vector mixture was added to HEK293 cells stably transfected with an inducible Ad E4 gene (ATCC). Addition of Ponasterone A (Invitrogen) to the media (1 ug/mL) was used to induce E4 expression 18 h before the addition of AAV. Cells were then incubated at 371C for 1 h before addition of 100 ml of complete medium and further incubated at 371C for 24 h. GFP-positive cells were counted and compared to those
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AAV Capsid-specific CD8 þ T Cells and AAV-transduced Hepatocytes
Figure 6 AAV2-specific CD8 þ T cells lyse AAV8 peptide-pulsed target cells. Groups of BALB/c mice were first immunized with Ad-GFP or Ad-AAV2capsid. After 9 days, half of the Ad-AAV2capsid immunized groups were treated with AAV8-F.IX. In vivo CTL assay were performed 13 days later. CFSE level of unpulsed (CFSEhigh) and AAV8-peptide pulsed (CFSElow) target cells in spleen, blood, and liver of these mice are shown.
of AAV2/8-GFP mixed with normal mouse serum to calculate the percentage of inhibition of AAV2/8-GFP transduction. The neutralization titer was determined as the highest serum dilution at which X50% inhibition occurred.
purchased from BD Pharmingen. The positive magnetic separation was performed with LS column (Miltenyi Biotec, Auburn, CA) according to the suggested protocol. The purity of the CD8 þ cells was about 75%. Then 1.5 107 CD8 þ T cells were transferred into each of the SCID mice, which were injected AAV8-F.IX for 24 h.
ELISA for human F.IX in mouse plasma. Nunc 96-well plates were
coated with monoclonal anti-human F.IX antibody (Sigma) diluted 1:600 in 0.1 M carbonate coating buffer (pH 9.6). Plates were washed three times with 0.05% Tween-20/PBS, blocked 2 h in room temperature with 1% bovine serum albumin, 0.05% Tween-20 in PBS, then incubated with diluted mouse plasma samples at 41C overnight, and detected by 1:500 dilution of horseradish peroxidase-conjugated goat anti-human F.IX antibody (Enzyme Research Laboratories, South Bend, IN). Levels of human F.IX were determined by OD450 and quantified against the linear standard curve generated with serially diluted normal human plasma. CD4 þ CD25 þ regulatory T-cell depletion in BALB/c mice. BALB/c
mice were treated twice with 0.5 mg of anti-CD25 antibody (clone PC61, BioExpress, West Lebanon, NH) or its isotype control antibody (rat IgG1, BioExpress, West Lebanon, NH) by intraperitoneal injection. The interval between two treatments was 3 days. The kinetics of depleted mice were studied 14 days after the second injection.
In vivo cytotoxicity assay. Splenocytes were isolated from naı¨ve BALB/
c mice and lysed with ACK lysing buffer (Invitrogen) to eliminate red blood cells. Half of the cells were pulsed with 5 mg/ml AAV2 peptide (VPQYGYLTL) or AAV8 peptide (IPQYGYLTL) for 1 h at 371C, and the remaining cells were treated with control peptide. Then all cells were washed twice with PBS. The pulsed cells were labeled with 0.2 mM CFSE (CFSElow), whereas the unpulsed cells were labeled with 2 mM CFSE (CFSEhigh) for 10 min. CFSE was obtained from Molecular Probes (Engene, OR). The cells were washed extensively and counted, and approximately equal numbers of the two different populations were mixed together and injected i.v. into the BALB/c mice, which had immunized with different vectors. Approximately 10 106 cells from each of the target groups were injected per mouse. The mice were euthanized 20 h later, and the various organs were harvested to test CFSE stained cells. The cell populations of CFSElow and CFSEhigh in different tissues of control mice (AdHu5-GFP immunized mice) were treated as 100%. The cell populations in other groups were normalized against the populations in control group correspondingly.
CD8 þ T-cell purification and adoptive transfer in SCID mice. Spleno-
cytes were isolated from BALB/c mice immunized with AdHu5-GFP or AdHu5-AAV2capsid 9 days ago. CD8 þ T cells were purified by incubating the splenocytes with rat anti-I-A/I-E MHC II, anti-CD45R/ B220, anti-CD4, anti-Ter-119/Ly-76, anti-CD19, anti-Ly-6G antibodies in PBS/1% fetal bovine serum for 30 min at 41C. All the antibodies were
Molecular Therapy vol. 15 no. 4, april 2007
ACKNOWLEDGMENTS This work was supported by a program project grant administered through the National Institutes of Health (P01HL078810) and a training grant T32-DK-007748 (SLM).
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