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Adenoviral Vector Expressing ICP47 Inhibits Adenovirus-Specific Cytotoxic T Lymphocytes in Nonhuman Primates
doi:10.1006/mthe.2000.0197, available online at http://www.idealibrary.com on IDEAL
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Adenoviral Vector Expressing ICP47 Inhibits AdenovirusSpecific Cytotoxic T Lymphocytes in Nonhuman Primates Abraham Scaria,1 Jennifer A. Sullivan, Judith A. St. George, Johanne M. Kaplan, Michael J. Lukason, James E. Morris, Malinda Plog, Charles Nicolette, Richard J. Gregory, and Samuel C. Wadsworth Genzyme Corporation, 31 New York Avenue, Framingham, Massachusetts 01701 Received for publication July 6, 2000, and accepted in revised form September 25, 2000; published online October 23, 2000
Studies from several laboratories have shown that administration of E1-deleted Ad vectors results only in transient transgene expression in the lungs of immunocompetent animals. This is due, at least in part, to destruction of vector-transduced cells by host cellular immune responses (predominantly CD8+ CTLs) directed against viral proteins and/or immunogenic transgene products. We have previously demonstrated that E1-deleted Ad vectors can lead to persistent expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) in the lungs of several strains of immunocompetent mice, despite the presence of Ad-specific CTLs. However, we found that these same vectors gave rise only to transient hCFTR expression in the lungs of rhesus monkeys. We have constructed new Ad vectors that coexpress both hCFTR and the ICP47 gene from herpes simplex virus. ICP47 has been shown to inhibit the transporter associated with antigen presentation, thus blocking major histocompatibility antigen I (MHC class I)-mediated antigen presentation to CD8+ T cells. The Ad/hCFTR/ICP47 vector decreased levels of cell-surface MHC class I molecules on infected monkey and human cell lines. Similar results were obtained with primary human cells and primary monkey airway epithelial cells. In vitro studies showed that the Ad/hCFTR/ICP47 vector decreased cytolysis by both monkey and human CTLs. When Ad/hCFTR/ICP47 was administered to the lungs of rhesus monkeys, it inhibited the generation of Ad-specific CTLs. However, natural killer cell activity was enhanced in monkeys treated with the Ad/hCFTR/ICP47 vector. Key Words: adenoviral vector; ICP47; MHC class I; CTLs; natural killer cells.
INTRODUCTION Adenovirus (Ad) is an attractive option for the development of gene therapy vectors for many reasons. Primary advantages include the ability of Ad vectors to transduce a wide variety of dividing and nondividing cells and the ability to manufacture vectors at high titer (1–6). Early Ad-based vectors were deleted of E1 sequences, a necessary step for rendering Ad vectors replication-defective, but it was also believed that deletion of E1 sequences would silence the remaining Ad genes present in the vector genome. Ad vectors with this basic design were developed for therapeutic use for cystic fibrosis (CF), a monogenic disorder in which the cystic fibrosis transmembrane conductance regulator (hCFTR) is mutated.
1To whom correspondence and reprint requests should be addressed. Fax: (508) 872 4091. E-mail: [email protected].
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We and others have used simple, E1-deleted Ad vectors to successfully transfer hCFTR cDNA into respiratory epithelium in animal models and to CF patients in vivo (1, 3, 7–10). In parallel, studies from several laboratories have suggested that administration of high doses of Ad vector results in only transient transgene expression in immunocompetent animal models (4, 11, 12–15). In contrast to the majority of published studies, we have recently demonstrated that an E1-deleted Ad vector, Ad2/CFTR-16, can give rise to persistent expression of hCFTR in the lungs of immunocompetent mice, despite the presence of Ad-specific cytotoxic T lymphocytes (CTLs) (16). In this report we show that the same vector, Ad2/CFTR-16, gives rise to transient transgene expression in the lungs of rhesus monkeys, and we describe a strategy designed to overcome this limitation. In rodent systems, transience of transgene expression has partly been attributed to the destruction of vector transduced cells by CD8+ CTLs directed against the
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ARTICLE transgene product and/or Ad viral proteins (4, 11–15, 17). Other potential reasons for the decline in transgene expression can include vector-induced cytotoxicity, complete loss of vector DNA, antibody response to secreted transgene products, promoter shut-off, and normal turnover of the transduced cell (18–20). Reduction of the cellular immune response and improved persistence have been reported using second-generation Ad vectors with modification or deletion of the E4, E2a, or E2b regions in addition to the E1 deletion (9, 11, 21, 22) and with the fully-deleted Ad vectors (23–26). Persistence of transgene expression can also be obtained by using transient immunomodulation strategies to block the in vivo interactions between antigen-presenting cells and T cells (27–31). To investigate the role of CTLs in the decline of hCFTR expression we observed in the monkey lung, we designed and constructed new Ad vectors that express both hCFTR and the immunomodulatory gene ICP47 from the herpes simplex virus. In vitro studies have shown that ICP47 inhibits the function of human transporter associated with antigen presentation (TAP), thus blocking major histocompatibility complex type I (MHC class I) mediated antigen presentation to CD8+ T cells (32–35). The Ad2/CFTR/ICP47 vector decreased levels of cell surface MHC I molecules on infected human and monkey cells in vitro. To date, there have been no reports on the effects of in vivo expression of ICP47. In this article, we describe the in vivo characterization of Ad2/CFTR/ICP47 in the lungs of rhesus monkeys and the effects of ICP47 expression on the generation of CTLs, natural killer (NK) cell responses, and persistence of hCFTR mRNA expression.
MATERIALS
AND
METHODS
Adenoviral vectors. All vectors used in these studies are Ad2 based and the transgenes are driven by the cytomegalovirus (CMV) promoterenhancer. All vectors contain a wt E2 region. (1) Ad2/CFTR-16 has been described previously (16). It contains a CMV enhancer-promoter-driven hCFTR cDNA followed by a bovine growth hormone poly(A) signal. The hCFTR expression cassette is inserted in the E1 region. The E3 region has a 1549-bp deletion in the E3B region corresponding to Ad2 nucleotides 29,292 to 30,840. The vector has a wt E4 region. (2) Ad2/CFTR/ICP47 has the same hCFTR expression cassette as Ad2/CFTR-16. The E3 region corresponding to Ad2 nucleotides 27,971 to 30,937 is deleted and is replaced with the ICP47 expression cassette. The ICP47 cDNA was cloned by PCR from herpes simplex virus I DNA obtained from Advanced Biotechnologies, Inc. (Columbia, MD). The ICP47 expression cassette has the CMV promoter-enhancer and an SV40 poly(A) signal. (3) Ad2/β-gal/∆E3 has been described previously (16) and has the CMV enhancer-promoter driving β-galactosidase. The E3 region has the identical deletion as Ad2/CFTR/ICP47. (4) Ad2/β-gal/ICP47 has the same β-galactosidase expression cassette as Ad2/β-gal/∆E3. The E3 region is deleted and contains the same ICP47 expression cassette as described above for Ad2/CFTR/ICP47. (5) Ad2/EV is an Ad2 based empty vector control that has no transgene cassette. Both E1 and E3 regions are deleted. It has wt E2 and E4 regions. (6) Ad2/hgp100v2 has been described before (36). The vector has the human gp100 expression cassette in the E1 region and has a wt E3
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region. The E4 region is modified by removal of all open reading frames (ORFs) and replacement with the E4 ORF6 and pIX has been moved from its original location to the junction between fiber and E4 genes. Cell lines. The HLA-A2-restricted T cell clone TIL1520 specific for the 209–217 epitope of the human melanoma antigen gp100 was obtained from Dr. M. Nishimura, Surgery Branch, NCI, and has been described previously (37). Cells were maintained in AIMV medium (Gibco) supplemented with 10% human AB serum (Sigma) and 1% penicillin/streptomycin (Life Technologies). Dendritic cell (DC) preparation. Normal donor monocytes were harvested by leukophoresis (Dana Farber Cancer Institute) in a volume of ~175 ml. The cells were diluted 1:1 with PBS (Mg2+, Ca2+ free) and 30 ml layered onto 20 ml Ficoll–Paque (Pharmacia Biotech.). Cells were collected at the interface by centrifugation (IEC, Model GP8, 1400 RPM/40 min). The cells were washed three times with PBS and resuspended in RPMI (Gibco) supplemented with P/S, glutamine, 5% human AB serum (Sigma), and 10 mM Hepes (complete medium) and rocked overnight on a Nutator in 50-ml conical tubes at 4°C. The cells were then plated at 1.5 × 108 cells/T-150 Flask (Corning). After 1 h, nonadherent cells were removed by washing three times with unsupplemented RPMI. The cells were then fed with complete RPMI plus 100 ng/ml GM-CSF (Genzyme) and 20 ng/ml IL-4 (Genzyme) in a volume of 20 ml/T150 flask. On day 3, 5 ml complete RPMI (plus cytokines) was added to each flask. On day 6, the cells were processed for assaying as described. Human CTL assay. DCs (1 × 106) were labeled overnight in 900 µl complete medium supplemented with 100 µl (100 µCi) 51 Cr (NEN/Dupont) in 1 well of a 24-well cluster plate (total volume = 1 ml). The DCs were transferred to a 1.5-ml Eppendorf tube and washed three times with 1 ml of serum-free AIM-V medium (Gibco BRL). Five thousand cells were transferred to each well of a 96-well V-bottom plate (Costar, polystyrene) in a volume of 100 µl/well. Effector cells were added to each well in AIM-V medium/10% human AB serum at the indicated E:T ratios in a volume of 100 µl for a total reaction volume of 200 µl. Medium (no effector cells) was added to the spontaneous release wells, and no additional media was added to total release wells. The plate was then spun at 1200 rpm for 3 min and returned to the incubator for 4 h. After the incubation, 100 µl of 1% Triton X-100 was added to the total release wells (total volume = 200 µl) and the plate was spun at 1000 rpm for 10 min. Fifty microliters of supernatant was removed from each well and added to a Wallac 96-well plate (polyethylene terepthalate) containing 150 µl scintillation fluid (Wallac Optiphase Supermix). This plate was sealed with an adhesive plate sealer and incubated at room temperature overnight. The radioactivity was measured using a MicroBeta Trilux Scintillation Counter (Wallac, Gaithersburg, MD). All reactions were performed in triplicate, and each graphed data point represents the average of the replicates. Percent specific killing was calculated according to the formula 100 × (experimental cpm − spontaneous cpm)/(total cpm − spontaneous cpm). FACS analysis. Approximately 106 cells were trypsinized and resuspended in FACS buffer (PBS containing 0.5% BSA). Cells were incubated at 4°C for 1 h in the presence of an antibody (W6/32, Accurate Chemical) which detects all haplotypes of MHC class I. Cells were washed and incubated at 4°C for 1 h with FITC goat anti-mouse IgG (Jackson Immunoresearch). The fluorescence profiles were then analyzed on a FACS flow cytometer (Becton–Dickinson, San Jose, CA). Aerosol study with Ad2/CFTR-16. Ad2/CFTR-16 was delivered to the lungs of rhesus monkeys (Macaca mulatta) through inhalation of aerosolized vector. A miniHEART low flow nebulizer (Vortran Medical Technology, Inc.) was filled with 10 ml of PBS/sucrose containing 5 × 1011 IU of vector. The aerosol was directed to a modified anesthesia mask fitted to anesthetized animals that had their nares occluded. This provided transoral delivery of the aerosol. The respired fraction of aerosol was determined for each animal based upon the animal’s weight and rate of respiration. By these criteria, approximately 7.6 × 1010 infectious units of Ad2/CFTR-16 was determined to be deposited in the lungs of each animal. MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
ARTICLE Vector administration via bronchoscope. Rhesus monkeys (M. mulatta) received 1.6 × 1010 infectious units of either Ad2/CMV/CFTR-ICP47 (4.9 × 1011 particles) or Ad2/CFTR-16 (4.0 × 1011 particles) administered to the left caudal lobe using a pediatric bronchoscope. The dose of virus was delivered in 1.0 ml of PBS containing 10% sucrose followed by a rinse with 1 ml of the PBS/sucrose solution. One animal from each group was sacrificed on day 3 and day 21 following the administration of the vectors. RNA extraction. The lungs were removed and portions of the conducting airways were dissected free of parenchyma for RT-PCR analysis. Pieces of the trachea, lobar bronchus, segmental, subsegmental bronchus (to a diameter of ≈1 mm) and alveolar tissue were placed in RNA Stat-60 solution (Tel-Test B, Inc., Friendswood, TX) and frozen on dry ice for subsequent storage at −80°C. Tissue samples were homogenized in RNA Stat60 solution and RNA was extracted using an acid guanidinium/phenol–chloroform method according to the manufacturer’s directions. Tissue was disrupted in a Mini-Bead Beater (Biospec Corp., Batlesville, OK). RT-PCR for aerosol experiment and experiment A. Ad2/CFTR-16 specific mRNA was detected in the aerosol administration experiment and experiment “A” (Fig. 8) using an isotopic, nested RT-PCR protocol. Total RNA (1 µg) from each sample was treated with RNase-free DNase I (Promega, Madison, WI) to remove any contaminating DNA in the RNA samples. Following DNase treatment, the RNA sample was divided into two tubes. The RT reaction was performed using components of Invitrogen’s cDNA cycle reverse transcription kit. 250 nM of a vector specific reverse primer B23∆1.6 (5′-TATGTTTACCGCCACACTCGCAGG-3′), was used to prime the 15 µl reverse transcriptase (RT) reaction. One aliquot from each sample was processed without RT enzyme to serve as a negative control. Following the RT reaction, 5 µl of cDNA was amplified in the 100-µl firstround reaction using 2.5 U Taq polymerase (Perkin–Elmer), in a mixture containing a final concentration of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.75 mM MgCl2, 0.01% gelatin, 200 mM each dTTP, dATP, dGTP, and dCTP, 500 nM each primer F12∆1.6 (5′-AGGATAGAAGCAATGCTGGAATGCC-3′) and B23∆1.6 (5′-TATGTTTACCGCCACACTCGCAGG-3′), and 0.5 µl [33P]dCTP (3000 Ci/mmol). After a denaturing step of 1 min at 94°C, 25 amplification cycles consisting of 60 s at 92°C, 60 s at 62.5°C, and 60 s at 72°C, with a final extension step of 5 min at 72°C was performed. First round amplification products were diluted 1:100 in DEPCtreated H2O and 10 µl of this dilution was used as template in the nested amplification reaction. The nested round consisted of 20 amplification cycles under conditions identical to the first round reaction using primers F20∆1.6 (5′-AGCCCCAGATTGCTGCTCTGAAAG-3′) and B22∆1.6 (5′CGTCAGACAATGCGATGCAATTTC-3′). Amplified products from both rounds were electrophoresed in 1.5% LE agarose gels, stained with ethidium bromide and visualized on a transilluminator. The gels were dried, placed in an exposure cassette and product bands quantitated using image analysis software on a phosphorimager (Molecular Dynamics).
Pixel volumes from the study samples were compared to those of a DNA standard curve containing log concentrations (from 1 × 101 to 1 × 105) of pAd2/CFTR-5 plasmid to quantitate the target molecules in the study samples. RT-PCR analysis for experiment B. In experiment B Ad2/CFTR-16 specific mRNA was detected using a real-time assay that employs the 5′–3′ exonuclease activity of Taq polymerase to cleave an oligonucleotide probe labeled with fluorescent reporter and quencher molecules during the extension phase of the amplification reaction. The fluorescence of the reactions are measured in real time on an ABI PRISM 7700 sequence detection system (PE Biosystems) instrument where fluorochromes are excited using a 488nm argon laser and fluorescent emission spectra measured using a spectrograph and a CCD camera. Total RNA (1 µg) from each sample was treated with RNase-free DNase I (Promega, Madison, WI) to remove any contaminating DNA in the RNA samples. Following DNase treatment, the RNA sample was divided into two tubes. One aliquot from each sample was processed without RT enzyme to serve as a negative control. Reverse transcription was performed using the Promega reverse transcription kit and 250 nM of a vector specific reverse primer JMTM99-04 (5′-GTGCGGGTCTCATCGTACCT-3′), according to the manufacturer’s protocol. Two and a half microliters of the RT reaction was run in the amplification reaction described below. cDNA samples were amplified in a 50-µl reaction containing 25 µl of TaqMan Universal PCR Master Mix (PE Biosystems) with 300 nM of each primer JMTM99-03 (5′-ATGAGGAAATTGCATCGCATT-3′) and JMTM99-04 (5′-GTGCGGGTCTCATCGTACCT-3′), 200 nM of TaqMan probe 77oJM-6 (5′-FAM-TCTGACGCGTTACGCGGGAAGGT-TAMRA-3′), 2.5 U AmpliTaq Gold polymerase, 0.5 U AmpErase UNG and 10% glycerol. The reactions were held at 50°C for 2 min to allow UNG activity to remove any dUTP containing contaminates present in the reactions followed by a 10-min incubation at 95°C for 10 min to activate AmpliTaq Gold. These incubations were followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. In addition to study samples, water negative controls and a DNA standard curve containing log concentrations (from 1 × 101 to 1 × 105) of pAd2/CFTR-5 were run as well. Sample and standard CT values are exported to an Excel spreadsheet (Microsoft Corp., Redmond, WA) and sample values were determined from a standard curve by interpolation. Cytotoxic T cell assay (monkey). Primary fibroblast cell lines were established from skin biopsies from individual monkeys as a source of MHCcompatible target cells. The fibroblasts were grown in DMEM (Gibco BRL) supplemented with 20% fetal calf serum, 1 mM Hepes buffer, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 30 µg/ml vancomycin and 10 µg/ml gentamycin. A portion of spleen as well as tracheobronchial lymph nodes were collected aseptically at the time of sacrifice and a single-cell suspension was prepared by mincing the specimens in PBS and shearing the fragments between the frosted edges of two sterile glass slides. To expand vector-specific cytotoxic T lymphocytes (CTLs), spleen and lymph node cells were restimulated in vitro with autologous fibroblasts transduced with Ad vector at a multiplicity of infection (m.o.i.) of 200. The
FIG. 1. Genomic structures of recombinant Ad vectors. The expression cassette for hCFTR is inserted in place of the E1 region in both vectors. The ICP47 expression cassette is inserted in the E3 region of Ad2/CFTR/ICP47. Both vectors have wt. E2 and E4 regions.
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B
FIG. 2. Persistence of Ad2/CFTR-16 in the monkey lung. Ad2/CFTR-16 (7.6 × 1010 IU per monkey) was administered to the lungs of rhesus monkeys by aerosolization. Tissue samples from lung were analyzed for hCFTR mRNA expression by RT-PCR at different time points as described under Materials and Methods. Percentages of samples positive for hCFTR mRNA are shown in A and the average mRNA expression levels in the positive samples are shown in B.
cells were cultured in the wells of 24-well plates containing 5 × 106 spleen or lymph nodes cells with 5–6 × 104 stimulator fibroblasts in 2 ml of AIMV medium (Gibco BRL) supplemented with 10% human AB serum. The cultures were incubated for 6 days at 37°C/5% CO2 and received 20 U/ml recombinant human IL-2 (Genzyme Corp.) on the fourth day of culture. Cytolytic activity was then tested against autologous or allogeneic fibroblasts transduced with Ad vector to assess specific MHC-restricted killing. The K-562 myelogenous leukemia cell line was also used as a target to evaluate levels of NK cell activity. Target cells were treated with 500 U/ml recombinant human γ-interferon (Genzyme Corp.) for 24 h (except for K562 cells) to enhance MHC Class I presentation and were labeled with
51chromium (New England Nuclear) overnight (25 µCi/105 cells). Effector
and target cells were added at various effector: target (E:T) ratios to triplicate wells of 96-well round-bottom plates in a 200-µl volume. After 5 h of incubation at 37°C/5% CO2, 25 µl of cell-free supernatant was collected from each well and counted in a MicroBeta Trilux scintillation counter (Wallac, Gaithersburg, MD). The amount of 51Cr spontaneously released was obtained by incubating target cells in medium alone and was typically below 20%. The total amount of 51Cr incorporated was determined by adding 1% Triton X-100 in distilled water, and the percentage lysis was calculated as % lysis = [(sample cpm − spontaneous cpm)/(total cpm − spontaneous cpm)] × 100.
FIG. 3. Downregulation of MHC class I. Human A549 cells were infected at an m.o.i. of 200 and monkey CV1 cells were infected at an m.o.i. of 100 with different Ad vectors. Cell surface expression of MHC class I was measured using FACS analysis at 24 h postinfection with Ad vectors. Results are shown as mean fluorescence intensities of staining for MHC class I.
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FIG. 4. MHC I expression on primary cells. Primary human dendritic cells were infected at an m.o.i. of 400 and monkey airway cells were infected at an m.o.i. of 150 with different Ad vectors. Cell surface MHC class I levels were measured using FACS analysis at 24 h postinfection. Results are shown as mean fluorescence intensities of staining for MHC class I.
RESULTS Ad2/CFTR-16 in the Monkey Lung We have previously shown that an E1-deleted Ad vector encoding human CFTR, namely Ad2/CFTR-16, gives rise to persistent transgene expression for 70 days in the lungs of several strains of immunocompetent mice (16). This persistence was achieved by the use of the CMV enhancer/promoter for hCFTR expression in conjunction with a wt E4 region (18). To examine the properties of the same vector in the lungs of nonhuman primates, we delivered approximately 7.6 × 1010 IU of Ad2/CFTR-16 into the lungs of rhesus monkeys by aerosol administration. One monkey was sacrificed at each time point (days 3, 21, and 71) after vector administration. At necropsy, tissue samples of airways, alveoli and parenchyma were dissected out from each lobe of the lung. Approximately 25 to 30 samples from each monkey lung were analyzed for expression of hCFTR mRNA by RT-PCR analysis. We found that on day 3, 90% of the samples were positive for hCFTR expression (Fig. 2). However, only about 50% of the samples were positive for transgene expression on day 21 and day 71. Also, the levels of hCFTR expression in the positive samples declined about 100-fold compared to the mRNA levels on day 3 (Fig. 2).
Construction of Ad2/CFTR/ICP47 and in Vitro Characterization We designed and constructed new Ad vectors that expressed hCFTR along with the ICP47 gene from herpes simplex virus (Fig. 1). ICP47 has been shown to bind to and inhibit the function of the transporter associated with antigen presentation (TAP), which normally functions to translocate antigenic peptides across the endoMOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
plasmic reticulum (ER) membrane (33–35). The result of this inhibition is that empty MHC I molecules accumulate in the ER and fail to reach the cell surface. Expression from the CMV promoter driven ICP47 cassette inserted in the E3 region of Ad2/CFTR/ICP47 was confirmed by immunoprecipitation analysis (data not shown) and fluorescence-activated cell sorter (FACS) analysis of cell surface MHC class I expression on human and monkey cell lines (Fig. 3) as well as primary human dendritic cells and monkey airway cells (Fig. 4). As shown in Figs. 3 and 4, cell surface expression of MHC class I was almost completely inhibited on cells transduced with Ad vectors expressing ICP47 compared to uninfected cells or cells infected with a control Ad vector.
Inhibition of Lysis by Human CTLs We used an in vitro model system to test the ability of an Ad vector expressing ICP47 to inhibit lysis by human cytotoxic T lymphocytes (CTLs). In the experiment shown (Fig. 5), human DCs from a donor expressing the HLA-A2 haplotype were infected with Ad2/hgp100v2 vector which expresses a human melanoma associated antigen gp100. A human CTL clone that specifically recognizes human gp100 in the context of HLA-A2 (37) was then used to lyse the gp100 expressing cells. Coinfection with a control vector Ad2/EV did not affect lysis by the CTL clone. However, coinfection with Ad2/ICP47 completely inhibited lysis of the human DCs expressing gp100.
ICP47 Inhibits Ad-Specific CTLs in Primates Ad2/CFTR/ICP47 (1.6 × 1010 IU per monkey) was instilled into the left caudal lobe of rhesus monkeys using a pediatric bronchoscope. Another group of mon-
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FIG. 5. Ad2/ICP47 protects against human CTLs. Human dendritic cells were infected with different Ad vectors at an m.o.i. of 400 for each Ad vector. Ad2/hgp100v2 vector expresses human melanoma antigen gp100. A human CTL clone TIL1520 that specifically recognizes human gp100 was then used to lyse gp100 expressing cells. Results shown are mean percentages of lysis from triplicate wells.
keys was instilled with the same dose of Ad2/CFTR-16. Spleens and tracheobronchial lymph nodes were collected at the time of sacrifice on day 21 to expand vector specific CTLs. As shown in Fig. 6A, administration of Ad2/CFTR-16 led to the generation of Ad specific CTLs. Infection of autologous fibroblast targets with Ad2/CFTR/ICP47 decreased the lysis by Ad specific CTLs
A
from the monkey, although the inhibition was not as dramatic as seen with the human CTLs (Fig. 5). In the monkeys instilled with Ad2/CFTR/ICP47, no adenovirusspecific CTL activity was detected (Fig. 6B). However, we detected higher levels of cytolytic activity against autologous fibroblasts that were infected with Ad2/CFTR/ICP47.
B
FIG. 6. CTL response to Ad vectors in primates. Ad2/CFTR-16 or Ad2/CFTR/ICP47 (1.6 × 1010 IU per monkey) vectors were instilled into the lungs of rhesus monkeys using a pediatric bronchoscope. On day 21, spleens were collected and Ad vector specific CTLs were expanded in vitro for 6 days. Cytolytic activity was then tested against autologous fibroblasts transduced with different Ad vectors. Results shown are mean percentages of lysis from triplicate wells.
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FIG. 7. NK cell response to Ad vectors in primates. Ad vectors were instilled into the lungs of rhesus monkeys as described for Fig. 6. Regional lymph nodes were collected, expanded in vitro and tested against allogeneic fibroblasts transduced with different Ad vectors and against K562 cells. Results are shown as mean percentages of lysis from triplicate wells.
Activation of Natural Killer Cells As shown in Figs. 3 and 4, expression of ICP47 inhibits cell-surface expression of MHC class I on monkey cells. Therefore, the cytolytic activity that we observed against Ad2/CFTR/ICP47 infected autologous fibroblasts was most likely mediated by NK cells, which preferentially lyse targets expressing low levels of MHC class I. The involvement of NK effector cells was confirmed by the ability of cells isolated from the regional lymph nodes of Ad2/CFTR/ICP47-treated monkeys to lyse traditional NK target K562 cells as well as allogeneic monkey fibroblasts infected with Ad2/CFTR/ICP47 to reduce MHC class I expression (Fig. 7A). Allogeneic fibroblasts infected with Ad2/CFTR-16 were not lysed in this assay since they express normal levels of MHC class I. No significant level of NK cell activity was detected in cultures of lymph node cells from Ad2/CFTR-16-treated monkeys (Fig. 7B).
Persistence of Transgene Expression in the Monkey Lung Two separate experiments (A and B) were conducted to evaluate persistence of transgene expression in the primate lung from Ad2/CFTR/ICP47 versus Ad2/CFTR-16. In each experiment, one monkey from each group was sacrificed on days 3 and 21, airway samples were dissected out and assayed for hCFTR mRNA by RT-PCR analysis. On day 3, almost all samples analyzed from the treated lobe (left caudal lobe) were positive for hCFTR expression in agreement with our previous results (Fig. 2). In the samples that were positive for hCFTR expression we found that the average hCFTR expression levels from the Ad2/CFTR-16-treated monkeys were not significantly different from the average expression levels in the positive MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
samples from the Ad2/CFTR/ICP47-treated monkeys. The expression levels declined over time in both groups (Fig. 8A). As expected, the percentage of samples that are positive for expression declined rapidly by day 21 in the Ad2/CFTR-16-treated monkey (Fig. 8B). However, in the Ad2/CFTR/ICP47-treated monkey, we found that almost all samples were positive for transgene expression even on day 21 in both experiments A and B (Fig. 8B).
DISCUSSION Adenoviral vectors have been used successfully to transfer genes to a variety of organs including the lung, liver, muscle and brain in normal animals and animal models of genetic diseases. However, in the majority of immunocompetent animal models, investigators have reported only transient expression of the transgene. In mice, this transience of transgene expression has been attributed primarily to the cellular immune response directed against the foreign transgene products and/or Ad viral proteins (4, 11–15). We (16, 38) and others (5, 6, 19, 39, 40) have demonstrated that Ad vectors can give rise to persistent transgene expression in immunocompetent mice provided the transgene product is nonimmunogenic in the particular strain of mouse tested. We have shown recently that an E1-deleted vector, Ad2/CFTR-16, gives rise to persistent expression in the lungs of three different strains of immunocompetent mice (16). The mice tested clearly generated Ad-specific CTL activity; however, a CTL response against hCFTR was not observed. In this article we demonstrate that the same vector Ad2/CFTR-16 gives rise to transient transgene expression in the lungs of rhesus monkeys. Therefore, the immune response generated to Ad2/CFTR-16 in
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FIG. 8. Persistence of transgene expression in the monkey lung. Ad vectors (1.6 × 1010 IU per monkey) were instilled into the lungs of rhesus monkeys as described for Fig. 6. Tissue samples from lung were analyzed for hCFTR mRNA expression by RT-PCR at different time points as described under Materials and Methods. The average mRNA expression levels in the positive samples are shown in A and the percentages of samples positive for hCFTR mRNA in the treated lobe are shown in B.
monkeys seems to be more effective in eliminating transgene expression compared to the immune response generated to the same Ad vector in the mouse model. One possibility is that the cells transduced with the Ad vector in the monkey lung are more efficient at antigen presentation to the CD8+ T cells compared to the cells transduced in the mouse lung. Another possibility is that an E1-deleted Ad vector, being derived from a human adenovirus, is more competent at viral gene expression in primate cells than in rodent cells. To investigate further the role of CD8+ CTLs in the elimination of transgene expression in the primate lung, we constructed Ad vectors expressing the herpes simplex virus ICP47 protein. We show here that the ICP47expressing vectors are capable of decreasing cell surface expression of MHC class I on primary human and monkey cells and offer protection in vitro from lysis by human and monkey antigen specific CTLs. These data are in agreement with published reports showing that ICP47 is capable of inhibiting TAP function in human and primate cell lines (32–35). The experiments
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described in this report extend the studies on effects of ICP47 expression to an animal model. We find that administration of Ad2/CFTR/ICP47 into the lungs of monkeys leads to an inhibition of the generation of the Ad specific CTL response. However, we find that inhibition of the Ad-specific CTL response using Ad/CFTR/ICP47 was not sufficient to give rise to prolonged high levels of hCFTR mRNA expression in the monkey lung, even though a higher proportion of the lung samples were positive for transgene expression on day 21 when compared to the lung samples from the monkeys treated with the Ad2/CFTR-16 vector. An important observation from our studies in the primate lung is the activation of NK cells in the regional lymph nodes of monkeys treated with Ad2/CFTR/ICP47. NK cells are of the lymphoid lineage, although distinct from T and B cells. NK cells are especially efficient in the rejection of tumors lacking host MHC I molecules, including those with defects in the TAP protein (41). In vivo, NK cells are recruited to sites of virus infection and are activated to become more cytotoxic and to produce
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ARTICLE INF-γ (42, 43). Recently, it has been shown that tumor cells lacking appropriate MHC I expression induce NK cell infiltration, cytotoxic activation and induction of transcription of INF-γ in NK cells (44). It is possible that the loss in transgene expression that we observed in the lungs of Ad2/CFTR/ICP47-treated monkeys is partly due to enhanced NK cytotoxicity or due to downregulation of the CMV promoter (20, 45) by cytokines secreted by NK cells and activated CD4+ T cells. In future experiments, detailed analysis of Ad vector DNA persistence in the lung samples and measurement of cytokine levels in the bronchoalveolar fluid (BAL) could address these different possibilities. NK cytotoxicity is less likely to be the cause of the loss in transgene expression, as all samples from the Ad2/CFTR/ICP47-treated monkey were positive for transgene expression on day 21. Use of cellular promoters that are unaffected by cytokines like INF-γ and TNF-α may help avoid the issue of promoter downregulation. Adenoviruses and several other viruses have evolved mechanisms to reduce MHC class I expression (46, 47). Other groups have tried overexpression of adenoviral E3-region proteins to prolong Ad vector persistence in immunocompetent rodent models. Expression of E3 gp19K alone has been shown to inhibit MHC I expression in vitro (48), decrease cytolysis by Ad specific CTLs (49), and provide minimal improvement in transgene expression for 2 weeks in one strain (B10.HTG) of mice (50). Overexpression of the entire E3 region has been reported to decrease both the humoral and cellular immune response to Ad vectors and provide long-term transgene expression in the livers of Gunn rats (51). It is known that Ad E3 gp19K binds with different affinities to different MHC haplotypes in mice and humans (46), while ICP47 should be active independently of MHC haplotype, since it inhibits TAP function and indirectly reduces cell surface expression of MHC class I. However, our demonstration that a drastic reduction of MHC I expression could potentially lead to an enhanced NK response in vivo suggests that alternate strategies to protect the Ad-transduced cell without interfering with MHC I expression should also be explored. Another promising approach to improve persistence of transgene expression from Ad vectors involves deletion of additional viral genes, which should lead to a decreased cellular immune response in vivo. Improved persistence has been reported with Ad vectors with modifications or deletions in the E4, E2a, or E2b regions (9, 11, 21, 22). Recently, it has been shown that Ad vectors lacking all viral genes can give rise to persistent transgene expression in the muscle (24) and liver (25, 26) of immunocompetent animals. This does not, however, address the issue of immunogenicity of the transgene product, which could potentially be a neoantigen in patients with genetic disease that we wish to treat with these vectors. Transient immunosuppression has been shown to effectively suppress the cellular and humoral immune responses to Ad vector and transgene products MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
(27–31) and could be used in conjunction with an advanced Ad vector with all viral genes deleted. ACKNOWLEDGMENTS All primate studies were conducted at the New England Primate Research Center in Southborough, Massachusetts. We thank the Genzyme Virus Production Unit for the purified Ad vectors used in these studies, Dr. Srinivas Shankara for the Ad2/hgp100v2 vector, and Dr. Michael I. Nishimura (National Cancer Institute) for the T cell clone TIL1520.
REFERENCES 1
Rosenfeld, M. A., et al. (1992). In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 68: 143–155. 2 Smith, T. A. G., et al. (1993). Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Gene Ther. 5: 397–402. 3 Zabner, J., et al. (1994). Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats. Nat. Genet. 6: 75–83. 4 Dai, Y, Schwarz, E. M., Gu, D., Zhang, W., Sarvetnick, N., and Verma, I. M. (1995). Cellular and humoral responses to adenoviral vectors containing factor IX gene: Tolerization of factor IX and vector antigens allows for long term expression. Proc. Natl. Acad. Sci. USA 92: 1401–1405. 5 Tripathy, S. K., Black, H. B., Goldwasser, E., and Leiden, J. M. (1996) Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus. Nat. Med. 2: 545–550. 6 Connelly, S., et al. (1998). Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy. Blood 91: 3273–3281. 7 Zabner, J., Couture, L. A., Gregory, R. J., Graham, S. M., Smith, A. E., and Welsh, M. J. (1993). Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelium of patients with cystic fibrosis. Cell 75: 207–216. 8 Crystal, R. G., et al. (1994). Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat. Genet. 8: 42–51. 9 Yang, Y., Nunes, F. A., Berencsi, K., Gonczol, E., Engelhardt. J. F., and Wilson, J. M. (1994). Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat. Genet. 7: 362–369. 10 Yei, S., Mittereder, N., Tang, K., O’Sullivan, C., and Trapnell, B. C. (1994). Adenovirus mediated gene transfer for cystic fibrosis: Quantitative evaluation of repeated in vivo vector administration to the lung. Gene Ther. 1: 192–200. 11 Engelhardt, J. F., Ye, X., Doranz, B., and Wilson, J. M. (1994). Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. USA 91: 6196–6200. 12 Yang, Y., Li, Q., Ertl, H. C. J., and Wilson, J. M. (1995). Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69: 2004–2015. 13 Yang, Y., Xiang, Z., Ertl, H. C. J., and Wilson, J. M. (1995). Upregulation of class I major histocompatibility complex antigens by interferon γ is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo. Proc. Natl. Acad. Sci. USA 92: 7257–7261. 14 Zsengeller, Z. K., et al. (1995). Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu-nu) mice. Hum. Gene Ther. 6: 457–467. 15 Yang, Y., Su, Q., and Wilson, J. M. (1996). Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J. Virol. 70: 7209–7212. 16 Scaria, A., St. George, J. A., Jiang, C., Kaplan, J. M., Wadsworth, S. C., and Gregory, R. J. (1998). Adenovirus-mediated persistent cystic fibrosis transmembrane conductance regulator expression in mouse airway epithelium. J. Virol. 72: 7302–7309. 17 Kaplan, J. M, et al. (1997). Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung. Hum. Gene Ther. 8: 45–56. 18 Armentano, D., et al. (1997). Effect of the E4 region on the persistence of transgene expression from adenovirus vectors. J. Virol. 71: 2408–2416. 19 Morral, N., O’Neal, W., Zhou, H., Langston, C., and Beaudet, A. L. (1997). Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors: Comparison of E2a wild type and E2a-deleted vectors. Hum. Gene Ther. 8: 1275–1286. 20 Quin, L., Ding, Y., Pahud, D. R., Chang, E., Imperiale, M. J., and Bromberg, J. S. (1997). Promoter attenuation in gene therapy: Interferon-γ and tumor necrosis factor-α inhibit transgene expression. Hum. Gene Ther. 8: 2019–2029. 21 Wang, Q., Greenburg, G., Bunch, D., Farson, D., and Finer, M. H. (1997). Persistent transgene expression in mouse liver following in vivo gene transfer with a ∆E1/∆E4 adenovirus vector. Gene Ther. 4: 393–400. 22 Hu, H., Serra, D., and Amalfitano, A. (1999). Persistence of an [E1-, polymerase-] adenovirus vector despite transduction of a neoantigen into immune-competent mice. Hum. Gene Ther. 10: 355–364. 23 Haecker, S. E., et al. (1996). In vivo expression of full-length human dystrophin from
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ARTICLE adenoviral vectors deleted of all viral genes. Hum. Gene Ther. 7: 1907–1914. 24 Chen, H. H., Mack, L. M., Kelly, R., Ontell, M., Kochanek, S., and Clemens, P. R. (1997). Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc. Natl. Acad. Sci. USA 94: 1645–1650. 25 Schiedner, G., et al. (1998). Genomic DNA transfer with high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat. Genet. 18: 180–183. 26 Morral, N., et al. (1999). Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc. Natl. Acad. Sci. USA 96: 12816–12821. 27 Kay, M. A., et al. (1995). Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat. Genet. 11: 191–197. 28 Poller, W., et al. (1996). Stabilization of transgene expression by incorporation of E3 region genes into an adenoviral factor IX vector and by transient anti-CD4 treatment of the host. Gene Ther. 3: 521–530. 29 Yang., Y., Su, Q., Grewal, I. S., Schilz, R., Flavell, R. A., and Wilson, J. M. (1996). Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung. J. Virol. 70: 6370–6377. 30 Scaria, A., et al. (1997). Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse lung. Gene Ther. 4: 611–617. 31 Kaplan, J. M., and Smith, A. E. (1997). Transient immunosuppression with deoxyspergualin improves longevity of transgene expression and ability to readminister adenoviral vector to the mouse lung. Hum. Gene Ther. 8: 1095–1104. 32 York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L., and Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77: 525–535. 33 Hill, A., et al. (1995). Herpes simplex virus turns off the TAP to evade host immunity. Nature 375: 411–415. 34 Fruh, K., et al. (1995). A viral inhibitor of peptide transporters for antigen presentation. Nature 375: 415–418. 35 Jugovic, P., Hill, A. M., Tomazin, R., Ploegh, H., and Johnson, D. C. (1998). Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47. J. Virol. 72: 5076–5084. 36 Kaplan, J. M., et al. (1999). Induction of anti-tumor activity with dendritic cells transduced with adenovirus vector-encoding endogenous tumor-associated antigens. J. Immunol. 163: 699–707. 37 Clay, T. M., et al. (1999). Changes in the fine specificity of gp100 (209–217).-reactive T cells in patients following vaccination with a peptide modified at an HLA-A2.1
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anchor residue. J. Immunol. 162: 1749–1755. 38 Wadsworth, S. C., Zhou, H., Smith, A. E., and Kaplan, J. M. (1997). Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo. J. Virol. 71: 5189–5196. 39 Barr, D., et al. (1995). Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: Comparisons between immunocompetent and immunodeficient inbred strains. Gene Ther. 2: 151–155. 40 Michou, A. I., Santoro, L., Christ, M., Julliard, V., Pavirani, A., and Mehtali, M. (1997). Adenovirus-mediated gene transfer: Influence of transgene, mouse strain and type of immune response on persistence of transgene expression. Gene Ther. 4: 473–482. 41 Franksson, L., George, E., Powis, S., Butcher, G., Howard, J., and Karre, K.(1993). Tumorigenicity conferred to lymphoma mutant by major histocompatibility complexencoded transporter gene. J. Exp. Med. 177: 201–205. 42 Natuk, R. J., and Welsh, R. M. (1987). Accumulation and chemotaxis of natural killer/large granular lymphocytes at sites of virus replication. J. Immunol. 138: 877–883. 43 Biron, C. A. (1997). Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9: 24–34. 44 Glas, R., et al. (2000). Recruitment and activation of natural killer cells in vivo determined by the target cell phenotype: An adaptive component of NK cell-mediated responses. J. Exp. Med. 191: 129–138. 45 Harms, J. S., and Splitter, G. A. (1995). Interferon-γ inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter. Hum. Gene Ther. 6: 1291–1297. 46 Wold, W. S. M., Tollefson, A. E., and Hermiston, T. W. (1994). Adenovirus proteins that subvert host defenses. Trends Microbiol. 2: 437–443. 47 Ploegh, H. L. (1998). Viral strategies of immune evasion. Science. 280: 248–253. 48 Schowalter, D. B., Tubb, J. C., Liu, M., Wilson, C. B., and Kay, M. A. (1997). Heterologous expression of adenovirus E3-gp19K in an E1a-deleted adenovirus vector inhibits MHC I expression in vitro, but does not prolong transgene expression in vivo. Gene Ther. 4: 351–360. 49 Lee, M. G., Abina, M. A., Haddada, H., and Perricaudet, M. (1995). The constitutive expression of the immunomodulatory gp19K protein in E1−, E3− adenoviral vectors strongly reduces the host cytotoxic T cell response against the vector. Gene Ther. 2: 256–262. 50 Bruder, J. T., Jie, T., McVey, D. L., and Kovesdi, I. (1997). Expression of gp19K increases the persistence of transgene expression from an adenovirus vector in the mouse lung and liver. J. Virol. 71: 7623–7628. 51 Ilan, Y., et al. (1997). Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long-term gene expression. Proc. Natl. Acad. Sci. USA 94: 2587–2592.
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Adenoviral Vector Expressing ICP47 Inhibits AdenovirusSpecific Cytotoxic T Lymphocytes in Nonhuman Primates Abraham Scaria,1 Jennifer A. Sullivan, Judith A. St. George, Johanne M. Kaplan, Michael J. Lukason, James E. Morris, Malinda Plog, Charles Nicolette, Richard J. Gregory, and Samuel C. Wadsworth Genzyme Corporation, 31 New York Avenue, Framingham, Massachusetts 01701 Received for publication July 6, 2000, and accepted in revised form September 25, 2000; published online October 23, 2000
Studies from several laboratories have shown that administration of E1-deleted Ad vectors results only in transient transgene expression in the lungs of immunocompetent animals. This is due, at least in part, to destruction of vector-transduced cells by host cellular immune responses (predominantly CD8+ CTLs) directed against viral proteins and/or immunogenic transgene products. We have previously demonstrated that E1-deleted Ad vectors can lead to persistent expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) in the lungs of several strains of immunocompetent mice, despite the presence of Ad-specific CTLs. However, we found that these same vectors gave rise only to transient hCFTR expression in the lungs of rhesus monkeys. We have constructed new Ad vectors that coexpress both hCFTR and the ICP47 gene from herpes simplex virus. ICP47 has been shown to inhibit the transporter associated with antigen presentation, thus blocking major histocompatibility antigen I (MHC class I)-mediated antigen presentation to CD8+ T cells. The Ad/hCFTR/ICP47 vector decreased levels of cell-surface MHC class I molecules on infected monkey and human cell lines. Similar results were obtained with primary human cells and primary monkey airway epithelial cells. In vitro studies showed that the Ad/hCFTR/ICP47 vector decreased cytolysis by both monkey and human CTLs. When Ad/hCFTR/ICP47 was administered to the lungs of rhesus monkeys, it inhibited the generation of Ad-specific CTLs. However, natural killer cell activity was enhanced in monkeys treated with the Ad/hCFTR/ICP47 vector. Key Words: adenoviral vector; ICP47; MHC class I; CTLs; natural killer cells.
INTRODUCTION Adenovirus (Ad) is an attractive option for the development of gene therapy vectors for many reasons. Primary advantages include the ability of Ad vectors to transduce a wide variety of dividing and nondividing cells and the ability to manufacture vectors at high titer (1–6). Early Ad-based vectors were deleted of E1 sequences, a necessary step for rendering Ad vectors replication-defective, but it was also believed that deletion of E1 sequences would silence the remaining Ad genes present in the vector genome. Ad vectors with this basic design were developed for therapeutic use for cystic fibrosis (CF), a monogenic disorder in which the cystic fibrosis transmembrane conductance regulator (hCFTR) is mutated.
1To whom correspondence and reprint requests should be addressed. Fax: (508) 872 4091. E-mail: [email protected].
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We and others have used simple, E1-deleted Ad vectors to successfully transfer hCFTR cDNA into respiratory epithelium in animal models and to CF patients in vivo (1, 3, 7–10). In parallel, studies from several laboratories have suggested that administration of high doses of Ad vector results in only transient transgene expression in immunocompetent animal models (4, 11, 12–15). In contrast to the majority of published studies, we have recently demonstrated that an E1-deleted Ad vector, Ad2/CFTR-16, can give rise to persistent expression of hCFTR in the lungs of immunocompetent mice, despite the presence of Ad-specific cytotoxic T lymphocytes (CTLs) (16). In this report we show that the same vector, Ad2/CFTR-16, gives rise to transient transgene expression in the lungs of rhesus monkeys, and we describe a strategy designed to overcome this limitation. In rodent systems, transience of transgene expression has partly been attributed to the destruction of vector transduced cells by CD8+ CTLs directed against the
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ARTICLE transgene product and/or Ad viral proteins (4, 11–15, 17). Other potential reasons for the decline in transgene expression can include vector-induced cytotoxicity, complete loss of vector DNA, antibody response to secreted transgene products, promoter shut-off, and normal turnover of the transduced cell (18–20). Reduction of the cellular immune response and improved persistence have been reported using second-generation Ad vectors with modification or deletion of the E4, E2a, or E2b regions in addition to the E1 deletion (9, 11, 21, 22) and with the fully-deleted Ad vectors (23–26). Persistence of transgene expression can also be obtained by using transient immunomodulation strategies to block the in vivo interactions between antigen-presenting cells and T cells (27–31). To investigate the role of CTLs in the decline of hCFTR expression we observed in the monkey lung, we designed and constructed new Ad vectors that express both hCFTR and the immunomodulatory gene ICP47 from the herpes simplex virus. In vitro studies have shown that ICP47 inhibits the function of human transporter associated with antigen presentation (TAP), thus blocking major histocompatibility complex type I (MHC class I) mediated antigen presentation to CD8+ T cells (32–35). The Ad2/CFTR/ICP47 vector decreased levels of cell surface MHC I molecules on infected human and monkey cells in vitro. To date, there have been no reports on the effects of in vivo expression of ICP47. In this article, we describe the in vivo characterization of Ad2/CFTR/ICP47 in the lungs of rhesus monkeys and the effects of ICP47 expression on the generation of CTLs, natural killer (NK) cell responses, and persistence of hCFTR mRNA expression.
MATERIALS
AND
METHODS
Adenoviral vectors. All vectors used in these studies are Ad2 based and the transgenes are driven by the cytomegalovirus (CMV) promoterenhancer. All vectors contain a wt E2 region. (1) Ad2/CFTR-16 has been described previously (16). It contains a CMV enhancer-promoter-driven hCFTR cDNA followed by a bovine growth hormone poly(A) signal. The hCFTR expression cassette is inserted in the E1 region. The E3 region has a 1549-bp deletion in the E3B region corresponding to Ad2 nucleotides 29,292 to 30,840. The vector has a wt E4 region. (2) Ad2/CFTR/ICP47 has the same hCFTR expression cassette as Ad2/CFTR-16. The E3 region corresponding to Ad2 nucleotides 27,971 to 30,937 is deleted and is replaced with the ICP47 expression cassette. The ICP47 cDNA was cloned by PCR from herpes simplex virus I DNA obtained from Advanced Biotechnologies, Inc. (Columbia, MD). The ICP47 expression cassette has the CMV promoter-enhancer and an SV40 poly(A) signal. (3) Ad2/β-gal/∆E3 has been described previously (16) and has the CMV enhancer-promoter driving β-galactosidase. The E3 region has the identical deletion as Ad2/CFTR/ICP47. (4) Ad2/β-gal/ICP47 has the same β-galactosidase expression cassette as Ad2/β-gal/∆E3. The E3 region is deleted and contains the same ICP47 expression cassette as described above for Ad2/CFTR/ICP47. (5) Ad2/EV is an Ad2 based empty vector control that has no transgene cassette. Both E1 and E3 regions are deleted. It has wt E2 and E4 regions. (6) Ad2/hgp100v2 has been described before (36). The vector has the human gp100 expression cassette in the E1 region and has a wt E3
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region. The E4 region is modified by removal of all open reading frames (ORFs) and replacement with the E4 ORF6 and pIX has been moved from its original location to the junction between fiber and E4 genes. Cell lines. The HLA-A2-restricted T cell clone TIL1520 specific for the 209–217 epitope of the human melanoma antigen gp100 was obtained from Dr. M. Nishimura, Surgery Branch, NCI, and has been described previously (37). Cells were maintained in AIMV medium (Gibco) supplemented with 10% human AB serum (Sigma) and 1% penicillin/streptomycin (Life Technologies). Dendritic cell (DC) preparation. Normal donor monocytes were harvested by leukophoresis (Dana Farber Cancer Institute) in a volume of ~175 ml. The cells were diluted 1:1 with PBS (Mg2+, Ca2+ free) and 30 ml layered onto 20 ml Ficoll–Paque (Pharmacia Biotech.). Cells were collected at the interface by centrifugation (IEC, Model GP8, 1400 RPM/40 min). The cells were washed three times with PBS and resuspended in RPMI (Gibco) supplemented with P/S, glutamine, 5% human AB serum (Sigma), and 10 mM Hepes (complete medium) and rocked overnight on a Nutator in 50-ml conical tubes at 4°C. The cells were then plated at 1.5 × 108 cells/T-150 Flask (Corning). After 1 h, nonadherent cells were removed by washing three times with unsupplemented RPMI. The cells were then fed with complete RPMI plus 100 ng/ml GM-CSF (Genzyme) and 20 ng/ml IL-4 (Genzyme) in a volume of 20 ml/T150 flask. On day 3, 5 ml complete RPMI (plus cytokines) was added to each flask. On day 6, the cells were processed for assaying as described. Human CTL assay. DCs (1 × 106) were labeled overnight in 900 µl complete medium supplemented with 100 µl (100 µCi) 51 Cr (NEN/Dupont) in 1 well of a 24-well cluster plate (total volume = 1 ml). The DCs were transferred to a 1.5-ml Eppendorf tube and washed three times with 1 ml of serum-free AIM-V medium (Gibco BRL). Five thousand cells were transferred to each well of a 96-well V-bottom plate (Costar, polystyrene) in a volume of 100 µl/well. Effector cells were added to each well in AIM-V medium/10% human AB serum at the indicated E:T ratios in a volume of 100 µl for a total reaction volume of 200 µl. Medium (no effector cells) was added to the spontaneous release wells, and no additional media was added to total release wells. The plate was then spun at 1200 rpm for 3 min and returned to the incubator for 4 h. After the incubation, 100 µl of 1% Triton X-100 was added to the total release wells (total volume = 200 µl) and the plate was spun at 1000 rpm for 10 min. Fifty microliters of supernatant was removed from each well and added to a Wallac 96-well plate (polyethylene terepthalate) containing 150 µl scintillation fluid (Wallac Optiphase Supermix). This plate was sealed with an adhesive plate sealer and incubated at room temperature overnight. The radioactivity was measured using a MicroBeta Trilux Scintillation Counter (Wallac, Gaithersburg, MD). All reactions were performed in triplicate, and each graphed data point represents the average of the replicates. Percent specific killing was calculated according to the formula 100 × (experimental cpm − spontaneous cpm)/(total cpm − spontaneous cpm). FACS analysis. Approximately 106 cells were trypsinized and resuspended in FACS buffer (PBS containing 0.5% BSA). Cells were incubated at 4°C for 1 h in the presence of an antibody (W6/32, Accurate Chemical) which detects all haplotypes of MHC class I. Cells were washed and incubated at 4°C for 1 h with FITC goat anti-mouse IgG (Jackson Immunoresearch). The fluorescence profiles were then analyzed on a FACS flow cytometer (Becton–Dickinson, San Jose, CA). Aerosol study with Ad2/CFTR-16. Ad2/CFTR-16 was delivered to the lungs of rhesus monkeys (Macaca mulatta) through inhalation of aerosolized vector. A miniHEART low flow nebulizer (Vortran Medical Technology, Inc.) was filled with 10 ml of PBS/sucrose containing 5 × 1011 IU of vector. The aerosol was directed to a modified anesthesia mask fitted to anesthetized animals that had their nares occluded. This provided transoral delivery of the aerosol. The respired fraction of aerosol was determined for each animal based upon the animal’s weight and rate of respiration. By these criteria, approximately 7.6 × 1010 infectious units of Ad2/CFTR-16 was determined to be deposited in the lungs of each animal. MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
ARTICLE Vector administration via bronchoscope. Rhesus monkeys (M. mulatta) received 1.6 × 1010 infectious units of either Ad2/CMV/CFTR-ICP47 (4.9 × 1011 particles) or Ad2/CFTR-16 (4.0 × 1011 particles) administered to the left caudal lobe using a pediatric bronchoscope. The dose of virus was delivered in 1.0 ml of PBS containing 10% sucrose followed by a rinse with 1 ml of the PBS/sucrose solution. One animal from each group was sacrificed on day 3 and day 21 following the administration of the vectors. RNA extraction. The lungs were removed and portions of the conducting airways were dissected free of parenchyma for RT-PCR analysis. Pieces of the trachea, lobar bronchus, segmental, subsegmental bronchus (to a diameter of ≈1 mm) and alveolar tissue were placed in RNA Stat-60 solution (Tel-Test B, Inc., Friendswood, TX) and frozen on dry ice for subsequent storage at −80°C. Tissue samples were homogenized in RNA Stat60 solution and RNA was extracted using an acid guanidinium/phenol–chloroform method according to the manufacturer’s directions. Tissue was disrupted in a Mini-Bead Beater (Biospec Corp., Batlesville, OK). RT-PCR for aerosol experiment and experiment A. Ad2/CFTR-16 specific mRNA was detected in the aerosol administration experiment and experiment “A” (Fig. 8) using an isotopic, nested RT-PCR protocol. Total RNA (1 µg) from each sample was treated with RNase-free DNase I (Promega, Madison, WI) to remove any contaminating DNA in the RNA samples. Following DNase treatment, the RNA sample was divided into two tubes. The RT reaction was performed using components of Invitrogen’s cDNA cycle reverse transcription kit. 250 nM of a vector specific reverse primer B23∆1.6 (5′-TATGTTTACCGCCACACTCGCAGG-3′), was used to prime the 15 µl reverse transcriptase (RT) reaction. One aliquot from each sample was processed without RT enzyme to serve as a negative control. Following the RT reaction, 5 µl of cDNA was amplified in the 100-µl firstround reaction using 2.5 U Taq polymerase (Perkin–Elmer), in a mixture containing a final concentration of 10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.75 mM MgCl2, 0.01% gelatin, 200 mM each dTTP, dATP, dGTP, and dCTP, 500 nM each primer F12∆1.6 (5′-AGGATAGAAGCAATGCTGGAATGCC-3′) and B23∆1.6 (5′-TATGTTTACCGCCACACTCGCAGG-3′), and 0.5 µl [33P]dCTP (3000 Ci/mmol). After a denaturing step of 1 min at 94°C, 25 amplification cycles consisting of 60 s at 92°C, 60 s at 62.5°C, and 60 s at 72°C, with a final extension step of 5 min at 72°C was performed. First round amplification products were diluted 1:100 in DEPCtreated H2O and 10 µl of this dilution was used as template in the nested amplification reaction. The nested round consisted of 20 amplification cycles under conditions identical to the first round reaction using primers F20∆1.6 (5′-AGCCCCAGATTGCTGCTCTGAAAG-3′) and B22∆1.6 (5′CGTCAGACAATGCGATGCAATTTC-3′). Amplified products from both rounds were electrophoresed in 1.5% LE agarose gels, stained with ethidium bromide and visualized on a transilluminator. The gels were dried, placed in an exposure cassette and product bands quantitated using image analysis software on a phosphorimager (Molecular Dynamics).
Pixel volumes from the study samples were compared to those of a DNA standard curve containing log concentrations (from 1 × 101 to 1 × 105) of pAd2/CFTR-5 plasmid to quantitate the target molecules in the study samples. RT-PCR analysis for experiment B. In experiment B Ad2/CFTR-16 specific mRNA was detected using a real-time assay that employs the 5′–3′ exonuclease activity of Taq polymerase to cleave an oligonucleotide probe labeled with fluorescent reporter and quencher molecules during the extension phase of the amplification reaction. The fluorescence of the reactions are measured in real time on an ABI PRISM 7700 sequence detection system (PE Biosystems) instrument where fluorochromes are excited using a 488nm argon laser and fluorescent emission spectra measured using a spectrograph and a CCD camera. Total RNA (1 µg) from each sample was treated with RNase-free DNase I (Promega, Madison, WI) to remove any contaminating DNA in the RNA samples. Following DNase treatment, the RNA sample was divided into two tubes. One aliquot from each sample was processed without RT enzyme to serve as a negative control. Reverse transcription was performed using the Promega reverse transcription kit and 250 nM of a vector specific reverse primer JMTM99-04 (5′-GTGCGGGTCTCATCGTACCT-3′), according to the manufacturer’s protocol. Two and a half microliters of the RT reaction was run in the amplification reaction described below. cDNA samples were amplified in a 50-µl reaction containing 25 µl of TaqMan Universal PCR Master Mix (PE Biosystems) with 300 nM of each primer JMTM99-03 (5′-ATGAGGAAATTGCATCGCATT-3′) and JMTM99-04 (5′-GTGCGGGTCTCATCGTACCT-3′), 200 nM of TaqMan probe 77oJM-6 (5′-FAM-TCTGACGCGTTACGCGGGAAGGT-TAMRA-3′), 2.5 U AmpliTaq Gold polymerase, 0.5 U AmpErase UNG and 10% glycerol. The reactions were held at 50°C for 2 min to allow UNG activity to remove any dUTP containing contaminates present in the reactions followed by a 10-min incubation at 95°C for 10 min to activate AmpliTaq Gold. These incubations were followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. In addition to study samples, water negative controls and a DNA standard curve containing log concentrations (from 1 × 101 to 1 × 105) of pAd2/CFTR-5 were run as well. Sample and standard CT values are exported to an Excel spreadsheet (Microsoft Corp., Redmond, WA) and sample values were determined from a standard curve by interpolation. Cytotoxic T cell assay (monkey). Primary fibroblast cell lines were established from skin biopsies from individual monkeys as a source of MHCcompatible target cells. The fibroblasts were grown in DMEM (Gibco BRL) supplemented with 20% fetal calf serum, 1 mM Hepes buffer, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 30 µg/ml vancomycin and 10 µg/ml gentamycin. A portion of spleen as well as tracheobronchial lymph nodes were collected aseptically at the time of sacrifice and a single-cell suspension was prepared by mincing the specimens in PBS and shearing the fragments between the frosted edges of two sterile glass slides. To expand vector-specific cytotoxic T lymphocytes (CTLs), spleen and lymph node cells were restimulated in vitro with autologous fibroblasts transduced with Ad vector at a multiplicity of infection (m.o.i.) of 200. The
FIG. 1. Genomic structures of recombinant Ad vectors. The expression cassette for hCFTR is inserted in place of the E1 region in both vectors. The ICP47 expression cassette is inserted in the E3 region of Ad2/CFTR/ICP47. Both vectors have wt. E2 and E4 regions.
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B
FIG. 2. Persistence of Ad2/CFTR-16 in the monkey lung. Ad2/CFTR-16 (7.6 × 1010 IU per monkey) was administered to the lungs of rhesus monkeys by aerosolization. Tissue samples from lung were analyzed for hCFTR mRNA expression by RT-PCR at different time points as described under Materials and Methods. Percentages of samples positive for hCFTR mRNA are shown in A and the average mRNA expression levels in the positive samples are shown in B.
cells were cultured in the wells of 24-well plates containing 5 × 106 spleen or lymph nodes cells with 5–6 × 104 stimulator fibroblasts in 2 ml of AIMV medium (Gibco BRL) supplemented with 10% human AB serum. The cultures were incubated for 6 days at 37°C/5% CO2 and received 20 U/ml recombinant human IL-2 (Genzyme Corp.) on the fourth day of culture. Cytolytic activity was then tested against autologous or allogeneic fibroblasts transduced with Ad vector to assess specific MHC-restricted killing. The K-562 myelogenous leukemia cell line was also used as a target to evaluate levels of NK cell activity. Target cells were treated with 500 U/ml recombinant human γ-interferon (Genzyme Corp.) for 24 h (except for K562 cells) to enhance MHC Class I presentation and were labeled with
51chromium (New England Nuclear) overnight (25 µCi/105 cells). Effector
and target cells were added at various effector: target (E:T) ratios to triplicate wells of 96-well round-bottom plates in a 200-µl volume. After 5 h of incubation at 37°C/5% CO2, 25 µl of cell-free supernatant was collected from each well and counted in a MicroBeta Trilux scintillation counter (Wallac, Gaithersburg, MD). The amount of 51Cr spontaneously released was obtained by incubating target cells in medium alone and was typically below 20%. The total amount of 51Cr incorporated was determined by adding 1% Triton X-100 in distilled water, and the percentage lysis was calculated as % lysis = [(sample cpm − spontaneous cpm)/(total cpm − spontaneous cpm)] × 100.
FIG. 3. Downregulation of MHC class I. Human A549 cells were infected at an m.o.i. of 200 and monkey CV1 cells were infected at an m.o.i. of 100 with different Ad vectors. Cell surface expression of MHC class I was measured using FACS analysis at 24 h postinfection with Ad vectors. Results are shown as mean fluorescence intensities of staining for MHC class I.
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FIG. 4. MHC I expression on primary cells. Primary human dendritic cells were infected at an m.o.i. of 400 and monkey airway cells were infected at an m.o.i. of 150 with different Ad vectors. Cell surface MHC class I levels were measured using FACS analysis at 24 h postinfection. Results are shown as mean fluorescence intensities of staining for MHC class I.
RESULTS Ad2/CFTR-16 in the Monkey Lung We have previously shown that an E1-deleted Ad vector encoding human CFTR, namely Ad2/CFTR-16, gives rise to persistent transgene expression for 70 days in the lungs of several strains of immunocompetent mice (16). This persistence was achieved by the use of the CMV enhancer/promoter for hCFTR expression in conjunction with a wt E4 region (18). To examine the properties of the same vector in the lungs of nonhuman primates, we delivered approximately 7.6 × 1010 IU of Ad2/CFTR-16 into the lungs of rhesus monkeys by aerosol administration. One monkey was sacrificed at each time point (days 3, 21, and 71) after vector administration. At necropsy, tissue samples of airways, alveoli and parenchyma were dissected out from each lobe of the lung. Approximately 25 to 30 samples from each monkey lung were analyzed for expression of hCFTR mRNA by RT-PCR analysis. We found that on day 3, 90% of the samples were positive for hCFTR expression (Fig. 2). However, only about 50% of the samples were positive for transgene expression on day 21 and day 71. Also, the levels of hCFTR expression in the positive samples declined about 100-fold compared to the mRNA levels on day 3 (Fig. 2).
Construction of Ad2/CFTR/ICP47 and in Vitro Characterization We designed and constructed new Ad vectors that expressed hCFTR along with the ICP47 gene from herpes simplex virus (Fig. 1). ICP47 has been shown to bind to and inhibit the function of the transporter associated with antigen presentation (TAP), which normally functions to translocate antigenic peptides across the endoMOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
plasmic reticulum (ER) membrane (33–35). The result of this inhibition is that empty MHC I molecules accumulate in the ER and fail to reach the cell surface. Expression from the CMV promoter driven ICP47 cassette inserted in the E3 region of Ad2/CFTR/ICP47 was confirmed by immunoprecipitation analysis (data not shown) and fluorescence-activated cell sorter (FACS) analysis of cell surface MHC class I expression on human and monkey cell lines (Fig. 3) as well as primary human dendritic cells and monkey airway cells (Fig. 4). As shown in Figs. 3 and 4, cell surface expression of MHC class I was almost completely inhibited on cells transduced with Ad vectors expressing ICP47 compared to uninfected cells or cells infected with a control Ad vector.
Inhibition of Lysis by Human CTLs We used an in vitro model system to test the ability of an Ad vector expressing ICP47 to inhibit lysis by human cytotoxic T lymphocytes (CTLs). In the experiment shown (Fig. 5), human DCs from a donor expressing the HLA-A2 haplotype were infected with Ad2/hgp100v2 vector which expresses a human melanoma associated antigen gp100. A human CTL clone that specifically recognizes human gp100 in the context of HLA-A2 (37) was then used to lyse the gp100 expressing cells. Coinfection with a control vector Ad2/EV did not affect lysis by the CTL clone. However, coinfection with Ad2/ICP47 completely inhibited lysis of the human DCs expressing gp100.
ICP47 Inhibits Ad-Specific CTLs in Primates Ad2/CFTR/ICP47 (1.6 × 1010 IU per monkey) was instilled into the left caudal lobe of rhesus monkeys using a pediatric bronchoscope. Another group of mon-
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FIG. 5. Ad2/ICP47 protects against human CTLs. Human dendritic cells were infected with different Ad vectors at an m.o.i. of 400 for each Ad vector. Ad2/hgp100v2 vector expresses human melanoma antigen gp100. A human CTL clone TIL1520 that specifically recognizes human gp100 was then used to lyse gp100 expressing cells. Results shown are mean percentages of lysis from triplicate wells.
keys was instilled with the same dose of Ad2/CFTR-16. Spleens and tracheobronchial lymph nodes were collected at the time of sacrifice on day 21 to expand vector specific CTLs. As shown in Fig. 6A, administration of Ad2/CFTR-16 led to the generation of Ad specific CTLs. Infection of autologous fibroblast targets with Ad2/CFTR/ICP47 decreased the lysis by Ad specific CTLs
A
from the monkey, although the inhibition was not as dramatic as seen with the human CTLs (Fig. 5). In the monkeys instilled with Ad2/CFTR/ICP47, no adenovirusspecific CTL activity was detected (Fig. 6B). However, we detected higher levels of cytolytic activity against autologous fibroblasts that were infected with Ad2/CFTR/ICP47.
B
FIG. 6. CTL response to Ad vectors in primates. Ad2/CFTR-16 or Ad2/CFTR/ICP47 (1.6 × 1010 IU per monkey) vectors were instilled into the lungs of rhesus monkeys using a pediatric bronchoscope. On day 21, spleens were collected and Ad vector specific CTLs were expanded in vitro for 6 days. Cytolytic activity was then tested against autologous fibroblasts transduced with different Ad vectors. Results shown are mean percentages of lysis from triplicate wells.
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FIG. 7. NK cell response to Ad vectors in primates. Ad vectors were instilled into the lungs of rhesus monkeys as described for Fig. 6. Regional lymph nodes were collected, expanded in vitro and tested against allogeneic fibroblasts transduced with different Ad vectors and against K562 cells. Results are shown as mean percentages of lysis from triplicate wells.
Activation of Natural Killer Cells As shown in Figs. 3 and 4, expression of ICP47 inhibits cell-surface expression of MHC class I on monkey cells. Therefore, the cytolytic activity that we observed against Ad2/CFTR/ICP47 infected autologous fibroblasts was most likely mediated by NK cells, which preferentially lyse targets expressing low levels of MHC class I. The involvement of NK effector cells was confirmed by the ability of cells isolated from the regional lymph nodes of Ad2/CFTR/ICP47-treated monkeys to lyse traditional NK target K562 cells as well as allogeneic monkey fibroblasts infected with Ad2/CFTR/ICP47 to reduce MHC class I expression (Fig. 7A). Allogeneic fibroblasts infected with Ad2/CFTR-16 were not lysed in this assay since they express normal levels of MHC class I. No significant level of NK cell activity was detected in cultures of lymph node cells from Ad2/CFTR-16-treated monkeys (Fig. 7B).
Persistence of Transgene Expression in the Monkey Lung Two separate experiments (A and B) were conducted to evaluate persistence of transgene expression in the primate lung from Ad2/CFTR/ICP47 versus Ad2/CFTR-16. In each experiment, one monkey from each group was sacrificed on days 3 and 21, airway samples were dissected out and assayed for hCFTR mRNA by RT-PCR analysis. On day 3, almost all samples analyzed from the treated lobe (left caudal lobe) were positive for hCFTR expression in agreement with our previous results (Fig. 2). In the samples that were positive for hCFTR expression we found that the average hCFTR expression levels from the Ad2/CFTR-16-treated monkeys were not significantly different from the average expression levels in the positive MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
samples from the Ad2/CFTR/ICP47-treated monkeys. The expression levels declined over time in both groups (Fig. 8A). As expected, the percentage of samples that are positive for expression declined rapidly by day 21 in the Ad2/CFTR-16-treated monkey (Fig. 8B). However, in the Ad2/CFTR/ICP47-treated monkey, we found that almost all samples were positive for transgene expression even on day 21 in both experiments A and B (Fig. 8B).
DISCUSSION Adenoviral vectors have been used successfully to transfer genes to a variety of organs including the lung, liver, muscle and brain in normal animals and animal models of genetic diseases. However, in the majority of immunocompetent animal models, investigators have reported only transient expression of the transgene. In mice, this transience of transgene expression has been attributed primarily to the cellular immune response directed against the foreign transgene products and/or Ad viral proteins (4, 11–15). We (16, 38) and others (5, 6, 19, 39, 40) have demonstrated that Ad vectors can give rise to persistent transgene expression in immunocompetent mice provided the transgene product is nonimmunogenic in the particular strain of mouse tested. We have shown recently that an E1-deleted vector, Ad2/CFTR-16, gives rise to persistent expression in the lungs of three different strains of immunocompetent mice (16). The mice tested clearly generated Ad-specific CTL activity; however, a CTL response against hCFTR was not observed. In this article we demonstrate that the same vector Ad2/CFTR-16 gives rise to transient transgene expression in the lungs of rhesus monkeys. Therefore, the immune response generated to Ad2/CFTR-16 in
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FIG. 8. Persistence of transgene expression in the monkey lung. Ad vectors (1.6 × 1010 IU per monkey) were instilled into the lungs of rhesus monkeys as described for Fig. 6. Tissue samples from lung were analyzed for hCFTR mRNA expression by RT-PCR at different time points as described under Materials and Methods. The average mRNA expression levels in the positive samples are shown in A and the percentages of samples positive for hCFTR mRNA in the treated lobe are shown in B.
monkeys seems to be more effective in eliminating transgene expression compared to the immune response generated to the same Ad vector in the mouse model. One possibility is that the cells transduced with the Ad vector in the monkey lung are more efficient at antigen presentation to the CD8+ T cells compared to the cells transduced in the mouse lung. Another possibility is that an E1-deleted Ad vector, being derived from a human adenovirus, is more competent at viral gene expression in primate cells than in rodent cells. To investigate further the role of CD8+ CTLs in the elimination of transgene expression in the primate lung, we constructed Ad vectors expressing the herpes simplex virus ICP47 protein. We show here that the ICP47expressing vectors are capable of decreasing cell surface expression of MHC class I on primary human and monkey cells and offer protection in vitro from lysis by human and monkey antigen specific CTLs. These data are in agreement with published reports showing that ICP47 is capable of inhibiting TAP function in human and primate cell lines (32–35). The experiments
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described in this report extend the studies on effects of ICP47 expression to an animal model. We find that administration of Ad2/CFTR/ICP47 into the lungs of monkeys leads to an inhibition of the generation of the Ad specific CTL response. However, we find that inhibition of the Ad-specific CTL response using Ad/CFTR/ICP47 was not sufficient to give rise to prolonged high levels of hCFTR mRNA expression in the monkey lung, even though a higher proportion of the lung samples were positive for transgene expression on day 21 when compared to the lung samples from the monkeys treated with the Ad2/CFTR-16 vector. An important observation from our studies in the primate lung is the activation of NK cells in the regional lymph nodes of monkeys treated with Ad2/CFTR/ICP47. NK cells are of the lymphoid lineage, although distinct from T and B cells. NK cells are especially efficient in the rejection of tumors lacking host MHC I molecules, including those with defects in the TAP protein (41). In vivo, NK cells are recruited to sites of virus infection and are activated to become more cytotoxic and to produce
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ARTICLE INF-γ (42, 43). Recently, it has been shown that tumor cells lacking appropriate MHC I expression induce NK cell infiltration, cytotoxic activation and induction of transcription of INF-γ in NK cells (44). It is possible that the loss in transgene expression that we observed in the lungs of Ad2/CFTR/ICP47-treated monkeys is partly due to enhanced NK cytotoxicity or due to downregulation of the CMV promoter (20, 45) by cytokines secreted by NK cells and activated CD4+ T cells. In future experiments, detailed analysis of Ad vector DNA persistence in the lung samples and measurement of cytokine levels in the bronchoalveolar fluid (BAL) could address these different possibilities. NK cytotoxicity is less likely to be the cause of the loss in transgene expression, as all samples from the Ad2/CFTR/ICP47-treated monkey were positive for transgene expression on day 21. Use of cellular promoters that are unaffected by cytokines like INF-γ and TNF-α may help avoid the issue of promoter downregulation. Adenoviruses and several other viruses have evolved mechanisms to reduce MHC class I expression (46, 47). Other groups have tried overexpression of adenoviral E3-region proteins to prolong Ad vector persistence in immunocompetent rodent models. Expression of E3 gp19K alone has been shown to inhibit MHC I expression in vitro (48), decrease cytolysis by Ad specific CTLs (49), and provide minimal improvement in transgene expression for 2 weeks in one strain (B10.HTG) of mice (50). Overexpression of the entire E3 region has been reported to decrease both the humoral and cellular immune response to Ad vectors and provide long-term transgene expression in the livers of Gunn rats (51). It is known that Ad E3 gp19K binds with different affinities to different MHC haplotypes in mice and humans (46), while ICP47 should be active independently of MHC haplotype, since it inhibits TAP function and indirectly reduces cell surface expression of MHC class I. However, our demonstration that a drastic reduction of MHC I expression could potentially lead to an enhanced NK response in vivo suggests that alternate strategies to protect the Ad-transduced cell without interfering with MHC I expression should also be explored. Another promising approach to improve persistence of transgene expression from Ad vectors involves deletion of additional viral genes, which should lead to a decreased cellular immune response in vivo. Improved persistence has been reported with Ad vectors with modifications or deletions in the E4, E2a, or E2b regions (9, 11, 21, 22). Recently, it has been shown that Ad vectors lacking all viral genes can give rise to persistent transgene expression in the muscle (24) and liver (25, 26) of immunocompetent animals. This does not, however, address the issue of immunogenicity of the transgene product, which could potentially be a neoantigen in patients with genetic disease that we wish to treat with these vectors. Transient immunosuppression has been shown to effectively suppress the cellular and humoral immune responses to Ad vector and transgene products MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy
(27–31) and could be used in conjunction with an advanced Ad vector with all viral genes deleted. ACKNOWLEDGMENTS All primate studies were conducted at the New England Primate Research Center in Southborough, Massachusetts. We thank the Genzyme Virus Production Unit for the purified Ad vectors used in these studies, Dr. Srinivas Shankara for the Ad2/hgp100v2 vector, and Dr. Michael I. Nishimura (National Cancer Institute) for the T cell clone TIL1520.
REFERENCES 1
Rosenfeld, M. A., et al. (1992). In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell 68: 143–155. 2 Smith, T. A. G., et al. (1993). Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Gene Ther. 5: 397–402. 3 Zabner, J., et al. (1994). Safety and efficacy of repetitive adenovirus-mediated transfer of CFTR cDNA to airway epithelia of primates and cotton rats. Nat. Genet. 6: 75–83. 4 Dai, Y, Schwarz, E. M., Gu, D., Zhang, W., Sarvetnick, N., and Verma, I. M. (1995). Cellular and humoral responses to adenoviral vectors containing factor IX gene: Tolerization of factor IX and vector antigens allows for long term expression. Proc. Natl. Acad. Sci. USA 92: 1401–1405. 5 Tripathy, S. K., Black, H. B., Goldwasser, E., and Leiden, J. M. (1996) Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus. Nat. Med. 2: 545–550. 6 Connelly, S., et al. (1998). Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy. Blood 91: 3273–3281. 7 Zabner, J., Couture, L. A., Gregory, R. J., Graham, S. M., Smith, A. E., and Welsh, M. J. (1993). Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelium of patients with cystic fibrosis. Cell 75: 207–216. 8 Crystal, R. G., et al. (1994). Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat. Genet. 8: 42–51. 9 Yang, Y., Nunes, F. A., Berencsi, K., Gonczol, E., Engelhardt. J. F., and Wilson, J. M. (1994). Inactivation of E2a in recombinant adenoviruses improves the prospect for gene therapy in cystic fibrosis. Nat. Genet. 7: 362–369. 10 Yei, S., Mittereder, N., Tang, K., O’Sullivan, C., and Trapnell, B. C. (1994). Adenovirus mediated gene transfer for cystic fibrosis: Quantitative evaluation of repeated in vivo vector administration to the lung. Gene Ther. 1: 192–200. 11 Engelhardt, J. F., Ye, X., Doranz, B., and Wilson, J. M. (1994). Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver. Proc. Natl. Acad. Sci. USA 91: 6196–6200. 12 Yang, Y., Li, Q., Ertl, H. C. J., and Wilson, J. M. (1995). Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69: 2004–2015. 13 Yang, Y., Xiang, Z., Ertl, H. C. J., and Wilson, J. M. (1995). Upregulation of class I major histocompatibility complex antigens by interferon γ is necessary for T-cell-mediated elimination of recombinant adenovirus-infected hepatocytes in vivo. Proc. Natl. Acad. Sci. USA 92: 7257–7261. 14 Zsengeller, Z. K., et al. (1995). Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu-nu) mice. Hum. Gene Ther. 6: 457–467. 15 Yang, Y., Su, Q., and Wilson, J. M. (1996). Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J. Virol. 70: 7209–7212. 16 Scaria, A., St. George, J. A., Jiang, C., Kaplan, J. M., Wadsworth, S. C., and Gregory, R. J. (1998). Adenovirus-mediated persistent cystic fibrosis transmembrane conductance regulator expression in mouse airway epithelium. J. Virol. 72: 7302–7309. 17 Kaplan, J. M, et al. (1997). Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung. Hum. Gene Ther. 8: 45–56. 18 Armentano, D., et al. (1997). Effect of the E4 region on the persistence of transgene expression from adenovirus vectors. J. Virol. 71: 2408–2416. 19 Morral, N., O’Neal, W., Zhou, H., Langston, C., and Beaudet, A. L. (1997). Immune responses to reporter proteins and high viral dose limit duration of expression with adenoviral vectors: Comparison of E2a wild type and E2a-deleted vectors. Hum. Gene Ther. 8: 1275–1286. 20 Quin, L., Ding, Y., Pahud, D. R., Chang, E., Imperiale, M. J., and Bromberg, J. S. (1997). Promoter attenuation in gene therapy: Interferon-γ and tumor necrosis factor-α inhibit transgene expression. Hum. Gene Ther. 8: 2019–2029. 21 Wang, Q., Greenburg, G., Bunch, D., Farson, D., and Finer, M. H. (1997). Persistent transgene expression in mouse liver following in vivo gene transfer with a ∆E1/∆E4 adenovirus vector. Gene Ther. 4: 393–400. 22 Hu, H., Serra, D., and Amalfitano, A. (1999). Persistence of an [E1-, polymerase-] adenovirus vector despite transduction of a neoantigen into immune-competent mice. Hum. Gene Ther. 10: 355–364. 23 Haecker, S. E., et al. (1996). In vivo expression of full-length human dystrophin from
513
ARTICLE adenoviral vectors deleted of all viral genes. Hum. Gene Ther. 7: 1907–1914. 24 Chen, H. H., Mack, L. M., Kelly, R., Ontell, M., Kochanek, S., and Clemens, P. R. (1997). Persistence in muscle of an adenoviral vector that lacks all viral genes. Proc. Natl. Acad. Sci. USA 94: 1645–1650. 25 Schiedner, G., et al. (1998). Genomic DNA transfer with high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat. Genet. 18: 180–183. 26 Morral, N., et al. (1999). Administration of helper-dependent adenoviral vectors and sequential delivery of different vector serotype for long-term liver-directed gene transfer in baboons. Proc. Natl. Acad. Sci. USA 96: 12816–12821. 27 Kay, M. A., et al. (1995). Long-term hepatic adenovirus-mediated gene expression in mice following CTLA4Ig administration. Nat. Genet. 11: 191–197. 28 Poller, W., et al. (1996). Stabilization of transgene expression by incorporation of E3 region genes into an adenoviral factor IX vector and by transient anti-CD4 treatment of the host. Gene Ther. 3: 521–530. 29 Yang., Y., Su, Q., Grewal, I. S., Schilz, R., Flavell, R. A., and Wilson, J. M. (1996). Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung. J. Virol. 70: 6370–6377. 30 Scaria, A., et al. (1997). Antibody to CD40 ligand inhibits both humoral and cellular immune responses to adenoviral vectors and facilitates repeated administration to mouse lung. Gene Ther. 4: 611–617. 31 Kaplan, J. M., and Smith, A. E. (1997). Transient immunosuppression with deoxyspergualin improves longevity of transgene expression and ability to readminister adenoviral vector to the mouse lung. Hum. Gene Ther. 8: 1095–1104. 32 York, I. A., Roop, C., Andrews, D. W., Riddell, S. R., Graham, F. L., and Johnson, D. C. (1994). A cytosolic herpes simplex virus protein inhibits antigen presentation to CD8+ T lymphocytes. Cell 77: 525–535. 33 Hill, A., et al. (1995). Herpes simplex virus turns off the TAP to evade host immunity. Nature 375: 411–415. 34 Fruh, K., et al. (1995). A viral inhibitor of peptide transporters for antigen presentation. Nature 375: 415–418. 35 Jugovic, P., Hill, A. M., Tomazin, R., Ploegh, H., and Johnson, D. C. (1998). Inhibition of major histocompatibility complex class I antigen presentation in pig and primate cells by herpes simplex virus type 1 and 2 ICP47. J. Virol. 72: 5076–5084. 36 Kaplan, J. M., et al. (1999). Induction of anti-tumor activity with dendritic cells transduced with adenovirus vector-encoding endogenous tumor-associated antigens. J. Immunol. 163: 699–707. 37 Clay, T. M., et al. (1999). Changes in the fine specificity of gp100 (209–217).-reactive T cells in patients following vaccination with a peptide modified at an HLA-A2.1
514
anchor residue. J. Immunol. 162: 1749–1755. 38 Wadsworth, S. C., Zhou, H., Smith, A. E., and Kaplan, J. M. (1997). Adenovirus vector-infected cells can escape adenovirus antigen-specific cytotoxic T-lymphocyte killing in vivo. J. Virol. 71: 5189–5196. 39 Barr, D., et al. (1995). Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: Comparisons between immunocompetent and immunodeficient inbred strains. Gene Ther. 2: 151–155. 40 Michou, A. I., Santoro, L., Christ, M., Julliard, V., Pavirani, A., and Mehtali, M. (1997). Adenovirus-mediated gene transfer: Influence of transgene, mouse strain and type of immune response on persistence of transgene expression. Gene Ther. 4: 473–482. 41 Franksson, L., George, E., Powis, S., Butcher, G., Howard, J., and Karre, K.(1993). Tumorigenicity conferred to lymphoma mutant by major histocompatibility complexencoded transporter gene. J. Exp. Med. 177: 201–205. 42 Natuk, R. J., and Welsh, R. M. (1987). Accumulation and chemotaxis of natural killer/large granular lymphocytes at sites of virus replication. J. Immunol. 138: 877–883. 43 Biron, C. A. (1997). Activation and function of natural killer cell responses during viral infections. Curr. Opin. Immunol. 9: 24–34. 44 Glas, R., et al. (2000). Recruitment and activation of natural killer cells in vivo determined by the target cell phenotype: An adaptive component of NK cell-mediated responses. J. Exp. Med. 191: 129–138. 45 Harms, J. S., and Splitter, G. A. (1995). Interferon-γ inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter. Hum. Gene Ther. 6: 1291–1297. 46 Wold, W. S. M., Tollefson, A. E., and Hermiston, T. W. (1994). Adenovirus proteins that subvert host defenses. Trends Microbiol. 2: 437–443. 47 Ploegh, H. L. (1998). Viral strategies of immune evasion. Science. 280: 248–253. 48 Schowalter, D. B., Tubb, J. C., Liu, M., Wilson, C. B., and Kay, M. A. (1997). Heterologous expression of adenovirus E3-gp19K in an E1a-deleted adenovirus vector inhibits MHC I expression in vitro, but does not prolong transgene expression in vivo. Gene Ther. 4: 351–360. 49 Lee, M. G., Abina, M. A., Haddada, H., and Perricaudet, M. (1995). The constitutive expression of the immunomodulatory gp19K protein in E1−, E3− adenoviral vectors strongly reduces the host cytotoxic T cell response against the vector. Gene Ther. 2: 256–262. 50 Bruder, J. T., Jie, T., McVey, D. L., and Kovesdi, I. (1997). Expression of gp19K increases the persistence of transgene expression from an adenovirus vector in the mouse lung and liver. J. Virol. 71: 7623–7628. 51 Ilan, Y., et al. (1997). Insertion of the adenoviral E3 region into a recombinant viral vector prevents antiviral humoral and cellular immune responses and permits long-term gene expression. Proc. Natl. Acad. Sci. USA 94: 2587–2592.
MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright The American Society of Gene Therapy