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Page 1: The Adenovirus Capsid Protein Hexon Contains a Highly Conserved Human CD4               +               T-Cell Epitope

HUMAN GENE THERAPY 13:1167–1178 (July 1, 2002)© Mary Ann Liebert, Inc.

The Adenovirus Capsid Protein Hexon Contains a HighlyConserved Human CD41 T-Cell Epitope

MELANIE OLIVE,1,2 LAURENCE EISENLOHR,3,4 NEAL FLOMENBERG,2–4 SUSAN HSU,5

and PHYLLIS FLOMENBERG1,2,4

ABSTRACT

The immunogenicity of adenovirus vectors remains a major obstacle to their safe and efficacious use for genetherapy. In order to identify T-cell epitopes directly from adenoviruses, four viral protein sequences werescreened for the well-characterized 9-mer HLA-A2 binding motif. Peripheral blood mononuclear cells (PBMC)from healthy adults were tested for responses to 17 selected viral peptides using a short-term interferon-gELISPOT assay. Memory T-cell responses were identified to a single peptide derived from the major capsidprotein hexon in 5 of 6 HLA-A2–positive donors. Unexpectedly, responses to this hexon peptide were also de-tected in 4 of 6 HLA-A2–negative donors, and responder cells were identified as CD41 T cells by immuno-magnetic depletion experiments. A longer 15-mer peptide, H910-924, was identified as the optimal CD41

T-cell epitope. This hexon epitope induces strong proliferative T-cell responses that can be blocked by a mono-clonal antibody against HLA-DR, and molecular HLA typing of donors suggests that the peptide response isrestricted by multiple HLA-DR alleles. Additionally, quantitative analysis of responses to H910-924 and wholeadenovirus reveals that the frequency of circulating CD41 T cells specific for this single hexon epitope (mean 561 per 106 PBMC) represents up to one third of the total adenovirus-specific T-cell response. Finally, com-parison of hexon sequences from over 20 different human adenovirus serotypes indicates that H910-924 ishighly conserved. In most individuals, therefore, T-cell responses to this hexon epitope will be induced by alladenovirus vectors, including “gutted” vectors packaged with capsid proteins and vectors based on differentserotypes.

1167

OVERVIEW SUMMARY

Although most adults exhibit memory T-cell responsesagainst adenoviruses as a result of prior exposure to theseubiquitous pathogens, the target proteins and their epi-topes remain unknown. In this study, the major capsidprotein hexon was found to contain a CD41 T-cell epitopethat: (1) is recognized by most healthy adults; (2) exhibitsrestriction by multiple HLA-DR alleles; (3) accounts forup to one third of the CD41 T-cell response to whole ad-enovirus; and (4) is present on all adenovirus vectors, in-cluding “gutted” or helper-dependent vectors and vectorsbased on different human serotypes. The significance ofthis finding in regard to the design of adenovirus gene ther-apy vectors is discussed.

INTRODUCTION

ADENOVIRUSES are under intensive investigation as genetherapy vectors for a broad range of applications as sum-

marized in recent reviews (Wivel and Wilson, 1998; Albeldaet al., 2000). However, one major obstacle is the immuno-genicity of adenovirus vectors. In animal models, administra-tion of adenovirus vectors is associated with an acute nonspe-cific immune response followed by an antigen-specific cellularimmune response that results in elimination of vector-trans-duced cells. The early phase response consists of both localizedand systemic inflammatory reactions that appear to be medi-ated by cytokine release from tissue macrophages that take upinput adenovirus vectors (Worgall et al., 1997; Zhang et al.,2001). Within 2 to 3 weeks, there is a marked reduction of re-

1Center of Human Virology, 2Department of Medicine, 3Department of Microbiology and Immunology, and 4Kimmel Cancer Center, ThomasJefferson University, and 5American Red Cross, Philadelphia, PA 19107.

Page 2: The Adenovirus Capsid Protein Hexon Contains a Highly Conserved Human CD4               +               T-Cell Epitope

combinant protein expression caused by cytotoxic T-cell(CTL)–mediated destruction of adenovirus-transduced cells(Yang et al., 1994, 1996). In addition, serotype-specific neu-tralizing antibodies block expression from vectors on rechal-lenge (Dai et al., 1995). These immune responses limit both thesafety and efficacy of adenovirus-mediated gene therapy.

Although nearly all adults are exposed to these ubiquitouspathogens in early childhood and remain seropositive (Schmitzet al., 1983; Chirmule et al., 1999), the cellular immune re-sponse against adenoviruses is poorly understood. We previ-ously identified the presence of both CD41 and CD81 T-cellresponses against adenoviruses in peripheral blood mononu-clear cells (PBMC) from healthy adults (Flomenberg et al.,1995, 1996). Using a quantitative, short-term interferon-g (IFN-g) ELISPOT assay, we recently confirmed the presence ofmemory adenovirus-specific CD41 T cells at frequencies rang-ing from 30 to 300 per 106 PBMC and identified an inverse re-lationship between the frequencies of virus-specific T cells anddonor age (Olive et al., 2001). In addition, adenovirus-specificCD41 T-cell responses exhibited cross-reactivity with diverseserotypes. Similarly, other investigators have demonstratedcross-reactivity of adenovirus-specific human CD81 T-cell re-sponses (Smith et al., 1998). Although specific targets have notyet been well defined, the presence of cross-reactive T-cell re-sponses indicates that some T-cell epitopes are conservedamong different adenovirus serotypes, in contrast to neutraliz-ing antibodies that are serotype-specific.

As an initial approach to identify specific adenovirus T-cellepitopes, we screened PBMC from healthy adults for responsesto HLA-A2 motif peptides derived from adenovirus protein se-quences. HLA-A2 is the most common class I allele in generalpopulation, and the well-defined HLA-A2 peptide motif can beused to directly screen proteins for sequences predicted to bindto HLA-A2 (Ruppert et al., 1993). This strategy has been usedsuccessfully to identify HLA-A2–restricted T-cell epitopesfrom several other human pathogens, including Mycobacteriumtuberculosis and human immunodeficiency virus (HIV) (Lal-vani et al., 1998; Larsson et al., 1999). PBMC were tested forresponses to the peptides using the IFN-g ELISPOT assay, ahighly sensitive and quantitative method for the detection ofpeptide-specific T cells (Lalvani et al., 1997). Detection of IFN-g–secreting cells in response to a peptide by the short-termELISPOT assay is specific for memory/effector T cells andavoids potential artifacts caused by prolonged incubation invitro. The results of this analysis using peptides derived fromthe sequences of four different adenovirus proteins are de-scribed.

MATERIALS AND METHODS

Study participants

One buffy coat collection was obtained from the Thomas Jef-ferson University Hospital Blood Donor Center from an anony-mous blood donor (donor A). Heparinized blood specimens (40ml) were obtained from 11 healthy volunteers ages 18 to 65(donors B–L) after written informed consent was obtained. Serafrom all 11 volunteers tested positive for antiadenovirus anti-bodies by a dot-blot assay (described elsewhere). The Thomas

Jefferson University Institutional Review Board approved theresearch protocol.

HLA typing

Molecular HLA-A, HLA-B, and HLA-DR typing was per-formed by sequence-based methods using ABI Prism Dye Ter-minator sequencing kits on a 377 automatic sequencer (A & B,Applied Biosystem, Inc., Foster City, CA). HLA-DQB1 typingwas performed by a reverse PCR–sequence specific oligonu-cleotide probe technique using reagents from Murex Inno-genetics (Abbott Laboratories, Abbott Park, IL).

Adenovirus sequences

Adenovirus protein sequences were obtained from a searchof the Entrez databases on the NCBI website (www.ncbi.nlm.nih.gov), and sequence comparisons were performed usingthe similarity search program BLAST.

Synthetic peptides

Peptides were synthesized by Research Genetics (Huntsville,AL) using Fmoc synthesis methods and purity assayed by massspectrometry analysis. Peptides were dissolved in dimethyl sul-foxide (DMSO) at 10 mg/ml and stored in small aliquots at270°C.

Ex vivo ELISPOT assay

PBMC were separated from heparinized whole blood orbuffy coat collections using Ficoll-Hypaque density gradientcentrifugation, and cells were used either fresh or thawed fromcryopreserved aliquots. The ex vivo ELISPOT assay for detec-tion of IFN-g was based on a previously described protocolwith minor modifications (Lalvani et al., 1997). Ninety-six–well polyvinylidene difluoride-backed plates (Millipore,Bedford, MA) were coated overnight at 4°C with 15 mg/ml ofanti-IFN-g monoclonal antibody (mAb) 1-D1K (Mabtech,Stockholm, Sweden). Wells were washed and blocked withRPMI supplemented with 10% pooled human AB sera (AtlantaBiologicals, Atlanta, GA), 100 U/ml of penicillin, 100 mg/mlof streptomycin, 2 mM of glutamine, and 10 mM of HEPES(R10) for 1 hr at 37°C. Next, 10 mM of each peptide or pep-tide pool was incubated with 250,000 PBMC in 100 ml of R10in quadruplicate wells for 20 hr at 37°C in 5% CO2. As con-trols, PBMC were incubated with media alone or phytohemag-glutinin (PHA) 10 mg/ml (Difco, Detroit, MI) plus phorbol es-ter (PMA) 0.01 mg/ml (Alexis Biochemicals, San Diego, CA).Wells were washed extensively with phosphate-buffered saline(PBS) containing 0.05% Tween 20 and incubated with 1 mg/mlof biotinylated anti-IFN-g mAb 7-B6-1 (Mabtech) for 3 hr atroom temperature. Next, wells were washed and incubated witha 1:1000 dilution of alkaline phosphatase conjugate (Mabtech)for 2 hr. Wells were washed again and incubated with alkalinephophatase substrate (Vector Labs, Burlingame, CA) for 30min; the reaction was stopped with tap water. After wells air-dried overnight, large spots with fuzzy borders were countedunder a dissecting microscope. The number of spot-formingcells (SFCs) per 106 PBMC was calculated as the mean num-ber of SFCs in quadruplicate microwells 3 4. Peptide-specificprecursor frequencies (per 106 PBMC) were calculated as the

OLIVE ET AL.1168

Page 3: The Adenovirus Capsid Protein Hexon Contains a Highly Conserved Human CD4               +               T-Cell Epitope

mean number of SFCs in the peptide-stimulated microwells mi-nus the mean number of SFCs in the control microwells 3 4.

Peptide-specific proliferation assay

PBMC suspended in R10 were plated in 96-well round-bot-tom plates at a concentration of 105 cells per well and incu-bated with 1 mM peptide or media alone in triplicate. For block-ing experiments, based on a titration analysis, class IIlocus-specific mAbs against monomorphic determinants onHLA-DR (clone L243), HLA-DP (clone B7/21), and HLA-DQ(clone Ia3) (Leinco Technologies, St. Louis, MO) were prein-cubated at 10 mg/ml with PBMC for 30 min at room tempera-ture and then diluted to 5 mg/ml with media 6 peptide. Afterincubation at 37°C in 5% CO2 for 7 days, cells were pulsed for6 hr with 1 mCi [3H]-thymidine (50 mCi/ml; ICN, Costa Mesa,CA) per well. DNA was harvested and incorporated thymidinewas measured in a Wallac 1205 Betaplate liquid scintillationcounter. Results were expressed as the mean counts per minute(cpm); Dcpm, and % inhibition were calculated as follows:Dcpm 5 mean cpm peptide 6 mAb 2 mean cpm without pep-tide; % inhibition 5 1 d (delta cpm peptide 1 mAb/delta cpmpeptide) 3 100.

Hexon peptide-specific T-cell lines

PBMC (4 3 106) PBMC were suspended in 200 ml of R10and incubated with 100 mM of hexon peptide for 1 hr. Cellswere diluted to 2 ml with R10 and incubated in a 24-well plateat 37°C in a 5% CO2 atmosphere. Human recombinant inter-leukin-2 (IL-2; Becton Dickinson, Bedford, MA) was added toa final concentration of 20 U/ml on days 7 and 11. At day 14,specificity was tested with the IFN-g ELISPOT assay by incu-bating the T-cell line (TCL) (10,000 per well) with B-lym-phoblastoid cell lines (B-LCL) (50,000 per well) preloaded 1hr with 10 mM H910-924 or mock-loaded as a control. In somecases, TCL (1 3 106 per nickel well) were restimulated every2 weeks with peptide-loaded autologous B-LCL (2 3 106), andcultures were supplemented with 20 U/ml of IL-2 every 3 to 4days. B-LCL were prepared from donor PBMC as previouslydescribed (Flomenberg et al., 1996).

Cytotoxic T-cell assay

Cytotoxicity was measured by a calcein release assay as de-scribed (Lichtenfels et al., 1994). Targets were suspended in100 ul of Hank’s balanced salt solution without phenol sup-plemented with 5% fetal bovine serum (FBS), 2 mM of gluta-mine, and 10 mM of HEPES (CTL assay media), labeled for30 min with 5 mg/ml of calcein (Molecular Probes, Eugene,OR) at 37°C, and washed twice. Targets were then plated intriplicate at 5 3 103 per well with varying concentrations of ef-fectors in a 200 ml final volume in 96-well round-bottom plates.Plates were spun at 200g for 1 min and incubated at 37°C for3 hr. Cells are pelleted at 700g for 5 min, 100 ml of each su-pernatant was transferred to new wells, and fluorescence wasmeasured on a Victor2 1420 multilabel counter (Wallace,Gaithersburg, MD). Spontaneous release (SR) was measuredfrom target cells incubated in medium alone. Maximal release(MR) was measured from target cells incubated with 0.1% Tri-ton in 50 mM of sodium borate, pH 9.0. Percent lysis was cal-

culated as follows: [(sample fluorescence 2 SR fluorescence)/(MR fluorescence 2 SR fluorescence)]3 100.

Preparation of adenovirus and control antigens

Adenovirus and control antigens were prepared from the sub-group C adenovirus type 2-infected and uninfected cell lysatesas previously described (Flomenberg et al., 1995). Briefly, thehuman lung carcinoma cell line A549 was inoculated with ad-enovirus and incubated in Dulbecco’s modified Eagle medium(DMEM) supplemented with 2% FBS. When the adenovirus-infected monolayer exhibited 31 cytopathic effect (CPE), cellswere harvested and suspended in PBS at a concentration of 107

cells per milliliter. Specimens were subjected to three freeze-thaw cycles to disrupt cells, and the supernatants were har-vested. Uninfected cell lysates were prepared similarly. Ade-novirus and control antigen preparations were UV inactivatedfor 20 min and stored at 270°C. The protein concentration ofeach antigen preparation was measured by the Bradford method(BioRad, Hercules, CA). The adenovirus fiber mutant AdZ.F(pK7) was kindly provided by Dr. T. Wickham (GenVec,Gaithersburg, MD) (Wickham et al., 1996).

Preparation of targets infected withvaccinia/adenovirus recombinants

Vaccinia vectors expressing the adenovirus type-5 hexon andthe type-2 fiber were obtained from Dr. J. Wilson (Wistar In-stitute, Philadelphia, PA) and Dr. J. Engler (University of Al-abama), respectively (Hong and Engler, 1991; Jooss et al.,1998). Vaccinia/adenovirus recombinants were propagated inTK2 cells, and titered stocks were prepared as previously de-scribed (Wysocka et al., 1994). For B-LCL infections, 6 3 105

B-LCL were adsorbed with 10 plaque-forming units (pfu) percell each vaccinia vector in 200 ml RPMI, 0.1% bovine serumalbumin (BSA) for 90 min, rinsed, resuspended in R10, and in-cubated overnight for use as targets.

Depletion of CD41 and CD81 T cells

PBMC were suspended at 107 cells per milliliter in PBS con-taining 2% FBS, and 2 3 107 Dynabeads M-450 coated withCD4-specific mAb (Dynal, Lake Success, NY) were added. Af-ter incubation for 30 min at 4°–8°C with continuous mixing,the tube was placed in a magnetic stand, and cells remaining insolution were harvested and washed before use in the ELISPOTassay. Similarly, CD81 T cells were depleted using DynabeadsM-450–coated with anti-CD8 mAb. The efficiency of each de-pletion was confirmed to be more than 98% by flow cytome-try.

Immunophenotyping

Analysis of cell surface antigens was done by two-color im-munofluorescence flow cytometry using standard direct stain-ing methods. Briefly, 5 3 105 PBMC were pelleted in 4-mlpolystyrene tubes and 10 ml of the appropriate mAb cocktailwas added to each tube. After incubation at 4°C for 30 min,cells were washed, fixed with 2% formaldehyde, and analyzedby a flow cytometer (FACScan; Becton Dickinson, San Jose,CA). All mAbs were purchased as fluorescein or phycoerythrinconjugates (Becton Dickinson). mAb cocktails used were

ADENOVIRUS HEXON T CELL EPITOPE 1169

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CD45/CD14, CD3/CD8, and CD3/CD4. Nonspecific bindingwas monitored using isotypic controls.

Statistical methods

Data from the ELISPOT assays were analyzed using a pairedt test to compare the mean number of SFCs in the quadrupli-cate control and experimental microwells.

RESULTS

Identification of HLA-A2 motif peptides fromadenovirus proteins

Adenovirus, a nonenveloped double-stranded DNA (ds-DNA) virus, is packaged with four core proteins and sevenouter capsid proteins (Shenk, 1996). Two of these structuralproteins were selected for analysis: hexon, the major capsidprotein and core protein VII, the major core protein. In addi-tion, two nonstructural proteins (early proteins expressed ininfected cells but not packaged into virions) were evaluated:the transactivating region E1A, an important CTL target inmice (Rawle et al., 1991), and DNA polymerase, a major CTLtarget in other viral models (Rehermann et al., 1995; Larssonet al., 1999). The closely related group C adenovirus type-2and type-5 sequences for each protein were screened for theHLA-A2 binding motif that consists of a 9-mer peptide withspecific anchor residues at position 2 (L or M) and position 9(L, V, or I). In addition, certain residues in other positions arepreferred or deleterious. The peptide motif search programavailable on the National Institutes of Health website (www-bimas.dcrt.nih.gov/molbio/hla_bind) was utilized to screen

protein sequences and calculate the predicted binding affinityof each peptide (Parker et al., 1994). Seventeen peptides withpredicted high affinity binding to HLA-A2 were identified andsynthesized (Table 1).

OLIVE ET AL.1170

TABLE 1. PANEL OF SEVENTEEN HLA-A2 MOTIF PEPTIDES

IDENTIFIED FROM ADENOVIRUSa

Adenovirus protein Residue(total residues) positions Sequence t1/2

b

Hexon (951) 50–58 RLTLRFIPV 132511–519 GLVDCYINL 131712–720 YLNHTFKKV 608736–744 LLTPNEFEI 103759–767 NMTKDWFLV 495913–921 TLLYVLFEV 3433916–925 YVLFEVFDVV c 1382

Core protein VII (174) 117–125 RLAAGIVTV 160DNA polymerase (1056) 75–83 NLVQDVQPV 160

352–360 VMVRDTFAL 152466–474 GLTDASFNV 1654591–599 RLLPGVFTV 3433642–650 TLHNRGWRV 522835–843 VLAWTRAFV 650932–940 FLAPKLYAL 226

E1A (289) 111–119 SMPNLVPEV 116272–280 LLNESGQPL 149

aThe adenovirus type 5 residue positions are listed. The protein sequencesare identical to those from adenovirus type 2.

bEstimate of half time of dissociation in minutes.cHexon peptide 916–925 contains 2 overlapping HLA-A2 motif 9-mer pep-

tides.

FIG. 1. Detection of T-cell responses to adenovirus HLA-A2motif peptides. Peripheral blood mononuclear cells (PBMC)from HLA-A21 donor D were incubated with each individualpeptide (10 mM each) from the single positive group of pep-tides (pool 2). After 20 hr, interferon-g (IFN-g)–secreting cellswere quantified by the ex vivo ELISPOT assay. SFCs, spot-forming cells.

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Identification of an IFN-g response to the hexon peptide H913-921

PBMC from six HLA A2-positive donors were tested for re-sponses to the above panel of peptides using the short-termELISPOT assay (see Materials and Methods). PBMC were firsttested against pools of four or five adenovirus peptides each.Controls included the HLA-A2–restricted influenza matrix pep-tide M58-66 (Bednarek et al., 1991), PHA/PMA, and mediaalone. PBMC from all donors responded to the T-cell mitogens(.1000 SFCs per 106 PBMC), and three of six donors (D, I,

and L) responded to the influenza peptide control (mean, 62;range, 49–88 SFCs per 106 PBMC). Analysis of responses tothe adenovirus peptides revealed that five of six donors (D, E,F, G, and I) responded to a single, identical adenovirus peptidepool. Next, testing of individual peptides from the one positivepool revealed that all five donors responded to a single peptidefrom the capsid protein hexon, H913-921 (mean, 40; range,8–97 SFCs per 106 PBMC). A representative assay is shown inFigure 1.

The hexon peptide H913-921 activates CD41 T cells

IFN-g may be secreted by natural killer (NK) cells, CD41

T cells, and CD81 T cells. In order to identify the cell type ac-tivated to secrete IFN-g by the hexon peptide, PBMC were de-pleted of either CD41 or CD81 T cells by an immunomagneticseparation technique that minimizes nonspecific cell activation(see Materials and Methods). Each cell population was testedfor a response to H913-921 in the IFN-g ELISPOT assay. Inthree of three HLA-A2–positive donors tested, CD41 T-cell de-pletion eliminated the peptide-specific response, whereas CD81

T-cell depletion did not inhibit the response. Next, PBMC from2 HLA-A2–negative donors (B and C) were tested againstH913-921 and found to exhibit peptide-specific responses (74and 72 SFCs per 106 PBMC, respectively). CD41/CD81 T-celldepletion studies with both HLA-A2–negative donors con-firmed that the peptide response was primarily mediated byCD41 T cells, as shown in Figure 2. The fact that the IFN-gresponse is mediated by CD41 T cells in both HLA-A2–posi-tive and HLA-A2–negative donors suggests that the hexon pep-tide response is HLA class II-restricted rather than class I A2-restricted as originally predicted.

The hexon peptide H910-924 is the optimal CD41 T-cell epitope

PBMC were tested against a series of overlapping peptidesin the IFN-g ELISPOT assay in order to define the optimal Tcell epitope (Table 2). Elimination of three residues from eitherend of the original peptide (amino acids 913–915 or 919–921)completely abrogated the T-cell response, suggesting that thesespecific residues may be important in binding to peptide. Ad-

ADENOVIRUS HEXON T CELL EPITOPE 1171

TABLE 2. COMPARISON BETWEEN THE IFN-g RESPONSE TO HEXON PEPTIDE

H913–921 AND A PANEL OF OVERLAPPING PEPTIDESa

Hexon peptide Sequence SFCs per 106 PBMC

913–921 TLLYVLFEV 41

916–924 YVLFEVFDVV 0

910–918 DEPTLLYVL 4

907–921 DPMDEP TLLYVLFEV 64

913–927 TLLYVLFEV FDVVRV 61

910–924 DEP TLLYVLFEV FDV 75

aPBMC from donor B were incubated with 10 mM of each peptide for 20 hr. IFN-g expression was measured by the ELISPOT assay. The H913–921 sequenceis outlined. SFCs, spot-forming cells; PBMC, peripheral blood mononuclear cells;IFN-g, interferon-g.

FIG. 2. Comparison of hexon peptide-specific responses fromCD41 or CD81 T-cell–depleted peripheral blood mononuclearcells (PBMC). Donor B (HLA-A2–negative) PBMC were de-pleted of either CD41 or CD81 T cells by immunomagneticmethods and incubated with 10 mM H913-921 or media alone.At 20 hr, interferon-g (IFN-g)–secreting cells were quantifiedby ELISPOT assay. SFCs, spot-forming cells.

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dition of three residues to each end of the H913-921 peptide tocreate the 15-mer H910-924 elicited the strongest CD41 T-cellresponse. Furthermore, the response to H910-924 was detectedat a lower peptide concentration compared to H913-921 (Fig.3). These data suggest that H910-924 has a higher binding affin-ity to the presenting HLA allele(s), consistent with the fact thatthe preferred length of HLA class II epitopes is 13–16 residues,whereas class I molecules optimally bind 9–10 residue peptides.

H910-924 is recognized by the majority of donors

The 15-mer peptide H910-924 was further evaluated for theability to stimulate responses in both HLA-A2–positive andHLA-A2–negative donors in the IFN-g ELISPOT assay. PBMCfrom five of six HLA-A2–positive donors exhibited CD41

T-cell responses to H910-924 that were comparable to the re-sponses against H913-921 (mean, 40; range, 16–84 per 106

PBMC). In addition, four of six HLA-A2–negative donors haddetectable responses to H910-924 (mean, 86; range, 16–158CD41 T cells per 106 PBMC). Thus, the majority of donorstested (9/12) exhibit memory/effector CD41 T-cell responsesto the adenovirus hexon epitope H910-924.

The T cell response to H910-924 is restricted by multiple HLA-DR alleles

To address the restriction of the hexon epitope further, theability of HLA class II locus-specific mAbs to block the T-cellresponse to H910-924 was evaluated in a proliferation assay.The H910-924 peptide induced strong proliferative T-cell re-sponses in PBMC from all 4 donors tested (Table 3). Additionof a mAb against HLA-DR efficiently blocked the proliferativeresponse to peptide in all donors in all experiments (.90% in-hibition). In contrast, partial blocking (40%–70%) of the pro-liferative response to peptide by mAb against HLA-DP or HLA-DQ was observed in 1 and 2 donors, respectively. Therefore,the hexon peptide response appears primarily HLA-DR–re-stricted. Next, molecular HLA typing was compared betweenall 12 donors. As shown in Table 4, there was no single sharedDRB1 allele among the 9 donors that respond to peptide. In ad-dition, there was no shared DRB3 or DRB5 allele among theresponders (data not shown). The fact that the majority ofdonors tested responded to the H910-924 peptide and have di-verse HLA-DR types suggests that the hexon peptide responseis restricted by multiple HLA-DR alleles.

Hexon peptide-specific T cell lines recognize antigen-presenting cells infected with replication-deficient adenovirus

In order to determine if the hexon H910-924 is naturallyprocessed from the intact hexon protein, T cells previously stim-ulated with the hexon peptide were tested for the ability to rec-ognize adenovirus-infected antigen-presenting cells (APCs). Ashort-term hexon peptide-specific TCL was prepared fromdonor A PBMC by stimulation with hexon peptide and IL-2 for2 weeks (see Materials and Methods). Flow cytometry analy-

OLIVE ET AL.1172

TABLE 3. EFFECTS OF HLA CLASS II LOCUS-SPECIFIC MONOCLONAL ANTIBODIES ON THE

PROLIFERATIVE T-CELL RESPONSE TO HEXON PEPTIDEa

[H3]Thymidine incorporation (cpm)

DQ Peptide 1 anti-DR Peptide 1 anti-DP Peptide 1 anti-DQDonor No peptide Peptide (% inhibition) (% inhibition) (% inhibition)

A 6,949 104,041 13,300 (93) 114,930 (0) 62,103 (43)B 774 65,254 1,212 (99) 58,024 (11) 20,639 (69)C 942 118,083 8,713 (93) 102,389 (14) 112,453 (6)D 1,261 82,892 4,625 (94) 50,327 (40) 64,636 (22)

aPBMC were incubated with 1 mM H910–924 peptide or media alone for 7 days, and proliferation was measured by [3H]thymi-dine incorporation. For blocking experiments, PBMC were preincubated with mAbs against HLA-DR, HLA-DP, or HLA-DQ(10 mg/ml) before addition of peptide.

Cpm, counts per minute; PBMC, peripheral blood mononuclear cells; mAbs, monoclonal antibodies.

FIG. 3. Comparison of T-cell responses to the 15-mer and 9-mer hexon peptides. Peripheral blood mononuclear cells(PBMC) from donor B were incubated with sequential 10-folddilutions of each peptide for 20 hr. Peptide-specific responseswere quantified by the interferon-g (IFN-g) ELISPOT assay.SFCs, spot-forming cells.

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sis revealed that the cell line was approximately 80%CD3/CD41. The Ad5 E1-deleted vector AdZ.F(pK7), whichcontains an altered fiber protein with a heparin/heparan sulfatebinding domain that enhances binding to hematopoietic cells,was utilized to infect B-LCL APCs (Wickham et al., 1996). Thereplication-deficient AdZ.F(pK7) binds and enters B-LCL to al-low presentation of input virion proteins but does not expressnew hexon proteins in B-LCL (P. Flomenberg, unpublisheddata). The peptide-specific TCL exhibited specific IFN-g re-sponses to virus-infected or peptide-loaded autologous B-LCLbut not HLA DR-mismatched B-LCL (Fig. 4). Similarly, a pep-tide-specific TCL from donor B responded to adenovirus vec-tor-infected APC (data not shown). These data suggest that thehexon epitope is naturally processed from input virions in ad-enovirus-infected APCs and is recognized by T cells in an HLA-restricted manner.

A hexon peptide-specific TCL is cytotoxic and killstargets expressing hexon

To evaluate the specificity of the T-cell response to H910-924 further, hexon peptide-stimulated TCL were tested for cy-totoxic activity based on the fact that cytotoxic CD41 T-cellclones have been described against other viruses such as in-fluenza and measles (van Binnendijk et al., 1993; Jameson etal., 1998). Donor C PBMC (HLA-DR7, -DR15) were stimu-lated with hexon peptide and IL-2 for 4 weeks, and flow cy-tometry analysis confirmed that over 90% of cells wereCD3/CD41. The CD41 TCL was incubated with a panel of B-LCL targets with and without H910-924 and cytotoxicitymeasured by a calcein release assay, as described in Materialsand Methods. The TCL killed peptide-loaded B-LCL matchedfor either HLA-DR7 or HLA-DR15 but did not kill HLA-DR–mismatched targets or mock-loaded targets (Fig. 5A). TCLfrom donor A and B also exhibited peptide-specific cytotoxic-ity (data not shown). Next, the donor C TCL was tested for theability to recognize the individual Ad5 hexon protein expressed

from a vaccinia vector (Vac/hexon) in B-LCL targets. As shownin Figure 5B, the TCL killed HLA-DR15–matched B-LCL in-fected with a vaccinia vector expressing hexon but not targetsinfected with a control vaccinia vector expressing the adenovi-rus fiber protein. Together with the above data, these studiesconfirm that the hexon peptide is naturally processed from theintact hexon protein. In addition, as an example of the HLA-DR restriction, this analysis suggests that the peptide responseis restricted by both HLA-DR7 and HLA-DR15.

ADENOVIRUS HEXON T CELL EPITOPE 1173

TABLE 4. COMPARISON OF MOLECULAR HLA TYPING AMONG DONORS WHO RESPOND TO

HEXON PEPTIDE H910–924 AND NONRESPONDERSa

Class I Alleles Class II Alleles

Donor A B DRB1 DQB1

A 01011 2402 0801 52011 03011 15021 0201 0601B 03011 2301 1402 4102 01021 03011 0201 0501C 3303 44032 5601 0701 15021 0202 06011D 02011 31012 1302 4901 0101 0701 0202 05011E 02011 03011 0801 51011 03011 1301 0201 0603/14F 02011 2902 1402 3801 03011 0701 0201 03032G 0206 2402 27052 4402 04011 1201 0302/07 0603/14H 6601 6802 1402 4102 01021 03011 0201 0501I 02011 31012 0801 3801 03011 1302 0201 0604J 03011 6802 1402 3801 01021 1301 0501 0603/14Kb 03 28 14 38 01 13 NA NAL 02011 2301 07021 4402 04011 15011 03011 0602/11

aDonors A to I respond to H910–924; donors J to L do not respond to the hexon peptide in the IFN-g ELISPOT assay.bDonors J and K are related. Donor K was typed serologically.NA, not available.

FIG. 4. Hexon peptide-specific T cells recognize adenovirusvector-infected cells. A hexon peptide-specific T-cell line pre-pared from donor A (HLA-DR3, 15) was tested against autol-ogous (auto) B-lymphoblastoid cell lines (B-LCL) infected withthe Ad5 E1-deletion mutant AdZ.F(pK7) or preloaded with 1mM H910-924. As negative controls, untreated auto B-LCL andHLA-DR mismatched (allo) donor J B-LCL (HLA-DR1, 13)with and without peptide were tested. Interferon-g (IFN-g)–ex-pressing cells were quantified by ELISPOT assay at 20 hr.SFCs, spot-forming cells.

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The CD41 T-cell response to H910-924 represents one third of the response to whole adenovirus

The IFN-g ELISPOT assay was used to quantitate the fre-quencies of CD41 T cells specific for whole adenovirus, as pre-viously described (Olive et al., 2001). PBMC were tested against

an inactivated adenovirus type 2-infected cell lysate (adenovirusantigen) that contains both early regulatory viral proteins andstructural viral proteins; uninfected cell lysate was used as a con-trol antigen. All 12 donors exhibited adenovirus-specific CD41

T-cell responses. Among the 9 donors with peptide-specific re-sponses, the mean frequency of adenovirus-specific CD41 T cellswas 185 per 106 PBMC (range, 34–439). A comparison of the T-cell responses against whole adenovirus antigen and the H910-924 peptide from each donor is shown in Figure 6. The frequencyof hexon peptide-specific CD41 T cells (mean, 61 per 106 PBMC;range, 16–158) represented approximately one third of eachdonor’s response to whole adenovirus. These quantitative data in-dicate that hexon is a major CD41 T cell target, and that reactiv-ity to the H910-924 determinant represents a significant propor-tion of the T-cell response to whole adenovirus. Among the 3donors without hexon peptide-specific responses, donors J and Khad moderate frequencies of adenovirus-specific CD41 T cells(142 and 55 per 106 PBMC, respectively) and share HLA-DR1and HLA-DR13, suggesting that the hexon peptide may not bindto these class II alleles. In contrast, donor L (HLA-DR4, -DR15)had the lowest response to adenovirus antigen among all 12 donors(33 CD41 T cells per 106 PBMC). Hence, the low total frequencyof adenovirus-specific CD41 T cells may explain the negative re-sponse to the hexon epitope in this donor.

The H910-924 epitope is highly conserved among adenovirus serotypes

There are 51 different human adenovirus serotypes that areclassified into 6 groups A to F based on several properties in-

OLIVE ET AL.1174

A

B

FIG. 5. Hexon peptide-specific T cells kill targets expressinghexon. A peptide-specific CD41 T cell line prepared from donorC (HLA-DR7, 15) was tested against B-lymphoblastoid celllines (B-LCL) targets from donor D (HLA-DR 7-matched) ordonor L (HLA-DR15-matched) preloaded with 1 mM H910-924or infected overnight with a vaccinia vector expressing hexon(Vac/hexon). As negative controls, untreated targets, targets in-fected with a vaccinia vector expressing fiber (Vac/fiber), andHLA-DR mismatched donor J targets (HLA-DR1, 13) with andwithout peptide were tested. A: Effectors were incubated withtargets with and without peptide at an E:T ratio of 5:1. B: Ef-fectors were incubated at varying concentrations with vaccinia-infected HLA-DR15 matched targets. Cytotoxicity was mea-sured by a calcein-release assay after 3 hr. FIG. 6. Comparison of the frequencies of CD41 T cells

against the hexon peptide and whole adenovirus. Peripheralblood mononuclear cells (PBMC) were incubated with 10mg/ml adenovirus-infected cell lysate, uninfected cell lysate(lysate control), 10 mM H910-924, or media alone (peptide con-trol). The number of interferon-g (IFN-g)–expressing T cellswas measured by ELISPOT assay at 20 hr. Results were cal-culated by subtracting spot-forming cells (SFCs) in controlwells from experimental wells.

Page 9: The Adenovirus Capsid Protein Hexon Contains a Highly Conserved Human CD4               +               T-Cell Epitope

cluding DNA homology and hemagglutination patterns (Shenk,1996). Although there are unique antigenic determinants onhexon proteins from each serotype, the hexon epitope H910-924 is located in the conserved C-terminal domain of the cap-sid protein. A comparison of hexon C-terminal protein se-quences from 22 different adenovirus serotypes representing all6 groups was performed. The hexon epitope is highly conservedamong adenovirus serotypes. Twenty of 22 serotypes share theidentical epitope or have a single conserved amino acid change(V to L) at position 917 within the H910-924 epitope. Twoserotypes had other relatively conserved substitutions similarlylocated in the middle of the epitope (Table 5). A peptide withthe V to L substitution at position 917 was tested in the IFN-gELISPOT assay and induced a CD41 T-cell response equiva-lent to that by H910-924 (data not shown). Therefore, this con-served amino acid change in the middle of the epitope did notaffect T-cell recognition. In conclusion, hexon epitope-specificT cells recognize most, if not all, human adenovirus serotypes.

DISCUSSION

We have identified the first human T-cell epitope from ad-enovirus, located in the major capsid protein hexon. As a strat-egy, we utilized an IFN-g ELISPOT assay to screen PBMCfrom healthy adults for memory T-cell responses to a panel ofadenovirus peptides based on the well-defined HLA-A2 bind-ing motif. Unexpectedly, the T-cell response to the hexon pep-tide H913-921 was found to be HLA class II-restricted ratherthan HLA-A2-restricted as predicted. Analysis of overlappingpeptides revealed that a 15-mer peptide H910-924 was the op-timal T-cell epitope, consistent with the preferred larger size ofHLA class II epitopes. Additionally, the hexon peptide inducedstrong proliferative T-cell responses from PBMC that wereblocked by a mAb against HLA-DR in both HLA-A2–positiveand HLA-A2–negative donors.

Overall, hexon epitope-specific memory/effector CD41 Tcells were detected in 9 of 12 healthy donors. Analysis of mo-

lecular HLA typing from the donors revealed that there was nosingle shared HLA-DR allele among the peptide responders.The fact that most of the donors have circulating peptide-spe-cific CD41 T cells and exhibit diverse HLA-DR types suggeststhat H910-924 binds to multiple HLA-DR alleles. As an ex-ample, a peptide-specific TCL from an HLA-DR7, HLA-DR15–positive donor recognized peptide presented by eitherHLA-DR7- or HLA-DR15–matched targets. These data areconsistent with the known promiscuity of class II-restricted epi-topes. The peptide-binding groove of HLA class II moleculesis much less restrictive than that of class I antigens (Madden,1995). Many HLA-DR alleles have overlapping peptide-bind-ing specificities (Southwood et al., 1998), and promiscuousHLA-DR and HLA-DQ epitopes have been identified from several other pathogens including malaria and hepatitis C(Diepolder et al., 1997). For instance, 11 T-cell epitopes thatbind multiple HLA-DR alleles were identified from malaria pro-teins by screening sequences for an HLA-DR supertype motif(Doolan et al., 2000). Furthermore, a “universal” malaria T-cellepitope that binds to multiple HLA-DR and HLA–DQ mole-cules was identified from the malaria circumsporozoite protein(Calvo-Calle et al., 1997). More sophisticated studies are on-going to evaluate the spectrum of HLA class II alleles that re-strict the adenovirus hexon epitope further.

Analysis of overlapping peptides reveals that the 9-mer pep-tide H913-921 represents the minimum CD41 T-cell epitope.This minimum epitope represents the original HLA-A2 peptidemotif used to screen PBMC. Overlapping class I- and class II-restricted epitopes have been described in other models such asinfluenza and malaria (Carreno et al., 1992). For example, theinfluenza HLA-A2–restricted matrix protein epitope M58-66overlaps with the HLA-DR4–restricted T cell epitope M62-70(Linnemann et al., 2000). In our study, although an HLA-A2–restricted response to the hexon peptide was not detectedin PBMC from HLA-A2–positive donors, its presence belowthe limit of detection of the ELISPOT assay cannot be excluded.

Studies of peptide-specific CD41 TCL confirmed that theH910-924 epitope is naturally processed from the intact hexonprotein. First, peptide-specific TCL recognized B-LCL APCstreated with an E1-deleted, replication-deficient adenovirusvector in an HLA-restricted manner. In this case, B-LCL likelyprocess the hexon epitope from input virion capsid proteins be-cause hexon expression from the E1-deleted vector was not de-tected in B-LCL. Second, peptide-specific TCL recognized theindividual hexon protein expressed from a vaccinia vector inB-LCL targets. In the course of these studies, peptide-specificCD41 TCL were also documented to exhibit cytotoxicity. Al-though CD41 CTLs have been described in other viral models,such as herpes simplex virus, the relevance of this activity invivo is not well understood (Doymaz et al., 1991).

Importantly, comparison of available hexon sequences frommore than 20 different serotypes reveals that this epitope ishighly conserved among human adenovirus serotypes. Adeno-virus is packaged with an outer protein capsid primarily com-posed of three proteins: hexon, penton base, and fiber. The 951amino acid (Ad5) hexon represents the major virion protein andassociates as trimers within the capsid. Based on analysis of theavailable x-ray crystallography structure of the Ad5 hexon, theH910-924 epitope is located within an 8-stranded b-barrel “vi-ral jellyroll” sequence at the base of hexon protein (Rux and

ADENOVIRUS HEXON T CELL EPITOPE 1175

TABLE 5. COMPARISON OF THE H910–924 EPITOPE AMONG

DIFFERENT ADENOVIRUS SEROTYPESa

Group Serotype Sequence

C 5 DEPTLLYVLFEVFDV

A 12 ---------------

B 3 -------L-------

7 -------L-------

16 ------SLV------

C 2 ---------------

D 48 -------L-------

E 4 --------V------

F 40 ---------------

41 ---------------

aRepresentative hexon sequences from 10 different adenovi-rus serotypes are shown in comparison to the Ad5 H910–924epitope. Only residues that are distinct from the Ad5 hexon epi-tope are listed.

-, residues identical to the H910–924 sequence.

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Burnett, 2000). The fact that this epitope is located within acrucial internal structure is consistent with the high degree ofconservation of the sequence from serotype to serotype.

In addition to the fact that most donors exhibit memory/ef-fector CD41 T-cell responses to this conserved hexon epitope,this response represents a significant proportion of the CD41

T-cell response to whole adenovirus. The frequency of hexonpeptide-specific T cells (mean, 61; range, 16–158 per 106

PBMC) represented approximately one third of the frequencyof T-cell responses to whole adenovirus detected by IFN-gELISPOT assay. Therefore, these quantitative data suggest thathexon is an important T-cell target and that H910-924 repre-sents a major CD41 T cell epitope. It is acknowledged, how-ever, that because the T-cell responses measured to whole ad-enovirus antigens require processing by APCs, this assay mayunderestimate the true frequencies of adenovirus-specific Tcells.

The fact that most adults exhibit memory CD41 T cells tothis conserved hexon epitope suggests that adenovirus genetherapy vectors will stimulate memory T-cell responses in mostpatients. Administration of adenovirus vectors is associatedwith rapid uptake of vector by macrophages and dendritic cells,a process that induces cytokine release (Zhang et al., 2001). Inaddition, these professional APCs may present input virion pro-teins via both major histocompatibility complex (MHC) class Iand class II antigens (Bhardwaj et al., 1994). We have docu-mented that adenovirus vector-infected APCs can process thehexon epitope from input virions. Moreover, hexon-specific T-cell responses are highly relevant to the use of all adenovi-rus vectors, including “gutted” or helper-dependent adenovirusvectors that are devoid of viral-coding sequences (Schiedner etal., 1998). The use of helper-dependent vectors, which are pack-aged with virion proteins, will still allow presentation of inputvirion proteins by APCs. Kafri and colleagues (1998) haveshown that psoralen-inactivation of an adenovirus vector didnot affect its immunogenicity in a mouse model, confirming theimportance of immune responses to input virion proteins. In ad-dition, T-cell responses specific for capsid proteins, includinghexon, were detected in 2 patients treated with adenovirus vec-tors (Molinier-Frenkel et al., 2000). Thus, activation of adeno-virus-specific memory T-cell responses to hexon may help explain the enhanced inflammatory responses to adenovirusvectors observed in human studies at lower vector doses com-pared to animal studies (Schnell et al., 2001).

In conclusion, the adenovirus H910-924 is a promiscuoushuman CD41 T-cell epitope that represents a highly conservedsequence from the major capsid protein hexon. The presence ofT-cell epitopes on virion proteins that are conserved acrossserotypes, such as this hexon epitope, suggests that the pro-posed use of adenovirus vectors based on uncommon humanserotypes will not evade memory T-cell responses (Mack et al.,1997). Other strategies to reduce the immune response to ade-novirus vectors need to be evaluated such as re-targeting of vec-tors to specific cell types and specific suppression of T-cell ac-tivation or induction of tolerance ( Ilan et al., 1997; Wickhamet al., 1997; Watkins et al., 1997; Zhang et al., 1998). Thesedata suggest another potential strategy, namely deletion of im-munodominant T-cell epitopes from adenovirus vectors. Fur-ther dissection of the human T-cell response to adenoviruseswill be important in order to improve the safety and efficacy ofadenovirus-mediated gene therapy.

ACKNOWLEDGMENTS

We thank all of the volunteers who donated blood for thisstudy, Dr. T. Wickham for the kind gift of the adenovirus mu-tant AdZ.F(pK7), and Dr. J. Engler for the kind gift of theVac/fiber recombinant. The Vac/hexon recombinant was pro-vided by the Institute for Human Gene Therapy (University ofPennsylvania). We thank Dr. R. Korngold for assistance withthe proliferation assay, and Don Choi for technical assistance.This work was supported by grants from the National Institutesof Health (RO1 AI42842) and GlaxoSmithKline.

REFERENCES

ALBELDA, S.M., WIEWRODT, R., and ZUCKERMAN, J.B. (2000).Gene therapy for lung disease: Hype or hope? Ann. Intern. Med. 132,649–660.

BEDNAREK, M.A., SAUMA, S.Y., GAMMON, M.C., PORTER, G.,TAMHANKAR, S., WILLIAMSON, A.R., and ZWEERINK, H.J.(1991). The minimum peptide epitope from the influenza virus ma-trix protein. Extra and intracellular loading of HLA-A2. J. Immunol.147, 4047–4053.

BHARDWAJ, N., BENDER, A., GONZALEZ, N., BUI, L. K., GAR-RETT, M.C., and STEINMAN, R.M. (1994). Influenza virus-in-fected dendritic cells stimulate strong proliferative and cytolytic re-sponses from human CD81 T cells. J. Clin. Invest. 94, 797–807.

CALVO-CALLE, J.M., HAMMER, J., SINIGAGLIA, F., CLAVIJO,P., MOYA-CASTRO, Z.R., and NARDIN, E.H. (1997). Binding ofmalaria T cell epitopes to DR and DQ molecules in vitro correlateswith immunogenicity in vivo: Identification of a universal T cell epi-tope in the Plasmodium falciparum circumsporozoite protein. J. Im-munol. 159, 1362–1373.

CARRENO, B.M., TURNER, R.V., BIDDISON, W.E., and COLI-GAN, J.E. (1992). Overlapping epitopes that are recognized byCD81 HLA class I-restricted and CD41 class II-restricted cytotoxicT lymphocytes are contained within an influenza nucleoprotein pep-tide. J. Immunol. 148, 894–899.

CHIRMULE, N., PROPERT, K., MAGOSIN, S., QIAN, Y., QIAN, R.,and WILSON, J. (1999). Immune responses to adenovirus and adeno-associated virus in humans. Gene Ther. 6, 1574–1583.

DAI, Y., SCHWARZ, E.M., GU, D., ZHANG, W.W., SARVETNICK,N., and VERMA, I.M. (1995). Cellular and humoral immune re-sponses to adenoviral vectors containing factor IX gene: Toleriza-tion of factor IX and vector antigens allows for long-term expres-sion. Proc..Natl. Acad. Sci. U.S.A. 92, 1401–1405.

DIEPOLDER, H.M., GERLACH, J.T., ZACHOVAL, R., HOFF-MANN, R.M., JUNG, M.C., WIERENGA, E.A., SCHOLZ, S., SAN-TANTONIO, T., HOUGHTON, M., SOUTHWOOD, S., SETTE, A.,and PAPE, G.R. (1997). Immunodominant CD41 T-cell epitopewithin nonstructural protein 3 in acute hepatitis C virus infection. J.Virol. 71, 6011–6019.

DOOLAN, D.L., SOUTHWOOD, S., CHESNUT, R., APPELLA, E.,GOMEZ, E., RICHARDS, A., HIGASHIMOTO, Y.I., MAEWAL,A., SIDNEY, J., GRAMZINSKI, R.A., MASON, C., KOECH, D.,HOFFMAN, S.L., and SETTE, A. (2000). HLA-DR-promiscuous Tcell epitopes from Plasmodium falciparum pre-erythrocytic-stageantigens restricted by multiple HLA class II alleles. J. Immunol. 165,1123–1137.

DOYMAZ, M.Z., FOSTER, C.M., DESTEPHANO, D., and ROUSE,B.T. (1991). MHC II-restricted, CD41 cytotoxic T lymphocytes spe-cific for herpes simplex virus-1: Implications for the development ofherpetic stromal keratitis in mice. Clin. Immunol. Immunopathol. 61,398–409.

FLOMENBERG, P., PIASKOWSKI, V., TRUITT, R.L., and CASPER,

OLIVE ET AL.1176

Page 11: The Adenovirus Capsid Protein Hexon Contains a Highly Conserved Human CD4               +               T-Cell Epitope

J.T. (1995). Characterization of human proliferative T cell responsesto adenovirus. J. Infect. Dis. 171, 1090–1096.

FLOMENBERG, P., PIASKOWSKI, V., TRUITT, R.L., and CASPER,J.T. (1996). Human adenovirus-specific CD81 T-cell responses arenot inhibited by E3-19K in the presence of gamma interferon. J. Vi-rol. 70, 6314–6322.

HONG, J.S., and ENGLER, J.A. (1991). The amino terminus of the ad-enovirus fiber protein encodes the nuclear localization signal. Virol-ogy 185, 758–767.

ILAN, Y., PRAKASH, R., DAVIDSON, A., JONA, DROGUETT, G.,HORWITZ, M.S., CHOWDHURY, N.R., and CHOWDHURY, J.R.(1997). Oral tolerization to adenoviral antigens permits long-termgene expression using recombinant adenoviral vectors. J. Clin. In-vest. 99, 1098–1106.

JAMESON, J., CRUZ, J., and ENNIS, F.A. (1998). Human cytotoxicT-lymphocyte repertoire to influenza A viruses. J. Virol. 72, 8682–8689.

JOOSS, K., ERTL, H.C., and WILSON, J.M. (1998). Cytotoxic T-lym-phocyte target proteins and their major histocompatibility complexclass I restriction in response to adenovirus vectors delivered tomouse liver. J. Virol. 72, 2945–2954.

KAFRI, T., MORGAN, D., KRAHL, T., SARVETNICK, N., SHER-MAN, L., and VERMA, I. (1998). Cellular immune response to ad-enoviral vector infected cells does not require de novo viral gene ex-pression: Implications for gene therapy. Proc. Natl. Acad. Sci. U.S.A.95, 11377–11382.

LALVANI, A., BROOKES, R., HAMBLETON, S., BRITTON, W.J.,HILL, A.V., and MCMICHAEL, A.J. (1997). Rapid effector func-tion in CD81 memory T cells. J. Exp. Med. 186, 859–865.

LALVANI, A., BROOKES, R., WILKINSON, R.J., MALIN, A.S.,PATHAN, A.A., ANDERSEN, P., DOCKRELL, H., PASVOL, G.,and HILL, A.V. (1998). Human cytolytic and interferon gamma-se-creting CD81 T lymphocytes specific for Mycobacterium tubercu-losis. Proc. Natl. Acad. Sci. U.S.A. 95, 270–275.

LARSSON, M., JIN, X., RAMRATNAM, B., OGG, G.S., ENGEL-MAYER, J., DEMOITIE, M.A., MCMICHAEL, A.J., COX, W.I.,STEINMAN, R.M., NIXON, D., and BHARDWAJ, N. (1999). A re-combinant vaccinia virus based ELISPOT assay detects high fre-quencies of Pol-specific CD8 T cells in HIV-1–positive individuals.AIDS 13, 767–777.

LICHTENFELS, R., BIDDISON, W.E., SCHULZ, H., VOGT, A.B.,and MARTIN, R. (1994). CARE-LASS (calcein-release-assay), animproved fluorescence-based test system to measure cytotoxic Tlymphocyte activity. J. Immunol. Methods 172, 227–239.

LINNEMANN, T., JUNG, G., and WALDEN, P. (2000). Detection andquantification of CD4(1) T cells with specificity for a new majorhistocompatibility complex class II-restricted influenza A virus ma-trix protein epitope in peripheral blood of influenza patients. J. Vi-rol. 74, 8740–8743.

MACK, C.A., SONG, W.R., CARPENTER, H., WICKHAM, T.J.,KOVESDI, I., HARVEY, B.G., MAGOVERN, C.J., ISOM, O.W.,ROSENGART, T., FALCK-PEDERSEN, E., HACKETT, N.R.,CRYSTAL, R.G., and MASTRANGELI, A. (1997). Circumventionof anti-adenovirus neutralizing immunity by administration of an ad-enoviral vector of an alternate serotype. Hum. Gene Ther. 8, 99–109.

MADDEN, D.R. (1995). The three-dimensional structure of peptide-MHC complexes. Annu. Rev. Immunol. 13, 587–622.

MOLINIER-FRENKEL, V., GAHERY-SEGARD, H., MEHTALI, M.,LE BOULAIRE, C., RIBAULT, S., BOULANGER, P., TURSZ, T.,GUILLET, J.G., and FARACE, F. (2000). Immune response to re-combinant adenovirus in humans: Capsid components from viral in-put are targets for vector-specific cytotoxic T lymphocytes. J. Virol.74, 7678–7682.

OLIVE, M., EISENLOHR, L.C., and FLOMENBERG, P. (2001).Quantitative analysis of adenovirus-specific CD41 T cell responsesfrom healthy adults. Viral Immunol. 14, 403–413.

PARKER, K.C., BEDNAREK, M.A., and COLIGAN, J.E. (1994).

Scheme for ranking potential HLA-A2 binding peptides based on in-dependent binding of individual peptide side-chains. J. Immunol.152, 163–175.

RAWLE, F.C., KNOWLES, B.B., RICCIARDI, R.P., BRAH-MACHERI, V., DUERKSEN-HUGHES, P., WOLD, W.S., andGOODING, L.R. (1991). Specificity of the mouse cytotoxic T lym-phocyte response to adenovirus 5. E1A is immunodominant in H-2b,but not in H-2d or H-2k mice. J. Immunol. 146, 3977–3984.

REHERMANN, B., FOWLER, P., SIDNEY, J., PERSON, J., RE-DEKER, A., BROWN, M., MOSS, B., SETTE, A., and CHISARI,F.V. (1995). The cytotoxic T lymphocyte response to multiple he-patitis B virus polymerase epitopes during and after acute viral he-patitis. J. Exp. Med. 181, 1047–1058.

RUPPERT, J., SIDNEY, J., CELIS, E., KUBO, R.T., GREY, H.M.,and SETTE, A. (1993). Prominent role of secondary anchor residuesin peptide binding to HLA-A2.1 molecules. Cell 74, 929–937.

RUX, J.J., and BURNETT, R.M. (2000). Type-specific epitope loca-tions revealed by X-ray crystallographic study of adenovirus type 5hexon. Mol. Ther. 1, 18–30.

SCHIEDNER, G., MORRAL, N., PARKS, R.J., WU, Y., KOOP-MANS, S.C., LANGSTON, C., GRAHAM, F.L., BEAUDET, A.L.,and KOCHANEK, S. (1998). Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expres-sion and decreased toxicity. Nat. Genet. 18, 180–183.

SCHMITZ, H., WIGAND, R., and HEINRICH, W. (1983). Worldwideepidemiology of human adenovirus infections. Am. J. Epidemiol.117, 455–466.

SCHNELL, M.A., ZHANG, Y., TAZELAAR, J., GAO, G.P., YU, Q.C.,QIAN, R., CHEN, S.J., VARNAVSKI, A.N., LECLAIR, C.,RAPER, S.E., and WILSON, J.M. (2001). Activation of innate im-munity in nonhuman primates following intraportal administrationof adenoviral vectors. Mol. Ther. 3, 708–722.

SHENK, T. (1996). Adenoviridae: The viruses and their replication. InFields Virology. B.N. Fields, D.M. Knipe, and P.M. Howley, eds.(Lippincott-Raven, Philadelphia, PA) pp. 2111–2148.

SMITH, C.A., WOODRUFF, L.S., ROONEY, C., and KITCHING-MAN, G.R. (1998). Extensive cross-reactivity of adenovirus-specificcytotoxic T cells. Hum. Gene Ther. 9, 1419–1427.

SOUTHWOOD, S., SIDNEY, J., KONDO, A., DEL GUERCIO, M.F.,APPELLA, E., HOFFMAN, S., KUBO, R.T., CHESNUT, R.W.,GREY, H.M., and SETTE, A. (1998). Several common HLA-DRtypes share largely overlapping peptide binding repertoires. J. Im-munol. 160, 3363–3373.

VAN BINNENDIJK, R.S., VERSTEEG-VAN OOSTEN, J.P., POE-LEN, M.C., BRUGGHE, H.F., HOOGERHOUT, P., OSTERHAUS,A.D., and UYTDEHAAG, F.G. (1993). Human HLA class I- andHLA class II-restricted cloned cytotoxic T lymphocytes identify acluster of epitopes on the measles virus fusion protein. J. Virol. 67,2276–2284.

WATKINS, S.J., MESYANZHINOV, V.V., KUROCHKINA, L.P.,and HAWKINS, R.E. (1997). The ‘adenobody’ approach to viral tar-geting: specific and enhanced adenoviral gene delivery. Gene Ther.4, 1004–1012.

WICKHAM, T.J., ROELVINK, P.W., BROUGH, D.E., andKOVESDI, I. (1996). Adenovirus targeted to heparan-containing re-ceptors increases its gene delivery efficiency to multiple cell types.Nat. Biotechnol. 14, 1570–1573.

WICKHAM, T.J., TZENG, E., SHEARS, L.L., ROELVINK, P.W., LI,Y., LEE, G.M., BROUGH, D.E., LIZONOVA, A., and KOVESDI,I. (1997). Increased in vitro and in vivo gene transfer by adenovirusvectors containing chimeric fiber proteins. J. Virol. 71, 8221–8229.

WIVEL, N.A., and WILSON, J.M. (1998). Methods of gene delivery.Hematol. Oncol. Clin. North Am. 12, 483–501.

WORGALL, S., LEOPOLD, P.L., WOLFF, G., FERRIS, B., VAN ROI-JEN, N., and CRYSTAL, R.G. (1997). Role of alveolar macrophagesin rapid elimination of adenovirus vectors administered to the epithe-lial surface of the respiratory tract. Hum. Gene Ther. 8, 1675–1684.

ADENOVIRUS HEXON T CELL EPITOPE 1177

Page 12: The Adenovirus Capsid Protein Hexon Contains a Highly Conserved Human CD4               +               T-Cell Epitope

WYSOCKA, M., EISENLOHR, L.C., OTVOS, L., Jr., HOROWITZ,D., YEWDELL, J.W., BENNINK, J.R., and HACKETT, C.J. (1994).Identification of overlapping class I and class II H-2d-restricted Tcell determinants of influenza virus N1 neuraminidase that requireinfectious virus for presentation. Virology 201, 86–94.

YANG, Y., NUNES, F.A., BERENCSI, K., FURTH, E.E., GONCZOL,E., and WILSON, J.M. (1994). Cellular immunity to viral antigenslimits E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad.Sci. U.S.A. 91, 4407–4411.

YANG, Y., SU, Q., and WILSON, J.M. (1996). Role of viral antigensin destructive cellular immune responses to adenovirus vector-trans-duced cells in mouse lungs. J. Virol. 70, 7209–7212.

ZHANG, H.G., LIU, D., HEIKE, Y., YANG, P., WANG, Z., WANG,X., CURIEL, D.T., ZHOU, T., and MOUNTZ, J.D. (1998). Induc-tion of specific T-cell tolerance by adenovirus-transfected, Fas li-gand-producing antigen presenting cells. Nat. Biotechnol. 16,1045–1049.

ZHANG, Y., CHIRMULE, N., GAO, G.P., QIAN, R., CROYLE, M.,

JOSHI, B., TAZELAAR, J., and WILSON, J.M. (2001). Acute cy-tokine response to systemic adenoviral vectors in mice is mediatedby dendritic cells and macrophages. Mol. Ther. 3, 697–707.

Address reprint requests to:Phyllis Flomenberg, M.D.

Thomas Jefferson University1020 Locust Street

Room 329Philadelphia, PA 19107

E-mail: [email protected]

Received for publication March 12, 2002; accepted after revi-sion May 24, 2002.

Published online: June 11, 2002.

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