JOURNAL OF VIROLOGY, Dec. 2006, p. 11899–11910
0022-538X/06/$08.00⫹0 doi:10.1128/JVI.00890-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Vol. 80, No. 24

Induction of Robust Immune Responses against Human Immunodeficiency
Virus Is Supported by the Inherent Tropism of Adeno-AssociatedVirus Type 5 for Dendritic Cells䌤
Ke-Qin Xin,1 Hiroaki Mizukami,2 Masashi Urabe,2 Yoshihiko Toda,1 Kaori Shinoda,1Atsushi Yoshida,1 Kenji Oomura,1 Yoshitsugu Kojima,1 Motohide Ichino,3Dennis Klinman,4 Keiya Ozawa,2 and Kenji Okuda1*
Departments of Molecular Biodefense Research1 and Immunology,3 Yokohama City University Graduate School of Medicine,
Yokohama 236-0004, Japan; Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School,
Tochigi-ken 329-0498, Japan2; and Center for Biologics Evaluation and Research, U.S. Food andDrug Administration, Bethesda, Maryland 208924

The ability of adeno-associated virus serotype 1 to 8 (AAV1 to AAV8) vectors expressing the humanimmunodeficiency virus type 1 (HIV-1) Env gp160 (AAV-HIV) to induce an immune response was evaluated in BALB/c mice. The AAV5 vector showed a higher tropism for both mouse and human dendritic cells(DCs) than did the AAV2 vector, whereas other AAV serotype vectors transduced DCs only poorly. AAV1,
AAV5, AAV7, and AAV8 were more highly expressed in muscle cells than AAV2. An immunogenicity studyof AAV serotypes indicates that AAV1, AAV5, AAV7, and AAV8 vectors expressing the Env gp160 geneinduced higher HIV-specific humoral and cell-mediated immune responses than the AAV2 vector did, with
the AAV5 vector producing the best responses. Furthermore, mice injected with DCs that had beentransduced ex vivo with an AAV5 vector expressing the gp160 gene elicited higher HIV-specific cellmediated immune responses than did DCs transduced with AAV1 and AAV2 vectors. We also found that
AAV vectors produced by HEK293 cells and insect cells elicit similar levels of antigen-specific immune
responses. These results demonstrate that the immunogenicity of AAV vectors depends on their tropism
for both antigen-presenting cells (such as DCs) and non-antigen-presenting cells (such as muscular cells)
and that AAV5 is a better vector than other AAV serotypes. These results may aid in the development of
AAV-based vaccine and gene therapy.
Recombinant adeno-associated viruses (AAVs) have been
widely used as gene delivery vectors in animal models (17, 18),
and these have entered human clinical trials (34). AAVs havebeen found in many animal species, including nonhuman primates, canines, fowls, and humans. An increasing number of
AAV serotypes have been reported. AAV2, AAV3, and AAV5
are found in humans, while AAV4, AAV7, and AAV8 arefound in nonhuman primates (21, 45, 50). The reservoir for
AAV1 is unclear because these viruses have not been primarily
isolated from tissues; however, reactive antibodies (Abs)against AAV1 have been found to exist in both humans andnonhuman primates (9). AAV6 appears to be a recombinant
between AAV1 and AAV2 (60). Most of the current studies
involving AAV are based on AAV2 since it was the first available infectious clone (51). The use of AAV2 as a vector to
introduce exogenous genes encoding immunogenic proteinsfor the purpose of vaccination has been explored in several
studies conducted by us (65, 66) and other researchers (17, 30,
46, 47). An ex vivo experiment demonstrated that the AAV2
vector can transduce dendritic cells (DCs) and that these cells* Corresponding author. Mailing address: Department of Molecular
Biodefense Research, Yokohama City University Graduate School of
Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan.
Phone: 81(45)787-2602. Fax: 81 (45)787-2851. E-mail: kokuda@med.yokohama-cu.ac.jp.
䌤Published ahead of print on 27 September 2006.

then present the AAV-encoded antigen to T cells (46, 65).
Other AAV serotypes may have advantages as vaccine vectors
because AAV serotypes differ in their tissue and/or cell tropism (23, 25, 30, 47). For example, AAV1 and AAV7 are more
efficient than AAV2 for the transduction of skeletal muscle(21, 60), while AAV3 is superior for the transduction of
megakaryocytes (27). AAV5 and AAV6 infect apical airway
cells more efficiently (24, 68). AAV2, AAV4, and AAV5 infectcells of the central nervous system; however, differences withregard to the distribution and target cell types exist among
these three serotypes (68).
AAV is a small, single-stranded DNA virus that lacks anenvelope. This virus requires a helper virus to facilitate efficient replication. The genome of wild-type AAV is known tointegrate into the human genome at a specific site on chromosome 19q (36). However, in nondividing cells, AAV vector
genomes mostly adopt the form of concatameric circular episomes that comprise active transcriptional units (16, 54).
AAV is currently the only nonpathogenic viral vector thathas been shown to mediate long-term gene expression without causing toxicity in vivo. Using this vector system, exogenous genes have been efficiently transferred into a numberof tissues, including brain (18), muscle (29), lung (19), gut(17), liver (55), and eye (37). A human clinical trial of AAV2
has been conducted (34, 41).
The objectives of the present study were (i) to compare theimmunogenicities of AAV serotypes (1 to 8) expressing human

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Received 2 May 2006/Accepted 11 September 2006

11900XIN ET AL.

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immunodeficiency virus (HIV) Env gp160 in BALB/c mice, (ii)to compare the immunogenicities of AAV serotype vectors
produced by HEK293 cells and insect cells, and (iii) to evaluate
the mechanisms involved in the observed responses.
MATERIALS AND METHODS

FIG. 1. Transduction of mouse muscle by AAV serotype vectors.
AAV LacZ-expressing vectors (1010 vp) were injected intramuscularly
into five mice/group. ␤-Galactosidase activity in the muscle was examined 2 weeks later using the Beta-Glo Assay System. An asterisk
indicates a significant difference between the two groups (P ⬍ 0.05).
RLU, relative light units.

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AAV production. AAV vectors were generated as described previously (65,66). In brief, a lacZ gene or a fragment containing HIV Env gp160 and Rev
coding genes, which were derived from the HIV IIIB strain, was subcloned intoa shuttle vector containing the cytomegalovirus (CMV) promoter, poly(A), and
the AAV2 inverted terminal repeat (the AAV5 inverted terminal repeat wasused for construction of the AAV5 vector; the AAV2 inverted terminal repeat
was used for other AAV serotype vectors). We included a Rev coding gene in theconstruct because expression of HIV Env gp160 is dependent on Rev protein.
This approach resulted in increased HIV Env gp160 protein expression in vitro
and enhanced immune responses against HIV Env gp160 in vivo (33). The
recombinant shuttle vector was packaged by triple transfection of HEK293 cellswith an adenovirus helper plasmid, a chimeric packaging construct in which the
AAV2 Rep gene was fused to the cap gene derived from either AAV serotype,and a shuttle vector plasmid to produce pseudotypes AAV2-based AAV1-LacZ,
AAV2-LacZ, AAV3-LacZ, AAV4-LacZ, AAV5-LacZ, AAV7-LacZ, andAAV8-LacZ or AAV1-HIV, AAV2-HIV, AAV3-HIV, AAV4-HIV, AAV5-

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HIV, AAV7-HIV, and AAV8-HIV vectors. The AAV vectors were purified by
the standard cesium chloride sedimentation method (65, 66). The titer wasdetermined by quantitative DNA dot blot hybridization.
The insect cell-produced AAV vectors (BacAAVs) were generated as described previously (58). The fragment containing the CMV promoter, HIV Env
gp160 and Rev coding genes, and poly(A) was excised from the AAV shuttle
vector by using NotI; the resulting fragment was inserted into the correspondingsite of a baculovirus transfer plasmid between the serotype 2 or 5 inverted
terminal repeats. Recombinant baculoviruses were generated by using the Bacto-Bac baculovirus expression system (Invitrogen, Carlsbad, CA). Recombinantbaculoviruses containing the HIV Env gp160 and Rev coding genes; an AAV
Rep of serotypes 1, 2, and 5; and an AAV1, AAV2, or AAV5 capsid were used
to infect insect cells in order to produce BacAAV1-HIV, BacAAV2-HIV, and
BacAAV5-HIV vectors, respectively. The AAV vectors produced were purified
by two rounds of ultracentrifugation with a standard cesium chloride density
gradient (65, 66). The titer was determined by quantitative DNA dot blot hybridization.
In vivo expression of ␤-galactosidase. The AAV-LacZ vector (1010 viral particles [vp]/mouse) was injected into mouse muscle. Two weeks after the administration, the mouse was sacrificed and the ␤-galactosidase activity in the muscle
was monitored periodically from 1 week through 6 months after administrationby using the Beta-Glo Assay System (Promega, Madison, WI).
Mouse DC preparation. DCs were isolated from BALB/c mouse bone marrow,as described previously (7, 62). In brief, the bone marrow was obtained from thetibia and femur of BALB/c mice. The DCs at a density of 5 ⫻ 105 cells/ml were

cultured in RPMI 1640 medium containing 10% fetal calf serum, 1 ng/ml recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF; Kirin
Beer Corp., Tokyo, Japan), and recombinant interleukin-4 (IL-4) for 6 days.
Transduction of AAV vectors to mouse cells. Immature mouse DCs were
stained with phycoerythrin (PE)-conjugated anti-mouse CD11c antibody (clone
N418; eBioscience, Boston, MA). The CD11c⫹ DCs were sorted using a MoFlo
Cell Sorter (Takara Bio Corp., Tokyo, Japan). The cells were transduced with
LacZ-expressing serotype AAV vectors at 37°C at 103 to 105 vp/cell for 2 days.
The transduced cells were washed with phosphate-buffered saline (PBS) andstained with 40 mM X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside)in staining buffer [5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 2 mM MgCl2 in PBS] at
37°C for 2 h. The ␤-galactosidase activity was detected by using the Beta-Glo
Assay System (Promega). To count LacZ-expressing cells, the sorted CD11c⫹
DCs were treated with 1 ␮M of fluorescein digalactoside (FDG; Molecular
Probes, Eugene, OR) followed by flow cytometric analysis (the data are shown in
Fig. 2b). The enriched, unsorted DCs were transduced with LacZ-expressing
serotype AAV vectors at 37°C at 105 vp/cell for 2 days. The cells were stainedwith anti-mouse CD11c antibody and treated with FDG followed by flow cytometric analysis (the data are shown in Fig. 2c).
The mouse DCs used in this study were derived from GM-CSF- and IL-4treated bone marrow cells. To explore the efficiencies of transduction of AAV
serotype vectors to hematopoietic cells, unpurified mouse splenocytes and bonemarrow cells were transduced with LacZ-expressing serotype AAV vectors at 105
vp/cell at 37°C for 2 days. The bone marrow cells were treated with 1 ␮M of
FDG, and splenocytes were treated with 1 ␮M of FDG and PE-conjugatedDownloaded from jvi.asm.org at YOKOHAMA CITY UNIVERSITY on December 7, 2008

FIG. 2. Transduction of mouse purified DCs by AAV serotype vectors. (a) Mouse CD11c⫹ DCs were transduced with LacZ-expressing AAV
vectors at 104 vp/cell (triplicate for each sample). Two days after infection, the cells were stained with X-Gal (upper panel), and the ␤-galactosidase
activity was measured using the Beta-Glo Assay System (bottom panel). The data presented were averaged from three separate experiments. The
asterisk indicates a significant difference when AAV2-LacZ-transduced cells were compared to AAV1-LacZ-, AAV3-LacZ-, AAV4-LacZ-,
AAV7-LacZ-, and AAV8-LacZ-transduced cells and mock-transduced cells; the double asterisk indicates a significant difference when AAV5LacZ-transduced cells were compared to AAV2-LacZ-transduced cells. RLU, relative light units. (b) Mouse CD11c⫹ DCs were transduced with
AAV-LacZ vectors at 103 to 105 vp/cell (triplicate for each sample). Two days after transduction, the cells were stained with X-Gal, and thepercentages of LacZ-expressing DCs (FDG⫹ CD11c⫹ DCs) were determined. The data presented were averaged from three separate experiments.
(c) Enriched immature DCs were transduced with 105 vp/cell of various AAV-LacZ vectors (triplicate for each sample). Two days after
transduction, the cells were treated with PE-conjugated anti-mouse CD11c Ab and FDG followed by flow cytometric analysis. The data provided
represent the fraction of FDG/CD11c dual-positive cells as a percentage of the total population of CD11c⫹ cells. The data presented were averaged
from three independent experiments.

11902XIN ET AL.

Gaithersburg, MD) at room temperature for 20 min, and the reaction was
terminated by holding the plate under running distilled water. The number ofspots was counted using a computer-assisted video image analyzer. The results
were expressed as spot-forming cells (SFC) per million cells.
Tetramer assay. The tetramer assay was performed as described previously(62–65). The H-2Dd/p18 tetramer (RGPGRAFVTI) labeled with PE was prepared by ProImmune Limited (Oxford, United Kingdom). In brief, mousesplenocytes were incubated with 4% healthy mouse serum in PBS for 15 min at
4°C. The cells were stained with fluorescein isothiocyanate (FITC)-labeled antimouse CD8␣ (Ly-2; PharMingen) at 0.5 ␮g/106 cells for 30 min at 4°C. After two
washes in staining buffer (3% fetal calf serum, 0.1% NaN3 in PBS), the cells wereincubated with the tetramer reagent for 30 min at 4°C and analyzed by flow
cytometry.
In vivo CTL assay. The assay was performed as previously described (52). Briefly,
cytotoxic T-lymphocyte (CTL) target cells were derived from naı̈ve splenocytes andthen pulsed with 10 ␮g/ml of the HIV V3 peptide (RGPGRAFVTI) or with nopeptide at 37°C for 1 h. Peptide-pulsed cells were labeled with a high concentrationof carboxyfluorescein diacetate succinimidyl ester (CFSE; 5 ␮M; Molecular Probes,
Eugene, Oreg.), or unpulsed cells were labeled with a low concentration of CFSE
(0.5 ␮M). Cells were then washed and enumerated. For the in vivo CTL assay, 5 ⫻106 pulsed and 5 ⫻ 106 unpulsed cells were combined into a final 200 ␮l of PBS andintravenously injected into vaccinated mice. Mice were killed 24 h after in vivo target
cell incubation. The mouse splenocytes were analyzed by flow cytometry. The percent in vivo killing of CFSE-labeled target cells was defined as the relative loss ofsuch cells after in vivo incubation and thus was taken as the measure of CTL. It wascalculated using the formula % killing ⫽ [1 ⫺ (unpulsed cells/peptide-pulsed cells
from unimmunized control)/(unpulsed cells/peptide-pulsed cells from immunizedgroup)] ⫻ 100.
Surface expression of HIV gp120 and ex vivo transduction. To investigate
whether the AAV-HIV-transduced DCs could present the HIV peptide to T
cells, mouse bone marrow-derived DCs were transduced with AAV-HIV vectors
at 105 vp/cell at 37°C for 2 days. To explore whether HIV gp160 is expressed on
the surface of DCs, the AAV-HIV-transduced DCs were stained with HIV gp120
MAb (hybridoma 902) followed by FITC-conjugated rabbit anti-mouse IgG andPE-conjugated anti-mouse CD11c antibody (clone N418; eBioscience). The cells
were analyzed by flow cytometry. Enriched mouse DCs were transduced with the
AAV-HIV vectors for 2 h and then washed with PBS, and 105 cells were intravenously administered to recipient BALB/c mice. One week after the administration, HIV-specific responses were detected by a tetramer assay.
Data analysis. All the values are expressed as means ⫾ standard errors.
Statistical analysis of the experimental data and controls was conducted using
one-way factorial analysis of variance. Statistical significance was defined as avalue of P ⬍ 0.05.

RESULTS
Different expression levels of AAV serotype vectors in mousemuscle. Muscular cells are one of the major targets transduced when viral vectors are delivered via the i.m. route. In
this study, the relative efficiency with which various AAV
serotype vectors were transduced and expressed in vivo was
explored. Mice were injected i.m. with AAV serotype vectors carrying the lacZ gene. ␤-Galactosidase activity wasmonitored periodically from 1 week through 6 months afteradministration. Results show that the AAV1 and AAV7
vectors elicited significantly higher ␤-galactosidase activity
than the AAV2, AAV3, and AAV4 vectors, with AAV5 andAAV8 yielding intermediate levels of expression at 2 weeks
after administration (Fig. 1). We found that AAV expression was maximal 2 to 4 weeks after AAV vector administration, although ␤-galactosidase activity remained elevatedfor up to 6 months (data not shown).
Strong tropism of AAV5 for mouse DCs. The ability of each
AAV serotype to transduce DCs was then examined. Enriched
CD11c⫹ DCs were purified and incubated for 2 days with 104
vp/cell of each AAV serotype. Some cells were stained with
X-Gal, and others were examined for ␤-galactosidase expression. As shown in Fig. 2a, very strong X-Gal staining was

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anti-mouse CD3e (clone145-2C11), PE-conjugated anti-mouse CD11b (clone
M1/70), or PE-conjugated anti-mouse CD19 (clone MB19-1) (all antibodies werefrom eBioscience) followed by flow cytometric analysis for staining T cells,
macrophages, and B cells, respectively.
Human DC preparation. To explore the transduction efficiencies of AAV
serotype vectors to human cells, human peripheral blood mononuclear cells(PBMCs) and an enriched population of PBMC-derived immature DCs wereprepared as previously described (67). Briefly, human PBMCs were isolated fromthe blood of a healthy person using Lymphoparu I (Immune Biological Laboratory, Gunma, Japan) according to the manufacturer’s instructions. Fresh human
PBMCs at 3 ⫻ 106 cells/ml in RPMI 1640 medium were dispensed into individual
wells of 12-well plates (1 ml/well), which had been previously coated with autologous plasma for 30 min at 37°C. The PBMC cultures were allowed to incubate
at 37°C for 1 h. After gentle washing with serum-free RPMI 1640 medium, theadherent cells were cultured in Iscove’s modification of Dulbecco modified Eagle
medium (2 ml/well) containing human GM-CSF (500 ng/ml) and IL-4 (200
␮g/ml) (both from Pierce Biotechnology, Rockford, IL) for 5 days.
Transduction of AAV vectors to human cells. The human PBMCs and anenriched population of PBMC-derived immature DCs were transduced with
LacZ-expressing serotype AAV vectors at 105 vp/cell at 37°C for 2 days. The
AAV-transduced DCs were treated with 1 ␮M of FDG, and AAV-transduced
PBMCs were treated with FDG and PE-conjugated anti-human CD11c (clone
3.9), PE-conjugated anti-human CD3 (clone HIT3a), PE-conjugated anti-humanCD11b (clone ICRF44), or PE-conjugated anti-human CD19 (J4-166) (all antibodies were from eBioscience) followed by flow cytometric analysis for staining
DCs, T cells, macrophages, and B cells, respectively.
Western blot analysis. To confirm the expression of HIV Env gp160, HEK293
cells were transduced with the AAV vectors encoding the HIV gene in a six-wellplate. Two days posttransduction, the cells were washed in PBS and lysed with 0.1
M Tris-HCl (pH 7.8) and 0.125% Nonidet P-40. The cell lysates were mixed withan equal volume of 2⫻ sodium dodecyl sulfate buffer (125 mM Tris-HCl, pH 6.8,
4% sodium dodecyl sulfate, 20% glycerol, 0.01% bromophenol blue, and 10%
␤-mercaptoethanol) and boiled for 10 min. Then the cell lysates were loaded on
an 8% polyacrylamide gel and transferred to a Hybond ECL nitrocellulose
membrane (Amersham Pharmacia Biotech, Buckinghamshire, England). The
HIV gp160 and ␤-actin proteins were detected using a mouse anti-HIV gp120
monoclonal Ab (MAb) (hybridoma 902; AIDS Research and Reference Reagent
Program, National Institutes of Health, Maryland) and an anti-human ␤-actin
MAb (Sigma), respectively. An affinity-purified horseradish peroxidase-labeledanti-mouse immunoglobulin (Ig; ICN Pharmaceuticals, Inc., Solon, OH) wasused as the secondary Ab. Protein was detected using the ECL Plus Western
Blotting Detection System (Amersham Pharmacia Biotech, Uppsala, Sweden).
Animals and immunization. Eight-week-old BALB/c female mice were purchased from Japan SLC, Inc. (Hamamatsu, Japan). The mice were housed in the
animal center located at Yokohama City University and maintained on a 12-h
day-night cycle. The mice were intramuscularly (i.m.) immunized three times
with 1010 particles of the AAV vector at 2-week intervals.
Enzyme-linked immunosorbent assay. The enzyme-linked immunosorbent assay was performed as described elsewhere (63, 65). To summarize, 96-well
microtiter plates were coated with 1 ␮g/ml of HIVSF2 gp120 protein (donated by
the AIDS Research and Reference Reagent Program, National Institutes of
Health) and incubated overnight at 4°C. The wells were blocked with PBS
containing 1% bovine serum albumin (BSA) for 2 h at room temperature. They
were then treated with 100 ␮l of serially diluted antisera and incubated for an
additional 2 h at 37°C. The bound immunoglobulin was quantified using anaffinity-purified horseradish peroxidase-labeled anti-mouse antibody (Sigma).
The mean antibody titer was expressed as the reciprocal of the serial serum
dilution that exceeded the assay background by 2 standard deviations.
IFN-␥ ELISPOT assay. Two weeks after the final immunization, a gammainterferon (IFN-␥) ELISPOT assay was performed as described previously (62).
In brief, MultiScreen-IP plates (Millipore, Bedford, MA) were coated with 50 ␮lof 10-␮g/ml purified rat anti-mouse IFN-␥ antibody (XMG1.2; PharMingen, San
Diego, CA) in PBS overnight at 4°C. The plate was then blocked with PBS
containing 5% BSA and 0.025% Tween 20 for 2 h at room temperature. Lymphocytes (1 ⫻ 105 to 10 ⫻ 105) isolated from the spleen were added to each wellin triplicate. The spleen cells were stimulated with or without 10 ␮g/ml of the
HIV V3 peptide (RGPGRAFVTI) for 24 h at 37°C. After incubation, the cells
were removed and incubated with 0.5 ␮g/ml of biotinylated anti-mouse IFN-␥
antibody (PharMingen) for 2 h at 37°C, followed by the addition of 100 ␮l/wellof 0.2% alkaline phosphatase-streptavidin (Vector Laboratories, Burlingame,CA) in PBS containing 0.05% Tween 20 and 5% BSA for 1.5 h. Finally, the plate
was treated with 50 ␮l/well of 5-bromo-4-chloro-3-indolylphosphate–nitroblue
tetrazolium membrane phosphatase (Kirkegaard and Perry Laboratories,

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observed in cells treated with AAV5 vector, weaker staining in
those treated with AAV2, and none in cells exposed to theother AAV serotypes. Similar results were obtained when purified CD11c⫹ DCs were incubated with 103 to 105 vp/cell, and
X-Gal expression was monitored by flow cytometry. As thedose of vector increased, up to 96.5% of DCs were transducedby the AAV5 vector and 45.7% by the AAV2 vector, but⬍10% for all other vectors (Fig. 2b). Similar results wereobserved using enriched bone marrow-derived immature DCs(Fig. 2c). Of note, the AAV5 vector strongly transduced bothbone marrow-derived CD11c⫹ cells and CD11c⫺ cells (Fig.
2c). Further study demonstrated that AAV5 transfected most
unpurified bone marrow cells, whereas AAV2 was much lesseffective and other AAV serotypes largely ineffective (Fig. 3).
AAV5 was also effective at transducing T cells (⬎85% ofCD3⫹ cells) and macrophages (⬎30% of CD11b⫹ cells), butless than 10% of T cells and macrophages were transduced byother AAV serotypes (Fig. 3). However, there was no significant difference in the abilities of various vectors to transduce B
cells (⬍15%).
Strong tropism of AAV5 for human DCs. To explore thetransduction efficiency of AAV5 for human target cells, DCs
were prepared from human PBMCs. At an infection dose of

105 vp/cell, 26.8% of enriched human CD11⫹ DCs were transduced by AAV5, but less than 5% of DCs were transduced byother serotypes (Fig. 4). The cellular tropism of AAV5 was
also examined by flow cytometry. AAV5 infected 56.4% of
CD3⫹ T cells (Fig. 4) and 23.5% of CD11b⫹ macrophages(Fig. 4). However, other serotype vectors were poorly transduced into both T cells and macrophages (⬍5%, Fig. 4). No
significant difference in transduction of CD19⫹ B cells wasobserved among AAV serotype vectors (⬍10% of B cells). In
general, human DCs, macrophages, and T cells were transduced less effectively by AAV5 than were murine cells, perhaps
reflecting differences in AAV5 receptor expression between
these two species.
Similar immunogenicities of AAV vectors produced by
HEK293 cells and insect cells. Difficulty in producing hightitered AAV stocks has limited the clinical utility of this classof vectors. To resolve the problem, AAV vectors have been
produced utilizing baculovirus in insect cells (42, 56, 58) andherpes simplex virus (8, 14). In this study, HEK293 cells were
infected with 105 vp/cell of AAV-HIV vectors and the expression of AAV-HIV vectors was confirmed by Western blotting(Fig. 5a). HIV gene expression by AAV1 and AAV2 vectors
was slightly stronger than that by other serotype vectors. The

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FIG. 3. Transduction of mouse unpurified bone marrow cells and splenocytes by AAV serotype vectors. Mouse bone marrow cells andsplenocytes were transduced with 105 vp/cell of AAV-LacZ vectors. Two days after transduction, the cells were treated with FDG and thesplenocytes were stained with anti-mouse CD3 (T cells), anti-mouse CD11b (macrophages), or anti-mouse CD19 (B cells) antibody followed byflow cytometric analysis. Panels show results from one of three independent experiments.

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abilities to produce three AAV serotypes (AAV1, AAV2, andAAV5) in insect cells and in HEK293 cells were compared.
Expression levels of insect cell-produced AAV vectors similar
to those of HEK293-produced corresponding AAV vectors
were observed. Again, as seen in Fig. 6, similar cell-mediatedimmune responses were elicited by the corresponding AAV
vectors. However, AAV vectors derived from insect cells wereslightly more effective at inducing humoral immune responses
than vectors derived from HEK293 cells (Fig. 5b). This may be
due to vector contamination by baculovirus components, sincebaculovirus itself can have an adjuvant effect on vaccine-induced immune responses (1, 22).
AAV-HIV immunization stimulates a humoral immune response. To explore the immunogenicity of the AAV vectors,
BALB/c mice were immunized three times with 1010 vp ofvarious AAV-HIV vectors. The resultant HIV-specific serum
Ab response was examined 1 month after the last boost. The
strongest HIV-specific serum IgG response was generated inmice immunized with the AAV5-HIV vector; the poorest
humoral responses were observed in mice immunized with
AAV3-HIV or AAV4-HIV (Fig. 5b). AAV1-HIV, AAV7HIV, and AAV8-HIV induced intermediate humoral responses (Fig. 5b).
AAV-HIV immunization stimulates a cellular immune response. The induction of HIV-specific IFN-␥-secreting cells
was then examined in the immunized mice. As observed forhumoral immunity, animals immunized with the AAV5-HIV

vector generated the strongest cellular immune response(1,500 SFC/million cells). The AAV1, AAV2, AAV7, andAAV8 HIV vectors also stimulated ⬎300 IFN-␥-secreting
SFC/million spleen cells, whereas the AAV3- and AAV4-HIV
vectors elicited significantly weaker cellular responses (P ⬍0.05, Fig. 6a). The AAV5-HIV produced by either HEK293
or insect cells induced significantly higher cellular immune
responses than the AAV1-HIV produced by corresponding
cells.
A tetramer binding assay was used to identify major histocompatibility complex class I-restricted HIV-specific T cells(3). Mice immunized with the AAV1-, AAV5-, and AAV7HIV vectors induced strong HIV-specific CD8⫹ T-cell responses (⬎2%, Fig. 6b), and AAV2-HIV was intermediate(1.2%), while AAV3- and AAV4-HIV elicited only 0.4% HIVspecific CD8⫹ T cells.
An in vivo CTL assay was used to detect vaccine-elicited
cell-mediated immune responses. Whereas the AAV3-,
AAV4-, and AAV8-HIV vectors elicited approximately 40%
CTL activity, the AAV1-, AAV5-, and AAV7-HIV vectors
were significantly more effective (generally ⬎50% activity),
with the AAV2-HIV vector being intermediate (Fig. 6c).
DCs present HIV peptide to T cells in vivo. To examine
whether DCs play a role in AAV-HIV-induced immune responses, DCs were enriched from the bone marrow of naı̈ve
mice. Enriched DCs were then treated with AAV5-LacZ,
AAV1-HIV, AAV2-HIV, or AAV5-HIV vectors in vitro (105

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FIG. 4. Transduction of enriched human DCs and unpurified PBMCs by AAV serotype vectors. Human enriched DCs or PBMCs weretransduced with 105 vp/cell of AAV-LacZ vectors. Two days after transduction, the DCs were treated with FDG and anti-human CD11c Ab, and
PBMCs were treated with FDG and anti-human CD3 (T cells), anti-human CD11b (macrophages), or anti-human CD19 (B cells) antibody
followed by flow cytometric analysis. Panels show results from one of three independent experiments.

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vp/cell). The expression of HIV gp160 protein on the surface ofmouse DCs was confirmed by anti-mouse HIV gp120 monoclonal antibody and anti-mouse CD11c antibody staining followed by flow cytometric analysis. HIV gp160 protein was
detected in 13.5%, 19.4%, and 65.2% of CD11c⫹ DCs whichwere transduced with AAV1-HIV, AAV2-HIV, and AAV5HIV vectors, respectively (Fig. 7a). On the other hand, enriched mouse DCs were transduced with AAV-HIV vectors
and injected into naı̈ve recipients. Seven days later, HIV-specific tetramer binding CD8⫹ cells were quantified using thetetramer assay. Recipients of the AAV2-HIV (0.6%)- andAAV5-HIV (2.1%)-transduced cells had significantly higher
HIV peptide-binding CD8⫹ T cells than the AAV-LacZtreated control (0.0%) (Fig. 7b). In contrast, a weak immune
response was observed in recipients of AAV1-HIV vectortransduced cells (0.1%).

DISCUSSION
The present study demonstrates that AAV5 is a superior
vector for achieving humoral and cellular immune responses,
and the immunogenicity of AAV vectors depends on their
tropism for both antigen-presenting cells (APCs) (such asDCs) and non-antigen-presenting cells (such as muscular
cells).
Consistent with previous studies, AAV-HIV vectors of different serotypes had distinct effects on the induction of HIVspecific humoral and cell-mediated immune responses (10, 17,
20, 28, 31, 38, 40, 53, 65, 66). However, most of these studies
examined the durability of transgene expression after i.m. administration of the AAV vector but did not typically monitor
cell-mediated immune responses against the encoded antigen(19, 32, 61). In contrast to studies in which the AAV vector
encoded a self protein (as in gene therapy) or in which a

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FIG. 5. Expression of HIV gp160 and HIV-specific serum IgG titer by AAV serotype vectors. (a) HEK293 cells were transduced with AAV
vectors carrying the HIV Env gp160 gene. Two days after infection, cell lysates were analyzed by Western blotting using anti-HIV Env MAb andanti-human ␤-actin MAb. (b) BALB/c mice (five mice/group) were immunized with AAV-HIV vectors on days 0, 14, and 28. HIV-specific serum
IgG was measured 1 month after the final immunization. The BacAAV vectors were generated in insect cells, whereas other vectors were produced
in HEK293 cells. The asterisks indicate significant differences between the two groups (P ⬍ 0.05).

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XIN ET AL.
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neoantigen was used to evaluate long-term expression (such as
␤-galactosidase), the immune response elicited by our HIV
Env gp160-encoding vector was quite strong (10, 17, 20, 28, 31,
38, 40, 53, 65, 66).
Most currently used AAV vectors for vaccine or gene therapy are based on the AAV2 serotype (10, 17, 20, 28, 31, 38, 40,
53, 65, 66). The current work compared the immunogenicity of
AAV2-HIV to that of other AAV serotype-based vectors andfound that the AAV5 vector induced the strongest cellular andhumoral immune responses. This finding may help in the development of novel AAV vector-based vaccines, particularly
because natural immunity to AAV5 is rare (30). Of note, while
we used a prime/boost regimen involving repeated administration of the same AAV serotype vector, it may be even more
effective to prime with one serotype and boost with another(since neutralizing Abs raised against the first vector are unlikely to cross-react with a different serotype) (48).

Following vaccination with the AAV vector, antigen-specificimmune responses can be induced through at least two pathways. In one of the pathways, the vaccine vector directly infects
APCs, such as DCs, where the encoded antigen is expressed.
The antigen is then processed and presented by APCs to T
cells (Fig. 7) (65). The percentage of HIV gp160⫹ CD11c⫹cells in Fig. 7a was lower than the percentage of FDG⫹
CD11c⫹ cells in Fig. 2c. This may be due to the presentationof HIV gp160 peptides by DCs present in culture. In theother pathway, the vaccine vector infects non-APCs, such asmuscle cells. APCs take up the antigen expressed by theinfected cells and present peptides from the exogenous antigen to T cells by major histocompatibility complex class
I-restricted cross-presentation. It has been known that adenovirus vectors may use both pathways; however, AAV2
relies on the latter pathway due to a low efficiency of transfection of DCs (53). Current findings indicate that AAV1,

FIG. 6. HIV-specific cell-mediated immune responses induced by AAV vector vaccination. BALB/c mice (five mice/group) were immunizedwith AAV-HIV vectors on days 0, 14, and 28. Five mice per group for the IFN-␥ ELISPOT and tetramer assay and five mice per group for thein vivo CTL assay were used. The HIV-specific IFN-␥ ELISPOT assay was performed (a), the percentage of HIV-specific tetramer binding CD8⫹
cells was determined (b), and the in vivo CTL assay was performed (c) 2 weeks after the final immunization. BacAAV vectors were generated in
insect cells, whereas other AAV vectors were generated in HEK293 cells. Asterisks indicate significant differences between the two groups (P ⬍0.05). Panels b and c show results from individual representative animals, while the data represent the averages of five mice/group.

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FIG. 7. Immune responses induced by ex vivo DC transduction.
Mouse enriched DCs were transduced with 105 vp/cell of AAV-HIV
vectors for 2 days. The cells were stained with PE-conjugated antimouse CD11c antibody plus mouse anti-HIV gp120 antibody followed
by staining with FITC-conjugated anti-mouse IgG. The HIV gp160
expression of DC surface was analyzed by flow cytometry (a). Enrichedmouse DCs were transduced with AAV-HIV vectors for 2 h, washedwith PBS (⫺), and injected intravenously into recipient BALB/c mice
(five mice/group). HIV-specific responses were detected using the tetramer assay 1 week after administration. Dot plots show results fromindividual representative animals, while the data shown in the upper
right quadrant represent the averages of five mice/group (b). Neg.
Con., negative control.

11908XIN ET AL.

ACKNOWLEDGMENTS
We are grateful to M. Kawano for her technical assistance and A.
De La Fuente for her secretarial assistance. We also extend our appreciation to J. M. Wilson and G. P. Gao for providing helper plasmidsof AAV7 and AAV8.

This work was partially supported by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and theJapanese National Institute of Biomedical Innovation (no. 05-1).
REFERENCES
1. Abe, T., H. Hemmi, H. Miyamoto, K. Moriishi, S. Tamura, H. Takaku, S.
Akira, and Y. Matsuura. 2005. Involvement of the Toll-like receptor 9
signaling pathway in the induction of innate immunity by baculovirus. J. Virol. 79:2847–2858.
2. Aldrich, W. A., C. Ren, A. F. White, S. Z. Zhou, S. Kumar, C. B. Jenkins,
D. R. Shaw, T. V. Strong, P. L. Triozzi, and S. Ponnazhagan. 2006. Enhanced
transduction of mouse bone marrow-derived dendritic cells by repetitive
infection with self-complementary adeno-associated virus 6 combined withimmunostimulatory ligands. Gene Ther. 13:29–39.
3. Altman, J. D., P. A. Moss, P. J. Goulder, D. H. Barouch, M. G. McHeyzerWilliams, J. I. Bell, A. J. McMichael, and M. M. Davis. 1996. Phenotypic
analysis of antigen-specific T lymphocytes. Science 274:94–96.
4. Babcock, A. M., D. Standing, K. Bullshields, E. Schwartz, C. M. Paden, and
D. J. Poulsen. 2005. In vivo inhibition of hippocampal Ca2⫹/calmodulindependent protein kinase II by RNA interference. Mol. Ther. 11:899–905.
5. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of
immunity. Nature 392:245–252.
6. Bantel-Schaal, U., H. Delius, R. Schmidt, and H. zur Hausen. 1999. Humanadeno-associated virus type 5 is only distantly related to other known primate
helper-dependent parvoviruses. J. Virol. 73:939–947.
7. Behboudi, S., D. Chao, P. Klenerman, and J. Austyn. 2000. The effects of
DNA containing CpG motif on dendritic cells. Immunology 99:361–366.
8. Booth, M. J., A. Mistry, X. Li, A. Thrasher, and R. S. Coffin. 2004. Transfection-free and scalable recombinant AAV vector production using HSV/AAV hybrids. Gene Ther. 11:829–837.
9. Boucher, D. W., W. P. Parks, and J. L. Melnick. 1970. A sensitive neutralization test for the adeno-associated satellite viruses. J. Immunol. 104:555–
559.
10. Brockstedt, D. G., G. M. Podsakoff, L. Fong, G. Kurtzman, W. MuellerRuchholtz, and E. G. Engleman. 1999. Induction of immunity to antigens
expressed by recombinant adeno-associated virus depends on the route ofadministration. Clin. Immunol. 92:67–75.
11. Chiorini, J. A., F. Kim, L. Yang, and R. M. Kotin. 1999. Cloning andcharacterization of adeno-associated virus type 5. J. Virol. 73:1309–1319.
12. Chiorini, J. A., L. Yang, Y. Liu, B. Safer, and R. M. Kotin. 1997. Cloning of
adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4
particles. J. Virol. 71:6823–6833.
13. Chiriva-Internati, M., Y. Liu, E. Salati, W. Zhou, Z. Wang, F. Grizzi, J. J.
Roman, S. H. Lim, and P. L. Hermonat. 2002. Efficient generation of cytotoxic T lymphocytes against cervical cancer cells by adeno-associated virus/
human papillomavirus type 16 E7 antigen gene transduction into dendritic
cells. Eur. J. Immunol. 32:30–38.
14. Conway, J. E., C. M. Rhys, I. Zolotukhin, S. Zolotukhin, N. Muzyczka, G. S.
Hayward, and B. J. Byrne. 1999. High-titer recombinant adeno-associatedvirus production utilizing a recombinant herpes simplex virus type I vector
expressing AAV-2 Rep and Cap. Gene Ther. 6:986–993.
15. Di Pasquale, G., B. L. Davidson, C. S. Stein, I. Martins, D. Scudiero, A.
Monks, and J. A. Chiorini. 2003. Identification of PDGFR as a receptor for
AAV-5 transduction. Nat. Med. 9:1306–1312.
16. Duan, D., P. Sharma, J. Yang, Y. Yue, L. Dudus, Y. Zhang, K. J. Fisher, andJ. F. Engelhardt. 1998. Circular intermediates of recombinant adeno-associated virus have defined structural characteristics responsible for long-term
episomal persistence in muscle tissue. J. Virol. 72:8568–8577.
17. During, M. J., R. Xu, D. Young, M. G. Kaplitt, R. S. Sherwin, and P. Leone.
1998. Peroral gene therapy of lactose intolerance using an adeno-associatedvirus vector. Nat. Med. 4:1131–1135.
18. Fan, D. S., M. Ogawa, K. I. Fujimoto, K. Ikeguchi, Y. Ogasawara, M. Urabe,
M. Nishizawa, I. Nakano, M. Yoshida, I. Nagatsu, H. Ichinose, T. Nagatsu,
G. J. Kurtzman, and K. Ozawa. 1998. Behavioral recovery in 6-hydroxydopamine-lesioned rats by cotransduction of striatum with tyrosine hydroxylase
and aromatic L-amino acid decarboxylase genes using two separate adenoassociated virus vectors. Hum. Gene Ther. 9:2527–2535.
19. 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 adenoassociated virus vector. Proc. Natl. Acad. Sci. USA 90:10613–10617.
20. Gallez-Hawkins, G., X. Li, A. E. Franck, L. Thao, S. F. Lacey, D. J. Diamond,and J. A. Zaia. 2004. DNA and low titer, helper-free, recombinant AAV
prime-boost vaccination for cytomegalovirus induces an immune response to
CMV-pp65 and CMV-IE1 in transgenic HLA A*0201 mice. Vaccine 23:819–
826.
21. Gao, G. P., M. R. Alvira, L. Wang, R. Calcedo, J. Johnston, and J. M. Wilson.
2002. Novel adeno-associated viruses from rhesus monkeys as vectors forhuman gene therapy. Proc. Natl. Acad. Sci. USA 99:11854–11859.
22. Gronowski, A. M., D. M. Hilbert, K. C. Sheehan, G. Garotta, and R. D.
Schreiber. 1999. Baculovirus stimulates antiviral effects in mammalian cells.
J. Virol. 73:9944–9951.

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AAV7, and AAV8 vectors preferentially infect muscle cells,
whereas the AAV5 vector optimally infected DCs (Fig. 1 to
4 and 7). Following i.m. administration, all four vectors induced
higher HIV-specific immunogenicity than the AAV2 vector (Fig.
5 and 6).
DCs are primarily responsible for stimulating resting naı̈ve T
lymphocytes and initiating a CTL response (5). Immature DCs
residing in the peripheral tissues capture foreign antigens, mature, and then migrate to secondary lymphoid organs, where
the processed antigen is presented. To determine whether DCs
might contribute to the immune responses induced by the
AAV-HIV vector, enriched DCs were transduced with the
AAV5-HIV vector and transferred into naı̈ve mice. As seen in
Fig. 7b, recipients generated HIV-specific tetramer binding
CD8⫹ T cells. These results demonstrate that DCs can present
vector-encoded antigen to naı̈ve T cells efficiently. In this context, the levels of immune response induced by the AAV1HIV, AAV2-HIV, and AAV5-HIV vectors correlated with
their tropism for DCs (70).
A number of studies show that recombinant AAV vectors
can be used to transduce DCs (2, 13, 32, 35, 39, 46, 49, 53, 70).
Current results document that AAV5 can transfect mouse andhuman DCs much more efficiently than other AAV serotype
vectors (Fig. 2 to 4). In addition, AAV5 was efficiently transduced into T cells and macrophages (Fig. 3 and 4), which may
be useful for gene therapy of diseases involving hematopoietic
cells (4, 26, 43, 57, 59, 69).
Eight AAV serotypes (AAV1 to AAV8) have been described, with each serotype having unique binding and cell
tropism characteristics (6, 11, 12, 21, 44, 50, 60). A primary
coreceptor for AAV5 is sialic acid, while the platelet-derived
growth factor receptor is involved in the binding and cell entryof AAV5 (15). Platelet-derived growth factor receptor ismainly expressed on fibroblasts, smooth muscle cells, glial cells,
and chondrocytes but not on hematopoietic cells. Surprisingly,
we found that AAV5 was efficiently transduced into hematopoietic cells (Fig. 3 and 4), suggesting that another receptor(s)may be used by AAV5 for cell entry.
The AAV2 vector can be produced in insect cells by using
baculovirus expression vectors in suspension culture, a strategy
that is amenable to easy scale-up (58). In this study, AAV1,
AAV2, and AAV5 vectors expressing the HIV gp160 gene
were prepared using either insect cells or HEK293 cells. Similar immune responses were obtained using vectors producedby either method (Fig. 5b and 6), indicating that insect cellproduced AAV vectors should be considered for the large-scale
preparation of AAV vectors.
In summary, this is the first report that systematically compares DC tropism and immunogenicity of AAV serotype vectors. Results show that AAV5 vectors can efficiently transduce
DCs and produce stronger antigen-specific immune responses
than other AAV vectors. Thus, AAV5-based vectors deserve
further consideration for clinical vaccine development and immune therapy.

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McClure. 2005. Stable inhibition of hepatitis B virus proteins by small interfering RNA expressed from viral vectors. J. Gene Med. 7:918–925.
44. Muramatsu, S., H. Mizukami, N. S. Young, and K. E. Brown. 1996. Nucleotide sequencing and generation of an infectious clone of adeno-associatedvirus 3. Virology 221:208–217.
45. Padron, E., V. Bowman, N. Kaludov, L. Govindasamy, H. Levy, P. Nick, R.
McKenna, N. Muzyczka, J. A. Chiorini, T. S. Baker, and M. AgbandjeMcKenna. 2005. Structure of adeno-associated virus type 4. J. Virol. 79:
5047–5058.
46. Ponnazhagan, S., G. Mahendra, D. T. Curiel, and D. R. Shaw. 2001. Adenoassociated virus type 2-mediated transduction of human monocyte-derived
dendritic cells: implications for ex vivo immunotherapy. J. Virol. 75:9493–
9501.
47. Rabinowitz, J. E., F. Rolling, C. Li, H. Conrath, W. Xiao, X. Xiao, and R. J.
Samulski. 2002. Cross-packaging of a single adeno-associated virus (AAV)
type 2 vector genome into multiple AAV serotypes enables transduction withbroad specificity. J. Virol. 76:791–801.
48. Rabinowitz, J. E., and R. J. Samulski. 2000. Building a better vector: themanipulation of AAV virions. Virology 278:301–308.
49. Ribas, A. 2005. Genetically modified dendritic cells for cancer immunotherapy. Curr. Gene Ther. 5:619–628.
50. Rutledge, E. A., C. L. Halbert, and D. W. Russell. 1998. Infectious clones andvectors derived from adeno-associated virus (AAV) serotypes other than
AAV type 2. J. Virol. 72:309–319.
51. Samulski, R. J., K. I. Berns, M. Tan, and N. Muzyczka. 1982. Cloning of
adeno-associated virus into pBR322: rescue of intact virus from the recombinant plasmid in human cells. Proc. Natl. Acad. Sci. USA 79:2077–2081.
52. Santosuosso, M., X. Zhang, S. McCormick, J. Wang, M. Hitt, and Z. Xing.
2005. Mechanisms of mucosal and parenteral tuberculosis vaccinations:
adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway
lumen. J. Immunol. 174:7986–7994.
53. Sarukhan, A., S. Camugli, B. Gjata, H. von Boehmer, O. Danos, and K.
Jooss. 2001. Successful interference with cellular immune responses to immunogenic proteins encoded by recombinant viral vectors. J. Virol. 75:269–
277.
54. Schnepp, B. C., K. R. Clark, D. L. Klemanski, C. A. Pacak, and P. R.
Johnson. 2003. Genetic fate of recombinant adeno-associated virus vector
genomes in muscle. J. Virol. 77:3495–3504.
55. Snyder, R. O., C. H. Miao, G. A. Patijn, S. K. Spratt, O. Danos, D. Nagy,
A. M. Gown, B. Winther, L. Meuse, L. K. Cohen, A. R. Thompson, and M. A.
Kay. 1997. Persistent and therapeutic concentrations of human factor IX in
mice after hepatic gene transfer of recombinant AAV vectors. Nat. Genet.
16:270–276.
56. Sollerbrant, K., J. Elmen, C. Wahlestedt, J. Acker, H. Leblois-Prehaud, M.
Latta-Mahieu, P. Yeh, and M. Perricaudet. 2001. A novel method using
baculovirus-mediated gene transfer for production of recombinant adenoassociated virus vectors. J. Gen. Virol. 82:2051–2060.
57. Tomar, R. S., H. Matta, and P. M. Chaudhary. 2003. Use of adeno-associated viral vector for delivery of small interfering RNA. Oncogene 22:5712–
5715.
58. Urabe, M., C. Ding, and R. M. Kotin. 2002. Insect cells as a factory to
produce adeno-associated virus type 2 vectors. Hum. Gene Ther. 13:1935–
1943.
59. Xia, H., Q. Mao, S. L. Eliason, S. Q. Harper, I. H. Martins, H. T. Orr, H. L.
Paulson, L. Yang, R. M. Kotin, and B. L. Davidson. 2004. RNAi suppresses
polyglutamine-induced neurodegeneration in a model of spinocerebellar
ataxia. Nat. Med. 10:816–820.
60. Xiao, W., N. Chirmule, S. C. Berta, B. McCullough, G. Gao, and J. M.
Wilson. 1999. Gene therapy vectors based on adeno-associated virus type 1.
J. Virol. 73:3994–4003.
61. 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.
62. Xin, K. Q., Y. Hoshino, Y. Toda, S. Igimi, Y. Kojima, N. Jounai, K. Ohba, A.
Kushiro, M. Kiwaki, K. Hamajima, D. Klinman, and K. Okuda. 2003.
Immunogenicity and protective efficacy of orally administered recombinant
Lactococcus lactis expressing surface-bound HIV Env. Blood 102:223–228.
63. Xin, K. Q., N. Jounai, K. Someya, K. Honma, H. Mizuguchi, S. Naganawa,
K. Kitamura, T. Hayakawa, S. Saha, F. Takeshita, K. Okuda, M. Honda, andD. M. Klinman. 2005. Prime-boost vaccination with plasmid DNA and achimeric adenovirus type 5 vector with type 35 fiber induces protective
immunity against HIV. Gene Ther. 12:1769–1777.
64. Xin, K. Q., Y. Kumamoto, N. Jounai, Y. Kojima, K. Hamajima, and K.
Okuda. 2002. Recombinant vaccinia virus (WR strain) may not be suitablefor ex vivo stimulation. Vaccine 21:5–6.
65. Xin, K. Q., T. Ooki, H. Mizukami, K. Hamajima, K. Okudela, K. Hashimoto,
Y. Kojima, N. Jounai, Y. Kumamoto, S. Sasaki, D. Klinman, K. Ozawa, andK. Okuda. 2002. Oral administration of recombinant adeno-associated virus
elicits human immunodeficiency virus-specific immune responses. Hum.
Gene Ther. 13:1571–1581.

Downloaded from jvi.asm.org at YOKOHAMA CITY UNIVERSITY on December 7, 2008

23. Hacker, U. T., L. Wingenfeld, D. M. Kofler, N. K. Schuhmann, S. Lutz, T.
Herold, S. B. King, F. M. Gerner, L. Perabo, J. Rabinowitz, D. M. McCarty,
R. J. Samulski, M. Hallek, and H. Buning. 2005. Adeno-associated virus
serotypes 1 to 5 mediated tumor cell directed gene transfer and improvementof transduction efficiency. J. Gene Med. 7:1429–1438.
24. Halbert, C. L., J. M. Allen, and A. D. Miller. 2001. Adeno-associated virus
type 6 (AAV6) vectors mediate efficient transduction of airway epithelial
cells in mouse lungs compared to that of AAV2 vectors. J. Virol. 75:6615–
6624.
25. Halbert, C. L., E. A. Rutledge, J. M. Allen, D. W. Russell, and A. D. Miller.
2000. Repeat transduction in the mouse lung by using adeno-associated virus
vectors with different serotypes. J. Virol. 74:1524–1532.
26. Han, W., M. Wind-Rotolo, R. L. Kirkman, and C. D. Morrow. 2004. Inhibition of human immunodeficiency virus type 1 replication by siRNA targetedto the highly conserved primer binding site. Virology 330:221–232.
27. Handa, A., S. Muramatsu, J. Qiu, H. Mizukami, and K. E. Brown. 2000.
Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic
cells not susceptible to transduction with AAV-2-based vectors. J. Gen.
Virol. 81:2077–2084.
28. Hara, H., A. Monsonego, K. Yuasa, K. Adachi, X. Xiao, S. Takeda, K.
Takahashi, H. L. Weiner, and T. Tabira. 2004. Development of a safe oral
Abeta vaccine using recombinant adeno-associated virus vector for Alzheimer’s
disease. J. Alzheimers Dis. 6:483–488.
29. 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 humanblood coagulation factor IX after intramuscular injection of recombinantadeno-associated virus. Proc. Natl. Acad. Sci. USA 94:5804–5809.
30. Hildinger, M., A. Auricchio, G. Gao, L. Wang, N. Chirmule, and J. M.
Wilson. 2001. Hybrid vectors based on adeno-associated virus serotypes 2
and 5 for muscle-directed gene transfer. J. Virol. 75:6199–6203.
31. Johnson, P. R., B. C. Schnepp, M. J. Connell, D. Rohne, S. Robinson, G. R.
Krivulka, C. I. Lord, R. Zinn, D. C. Montefiori, N. L. Letvin, and K. R.
Clark. 2005. Novel adeno-associated virus vector vaccine restricts replicationof simian immunodeficiency virus in macaques. J. Virol. 79:955–965.
32. Jooss, K., Y. Yang, K. J. Fisher, and J. M. Wilson. 1998. Transduction ofdendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J. Virol. 72:4212–4223.
33. Jounai, N., K. Okuda, Y. Kojima, Y. Toda, K. Hamajima, K. Ohba, D.
Klinman, and K. Q. Xin. 2003. Contribution of the rev gene to the immunogenicity of DNA vaccines targeting the envelope glycoprotein of HIV.
J. Gene Med. 5:609–617.
34. Kay, M. A., C. S. Manno, M. V. Ragni, P. J. Larson, L. B. Couto, A.
McClelland, B. Glader, A. J. Chew, S. J. Tai, R. W. Herzog, V. Arruda, F.
Johnson, C. Scallan, E. Skarsgard, A. W. Flake, and K. A. High. 2000.
Evidence for gene transfer and expression of factor IX in haemophilia B
patients treated with an AAV vector. Nat. Genet. 24:257–261.
35. Koppold, B., G. Sauer, H. Buning, M. Hallek, R. Kreienberg, H. Deissler,
and C. Kurzeder. 2005. Efficient gene transfer of CD40 ligand into ovariancarcinoma cells with a recombinant adeno-associated virus vector. Int. J.
Oncol. 26:95–101.
36. Kotin, R. M., M. Siniscalco, R. J. Samulski, X. D. Zhu, L. Hunter, C. A.
Laughlin, S. McLaughlin, N. Muzyczka, M. Rocchi, and K. I. Berns. 1990.
Site-specific integration by adeno-associated virus. Proc. Natl. Acad. Sci.
USA 87:2211–2215.
37. Lewin, A. S., K. A. Drenser, W. W. Hauswirth, S. Nishikawa, D. Yasumura,
J. G. Flannery, and M. M. LaVail. 1998. Ribozyme rescue of photoreceptor
cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.
Nat. Med. 4:967–971.
38. Liu, D. W., J. L. Chang, Y. P. Tsao, C. W. Huang, S. W. Kuo, and S. L. Chen.
2005. Co-vaccination with adeno-associated virus vectors encoding humanpapillomavirus 16 L1 proteins and adenovirus encoding murine GM-CSF
can elicit strong and prolonged neutralizing antibody. Int. J. Cancer 113:93–
100.
39. Liu, Y., A. D. Santin, M. Mane, M. Chiriva-Internati, G. P. Parham, A.
Ravaggi, and P. L. Hermonat. 2000. Transduction and utility of the granulocyte-macrophage colony-stimulating factor gene into monocytes and dendritic cells by adeno-associated virus. J. Interferon Cytokine Res. 20:21–30.
40. Manning, W. C., X. Paliard, S. Zhou, M. P. Bland, A. Y. Lee, K. Hong, C. M.
Walker, J. A. Escobedo, and V. Dwarki. 1997. Genetic immunization withadeno-associated virus vectors expressing herpes simplex virus type 2 glycoproteins B and D. J. Virol. 71:7960–7962.
41. Manno, C. S., A. J. Chew, S. Hutchison, P. J. Larson, R. W. Herzog, V. R.
Arruda, S. J. Tai, M. V. Ragni, A. Thompson, M. Ozelo, L. B. Couto, D. G.
Leonard, F. A. Johnson, A. McClelland, C. Scallan, E. Skarsgard, A. W.
Flake, M. A. Kay, K. A. High, and B. Glader. 2003. AAV-mediated factor IX
gene transfer to skeletal muscle in patients with severe hemophilia B. Blood
101:2963–2972.
42. Meghrous, J., M. G. Aucoin, D. Jacob, P. S. Chahal, N. Arcand, and A. A.
Kamen. 2005. Production of recombinant adeno-associated viral vectors
using a baculovirus/insect cell suspension culture system: from shake flasks toa 20-L bioreactor. Biotechnol. Prog. 21:154–160.
43. Moore, M. D., M. J. McGarvey, R. A. Russell, B. R. Cullen, and M. O.

AAV-HIV VACCINE

11910XIN ET AL.

66. Xin, K. Q., M. Urabe, J. Yang, K. Nomiyama, H. Mizukami, K. Hamajima,
H. Nomiyama, T. Saito, M. Imai, J. Monahan, K. Okuda, and K. Ozawa.
2001. A novel recombinant adeno-associated virus vaccine induces a longterm humoral immune response to human immunodeficiency virus. Hum.
Gene Ther. 12:1047–1061.
67. Yoshida, A., R. Tanaka, T. Murakami, Y. Takahashi, Y. Koyanagi, M.
Nakamura, M. Ito, N. Yamamoto, and Y. Tanaka. 2003. Induction of protective immune responses against R5 human immunodeficiency virus type 1(HIV-1) infection in hu-PBL-SCID mice by intrasplenic immunization with
HIV-1-pulsed dendritic cells: possible involvement of a novel factor of human CD4⫹ T-cell origin. J. Virol. 77:8719–8728.

J. VIROL.
68. Zabner, J., M. Seiler, R. Walters, R. M. Kotin, W. Fulgeras, B. L. Davidson,
and J. A. Chiorini. 2000. Adeno-associated virus type 5 (AAV5) but not
AAV2 binds to the apical surfaces of airway epithelia and facilitates genetransfer. J. Virol. 74:3852–3858.
69. Zhang, W., R. Singam, G. Hellermann, X. Kong, H. S. Juan, R. F. Lockey,
S. J. Wu, K. Porter, and S. S. Mohapatra. 2004. Attenuation of dengue virus
infection by adeno-associated virus-mediated siRNA delivery. Genet. Vaccines Ther. 2:8.
70. Zhang, Y., N. Chirmule, G. Gao, and J. Wilson. 2000. CD40 ligand-dependent activation of cytotoxic T lymphocytes by adeno-associated virus vectors
in vivo: role of immature dendritic cells. J. Virol. 74:8003–8010.

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