CD8+ T-cell responses to adeno-associated virus capsid in humansFederico Mingozzi1,11, Marcela V Maus1,2,11, Daniel J Hui1,11,Denise E Sabatino1, Samuel L Murphy1, John E J Rasko3,4,Margaret V Ragni5, Catherine S Manno1,6, Jurg Sommer7,10,Haiyan Jiang7,10, Glenn F Pierce7,10, Hildegund C J Ertl8 &Katherine A High1,6,9

Hepatic adeno-associated virus (AAV)-serotype 2 mediated

gene transfer results in transgene product expression that is

sustained in experimental animals but not in human subjects.

We hypothesize that this is caused by rejection of transduced

hepatocytes by AAV capsid–specific memory CD8+ T cells

reactivated by AAV vectors. Here we show that healthy

subjects carry AAV capsid–specific CD8+ T cells and that

AAV-mediated gene transfer results in their expansion.

No such expansion occurs in mice after AAV-mediated gene

transfer. In addition, we show that AAV-2 induced human

T cells proliferate upon exposure to alternate AAV serotypes,

indicating that other serotypes are unlikely to evade

capsid-specific immune responses.

In the first clinical trial of hepatic AAV-2 factor IX (AAV-2-F.IX)gene transfer into subjects with hemophilia B, transgene expressionwas short lived and after 4–6 weeks started to decline, accompanied byan asymptomatic and reversible transaminitis1. Concomitantly,AAV-2 capsid–specific T cells became detectable in peripheral blood,implying T cell–mediated rejection of AAV-transduced hepatocytes.These findings raise safety questions for the use of AAV in genetransfer to humans who, owing to previous infections, harbormemory T cells to AAVs.

To test if hepatic AAV-2 gene transfer caused expansion of AAVcapsid–specific T cells in gene transfer subjects, we used a humanleukocyte antigen (HLA)-B*0702 pentamer loaded with the AAV-2capsid–derived immunodominant peptide VPQYGYLTL to testperipheral blood mononuclear cells (PBMCs) from one of ourHLA-B*0702+ subjects for frequencies of circulating AAV-2 capsid–specific CD8+ T cells. Direct staining of PBMCs drawn at week 2 aftervector infusion (preinfusion cells were not available) showed that0.14% of circulating CD8+ T cells were capsid specific (Fig. 1a). Threeweeks later, the capsid-specific population had expanded to 0.5% ofthe circulating CD8+ T cells, indicating proliferation of this T-cell

subset (Fig. 1a). By 20 weeks after vector infusion, the capsid-specific

CD8+ T-cell population had contracted to the level seen at 2 weeks

(Fig. 1b). The expansion and contraction of the capsid-specific CD8+

T-cell population paralleled the rise and fall of serum transaminases in

this subject (Fig. 1b).We cultured PBMCs drawn at 20 weeks after vector infusion with

the VPQYGYLTL peptide. A single round of in vitro stimulationincreased the pool of antigen-specific CD8+ T cells by tenfold, and3 rounds of stimulation increased the percentage to 25% (Fig. 1c).Similar robust expansion of capsid-specific CD8+ T cells responsive toa peptide carrying the AAV capsid sequence SADNNNSEY (identifiedby intracellular cytokine staining in healthy donors, using capsid-derived peptides, as previously described2) was observed in PBMCsfrom another subject with a HLA-A*0101 haplotype who had beeninfused with AAV-2-F.IX 2.5 years earlier (Fig. 1d). Expanded CD8+

T cells were functional, as evidenced by specific lysis of HLA-matchedtarget cells (Fig. 1e) and by interferon (IFN)-g production in responseto AAV epitopes (Fig. 1f). Capsid-specific CD8+ T cells from theHLA-B*0702+ hemophilic subject functionally cross-reacted with thecorresponding peptide (IPQYGYLTL) contained in AAV serotypes 1,6, 7 and 8 (Fig. 1f), which was expected considering the highconservation of this epitope among multiple serotypes of AAV(ref. 3; Supplementary Table 1 online). To determine whether AAVinfusion alone results in expansion of capsid-specific CD8+ T cells innaive mice, we infused mice transgenic for human HLA-B*0702 withAAV-2 vector over a range of doses and routes, and analyzed frequencyof capsid-specific CD8+ T cells after infusion (Fig. 1g). In contrast tothe human response, hepatic AAV-2 vector infusion at a range of dosesfailed to elicit capsid-specific CD8+ T cells in mice, although such cellswere readily elicited in mice immunized with an adenoviral vectorexpressing AAV capsid (Fig. 1g and ref. 2). Thus, hepatic AAV vectorinfusion in a naive mouse failed to elicit expansion of capsid-specificCD8+ T cells, supporting our hypothesis that the response in humansubjects was mediated by preexisting memory CD8+ T cells.

We analyzed the prevalence of AAV-specific memory T cells in thegeneral human population by testing 46 adults for AAV-2 neutralizingantibodies and for frequencies of circulating capsid-specific T cells bya screening ELISpot assay (see Supplementary Methods online).Approximately half of the subjects (25/46) had readily detectableserum titers of AAV-2 neutralizing antibodies (SupplementaryTable 2 online) while only 2/46 subjects had detectable T-cellresponses in blood (Supplementary Table 2). We identified peptideGSGAPMADNNEGADG as containing the immunodominant T-cellepitope in one of these subjects (Supplementary Fig. 1 online).

Received 6 December 2006; accepted 17 January 2007; published online 18 March 2007; doi:10.1038/nm1549

1The Children’s Hospital of Philadelphia, 3615 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, USA. 2Hospital of the University of Pennsylvania,Department of Medicine, 3400 Spruce Street, Philadelphia, Pennsylvania 19104, USA. 3Gene and Stem Cell Therapy Program, Centenary Institute of Cancer Medicineand Cell Biology, University of Sydney, New South Wales, Australia. 4Cell & Molecular Therapies, Sydney Cancer Centre, Royal Prince Alfred Hospital, Camperdown,New South Wales, Australia. 5University of Pittsburgh Medical Center, Hemophilia Center of Western Pennsylvania, 3636 Boulevard of the Allies, Pittsburgh,Pennsylvania 15213, USA. 6University of Pennsylvania School of Medicine, 3615 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, USA. 7Avigen, Inc.,1301 Harbor Bay Parkway, Alameda, California 94502, USA. 8The Wistar Institute, 3601 Spruce Street, Philadelphia, PA, 19104, USA. 9Howard Hughes MedicalInstitute, 3615 Civic Center Boulevard, Philadelphia, Pennsylvania 19104, USA. 10Present address: Bayer HealthCare LLC, 800 Dwight Way, Berkeley, California94710, USA. 11These authors contributed equally to this work. Correspondence should be addressed to K.A.H. (high@email.chop.edu).

NATURE MEDICINE VOLUME 13 [ NUMBER 4 [ APRIL 2007 419

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AAV-specific CD8+ T cells that produced IFN-g in response to thepeptide were CD45RA+CCR7–CD27+, a phenotype consistent with asubset of resting central memory CD8+ T cells4.

To increase the sensitivity of the T-cell detection method, weexpanded PBMCs from seven HLA-B*0702+ healthy donors with

peptide VPQYGYLTL and tested for AAV capsid–specific CD8+

T cells after 1–3 rounds of expansion (Fig. 2a). In two subjects thatscored negative for AAV-specific CD8+ T cells after direct ex vivotesting, such T cells could be detected after in vitro expansion (Fig. 2aand data not shown), whereas five remained negative. Two of these

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Figure 1 Cellular immune responses to AAV capsid in subjects previously infused with AAV vector. (a) AAV-specific T cells in the peripheral blood of gene

transfer subject (sub) G at weeks 2 (top row) and 5 (bottom row) after vector infusion. PBMCs from subject G were stained with three different phycoerythrin-

labeled peptide-bound HLA-B*0702 pentamers. Capsid-specific CD8+ T cells were stained with the HLA-B*0702-p74 (VPQYGYLTL) pentamer. The negative

control was a stain with an HIV-gag HLA-B*0702-restricted peptide-bound pentamer (subject HIV-negative). Epstein-Barr virus (EBV)-pentamer staining (with

an HLA-B*0702-restricted peptide) is also shown for comparison. Plots are gated on forward and side scatter, single-cell events, CD4–CD8+ cells. Numbers

in the plots indicate percentage of pentamer+ events within the plot. (b) Time course of serum transaminases, and frequency of AAV-2 peptide-specific CD8+

T cells in PBMCs, in subject G after vector infusion. Time 0, day of vector infusion. Shaded gray area, upper limit of normal for transaminases. (c,d) AAV-

specific T cells from two subjects infused with AAV-2 vector undergo robust expansion after in vitro stimulation with a major histocompatibility (MHC) class

I–restricted peptide derived from AAV capsid. (c) First panel, HLA-B*0702-p74 (VPQYGYLTL) pentamer staining of unexpanded PBMCs from subject G at

20 weeks after vector infusion. Second panel, pentamer staining after one round of in vitro stimulation (IVS1) with peptide VPQYGYLTL. Third panel,

pentamer staining after three rounds of in vitro stimulation (IVS3) with peptide. (d) HLA-A*0101-p99 (SADNNNSEY) pentamer staining of PBMCs from

subject E at 2.5 years after vector infusion (left panel) and after two rounds of in vitro stimulation with peptide 99 (right panel). The same populations of

cells stained with an HLA-A*0101 pentamer for influenza are negative (data not shown). Plots shown are gated on size, scatter, pulse width, CD4– and CD8+

cells. Numbers shown indicate percentage of AAV-specific CD8+ cells. (e) AAV-specific T cells kill MHC-matched peptide-pulsed target cells. Expanded AAV-2

(peptide VPQYGYLTL)-specific T cells from subject G were tested as effector cells against the HLA-B*0702 lymphoblastoid cell line (JY) pulsed with the same

AAV-2 peptide or HIV peptide in a standard chromium-release assay. The x axis shows four effector:target ratios, and the y axis shows the calculated specific

lysis of target cells. Data points indicate the mean of triplicates +/– s.e.m. (f) Human CD8+ T-cell cross-reactivity for AAV-2 and AAV-1, 6, 7, 8 antigens insubject previously infused with AAV-2. Cells from subject G were expanded, then stimulated with peptides as shown, and CD8+pentamer+ T cells secreting

IFN-g were identified by intracellular cytokine staining (Supplementary Methods). These studies were reviewed and approved by the Institutional Review

Board of the Children’s Hospital of Philadelphia. Informed consent was obtained from all human subjects. (g) Frequency of capsid-specific CD8+ T cells in

blood of mice transgenic for HLA-B*0702, measured by pentamer described above. Mice were previously injected with an adenoviral vector expressing AAV-2

capsid (triangles), or with an AAV-2-F.IX by intramuscular (IM) injection (squares), or with the same vector by intravenous (IV) injection (circles). These

experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Children’s Hospital of Philadelphia. AAV doses are

reported in vector genomes per kilogram body weight (vg/kg) and adenovirus doses in vector particles per kilogram body weight (vp/kg).

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five subjects had high-titer neutralizing antibodies to AAV-2, docu-menting prior exposure to AAV-2 (data not shown). Healthy-donor,capsid-specific CD8+ T cells that were expanded produced IFN-gupon stimulation with the epitopic peptide (Fig. 2b): healthy-donorcells that were expanded with AAV-2 vector capsids produced IFN-gupon stimulation with AAV-2 or AAV-8 capsids (Fig. 2c,d); similarly,healthy-donor cells that were expanded with AAV-8 capsid producedIFN-g upon stimulation with AAV-1, AAV-2 or AAV-8 capsids,demonstrating that all three vectors could be processed appropriatelyin vitro to present the epitopic peptide to capsid-specific T cells (seeSupplementary Note online).

As resting memory T cells may reside preferentially in lymphatictissues, we tested splenocytes from 18 children and 10 adults(Supplementary Table 3 online). AAV-specific T cells were detectedin only two subjects when cells were analyzed directly ex vivo. Uponexpansion of cells with single AAV capsid peptides, however, responseswere elicited in splenic samples of 5/8 children and 4/7 adults. Epitopemapping of the 5 pediatric samples and 1 adult sample showed thatmost subjects responded to one epitope; responses to two or moreepitopes were detected in only one subject each (for example,Supplementary Fig. 1). Epitopes differed depending on the subjects’HLA type, and whereas some epitopes were highly conservedamong different AAV serotypes, others were variable (SupplementaryTable 1). Ongoing analysis of human splenocytes will allow definitionof capsid epitopes for the most common HLA alleles.

Our data show that human subjects carry AAV-2–specific memoryCD8+ T cells at frequencies that are commonly too low for detectionby conventional direct ex vivo methods. The in vitro expansion methodwe employed to detect circulating AAV capsid–specific T cells maystill not be sensitive enough to fully appreciate the prevalence ofsuch T cells in humans, as indicated by the discrepancy between thepresence of AAV neutralizing antibodies and circulating CD8+ T cells.In vitro stimulation with a single peptide requires a frequency of

memory T cells at or above 1 in 106; lower levels will escape detectioneven upon in vitro expansion, since expansion typically beginswith 106 cells.

Based on studies in experimental animals that have resulted insustained transgene expression5–7, it has been assumed that AAVs failto induce cellular immune responses because they are unable to triggerinflammatory reactions needed for differentiation of dendritic cellsinto professional antigen-presenting cells. Indeed, using immaturehuman dendritic cells, we showed that AAV vectors, even if used athigh doses, fail to induce dendritic cell maturation (data not shown).In humans, initial exposure to AAV-2 occurs in the context of a helpervirus infection8, in which the robust inflammatory response to thehelper virus most likely insures that CD8+ T cells to reactive AAV andthe helper virus are primed concomitantly. It could be this preexistingmemory CD8+ T-cell response that accounts for the difference invector-infusion outcome between humans, the only natural hosts forAAV-2 infection, and other species.

It has recently been proposed that infusion of AAV-8 will leadto a different outcome, based on studies in nonhuman primatesdemonstrating a difference in the interactions of AAV-2 andAAV-8 with dendritic cells9. However, our data on PBMCs expandedfrom healthy human subjects by exposure to whole capsid(Fig. 2c,d) imply that alternative serotypes currently being consideredfor clinical applications (for example, AAV-1, AAV-8) are processedin a manner appropriate for presentation of the same epitope(s),at least by human cells, resulting in expansion of functionalCD8+ T cells that are indistinguishable from those elicited by AAV-2capsid. Moreover, and perhaps more to the point, since presentationby dendritic cells is not essential for activation of memory CD8+

T cells (see Supplementary Note), differential uptake of vectors bydendritic cells would be irrelevant to humans with immunologicalmemory to AAV undergoing a second exposure to AAV capsid as aconsequence of vector infusion. Our data would thus predict that

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Figure 2 Cellular immune responses to AAV capsid in healthy donors. (a,b) Healthy donors negative by ELISpot

carry a pool of expandable AAV-specific T cells. (a) PBMCs from a healthy donor (ND1) with the HLA-B*0702

haplotype were stained with the HLA-B*0702-p74 (VPQYGYLTL) pentamer either directly (unexpanded) or after

1, 2 or 3 in vitro stimulations (IVS) with the p74 (VPQYGYLTL) peptide. Population of AAV-2–specific CD8+ T cells

progressively rises. Plots shown are gated on forward and side scatter, pulse width and CD4–CD8+ cells. Numbers

shown indicate percentage of pentamer+ cells within the plot. (b) Intracellular cytokine stain of PBMCs from a

healthy HLA-A*0101 donor (ND2) previously expanded three times in vitro with peptide 99 (SADNNNSEY). Cells

were stimulated with medium or peptide 99, surface stained for CD8 and CD4 antigens and stained intracellularly

for IFN-g. Plots shown are gated on forward and side scatter, pulse width and CD4–CD8+ cells. Numbers indicate

percentage of IFN-g–producing CD8+ cells. (c,d) CD8+ T cells from healthy donors cross-react with alternative

serotypes. (c) Cells from a healthy donor (ND3) were expanded with AAV-2 whole capsid and restimulated with

either AAV-2 or AAV-8 whole capsid, and IFN-g production was measured by intracellular cytokine staining. Results

reported as fold increase over medium control of percentage of CD8+ T cells producing IFN-g. (d) Cells from ahealthy donor (ND4) were expanded with AAV-8 whole capsid, then restimulated with whole capsid of AAV-8, AAV-2

or AAV-1, and IFN-g synthesis was measured by intracellular cytokine staining. Results reported as fold increase over

medium control of percentage of CD8+ T cells producing IFN-g. Note that IFN-g+ cells for subject G (Fig. 1f)

represent a subset of CD8+pentamer+ T cells, whereas for ND3 and ND4 (Fig. 2c,d), IFN-g+ cells are a subset of all CD8+ T cells. These studies were

reviewed and approved by the Institutional Review Board of the Children’s Hospital of Philadelphia. Informed consent was obtained from all human subjects.

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alternate AAV serotypes will fare no better in hepatic gene transfertrials than AAV-2 vectors. Other target tissues may escape destructionby capsid-specific CD8+ T cells (see Supplementary Note).

In summary, we have documented a CD8+ T-cell response to AAVcapsid in humans, and have presented evidence suggesting thatmemory CD8+ T cells become reactivated upon AAV gene transfer (seeSupplementary Note). We have shown that this population of cells canrecognize other AAV serotypes, whether processed from whole capsid,or presented on peptide-loaded cells. Management of these CD8+

T-cell responses to capsid will ultimately be required for optimal use ofAAV vectors in liver, and transient immunosuppressive regimens maybe needed to achieve sustained AAV-mediated gene transfer in humans.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTSThis work was supported by US National Institutes of Health (NIH) grantsP01 HL078810, M01-RR00240 (NIH GCRC award to Children’s Hospital ofPhiladelphia), M01-RR000056 (NIH GCRC award to University of Pittsburgh),a grant to the Penn Center for AIDS Research P30 AI045008 and the HowardHughes Medical Institute. D.J.H. was supported by training grant NIH T32HL07439, S.L.M. by training grant NIH T32 DK07748 and D.E.S. by NIH F32HL69647. We thank F. Lemonnier (Institute Pasteur) for kindly providing HLA-B*0702 transgenic mice. We thank M. Lasaro and M. Tigges for scientific input,and J. Sun for assistance in manuscript preparation.

AUTHOR CONTRIBUTIONSF.M. and M.V.M. performed the in vitro expansion experiments on normaldonors and expansions on subjects with hemophilia B. They performedintracellular cytokine assays, the cytotoxic T lymphocyte (CTL) assay, the

pentamer staining, the immunophenotyping of memory CD8+ T cells specificto the AAV capsid and the cross-reactivity experiments. D.J.H. performedexperiments on human splenocytes and the AAV epitope characterization. D.E.S.performed in vitro expansion experiments on subjects with hemophilia B enrolledin the gene transfer study and part of the ELISpot studies on healthy donors.S.L.M. performed the experiments on HLA-B*0702 transgenic mice. H.J. and J.S.performed part of the ELISpot studies and AAV antibody titer determination onnormal donors. C.S.M., M.V.R. and J.E.J.R. directed and/or participated in theclinical gene transfer study, including the care of hemophilic subjects, andcollection of PBMCs used in the study. They provided assistance in draftingthe manuscript. H.C.J.E. and G.F.P. collaborated on experimental design andinterpretation, and helped to draft the manuscript. K.A.H. directed experimentaldesign, conduct, data analysis and interpretation, and drafted the manuscript.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-textHTML version of the paper at www.nature.com/naturemedicine/.

Published online at http://www.nature.com/naturemedicine

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions

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154–158 (2000).7. Snyder, R.O. et al. Nat. Med. 5, 64–70 (1999).8. Muzyczka, N. & Berns, K.I. Parvoviridae: The Viruses and Their Replication, 2327–

2359 (Lippincott Williams & Wilkins, Philadelphia, 2001).9. Vandenberghe, L.H. et al. Nat. Med. 12, 967–971 (2006).

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