Transduction of Dendritic Cells by DNA Viral Vectors Directs the Immune Response to Transgene Products in Muscle Fibers

Copyright © 1998, American Society for Microbiology

Transduction of Dendritic Cells by DNA Viral Vectors Directs the

Immune Response to Transgene Products in Muscle Fibers

KARIN JOOSS, YIPING YANG, KRISHNA J. FISHER,

JAMES M. WILSON*

Institute for Human Gene Therapy and Departments of Medicine and Molecular and Cellular Engineering,

University of Pennsylvania Health System, and The Wistar Institute,

Philadelphia, Pennsylvania 19104

Received 15 September 1997/Accepted 26 January 1998

Immune responses to vector-corrected cells have limited the application of gene therapy for treatment of

chronic disorders such as inherited deficiency states. We have found that recombinant adeno-associated virus

(AAV) efficiently transduces muscle fibers in vivo without activation of cellular and humoral immunity to

neoantigenic transgene products such as

b

-galactosidase, which differs from the experience with recombinant

adenovirus, where vibrant T-cell responses to the transgene product destroy the targeted muscle fibers. T cells

activated following intramuscular administration of adenovirus expressing lacZ (AdlacZ) can destroy

AAVlacZ-transduced muscle fibers, indicating a prior state of immunologic nonresponsiveness in the context of AAV

gene therapy. Adoptive transfer of dendritic cells infected with AdlacZ leads to immune mediated elimination

of AAVlacZ-transduced muscle fibers. AAVlacZ-transduced antigen-presenting cells fail to demonstrate

b

-ga-lactosidase activity and are unable to elicit transgene immunity in adoptive transfer experiments. These studies

indicate that vector-mediated transduction of dendritic cells is necessary for cellular immune responses to

muscle gene therapy, a step which AAV avoids, providing a useful biological niche for its use in gene therapy.

Somatic gene transfer is a powerful way to elicit cellular and

humoral immune responses to a foreign protein. While this has

been exploited for the development of vaccines for cancer (5,

29, 32–35, 43) and infectious disease (25, 37, 40), it is a

sub-stantial problem in the treatment of chronic diseases, such as

autosomal recessive disorders, where prolonged transgene

ex-pression may be desired (4, 44). This problem has been most

extensively documented following in vivo gene transfer with

recombinant adenoviruses. Adenoviruses expressing the lacZ

gene elicit vibrant cellular and humoral immune responses to

cytosolic

b

-galactosidase following delivery to liver, lung,

mus-cle, and joint that often contribute to destruction of the

genet-ically corrected target cells and lead to inflammation and loss

of transgene (14, 41, 46, 48, 52). Similar problems were

en-countered following low-density lipoprotein receptor gene

transfer in a murine model of familial hypercholesterolemia

(27). Target cell destruction is mediated, in part, by

antigen-specific class I-restricted cytotoxic T lymphocytes (CTL) to

both transgene product and newly expressed viral protein (39,

46, 47, 51, 52, 54). Activation of CD4

_{T cells, presumably of}

the TH

subset, is required for the full realization of the

de-structive CTL effect (22, 28, 48, 50).

Initial studies with recombinant adeno-associated virus

(AAV) delivered to skeletal muscle have yielded unexpected

results in terms of the stability of gene transfer and ensuing

immune responses. This human parvovirus can be rendered

defective by completely eliminating all viral open reading

frames, leaving the viral capsid proteins and the product of the

transgene as the only sources of antigen (26). In most cases,

gene transfer with AAV has been good; however, transgene

expression is often poor (9, 10, 12, 31, 36, 42). Two exceptions

are skeletal muscle (11, 23, 45) and the central nervous system

(21), where postmitotic, differentiated cells such as muscle

fibers and neurons are efficiently targeted with AAV, leading

to high-level and stable transgene expression. It was

particu-larly surprising that AAV failed to elicit immune responses to

highly expressed neoantigenic transgene products when

in-jected into muscle (11, 23, 45) whereas other vector systems

expressing the identical transgene, such as adenovirus (52) and

naked DNA, do. We have evaluated the mechanisms by which

AAV evades immunologic responses following injection into

muscle in the context of rate-limiting steps of immune

activa-tion by adenovirus.

MATERIALS AND METHODS

Animals.C57BL/6 mice were purchased from Jackson Laboratory (Bar Har-bor, Maine). In this study, 4- to 5-week-old male mice were used.

* Corresponding author. Mailing address: The Wistar Institute,

Room 204, 3601 Spruce St., Philadelphia, PA 19104-4268. Phone:

(215) 898-3000. Fax: (215) 898-6588. E-mail: [email protected]

.upenn.edu.

TABLE 1. Overview of experimental strategy

Group Left leg Right leg Group Left leg Adoptive_transfer

1 AAVlacZ 5 AAVlacZ APC AdlacZ

2 AAVlacZ AdlacZ 6 AAVlacZ APC naive 3 AAVlacZ1

AdBglII 7 AAVlacZ APC AdBglII

4 AAVlacZ1

AdALP

8 AAVlacZ APC AAVlacZ 9 AAVlacZ APC AAVlacZ/

AdBglII 10 AAVlacZ APC AdlacZ/

AAV ApoE a_{Animals were injected with AAVlacZ into the left tibialis anterior alone}

(group 1) and either coinjected into the same muscle with an E1-deleted ade-novirus not expressing any transgene (i.e., AdBglII [group 3]) or expressing the

ALP transgene (group 4), or injected separately with an adenovirus expressing

the lacZ transgene into the right leg (group 2). The second set of experiments (groups 5 to 9) were based on adoptive transfer into C57BL/6 mice of 53106

splenic APCs infected with various combinations of vectors ex vivo; all animals were injected with AAVlacZ in the left tibialis anterior at the time of adoptive transfer. These include APCs infected with AdlacZ (group 5), AdBglII (group 7), AAVlacZ (group 8), AAVlacZ and AdBglII (group 9), and AdlacZ and AAV ApoE (group 10). Group 6 received nontransduced APCs. The left tibialis anterior was harvested 10, 28, or 60 days later, cryosectioned, and stained for

b-galactosidase activity.

4212

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Production of recombinant AAV.Recombinant AAV expressing lacZ

(AAV-lacZ) was generated by plasmid transfection in 293 cells infected with E1-deleted

adenovirus as described previously (10). The cytomegalovirus promoter drives expression of lacZ in this vector. A brief description of the method is provided. 293 cells were infected with AdALP (multiplicity of infection [MOI] of 10) for 2 h in Dulbecco modified Eagle medium (DMEM)–2% fetal bovine serum (FBS). At 2 h postinfection, the transfection cocktail (containing, per 15-cm-diameter plate, 0.125 ml of 2.5 M CaCl, 37.5mg of trans plasmid [providing Rep/Cap], 12.5mg of cis plasmid [pAV.CMVlacZ], and 1.25 ml of 23HEPES) was added. The cells were incubated for 12 to 16 h at 37°C. Medium was replaced with fresh DMEM–2% FBS, and the infected/transfected cells were harvested 40 to 48 h postinfection.

Purification of recombinant AAV.Frozen cell suspensions were subjected to three rounds of freeze-thaw cycles to release virus from the cells. On completion of the final thaw, bovine pancreatic DNase (1,000 U [0.5 mg] per 15-cm plate) and RNase (0.2 mg/ml, final concentration) were added, and the extract was incubated at 37°C for 30 min. Sodium deoxycholate (10% stock) was added to the

sample to a final concentration of 0.5%, and the sample was incubated for 10 min at 37°C. CsCl (0.454 g/ml of sample) was added, and the sample was applied to a step gradient composed of equal volumes of CsCl in 10 mM Tris-Cl at 1.6 g/ml (bottom tier) and 1.45 g/ml (middle tier). Viral particles were banded at 25,000 rpm in a Beckman SW28 rotor for 8 h at 4°C. Fractions (1 ml) were collected from the bottom of the tube. Peak fractions that contained AAVlacZ (r51.41 g/ml) were combined and banded to equilibrium overnight in CsCl, using a Beckman Ty-70.1 rotor. Peak fractions (0.5 ml/fraction) of AAVlacZ were col-lected (r51.41 g/ml) and loaded onto a three-tier gradient consisting of 3 ml of 1.6-g/ml CsCl (bottom tier), 3 ml 1.45-g/ml CsCl (middle tier) and 3 ml of 1.33-g/ml CsCl (upper tier). Samples were spun in a Beckman SW41 rotor for 24 h at 35,000 rpm. Peak fractions (0.5 ml) were again collected (r51.41 g/ml) and loaded onto a three-tier gradient as described above. Peak fractions were dialyzed against 20 mM HEPES (pH 7.6)–150 mM NaCl.

Intramuscular injections.Mice were anesthetized with ketamine-xylazine (70 and 10 mg/kg of body weight, respectively). Recombinant AAVlacZ (731011

particles/ml) or E1-deleted adenovirus (H5.010CMVlacZ, 1012_{particles/ml) was}

FIG. 1. Impact of adenovirus on intramuscular AAVlacZ. C57BL/6 mice were injected in the left tibialis anterior with AAVlacZ in combination with adenovirus vectors. Representative macrographs of X-Gal histochemical stains of the left tibialis anterior harvested 10, 28, and 60 days after gene transfer are presented. Group 1, AAVlacZ alone; group 2, AAVlacZ with AdlacZ in the contralateral leg; group 3, AAVlacZ mixed with AdBglII; group 4, AAVlacZ mixed with AdALP. Magnification is345.

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injected into the tibialis anterior in a volume of 25ml after a small incision was made to lay open the muscle. Incisions were closed with Vicryl suture. This E1-deleted adenovirus will subsequently be called AdlacZ. Muscle was harvested on days 10, 28, and 60 by placing the tissue on OCT embedding compound, freezing it in nitrogen-cooled isopentane for 7 s, and transferring it to liquid nitrogen. Frozen sections were analyzed forb-galactosidase activity by 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-Gal) histochemistry.

Morphological analyses.For X-Gal staining, muscle sections were fixed in 0.5% glutaraldehyde for 10 min, washed three times with phosphate-buffered saline (PBS) containing 1 mM MgCl2, and incubated in 1 mg of X-Gal per ml–5

mM K3Fe(CN)6–5 mM K4Fe(CN)6–1 mM MgCl2in PBS for 6 h. Tissue was

counterstained with neutral red.

Cytotoxicity assay.Splenocytes and cells from regional lymph node were isolated from C57BL/6 animals 10 days after intramuscular injection of AdlacZ and/or AAVlacZ and restimulated for 5 days at 53106_{cells/well in a 24-well}

plate with AdlacZ (MOI of 0.8). These cells were assayed on MC57 target cells at different effector/target cell ratios (starting at 25:1) in a 6-h51_{Cr release assay.}

As target cells, MC57 cells were infected with AdlacZ at an MOI of 100 for 12 h, or a lacZ-expressing cell line was used (53). Uninfected MC57 cells were used as

a negative control. Before incubation with the effectors, target cells were labeled with 100mCi of51_{Cr (Na}

251CrO4; NEN) for 1 h and then washed three times

with DMEM without FBS. After the 6-h incubation of effector and target cells, 100ml of supernatant was removed from each well and counted in a Packard Cobra II gamma counter. Percentage of51_{Cr release was calculated as following:}

[(cpm of sample2cpm of spontaneous release)/(cpm of maximal release2cpm of spontaneous release)]3100. All samples were measured as quadruplicates; for maximum release, 5% sodium dodecyl sulfate was added to the target cells and spontaneous release was determined from target cells incubated without effector cells.

Cytokine ELISA.Lymphocytes (63106_{cells) were restimulated for 48 h with}

either UV-inactivated AdlacZ (53109_{particles/well), AAVlacZ (2310}8

par-ticles/well), purifiedb-galactosidase protein (10mg/well), or medium in 24-well Costar plates. Cell-free supernatants (100ml) were assayed for the secretion of interleukin-10 (IL-10) or gamma interferon (IFN-g) by enzyme-linked immu-nosorbent assay (ELISA) as recommended by the manufacturer of the ELISA kit (Pharmingen).

Enrichment of antigen-presenting cells (APCs) from spleen. A single-cell suspension of spleen was washed three times and then incubated for 2 h at 37°C

FIG. 2. CTL responses to intramuscular gene transfer. Lymphocytes were harvested from inguinal lymph nodes and spleen, restimulated in vitro for 5 days with AdlacZ, and analyzed for specific lysis using mock-infected (diamonds), AdlacZ-infected (squares), and pLJ-lacZ-infected (retrovirus transduced and selected to express

lacZ; circles) syngeneic target cells (MC57). Groups studied: (A) AAVlacZ (left leg); (B) AAVlacZ (left leg) and AdlacZ (right leg); (C) mixture of AAVlacZ and

AdBglII (left leg); (D) mixture of AAVlacZ and AdALP (left leg).

TABLE 2. Cytokine secretion upon intramuscular administration of AAVlacZ and various adenoviruses

Group Left leg Right leg IFN-g(pg/ml) IL-10 (pg/ml)

Mock Ad5 b-Gal AAV Mock Ad5 b-Gal AAV

1

AAVlacZ

0

0

0

0

0.5

0.5

3.1

86

2

AAVlacZ

AdlacZ

0

279

504

0

0.8

310

770

9

3

AAVlacZ

1

AdBglII

0

170

0

1.7

0.7

480

34

15

4

AAVlacZ

1

AdALP

9

699

0

5.1

0.8

860

25

101

a_{Lymphocytes from C57BL/6 animals harvested 10 days after infection were restimulated with purifiedb-galactosidase (b-Gal), AAVlacZ (AAV), UV-inactivated}

adenovirus type 5 (Ad5), or medium (Mock) for 48 h, and the supernatants were assayed for the secretion of IL-10 and IFN-gby cytokine ELISA. The data are presented as the amount of cytokine secreted following stimulation with antigen. This experiment was repeated three times with identical results; representative data are presented.

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(cells from two spleens/75-cm2_{flask). All nonadherent cells were removed and}

discarded, and fresh DMEM–5% FBS and granulocyte/macrophage colony-stim-ulating factor (GM-CSF) (5 ng/ml) were added to the adherent cell population. The next day, nonadherent cells were harvested, washed, and used for infection. For adenovirus infection, cells were infected with an MOI of 100, whereas cells were exposed to purified AAVlacZ at an MOI of 5 based on LacZ-forming units. After 2 h of infection, cells were extensively washed and 53106_{cells were}

adoptively transferred into the tail vein of each animal.

Enrichment of dendritic cells from mouse spleen.Splenocytes isolated from C57BL/6 mice were treated with ammonium chloride-Tris buffer for 2 min at 37°C to deplete erythrocytes. Cells were incubated for 90 min at 37°C (four spleens/150-cm2_{flask). After the incubation, nonadherent cells were removed}

and discarded, fresh DMEM–10% FBS and GM-CSF (5 ng/ml) were added, and the sample was incubated overnight at 37°C. Nonadherent cells were pooled, and 3 ml at 53107_{cells/ml was layered over a 3-ml metrizamide gradient (14.5% in}

PBS) or a 30% Percoll gradient and centrifuged at 5003g for 10 min. The

dendritic cell-enriched fraction was obtained from the interface. The purity of the fraction was determined by immunohistochemistry using antibody 33D1.

Purification of B cells from mouse spleen.A single-cell suspension from spleen of C57BL/6 animals was prepared at a density of 107_{cells/ml in DMEM–10%}

FBS, 4-ml volumes of cell suspensions were underlaid with 3 ml of Histopaque 1083 (Sigma), and gradients were spun at 7003g for 15 min at room

temper-ature. The lymphocytes banded at the interface were harvested, pooled, washed once, and resuspended to a cell density of 107_{cells/ml in DMEM–10% FBS.}

Cells were treated with a 1/1,000 dilution of anti-mouse Thy1.2 monoclonal antibody ascites plus 20 mg of anti-CD11b per ml for 30 to 45 min on ice. Cells were pelleted, resuspended in medium, and treated with rabbit complement for 60 min at 37°C. Purified B cells were pelleted and resuspended in medium to the appropriate cell density.

Isolation of macrophages.A single-cell suspension of splenic cells was allowed to adhere onto plastic tissue culture flasks for 90 min at 37°C. The adherent cell population was scraped off and allowed to readhere for another 60 min at 37°C. The readhered population was dislodged and resuspended in DMEM–10% FBS. Flow cytometric analysis using a monoclonal antibody to MacI revealed that this population contained.70% macrophages.

Adoptive transfer.Subpopulations of APCs were purified as described above, infected ex vivo with AdlacZ at an MOI of 100 for 2 h, washed six to eight times with DMEM-FBS, and adoptively transferred (106_{cells/animal) into animals}

which had been infected intramuscularly with AAVlacZ the same day. Control animals received the same number of uninfected purified APCs. Muscles were isolated 10 or 28 days after adoptive transfer and stained forb-galactosidase activity.

Cytospins.Purified populations of APCs were infected ex vivo with AdlacZ at an MOI of 100 for 2 h and washed six to eight times with DMEM-FBS. After 48 h, infected cells were washed once with PBS, and 100ml of cell suspension (106_{to 10}7_{cells/ml) was cytospun onto glass slides and then fixed and stained for}

b-galactosidase activity as described above.

FISH.Fixed specimens were washed briefly in PBS and then rinsed three times in 23SSC (13SSC is 150 mM NaCl plus 30 mM Na citrate) for 10 min at room temperature. They were placed directly into 50% formamide–23SSC for 10 min at room temperature and transferred to prehybridization solution (43SSC, 0.4% bovine serum albumin, 0.05% Tween 20, 50% formamide, 10mg of tRNA per ml [pH 7.0]) for 1 h at 37°C. Specimens were denatured at 95°C for 10 min on a heat block, plunged into ice-cold 70% ethanol, dehydrated, and air dried. Fluorescent in situ hybridization (FISH) was carried out with digoxygenin-labeled and biotin-labeled DNA probes (biotin-labeled by using a Random prime labeling kit from Boehr-inger Mannheim). Labeled probe was denatured at 70°C for 5 min. Hybridization was allowed to proceed overnight at 37°C in a humid environment with 20ml of probe solution per 22- by 22-mm coverslip sealed with rubber cement. After hybridization, specimens were washed as follows: 50% formamide–23SSC for

15 min at 40°C, 23SSC at 40°C for 15 min, 43SSC containing 0.05% Tween 20 (SSC-Tween buffer) for 10 min at room temperature, and 3% bovine serum albumin in 43SSC with 0.5% Tween 20 to block nonspecific binding of detection reagents. Detection was performed with streptavidin-fluorescein isothiocyanate and rhodamine antibodies for 20 min at 37°C. After three washes in 43 SSC-Tween buffer, coverslips were mounted in Vectashield antifade mounting solu-tion (Vector Laboratories, Burlingame, Calif.) containing 200 ng of DAPI (49, 6-diamidine-2-phenylindole) as a counterstain.

Immunoperoxidase staining method.Frozen sections were fixed with acetone for 10 min, air dried, and rehydrated in PBS. Sections were then blocked in 20% goat serum in PBS for 30 min at room temperature, incubated for 1 h in primary antibody at room temperature, washed three times in 2% goat serum, and then placed in a biotinylated secondary antibody complementary to the species the primary antibody was produced in for 45 min at room temperature. The sections were washed three times, incubated for 25 min in ABC (avidin-biotin-chroma-gen) solution (Vector Laboratories), and washed three times in PBS before being immersed in diaminobenzidine (Sigma) for 2 min, after which they were washed three times in water, counterstained with hematoxylin, dehydrated, and cover-slipped.

RESULTS

AAV-transduced muscle fibers expressing a neoantigen are

nonresponsive to immune activation.

One approach used in

this study for understanding the mechanism(s) by which

ani-mals respond to AAV- and adenovirus-encoded neoantigens in

muscle was to inject AAVlacZ into the tibialis anterior of

C57BL/6 mice and reconstitute components of the

immuno-logic responses to AdlacZ into the AAVlacZ-treated animals

(experimental groups are listed in Table 1). This will define

critical steps that occur in response to adenovirus vectors that

are not associated with AAV vectors. Injection of AAVlacZ

alone (group 1) confers stable transgene expression (Fig. 1A to

C), without activating CTL (Fig. 2A) or CD4

_{T helper cells}

(Table 2) to

b

-galactosidase; CD4

_{T cells secrete IL-10 in}

response to AAV virions (Table 2) and neutralizing antibodies

to AAV are elicited (Table 3), suggesting activation of TH

and B cells to viral capsid proteins. Several hypotheses were

considered to explain the T-cell activation to

adenovirus-en-coded

b

-galactosidase in muscle that is not observed with

AAV-derived

b

-galactosidase, including (i) cytokine-induced

expression of major histocompatibility complex (MHC) class I

and II in muscle fibers from adenovirus leading to direct

acti-vation of T cells by the transduced muscle fiber, (ii)

adenovi-rus-induced lysis of muscle fibers and third-party presentation

of leaked

b

-galactosidase, and (iii) preferential transduction or

activation of APC with adenovirus but not AAV.

Transduction of dendritic cells by adenovirus vectors is

re-quired for immunity against the transgene product.

Muscle

fibers normally express little MHC class I and no MHC class II,

raising questions as to their suitability as CTL targets or

po-tential as APC in the activation of CTL. Injection of AdlacZ

TABLE 3. Antibody responses to vector capsid proteins and

b

-galactosidase

Group Left leg Right leg

Adenovirus AAV b-Galactosidase, IgG-ELISA IgG-ELISA (absorbance) Neutralizing antibody (dilution) IgG-ELISA (absorbance) Neutralizing antibody (dilution)

1

AAVlacZ

0.10

0

0.66

1:1,280

0.05

2

AAVlacZ

AdlacZ

0.49

1:320

0.42

1:1,280

0.55

3

AAVlacZ

1

AdBglII

0.32

1:160

0.39

1:1,280

0.26

4

AAVlacZ

1

AdALP

0.38

1:160

0.35

1:1,280

0.2

Uninfected

ND

0

0.09

0

0.05

a_{Serum samples harvested 28 days after intramuscular infection of C57BL/6 mice with various combinations of AAV and adenovirus as indicated were tested for}

the presence of neutralizing antibodies (anti-AAV or antiadenovirus) or serum IgG (specific for AAV, adenovirus, orb-Gal). The neutralizing antibody titers are presented as reciprocal dilutions which inhibit infection of AdlacZ (HeLa cells) or AAVlacZ (E1- and E4-expressing cell line derived from 293 cells) by 50% based on number of lacZ-positive cells. Serum IgG (IgG-ELISA) measures the relative amounts (absorbance at 405 nm) of antibodies in the samples at a serum dilution of 1:100. ND, not determined.

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contralateral to the leg that received AAVlacZ (group 2)

re-sulted in substantial inflammation and loss of transgene

ex-pression in the AAVlacZ-transduced leg (Fig. 1D to F)

asso-ciated with infiltration of CD8

_{and CD4}

_{T cells (data not}

shown), as well as full CTL (Fig. 2B), TH

/TH

(Table 2), and

B-cell (Table 3) responses to both adenovirus and

b

-galacto-sidase. This finding suggests that AAV-transduced fibers are

suitable T-cell targets when CTL are appropriately activated

Group

FIG. 3. Impact of APC adoptive transfer on intramuscular AAVlacZ. C57BL/6 mice were injected with AAVlacZ in the left tibialis anterior in combination with adoptive transfer of APCs exposed to a variety of vectors. Representative macrographs of X-Gal histochemical stains of the left tibialis anterior harvested 10, 28, and 60 days after gene transfer are presented. APCs were mock infected (group 6) or infected with the following vectors prior to adoptive transfer: group 5, AdlacZ; group 7, AdBglII; group 8, AAVlacZ; group 9, AAVlacZ and AdBglII. Magnification is345.

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to antigen, a situation that does not appear to occur

follow-ing AAV injection into muscle. These experiments were

re-peated with an extended interval between the initial AAVlacZ

transduction and subsequent contralateral administration of

AdlacZ to determine if AAVlacZ induced tolerance to

b

-ga-lactosidase. The same result was achieved (i.e., apparent

de-struction of AAVlacZ- and AdlacZ-expressing muscle fibers),

suggesting that the animals are nonresponsive to

b

-galactosi-dase when it is provided by an AAV vector rather than being

tolerant to it (data not shown).

Inflammatory cytokines, such as IFN-

g

, induce MHC

ex-pression in cultured human myoblasts, suggesting that the

in-flammation associated with intramuscular injection of

adeno-virus may convert the muscle fiber to a bona fide APC, leading

to direct activation of CTL (15, 18, 30, 38). This was ruled out

by mixing an empty E1-deleted adenovirus (i.e., transgene

neg-ative called AdBglII) with AAVlacZ prior to injection into

muscle (group 3). The localized presence of adenovirus elicited

inflammation (Fig. 1G and H) but did not lead to CTL (Fig.

2C) or CD4

_{T-cell (Table 2) responses to}

_b

_{-galactosidase,}

and transgene expression was stable (Fig. 1G to I). Low-level

antibody to

b

-galactosidase was detected (Table 3).

Adenovirus-induced damage to the transduced fiber may

leak

b

-galactosidase to APCs, facilitating activation of both

CD4

_{and CD8}

_{T cells. Furthermore, adenovirus could}

func-tion as an adjuvant mobilizing and activating the cells

neces-sary for T-cell activation. In this experiment, AAVlacZ was

mixed with an adenovirus expressing a different neoantigenic

reporter gene, human alkaline phosphatase, which is a target

of destructive immune responses and should enhance leakage

and third-party presentation of

b

-galactosidase in

cotrans-duced fibers (group 4). This was associated with substantial

inflammation at the site of injection (Fig. 1J), infiltration of

CD4

_{and CD8}

_{T cells (data not shown), stable transgene}

expression (Fig. 1J to L), and T- and B-cell activation to

ade-novirus without significant immune responses to

b

-galactosi-dase (Fig. 2D; Tables 2 and 3), suggesting that third-party

antigen presentation or adjuvant effect of the vector is not

significant.

The remaining hypothesis is that vector transduction of

APCs and presentation of endogenously produced

b

-galacto-sidase is necessary for CTL activation; this would predict that

adenovirus transduces APCs. A mixture of APCs purified from

spleen of naive C57BL/6 mice was mock infected (group 6) or

infected ex vivo with AdlacZ (group 5) or AdBglII (group 7)

prior to adoptive transfer into C57BL/6 mice that received an

intramuscular injection of AAVlacZ. Animals receiving

Ad-lacZ-infected APCs quickly exhibited an impressive

mononu-clear inflammatory response localized to the

AAVlacZ-trans-duced muscle fibers (Fig. 3A to C) that was associated with

activation of CD4

_{T cells (i.e., TH}

1

and TH

) to

b

-galacto-sidase (Table 4) and precipitous loss of transgene (Fig. 3A to

C). Transgene expression was stable without detectable

inflam-matory or

b

-galactosidase specific T-cell responses following

adoptive transfer of mock-infected APCs (Fig. 3D to F; Table

4); adoptive transfer of APCs infected with AdBglII resulted in

modest activation of IL-10 secretion from T helper cells to

b

-galactosidase without detectable inflammation or diminution

in lacZ expression (Fig. 3G to I; Table 4). These data are

consistent with an essential role for transduction of the APCs

and presentation of endogenously produced antigen following

intramuscular injection of adenovirus vectors.

Additional experiments were performed to determine the

specific type of APC required for immune activation. Three

types of APCs (macrophages, B cells, and dendritic cells) were

isolated from spleens of naive mice. Immunophenotype

anal-ysis was performed on the enriched fractions of cells to assess

their level of purity (Table 5). The B-cell fraction

demon-strated the B-cell-specific marker B220

_{on 89% of cells; the}

macrophage fraction had the macrophage-specific markers

MacI and F4-80 in

.

70% of cells, with B and T cells

constitut-ing the remainconstitut-ing cells; and the dendritic cell fraction showed

positive staining with dendritic cell marker 33D1 on

.

70% of

cells, with contamination by 5% macrophages and 15% B cells.

Cultured APC fractions were analyzed directly for AdlacZ

transduction. The enriched fractions were evaluated by X-Gal

histochemistry 2 days after exposure to AdlacZ (Fig. 4). No

transduction was observed in the B-cell fraction (Fig. 4B), and

rare lacZ-positive cells (

,

0.19%) were found in the

macro-phage fraction (Fig. 4A). In contrast, the dendritic cell fraction

revealed lacZ expression in

.

10% of the cells (Fig. 4C).

Adop-TABLE 4. Cytokine secretion following adoptive transfer of virally infected APCs into animals transduced intramuscularly with AAVlacZ

Group Left leg Adoptive transfer IFN-g(pg/ml) IL-10 (pg/ml)

Mock Ad5 b-Gal AAV Mock Ad5 b-Gal AAV

5

AAVlacZ

APC AdlacZ

113

423

418

265

54

850

1,020

230

6

AAVlacZ

APC naive

1.5

4.5

70

9.2

24

23

66

59

7

AAVlacZ

APC AdBglII

34

219

80

120

54

813

340

320

8

AAVlacZ

APC AAVlacZ

2

3.4

5.8

9.2

34

35

70

160

9

AAVlacZ

APC AAVlacZ/AdBglII

51

195

92

95

57

605

291

214

a_{Lymphocytes from regional lymph nodes and spleen were harvested 10 days after intramuscular infection with AAVlacZ and adoptive transfer of 5310}6_APCs

infected ex vivo with AdlacZ (group 5), AdBglII (group 7), AAVlacZ (group 8), or AAVlacZ plus AdBglII (group 9) and mock infected (group 6). Cells were restimulated with purifiedb-galactosidase (b-Gal), AAVlacZ (AAVlacZ), UV-inactivated adenovirus type 5 (Ad5), or medium (Mock) for 48 h, and the supernatants were assayed for the secretion of IL-10 and IFN-gby cytokine ELISA. A representative experiment is shown; this study was repeated twice with identical results.

TABLE 5. Immunophenotype analysis of enriched fraction of cells

Fraction

% of total T cell,

CD31 B cell,_B2201 Macrophage,_F4-80/Mac1 Dendritic cell,_33D1

APC

12

55

25

ND

B cell

8

89

3

15

Macrophage

10

15

.

70

,

5

Dendritic cell

0

15

5

70–80

a_{A mixed population of APCs or enriched populations of B cells,}

macro-phages, and dendritic cells was isolated from naive spleen. The purity of the APC, B-cell, and macrophage fractions was determined in performing FACS analysis by staining the cells with anti-CD31_{for T cells, anti-B220}1_{for B cells,}

and anti-F4-80 or anti-MacI for macrophages. The purity of the dendritic cell population was determined in cytospinning the cells onto slides and subsequently performing immunohistochemical staining, utilizing the dendritic cell-specific antibody 33D1. ND, not determined.

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tive transfer of the enriched APC fractions, infected with

Ad-lacZ, was performed in C57BL/6 mice that were stably

express-ing lacZ in muscle followexpress-ing injection with AAVlacZ. The

AdlacZ-transduced B-cell fraction failed to activate CD4

_T

cells to either adenovirus or

b

-galactosidase (Table 6);

AAV-lacZ-transduced muscle fibers stably expressed

b

-galactosidase

without evidence of inflammation (Fig. 5A and B). Adoptive

transfer of AdlacZ-transduced macrophages did activate T

cells to adenovirus and

b

-galactosidase (Table 6);

mononu-clear cell infiltrate was noted in the AAVlacZ-transduced

mus-cle, although lacZ expression was stable (Fig. 5I and J).

In-flammation or CD4

_{T-cell activation did not occur when}

naive macrophages were adoptively transferred (Table 6; Fig.

5K and L). The results for AdlacZ-transduced dendritic cells

were qualitatively different with activation of TH

cells to

b

-ga-lactosidase (Table 6), as well as destruction of fibers and loss of

transgene expression (Fig. 5E and F). Naive dendritic cells

produced none of these findings (Table 6; Fig. 5G and H).

These data indicate that adenovirus-transduced dendritic cells

are capable of eliciting a vibrant T-cell response capable of

destroying lacZ-expressing muscle fibers. Activation of T cells

to

b

-galactosidase observed with adenovirus-transduced

mac-rophages was insufficient to destroy lacZ-expressing fibers by

day 28. We cannot rule out the possibility that the partial T-cell

activation noted with the macrophage fraction is due to the

small amount of contaminating dendritic cells. Previous

exper-iments with purified fractions of activated T cells indicated that

the number of T cells adoptively transferred in the dendritic

cell fraction are insufficient to target AAVlacZ-transduced

muscle fibers (data not shown). It is unlikely that the effect

observed with the dendritic cell fraction is due to

contaminat-ing B cells, T cells, and macrophages.

Inefficient transduction of APCs allows AAV to evade

im-munity.

The lack of detectable immune responses to

AAV-encoded neoantigens is of interest in terms of basic

mecha-nisms of T-cell activation as well as applications to gene

therapy. The experience with recombinant adenovirus is that

transduction of APCs is required for activation of T cells to

b

-galactosidase. We hypothesize that this step is defective in

the context of AAV, which was confirmed in the following

experiments. Naive APCs purified from spleen were exposed

to high-titer AAVlacZ prior to adoptive transfer into C57BL/6

mice previously injected intramuscularly with AAVlacZ (group

8). Direct analysis of the APCs failed to reveal evidence of

b

-galactosidase expression (data not shown). Furthermore,

re-cipient animals failed to activate T cells to

b

-galactosidase, and

expression of the transgene was stable (Fig. 3J to L; Table 4);

a delayed and self-limited CD4

_{T-cell inflammatory response}

was noted at day 60 (Fig. 3L), resolving by day 120 (data not

shown).

In an attempt to further evaluate mechanisms by which

AAV evades immunity, skeletal muscle was characterized for

indices of inflammation and immune activation, using

tech-niques of immunocytochemistry. Representative micrographs

for several markers are presented in Fig. 6; the relative

abun-dance of each marker quantified by morphometry is shown in

Table 7. Recombinant adenovirus elicits a substantial and

mixed infiltrate (T cells [CD4 and CD8], B cells [B220], and

dendritic cell and macrophages [CD11c]), with evidence for

activated lymphocytes (CD25) and activated macrophages/

dendritic cells (MHC class II, B7-2, and CD11c). AAV elicited

substantially less inflammation, with modest, if any,

lympho-cyte or APC activation markers.

From these experiments, we propose several mechanisms by

which AAV may avoid the productive presentation of antigen

TABLE 6. Cytokine secretion following adoptive transfer of

purified APCs into AAVlacZ-infected animals

APCs IFN-g(pg/ml) IL-10 (pg/ml) Mock Ad5 b-Gal Mock Ad5 b-Gal

B cell

lacZ

3.3

0.7

0.3

15

4.8

9.8

Naive

0.2

0.2

1

19

23

4

Macrophages

lacZ

76

291

247

2

34

16

Naive

3.7

4.8

8.3

0.9

3.6

3.4

DC

lacZ

14

274

268

0.7

9.5

1.7

Naive

6.7

12.5

13.3

2.6

8.4

2.6

a_{Lymphocytes from regional lymph nodes and spleen were harvested 10 days}

after adoptive transfer of 106_{purified B cells, macrophages, or dendritic cells}

(DC) either infected ex vivo with H5.010.CMVlacZ (lacZ) or uninfected (naive). Cells were restimulated with UV-inactivated adenovirus type 5 (Ad5), purified

b-galactosidase protein (b-Gal), or medium (Mock) for 48 h, and the superna-tants were assayed for the secretion of IL-10 or IFN-gby cytokine ELISA. FIG. 4. AdlacZ infectivity of various subpopulations of APCs. Purified or enriched populations of APCs were infected with AdlacZ (MOI of 100) for 48 h, cytospun on slides, and X-Gal stained. MØ, macrophages; DC, dendritic cells. Magnification is350.

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by APCs. These hypotheses include insufficient activation of

the APC for presentation of antigen, suppression of the

anti-gen presentation, or inability of AAV to transduce APC.

Ad-ditional in vitro and in vivo experiments were performed to

evaluate these positive mechanisms.

Adenovirus-injected muscle demonstrated higher levels of

APC-derived antigens and markers of APC and T-cell

activa-tion than what was observed with AAV (Fig. 6; Table 7). It is

possible that the recombinant adenovirus activates the

trans-duced APC more effectively than AAV, explaining differences

in immune activation to these vectors. To study this hypothesis,

naive APCs were infected with both AAVlacZ and AdBglII (an

E1-deleted adenovirus not expressing a transgene) prior to

adoptive transfer (group 9). T cells were not activated to

b

-ga-lactosidase (Table 4), nor was there an effect on transgene

stability (Fig. 3M and N). Another explanation is that AAV

efficiently transduces cells but also actively suppresses the

pre-sentation of antigens. How this could occur is unclear since the

only open reading frame in the recombinant genome is the

transgene (26); it is possible that viral capsid proteins or

pack-aged viral proteins (i.e., Rep) either are toxic to APCs or

suppress APC activation. To formally evaluate this mechanism,

FIG. 5. Adoptive transfer of purified APC fractions. C57BL/6 mice were injected with AAVlacZ in the left tibialis anterior in combination with adoptive transfer of purified fractions of APCs infected with AdlacZ (columns 1 and 2) or mock infected (columns 3 and 4). In each case, the left tibialis anterior was removed for X-Gal histochemistry 10 and 28 days after adoptive transfer. The following enriched fractions were adoptively transferred: B cells; dendritic cells (DC); and macrophages (MØ). Magnification is345.

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naive APCs were infected with both AdlacZ and a recombinant

AAV expressing an irrelevant gene (i.e., Apo E) prior to

adop-tive transfer (group 10). The presence of high-titer

recombi-nant AAV during the APC infection did not suppress the

ability of AdlacZ to activate APCs so that following adoptive

transfer, lacZ-expressing muscle fibers were destroyed in the

same manner as occurred with AdlacZ-infected APCs (data

not shown).

FIG. 6. Characterization of cells infiltrating virus-infected muscle. Muscle tissues from C57BL/6 animals were harvested 10 days after injection of either AAVlacZ (middle column) or AdlacZ (right column) in the left tibialis anterior. Uninfected muscle tissue (first column) served as a baseline staining. Sections were stained with the primary antibodies indicated on the left. The signal of the primary antibodies was amplified by using an ABC-peroxidase kit. Magnification is345.

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The most likely explanation for the difference between AAV

and Ad in eliciting transgene immunity is selective

transduc-tion of the APCs. Transductransduc-tion of fractransduc-tionated populatransduc-tions of

APCs with AdlacZ demonstrated lacZ expression in dendritic

cells and some macrophages (Fig. 4), which effectively

acti-vated T cells to

b

-galactosidase following adoptive transfer

(Fig. 5). LacZ expression was not detected in APCs following

exposure to AAVlacZ (data not shown), nor were these cells

capable of activating T cells in vivo (Fig. 3J to L). This finding

is consistent with the notion that AAV efficiently enters a

number of cell types, most of which are not permissive for

transduction due to postentry blocks. The block to AAV

trans-duction in APCs was studied in vitro by using FISH to localize

the vector genome within the cell. Previous studies in cultured

epithelial cell lines indicated that AAV vectors efficiently enter

the cell but do not localize to the nucleus and do not replicate

to form transcriptionally active replicated or integrated forms

(9, 10). Coinfection with an E1- and E4-expressing adenovirus

mobilized the AAV genome into intranuclear replication

cen-ters which are transcriptionally active. FISH analysis of

den-dritic cells infected with AAVlacZ demonstrated vector

ge-nome in a perinuclear distribution (Fig. 7A) that was not seen

in adenovirus-infected cells hybridized with the AAV probe

(data not shown). Exposure of dendritic cells with the wild-type

adenovirus and AAVlacZ resulted in the formation of

replica-tion centers within the nucleus (Fig. 7B). Toxicity of the

wild-type adenovirus to the dendritic cells precluded their adoptive

transfer. These data indicate that AAV enters APCs in a

non-productive transduction.

DISCUSSION

This study underscores the critical role of antigen

presenta-tion, and the context in which it occurs, as defining the nature

of the ensuing immune responses to gene therapy. Our data

indicate that presentation of Escherichia coli

b

-galactosidase

expressed in the APC is necessary for both CTL and CD4

T-cell responses following intramuscular injection of vector.

Fractionation experiments indicated that transduction of

den-dritic cells may be a limiting step in the activation of T cells to

vector-encoded antigens.

Immune responses to tumor-associated antigens provide a

substantially different biological context in which similar

ques-tions have been asked. Potential mechanisms of T-cell

activa-tion are simplified in that endogenous producactiva-tion of

tumor-associated antigens in professional APCs is not possible. The

most informative results have emerged from experiments in

which bone marrow chimeras that had APCs expressing MHC

molecules of a separate haplotype from those on the

immu-nizing tumor cells were created (20). These studies supported

the “cross priming” hypothesis in which tumor antigens are

taken up by APCs, processed, and presented on the MHC class

I molecule. For this to be operative, exogenous antigens must

be shunted into the transporter-associated protein

(TAP)-de-pendent pathways for MHC class I processing (19).

Direct intramuscular injection of plasmid based DNA is a

well-characterized system for eliciting immune responses to a

recombinant gene product (7). This approach is being

consid-ered for a number of vaccines for viral diseases. Injection of

DNA into muscle results in low-level transduction of muscle

fibers and activation of both cellular and humoral immune

responses to the transgene product (7). Expression of the

transgene is usually transient, although stable gene expression

has been reported, in some cases, despite immune activation.

Several mechanisms have been considered. The classic bone

marrow chimera experiments, developed to study

tumor-asso-ciated antigens, suggest that cross priming from the transfected

muscle fiber to the APC contributes to T-cell activation (6, 13).

TABLE 7. Immunohistochemical analysis of muscle for markers of

inflammation and immune activation

Marker Naive AAV Adenovirus

CD4

1

4

40

CD8

1

2

55

B220

1

6

12

CD25

1

8

44

MHC II

1

12

52

B7-2

1

1

9

CD11c

1

2.1

4.6

a_{C57BL/6 mice were uninfected (mock) or injected with AAVlacZ or AdlacZ}

into the tibialis anterior. Ten days later, muscle was harvested and evaluated for the presence of the markers listed, using techniques of immunohistochemistry. Expression of the markers was quantified from multiple samples using morpho-metric techniques as described in Materials and Methods. Each sample is nor-malized to the level found in naive animals.

FIG. 7. Localization of AAV in virus-infected dendritic cells. Purified dendritic cells were either infected with AAVlacZ (A) or coinfected with AAVlacZ and wild-type adenovirus (B) for 48 h. The cells were cytospun on slides, and FISH analysis was performed with a biotinylated AAV-specific probe. Magnification is3168.

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A recent study noted that cotransfection of the immunizing

plasmid with one expressing B7-2 was associated with

en-hanced T- and B-cell responses, indicating that the muscle

fiber may function as an APC under the right conditions (24).

Direct transfection of the APC has not been ruled out in any of

these studies.

Of potential relevance to the present study is previous work

on the nature of antigen presentation to viral infection in

animal models. Intratracheal infection of mice with influenza

virus was associated with infection of dendritic cells from

me-diastinal lymph nodes at efficiencies sufficient to activate naive

T cells in vitro (16). Analysis of influenza virus-infected APCs

from humans demonstrated that dendritic cells are 20-fold

more effective than macrophages in activating T cells (2).

Sim-ilar results have been obtained with vaccinia virus as an

anti-tumor vaccine in murine models. In vivo activation of T cells to

the tumor-associated antigen was dependent on the promoter

driving its expression in the virus, which correlated with the

ability of infected dendritic cells to activate T cells in vitro (3).

The critical role of APC transduction in gene therapy with

DNA viral vectors has important implications in the

develop-ment of safe and effective adenovirus vectors. This hypothesis

would predict that the transcriptional unit responsible for

ex-pression of the transgene in an adenovirus could have a

sub-stantial effect on the nature of the ensuing immune response.

Most experiments have used active constitutive promoters,

which may express very efficiently in dendritic cells. A blunted

immune response may be achieved with vectors that contain

more specific promoters, which are not active in APCs.

Vector-specific differences in transgene expression within APCs due to

dose, route of administration, promoter, etc., could explain

some of the variation in immune responses that have

charac-terized in vivo applications (1, 8, 49). A similar outcome could

be achieved with adenovirus vectors whose tropism is restricted

through modifications in the capsid proteins.

The ability of AAV injected into muscle to evade immune

responses is of immediate value to the use of this vector in gene

therapy where stable expression is desired. Stable transgene

expression has been achieved in murine models with a number

of constructs, including those that express factor IX (17) and

Apo E (unpublished data), despite the fact that antibodies

were generated to these secreted human proteins. We

con-clude that intramuscular injection of AAV evades destructive

immune responses to vector-encoded antigens although a

hu-moral immune response may develop if the antigen is secreted.

The low efficiency of APC transduction by AAV observed in

this study is consistent with previous work that identified a

postentry block of AAV-mediated gene transfer in most cell

types except muscle fibers and neurons (9, 10). The exquisite

cell specific restriction of AAV transduction appears to have

created a biological niche for gene therapy in which muscle

fibers are efficiently transduced without activating APCs.

ACKNOWLEDGMENTS

The first two authors contributed equally to this work.

The technical support of Qin Su and the Cell Morphology and

Vector Cores of the Institute for Human Gene Therapy was

appreci-ated. The following antibodies were kindly provided by Ellen Pure:

B220

_{, 33D1, and CD11C.}

Support was derived from the National Institutes of Health (P30

DK47757 and P01 AR/NS43648) and Genovo, Inc. Karin Jooss is

supported by the Human Frontier Science Program Organization,

Strasbourg, France.

REFERENCES

1. Armentano, D., J. Zabner, C. Sacks, C. S. Sookedo, M. P. Smith, J. A. St.

George, S. C. Wadsworth, A. E. Smith, and R. J. Gregory.1997. Effect of the

E4 region on the persistence of transgene expression from adenovirus vec-tors. J. Virol. 71:2408–2416.

2. Bhardwaj, N., A. Bender, N. Gonzalez, L. K. Bui, M. C. Garrett, and R. M.

Steinman.1994. Influenza virus-infected dendritic cells stimulate strong pro-liferative and cytolytic responses from human CD81T cells. J. Clin. Invest.

94:797–807.

3. Bronte, V., M. W. Carroll, T. J. Goletz, M. Wang, W. W. Overwijk, F.

Marincola, S. A. Rosenberg, B. Moss, and N. P. Restifo.1997. Antigen expression by dendritic cells correlates with the therapeutic effectiveness of a model recombinant poxvirus tumor vaccine. Proc. Natl. Acad. Sci. USA

94:3183–3188.

4. Crystal, R. 1995. Transfer of genes to humans: early lessons and obstacles to success. Science 270:404–410.

5. Descamps, V., M. T. Duffour, M. C. Mathieu, N. Fernandez, L. Cordier,

M. A. Abina, E. Kremer, M. Perricaudet, and H. Haddada.1996. Strategies for cancer gene therapy using adenoviral vectors. J. Mol. Med. 74:183–189. 6. Doe, B., M. Selby, S. Barnett, J. Baenziger, and C. M. Walker. 1996. Induc-tion of cytotoxic T lymphocytes by intramuscular immunizaInduc-tion with plasmid DNA is facilitated by bone marrow-derived cells. Proc. Natl. Acad. Sci. USA

93:8578–8583.

7. Ertl, H. C., and Z. Xiang. 1996. Novel vaccine approaches. J. Immunol.

156:3579–3582.

8. Fang, B., H. Wang, G. Gordon, D. A. Bellinger, M. S. Read, K. M. Brinkhous,

S. L. C. Woo, and R. C. Eisensmith. 1996. Lack of persistence of E1-recombinant adenoviral vectors containing a temperature-sensitive E2a mu-tation in immunocompetent mice and hemophilia B dogs. Gene Ther. 3:217– 222.

9. Ferrari, F. K., T. Samulski, T. Shenk, and R. J. Samulski. 1996. Second-strand synthesis is a rate-limiting step for efficient transduction by recombi-nant adeno-associated virus vectors. J. Virol. 70:3227–3234.

10. Fisher, K. J., G.-P. Gao, M. D. Weitzman, R. DeMatteo, J. F. Burda, and

J. M. Wilson.1996. Transduction with recombinant adeno-associated virus for gene therapy is limited by leading-strand synthesis. J. Virol. 70:520–532. 11. Fisher, K. J., K. Jooss, J. Alston, Y. Yang, S. E. Haecker, K. High, R. Pathak,

S. E. Raper, and J. M. Wilson.1997. Recombinant adeno-associated virus for muscle directed gene therapy. Nat. Med. 3:306–312.

12. Flotte, T. R., S. A. Afione, C. Conrad, S. A. McGrath, R. Solow, H. Oka, P. L.

Zeitlin, W. B. Guggino, and B. J. Carter.1993. Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc. Natl. Acad. Sci. USA 90:10613–10617. 13. Fu, T.-M., J. B. Ulmer, M. J. Caulfield, R. R. Deck, A. Friedman, S. Wang,

X. Liu, J. J. Donnelly, and M. A. Liu.1997. Priming of cytotoxic T lympho-cytes by DNA vaccines: requirement for professional antigen presenting cells and evidence for antigen transfer from myocytes. Mol. Med. 3:362–371. 14. Gao, G.-P., Y. Yang, and J. M. Wilson. 1996. Biology of adenovirus vectors

with E1 and E4 deletions for liver-directed gene therapy. J. Virol. 70:8934– 8943.

15. Goebels, N., D. Michaelis, H. Wekerly, and R. Hohlfeld. 1992. Human myoblasts as antigen-presenting cells. J. Immunol. 149:661–667.

16. Hamilton-Easton, A. M., and M. Eichelberger. 1995. Virus-specific antigen presentation by different subsets of cells from lung and mediastinal lymph node tissues of influenza virus-infected mice. J. Virol. 69:6359–6366. 17. Herzog, R. W., J. N. Hagstrom, S.-H. Kung, S. J. Tai, J. M. Wilson, K. J.

Fisher, and K. A. High.1997. Stable gene transfer and expression of human factor IX following intramuscular injection of recombinant AAV. Proc. Natl. Acad. Sci. USA 94:5804–5809.

18. Hohlfeld, R., and A. G. Engel. 1990. Induction of HLA-DR expression on human myoblasts with interferon-g. Am. J. Pathol. 136:503–508. 19. Huang, A. Y. C., A. T. Bruce, D. M. Pardoll, and H. Z. Levitsky. 1996. In vivo

cross-priming of MHC class I-restricted antigens requires the TAP trans-porter. Immunity 4:349–355.

20. Huang, A. Y. C., P. Golumbek, M. Ahmadzadeh, E. Jaffee, D. Pardoll, and H.

Levitsky.1994. Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens. Science 264:961–965.

21. Kaplitt, M. G., P. Leone, R. J. Samulski, X. Xiao, D. W. Pfaff, K. L. O’Malley,

and M. J. During.1994. Long-term gene expression and phenotypic correc-tion using adeno-associated virus vectors in the mammalian brain. Nat. Genet. 8:148–153.

22. Kay, M. A., A.-X. Holterman, L. Meuse, A. Gown, H. D. Ochs, P. S. Linsley,

and C. B. Wilson.1995. Long-term hepatic adenovirus-mediated gene ex-pression in mice following CTLA4Ig administration. Nat. Genet. 11:191–197. 23. Kessler, P. D., G. Podsakoff, X. Chen, S. A. McQuiston, P. C. Colosi, L. A.

Matelis, G. J. Kurtzman, and B. J. Byrne.1996. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc. Natl. Acad. Sci. USA 93:14082–14087.

24. Kim, J. J., M. L. Bagarazzi, N. Trivedi, Y. Hu, K. Kazahaya, D. M. Wilson,

R. Ciccarelli, M. A. Chattergoon, K. Dang, S. Mahalingam, A. A. Chalian, M. G. Agadjanyan, J. D. Boyer, B. Wang, and D. B. Weiner.1997. Engineer-ing of in vivo immune responses to DNA immunization via co-delivery of costimulatory molecule genes. Nat. Biotechnol. 15:641–646.

25. Kohn, D. B., and N. Sarver. 1996. Gene therapy for HIV-1 infection. Adv. Exp. Med. Biol. 394:421–428.

on November 9, 2019 by guest

http://jvi.asm.org/

26. Kotin, R. M. 1994. Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum. Gene Ther. 5:793–801.

27. Kozarsky, K. F., K. Jooss, M. Donahee, J. F. Strauss, and J. M. Wilson. 1996. Effective treatment of familial hypercholesterolaemia in the mouse model using adenovirus-mediated transfer of the VLDL receptor gene. Nat. Genet.

13:54–61.

28. Lei, D., M. Lehmann, J. E. Shellito, S. Nelson, A. Siegling, H.-D. Volk, and

J. K. Kolls.1996. Nondepleting anti-CD4 antibody treatment prolongs lung-directed E1-deleted adenovirus-mediated gene expression in rats. Hum. Gene Ther. 7:2273–2279.

29. Lew, D., S. E. Parker, T. Latimer, A. M. Abai, A. Kuwahara-Rundell, S. G.

Doh, Z.-Y. Yang, D. Laface, S. H. Gromkowski, G. J. Nabel, M. Manthorpe, and J. Norman.1995. Cancer gene therapy using plasmid DNA: pharmaco-kinetic study of DNA following injection in mice. Hum. Gene Ther. 6:553– 564.

30. Mantegazza, R., S. M. Hughes, D. Mitchell, M. Travis, H. M. Blau, and L.

Steinman.1991. Modulation of MHC class II antigen expression in human myoblasts after treatment with IFN-g. Neurology 41:1128–1132.

31. Miller, J. L., R. E. Donahue, S. E. Sellers, R. J. Samulski, N. S. Young, and

A. W. Nienhuis.1994. Recombinant adeno-associated virus (rAAV)-medi-ated expression of a human g-globin gene in human progenitor-derived erythroid cells. Proc. Natl. Acad. Sci. USA 91:10183–10187.

32. Mullen, C. A., and R. M. Blaese. 1996. Gene therapy of cancer. Cancer Chemother. Biol. Response Modifiers 16:285–294.

33. Nabel, G. J., Z. Y. Yang, E. G. Nabel, K. Bishop, M. Marquet, M. Felgner,

D. Gordon, and A. E. Chang.1995. Direct gene transfer for treatment of human cancer. Ann. N.Y. Acad. Sci. 772:227–231.

34. Parker, S. E., H. L. Vahlsing, L. M. Serfilippi, C. L. Franklin, S. G. Doh,

S. H. Gromkowski, D. Lew, M. Manthorpe, and J. Norman.1995. Cancer gene therapy using plasmid DNA: safety evaluation in rodents and non-human primates. Hum. Gene Ther. 6:575–590.

35. Plautz, G. E., Z.-Y. Yang, B.-Y. Wu, X. Gao, L. Huang, and G. J. Nabel. 1993. Immunotherapy of malignancy by in vivo gene transfer into tumors. Proc. Natl. Acad. Sci. USA 90:4645–4649.

36. Podsakoff, G., K. K. Wong, and S. Chatterjee. 1994. Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors. J. Virol. 68: 5656–5666.

37. Poeschla, E. M., and F. Wong-Staal. 1996. Gene therapy and HIV disease, p. 1–45. In P. Volberg and M. A. Jacobson (ed.), AIDS clinical review, 1995/ 1996. Marcel Dekker, Inc., New York, N.Y.

38. Roy, R., G. Danserau, J. B. Tremblay, M. Belles-Isles, J. Huard, C.

La-brecque, and J. P. Bouchard.1991. Expression of major histocompatibility complex antigens on human myoblasts. Transplant. Proc. 23:799–801. 39. Tripathy, S. K., H. B. Black, E. Goldwasser, and J. M. Leiden. 1996. Immune

responses to transgene-encoded proteins limit the stability of gene expres-sion after injection of replication-defective adenovirus vectors. Nat. Med.

2:545–550.

40. Ulmer, J. B., J. J. Donnelley, S. E. Parker, G. H. Rhodes, P. L. Felgner, V. J.

Dwarki, S. J. Gromkwoski, R. R. Deck, C. M. DeWitt, A. Friedman, L. A.

Hawe, K. R. Leander, D. Martinez, H. C. Perry, J. W. Shiver, D. L. Mont-gomery, and M. A. Liu.1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259:1745–1749. 41. VanGinkel, F. W., C. Liu, J. W. Simecka, J.-Y. Dong, T. Greenway, R. A.

Frizzell, H. Kiyono, J. R. McGhee, and D. W. Pascual.1995. Intratracheal gene delivery with adenoviral vector induces elevated systemic IgG and mucosal IgA antibodies to adenovirus and b-galactosidase. Hum. Gene Ther. 6:895–903.

42. Walsh, C. E., A. W. Neinhuis, R. J. Samulski, M. G. Brown, J. L. Miller, N. S.

Young, and J. M. Liu.1994. Phenotypic correction of fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vec-tor. J. Clin. Invest. 94:1440–1448.

43. Whartenby, K. A., C. N. Abbound, A. J. Marrogi, R. Ramesh, and S. M.

Freeman.1995. The biology of cancer gene therapy. Lab. Invest. 72:131–145. 44. Wilson, J. M. 1995. Gene therapy for cystic fibrosis: challenges and future

directions. J. Clin. Invest. 96:2574–2554.

45. Xiao, X., J. Li, and R. J. Samulski. 1996. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J. Virol. 70:8098–8108.

46. Yang, Y., H. C. J. Ertl, and J. M. Wilson. 1994. MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice in-fected with E1-deleted recombinant adenoviruses. Immunity 1:433–442. 47. Yang, Y., Z. Xiang, H. C. J. Ertl, and J. M. Wilson. 1995. Upregulation of

class I major histocompatibility complex antigens by interferongis necessary for T-cell-mediated elimination of recombinant adenoviruses-infected hepa-tocytes in vivo. Proc. Natl. Acad. Sci. USA 92:7257–7261.

48. Yang, Y., Q. Li, H. C. J. Ertl, and J. M. Wilson. 1995. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69:2004–2015.

49. Yang, Y., F. A. Nunes, K. Berencsi, E. Gonczol, J. F. Engelhardt, and J. M.

Wilson.1995. Inactivation of E2a in recombinant adenovirus improves the prospect for gene therapy in cystic fibrosis. Nat. Genet. 7:362–369. 50. Yang, Y., G. Trinchieri, and J. M. Wilson. 1995. Recombinant IL-12 prevents

formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to mouse lung. Nat. Med. 1:890–893.

51. Yang, Y., Q. Su, and J. M. Wilson. 1996. Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J. Virol. 70:7209–7212.

52. Yang, Y., S. E. Haecker, Q. Su, and J. M. Wilson. 1996. Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle. Hum. Mol. Gen.

5:1703–1712.

53. Yang, Y., K. Jooss, Q. Su, H. C. J. Ertl, J. M. Wilson. 1996. Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo. Gene Ther. 3:137–144. 54. Zsengeller, Z. K., S. E. Wert, W. M. Hull, X. Hu, S. Yei, B. C. Trapnell, and

J. A. Whitsett.1995. Persistence of replication-deficient adenovirus-medi-ated gene transfer in lungs of immune-deficient (nu/nu) mice. Hum. Gene Ther. 6:457–467.

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