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A chimeric adenovirus vector encoding reovirus
attachment protein
1 targets cells expressing
junctional adhesion molecule 1
George T. Mercier*, Jacquelyn A. Campbell
†‡
, James D. Chappell
‡§
, Thilo Stehle
¶
, Terence S. Dermody
†‡储
**,
and Michael A. Barry*
,
**
††‡‡§§
*Department of Bioengineering, Rice University, Houston, TX 77005; Departments of
†
Microbiology and Immunology,
§
Pathology, and
储
Pediatrics and
‡
Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, TN 37232;
¶
Laboratory of Developmental
Immunology, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114; and Departments of
††
Molecular and Human Genetics and
‡‡
Immunology and
§§
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX 77030
Edited by Peter Palese, Mount Sinai School of Medicine, New York, NY, and approved February 23, 2004 (received for review January 23, 2004)
The utility of adenovirus (Ad) vectors for gene transduction can be
limited by receptor specificity. We developed a gene-delivery
vehicle in which the potent Ad5 vector was genetically reengi-
neered to display the mucosal-targeting
1 protein of reovirus
type 3 Dearing (T3D). A
1 construct containing all but a small
virion-anchoring domain was fused to the N-terminal 44 aa of Ad5
fiber. This chimeric attachment protein Fibtail-T3D
1 forms trimers
and assembles onto Ad virions. Fibtail-T3D
1 was recombined into
the Ad5 genome, replacing sequences encoding wild-type fiber.
The resulting vector, Ad5-T3D
1, expresses Fibtail-T3D
1 and
infects Chinese hamster ovary cells transfected with human or
mouse homologs of the reovirus receptor, junctional adhesion
molecule 1 (JAM1), but not the coxsackievirus and Ad receptor.
Treatment of Caco-2 intestinal epithelial cells with either JAM1-
specific antibody or neuraminidase reduced transduction by Ad5-
T3D
1, and their combined effect decreased transduction by 95%.
Ad5-T3D
1 transduces primary cultures of human dendritic cells
substantially more efficiently than does Ad5, and this transduction
depends on expression of JAM1. These data provide strong evi-
dence that Ad5-T3D
1 can be redirected to cells expressing JAM1
and sialic acid for application as a vaccine vector.
A
denovirus (Ad) vectors are potent gene-delivery vehicles
capable of eliciting both mucosal and systemic immune
responses (1). Human Ad serotypes 2 and 5 (Ad2 and Ad5) bind
and enter cells by using the combined interactions of the fiber
and penton base proteins with cellular receptors. The fiber
protein is an elongated trimer with an N-terminal fibrous tail
domain (shaft) and a C-terminal globular head domain (knob).
Ad2 and Ad5 engage the coxsackievirus and Ad receptor (CAR)
(2, 3) via a binding site located in the knob (4). CAR is a member
of the Ig superfamily (2, 3) expressed at regions of cell–cell
contact (5). After fiber-mediated attachment, the penton base
binds to cell surface
␣
v
integrins, which mediate internalization (6).
Although Ad5 vectors transduce many types of cells, the
efficiency of these vectors is limited if cells lack one or more of
its receptors (7). For example, dendritic cells (DCs) do not
express CAR and are poorly transduced by Ad5 (8). This
relatively poor transduction of DCs can be enhanced by reengi-
neering the vector to target alternative receptors (9, 10). Ad
serotypes that bind to other receptors [e.g., CD46 (11)]
mediate increased transduction of immunologically relevant
cells (12), but these vectors are more promiscuous than Ad5
and deliver genes into cells that may not contribute to vacci-
nation and thus may increase toxicity. Therefore, although
potent, current Ad vectors lack sufficient specificity to func-
tion in some applications.
Mammalian reoviruses are nonenveloped, double-stranded
RNA viruses with a broad host range (13). Reovirus infections
are common, but most are asymptomatic. Reovirus enters the
host by either the respiratory or enteric routes and infects
epithelium and associated lymphoid tissue (14). The reovirus
attachment protein,
1, plays a key role in targeting the virus to
distinct cell types, including those at mucosal surfaces (15–18).
Similar to the Ad fiber, reovirus
1 is an elongated trimer with
head-and-tail morphology (19–21). A domain in the fibrous tail
of serotype 3 Dearing (T3D)
1 binds to
␣
-linked sialic acid
(22–25), whereas the head binds to junctional adhesion molecule
1 (JAM1) (26). JAM1 is an Ig-superfamily member expressed by
a variety of cells including DCs (27) and epithelial and endo-
thelial barriers (28–30).
The structures of the Ad fiber (31) and reovirus
1 (32)
proteins are strikingly similar (Fig. 1). The two proteins are the
only structures known to date to form trimers by using triple

-spiral motifs. The fiber shaft most likely is composed entirely
of

-spiral repeats (31), whereas the
1 tail is predicted to also
contain an
␣
-helical coiled-coil N-terminal to the

-spiral region
(32). The head domains of both proteins are formed by eight
antiparallel

-strands with identical interstrand connectivity.
Therefore, although Ad and reovirus belong to different virus
families and have few overall properties in common, the ob-
served similarities between the attachment proteins and recep-
tors of these viruses suggest a conserved mechanism of binding.
Based on the structural similarities between Ad fiber and
reovirus
1, we engineered chimeric fiber-
1 attachment pro-
teins to exploit the JAM1- and sialic acid-binding properties of
1. Of those tested, only a near-full-length version of
1 grafted
onto the virion-insertion domain of Ad fiber (Fibtail-T3D
1)
formed trimers and assembled onto Ad particles. We show here
that when the fiber gene in the Ad5 genome is replaced with
Fibtail-T3D
1, the resulting virus, Ad5-T3D
1, is capable of
infecting intestinal epithelial cells expressing JAM1 and sialic
acid and primary human DCs expressing JAM1. These data
provide proof of principle for the development of chimeric Ad
vectors encoding reovirus
1 for gene delivery to mucosal
surfaces. This work also establishes a foundation for the use of
Ad-
1 chimeric viruses as a template to enable facile reverse
genetic manipulation of the reovirus attachment protein for
studies of virus–cell and virus–host interactions.
Methods
Cells, Antibodies, and Viruses. 293A (Q-BIOgene, Carlsbad, CA)
and Chinese hamster ovary (CHO) cells (American Type Cul-
ture Collection) were maintained as described (10). 633 cells, a
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: Ad, adenovirus; CAR, coxsackievirus and Ad receptor; DC, dendritic cell; T3D,
type 3 Dearing; JAM1, junctional adhesion molecule 1; CHO, Chinese hamster ovary; h,
human; CMV, cytomegalovirus; m, murine.
**To whom correspondence may be addressed. E-mail: terry.dermody@vanderbilt.edu or
mab@bcm.tmc.edu.
© 2004 by The National Academy of Sciences of the USA
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derivative of A549 cells expressing E1, E2A, and Ad5 fiber, were
provided by D. Von Seggern (The Scripps Research Institute, La
Jolla, CA) and maintained as described (33). Caco-2 cells
(American Type Culture Collection) were maintained in Alpha
minimum essential medium (GIBCO) with 20% FBS. Primary
human DCs (NHDC, Cambrex, Baltimore) were maintained
according to vendor protocol.
The human (h)CAR-specific mAb RmcB was purified from
CRL-2379 hybridoma cells (American Type Culture Collection).
The hJAM1-specific mAb J10.4 was provided by Chuck Parkos
(Emory University School of Medicine, Atlanta). Rabbit poly-
clonal serum 1561 was raised against the N-terminal region of
Ad5 fiber (peptide ARPSEDTFNPVY). The c-Myc-specific
mAb was purchased from PharMingen.
Ad vectors used in this study are based on the AdEasy system
(Q-BIOgene) and carry the full E1- and E3-deleted Ad5 genome
with the firefly luciferase gene, an internal ribosome entry site,
and the humanized Renilla GFP expressed from a cytomegalo-
virus (CMV) immediate-early promoter in the E1 region.
Generation of Chimeric Fiber-
1 Attachment Proteins. Fiber-
1 fu-
sion constructs were generated by using
phage red recombi-
nase (34) expressed in Escherichia coli strain BW25113兾pKD46
(35) obtained from the E. coli Genetic Stock Center (http:兾兾
cgsc.biology.yale.edu) as follows: Fibshaft-T3D
1, consisting of
the N-terminal 396 aa of Ad5 fiber fused to amino acid 292 of
T3D
1; Fib8-T3D
1, consisting of the N-terminal 170 aa of Ad5
fiber fused to amino acid 167 of T3D
1; and Fibtail-T3D
1,
consisting of the N-terminal 44 aa of Ad5 fiber fused to amino
acid 18 of T3D
1. Sequences encoding the reovirus T3D
1
protein flanked by a bovine growth hormone polyadenylation
signal and a zeocin-resistance gene were amplified by using Pfu
polymerase (Stratagene) and primers containing 39-nt over-
hangs homologous to the pCMVfiber plasmid. The pCMVfiber
plasmid, containing the Ad5 fiber gene expressed from a CMV
immediate-early promoter, was cotransformed with the PCR
product into the
phage red strain BW25113兾pKD46. Recom-
binants were selected by using zeocin-containing agar plates.
Fibtail-T3D
1 was subcloned into a plasmid containing se-
quences homologous to E4 and then recombined into the Ad5
genome to replace the fiber gene using red recombinase. To aid
in detection of the chimeric protein, two c-Myc tags (C2), and
one hexahistidine tag (H6) were added to the C terminus of the
chimera (Fibtail-T3D
1C2H6) before recombination. The re-
combinants were screened for loss of the fiber gene by restriction
endonuclease mapping and sequencing.
Protein Expression and Characterization. CHO cells were trans-
fected with plasmids encoding fiber-
1 chimeras by using Lipo-
fectamine-PLUS (Invitrogen), and cell extracts were harvested
for SDS兾PAGE. Immunoblots were performed as described (10).
Generation of a Chimeric Ad Vector. Linearized Ad genome en-
coding the Fibtail-T3D
1C2H6 chimera was transfected into 633
cells and maintained in the presence of 0.3
M dexamethasone
and 4
g兾ml polybrene. Virus was propagated, purified by CsCl
gradient centrifugation, and quantitated as described (36). The
resultant recombinant virus, Ad5-T3D
1, was amplified for a
final round by using 293A cells to remove any residual fiber from
newly assembled virions.
CsCl-banded Ad5, CAR-ablated biotinylated Ad [Ad5-BAP-
TR (10)], and Ad5-T3D
1 were precipitated with trichloroacetic
acid. Pellets were resuspended in loading buffer, and 4 ⫻ 10
10
particles per lane were resolved by SDS兾PAGE and immuno-
blotting. For total protein analysis, precipitated virus (1.5 ⫻ 10
11
particles per lane) was resolved by SDS兾PAGE, and gels were
stained with Coomassie blue.
Transduction of CHO Cells Transfected with Receptor Constructs.
CHO cells were transfected with plasmids expressing hCAR,
hJAM1, or murine (m)JAM1 (37, 38). After 48 h, the cells were
washed once with Hanks’ balanced salt solution (GIBCO) with
1% BSA (HBSS-BSA) and adsorbed with 5,000 particles per cell
of Ad5-T3D
1at4°C for 30 min. Cells were washed twice with
HBSS-BSA, and fresh medium was added. After incubation at
37°C for 24 h, cells were lysed, and luciferase activity (in lumens)
was measured as described (10).
Transduction of Caco-2 Cells and Primary DCs After Receptor Blockade.
Cells were harvested, washed with HBSS-BSA, and incubated in
suspension with 10
g兾ml of either hCAR-specific mAb RmcB
or hJAM1-specific mAb J10.4 at 4°C for 30 min. Alternatively,
cells were treated with 333 milliunits兾ml of Clostridium perfrin-
gens neuraminidase type X (Sigma) at 37°C for 30 min to remove
cell-surface sialic acid, followed by two washes with HBSS-BSA.
Cells then were adsorbed with 5,000 particles per cell of Ad5-
Fig. 1. Full-length models of Ad5 fiber (Upper) and reovirus
1(Lower). The three monomers within each trimer are shown in red, orange, and blue. Both
proteins have head-and-tail morphology, with an eight-stranded

-barrel domain forming the head. The Ad5 fiber shaft is predicted to consist of 21

-spiral
repeats (31). The Ad5 fiber model was generated by adding 17

-spiral repeats to the four present in the crystal structure of an Ad2 fragment, which also has
21

-spiral repeats (31). Sequence predictions suggest that
1 contains an N-terminal ⬇135-residue
␣
-helical coiled coil followed by eight

-spiral repeats and
the globular head domain (32, 49). The
1 model was generated by first adding five

-spiral repeats to the N terminus of the crystallized fragment (32). This model
then was joined with a 135-residue trimeric coiled coil formed by elongating an existing coiled-coil structure (50). The N-terminal 45 and 39 residuesoffiber and
1, respectively, are not included in the model, because they form a virion-anchoring structure (indicated by gray lines). The overall lengths of the fiber and
1
models are ⬇325 and 385 Å, respectively, which is consistent with data from electron microscopy studies. This figure was prepared by using
RIBBONS (51).
Mercier et al. PNAS
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T3D
1at4°C for an additional 30 min, washed twice, and seeded
onto 24-well plates in fresh medium. After incubation at 37°C for
24 h, cells were harvested for determination of luciferase activity.
Results
Design and Characterization of a Functional Fiber-
1 Chimera. Based
on the structural similarities between Ad5 fiber and reovirus
1
(Fig. 1), we engineered three Ad fiber-reovirus
1 chimeras with
increasingly larger portions of
1 protein replacing structurally
homologous regions of fiber (Fig. 2A). Fibshaft-T3D
1 contains
the N-terminal 21

-spiral repeats of fiber fused to the head
domain of T3D
1. Fib8-T3D
1 contains the N-terminal eight

-spiral repeats of fiber fused to the T3D
1

-spiral and head
domains. Fibtail-T3D
1 contains the N-terminal 44 aa virion-
anchoring domain (39) fused to T3D
1 lacking only the
N-terminal 17 amino acids. After transfection of CHO cells, each
of the chimeric attachment proteins was expressed, but only
Fibtail-T3D
1 formed trimers (Fig. 2B and data not shown),
suggesting that only this chimera maintains native folding.
Production and Characterization of an Ad Vector Expressing a Chi-
meric Fiber-
1 Attachment Protein. The Fibtail-T3D
1 gene was
recombined into an Ad5 genome lacking E1 and E3 to replace
the fiber gene by using
phage red recombinase (34). During the
cloning process, two c-Myc tags (C2) and one hexahistidine tag
(H6) were added to the C terminus of Fibtail-T3D
1 (Fibtail-
T3D
1C2H6) to facilitate protein detection. The resulting virus,
Ad5-T3D
1, was rescued by transfection and production in 633
fiber-expressing cells (33). After amplification in 633 cells, the
virus was passaged in 293A cells to eliminate fiber from the
virions and allow only Fibtail-T3D
1C2H6 to be encapsidated.
To determine whether Fibtail-T3D
1C2H6 was encapsidated
onto Ad5 virions, CsCl-purified Ad5, Ad5-BAP-TR, which
displays biotinylated fibers (10), and Ad5-T3D
1 were analyzed
by immunoblotting with antibodies specific for either the fiber N
terminus or the c-Myc epitope tag (Fig. 3A). Comparison of the
immunoblots demonstrated that Fibtail-T3D
1C2H6 was en-
capsidated onto Ad5 virions at levels similar to those of fiber on
Ad5 and Ad5-BAP-TR. As anticipated, the anti-c-Myc antibody
recognized both Ad5-BAP-TR and Ad5-T3D
1, which contain
c-Myc tags but not wild-type fiber. Coomassie blue staining
demonstrated that relative amounts of the capsid proteins of
wild-type Ad5 and Ad5-T3D
1 were indistinguishable (Fig. 3B).
Thus, Fibtail-T3D
1C2H6 is encapsidated onto Ad virions and
enables normal virion maturation.
Transient Transfection of CHO Cells with JAM1 Rescues Infection by
Ad5-T3D
1. To determine whether the chimeric Fibtail-T3D
1
attachment protein could bind to JAM1, CHO cells were
transfected with plasmids expressing hCAR, hJAM1, and
mJAM1 and tested for infection by luciferase-expressing Ad5-
T3D
1. CHO cells were chosen for these studies, because they
lack both CAR and JAM1 and are poorly infected by both Ad
and reovirus (38). Transduction of CHO cells by Ad5-T3D
1
was increased substantially by expression of either hJAM1 or
mJAM1 but not by expression of hCAR (Fig. 4A), the receptor
for Ad5 (2, 3). These data indicate that the JAM1-binding
domain of Ad5-T3D
1 is functional and can target JAM1-
expressing cells in a species-independent fashion.
Inhibition of Binding to JAM1 and Sialic Acid Blocks Ad5-T3D
1
Infection of Caco-2 Cells.
We next tested the capacity of hJAM1-
specific mAb J10.4 and C. perfringens neuraminidase to inhibit
transduction by Ad5-T3D
1. Caco-2 intestinal epithelial cells, a
model for enteric mucosal surfaces (40, 41), were used for these
experiments, because these cells express CAR, JAM1, and sialic
acid (26, 42). Transduction by Ad5-T3D
1 was inhibited 50% by
JAM1-specific mAb J10.4 and 80% by neuraminidase (Fig. 4B).
Combined treatment with both mAb J10.4 and neuraminidase
reduced transduction nearly 95%. In contrast, isotype-matched
hCAR-specific mAb RmcB, used as a negative control, did not
diminish luciferase transduction (Fig. 4B).
To ensure that JAM1-dependent transduction by Ad5-T3D
1
depends on
1 and not another Ad protein, we tested the
capacity of the T3D
1-specific mAb 9BG5 (24) to block
infection of Caco-2 cells. In contrast to T1L
1-specific mAb 5C6
(24), mAb 9BG5 inhibited transduction in a dose-dependent
fashion (data not shown). We noted a similar decrease in
transduction efficiency after incubation of Ad5-T3D
1 with
sialoglycophorin, which is known to interact with reovirus T3D
1 (22), before infection (data not shown). These results dem-
onstrate that transduction by Ad5-T3D
1 requires
1 and its
receptors, JAM1 and sialic acid.
Ad5-T3D
1 Transduces Primary Human DCs. DCs play important
roles in induction of adaptive immune responses (43). To
determine whether Ad5-T3D
1 is capable of transducing DCs,
we infected primary cultures of human DCs with Ad5 and
Ad5-T3D
1. DCs express JAM1 but not CAR (Fig. 5A), which
is consistent with previous observations (27). Transduction of
DCs by Ad5-T3D
1 was substantially more efficient than by Ad5
(Fig. 5B). Moreover, transduction was eliminated almost com-
pletely by treatment with hJAM1-specific mAb J10.4 (Fig. 5B).
These findings suggest that Ad5-T3D
1 may have utility for
transducing CAR-negative DCs at mucosal and other sites.
Fig. 2. Design and expression of chimeric fiber-
1 attachment proteins. (A)
Schematic diagram of the chimeric fiber-
1 attachment proteins described in
the text. Regions corresponding to fiber and
1 in the diagrams are shaded
black and gray, respectively (not drawn to scale). Fiber tail, which mediates
virion anchoring, is represented as a small cylinder, the
␣
-helical coiled coils as
small ovals, the

-spiral repeats as large cylinders, and the head domain as
three large ovals. (B) Immunoblots of denatured (boiled) and native (un-
boiled) lysates of CHO cells transfected with plasmid expressing Fibtail-T3D
1
probed with a serum (1561) that recognizes the N-terminal region of Ad5
fiber.
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Discussion
In this study, we fused two structurally homologous viral attach-
ment proteins, Ad fiber and reovirus
1, to produce a functional
chimeric virus, Ad5-T3D
1. Of the three fiber-
1 chimeras tested,
only Fibtail-T3D
1 bearing the Ad5 fiber virion-insertion domain
fused to an almost-full-length version of T3D
1 protein formed
trimers and assembled onto Ad virions. The lack of trimerization of
Fib8-T3D
1 and Fibshaft-T3D
1 was surprising, because both the
head and tail regions of
1 contain trimerization domains (44),
whereas the fiber knob domain initiates and maintains trimeriza-
tion (45). Because only Fibtail-T3D
1 formed trimers, it is likely
that the C-terminal trimerization domain of
1 is insufficient for
trimerization of the fiber shaft. Alternatively, it is possible that the
chimeric Fib8-T3D
1 and Fibshaft-T3D
1 proteins do not form
trimers, because the fused

-spiral junctions are imperfectly
matched.
In Ad5-T3D
1 virions, Fibtail-T3D
1 was encapsidated at levels
comparable with wild-type fiber. Furthermore, the capsid protein
profile of Ad5-T3D
1 is identical to that of wild-type Ad5. Most
importantly, experiments using receptor-transfected cells, anti-
bodies, and reagents that block
1–sialic acid interactions provide
Fig. 3. Characterization of Ad5-T3D
1. Ad5 virions expressing wild-type fiber (Fiberwt), CAR-ablated biotinylated fiber (Fiber-BAP-TR) (10), and Fibtail-
T3D
1C2H6 were precipitated with trichloroacetic acid. (A) Precipitated particles (4 ⫻ 10
10
per lane) were resolved by SDS兾PAGE and immunoblotted with
anti-c-Myc mAb 9E10 or antiserum 1561, which recognizes the N-terminal region of Ad5 fiber. (B) Precipitated particles (1.5 ⫻ 10
11
per lane) were resolved by
SDS兾PAGE and stained with Coomassie blue.
Fig. 4. Ad5-T3D
1 transduction is mediated by JAM1 and sialic acid. (A) CHO cells weretransiently transfected with plasmids encoding hCAR, hJAM1, or mJAM1.
After 48 h of incubation to permit receptor expression, cells were adsorbed with 5,000 particles per cell of Ad5-T3D
1 and harvested 24 h later for luciferase assay.
Transduction was measured in lumens. (B) Caco-2 cells were either untreated or treated with 10
g兾ml hCAR-specific mAb RmcB (CAR mAb), 10
g兾ml
hJAM1-specific mAb J10.4 (JAM1 mAb), 333 milliunits兾ml C. perfringens neuraminidase (NM), or both JAM1 mAb and neuraminidase. Cells were adsorbed with
5,000 particles per cell of Ad5-T3D
1 and harvested 24 h later for luciferase assay. Transduction was measured in lumens. The results are presented as the means
for three independent experiments. Error bars indicate SD. A paired Student’s t test was performed to compare transduction of transfected or treated cells versus
mock or untreated cells (
*
, P ⬍ 0.01;
**
, P ⬍ 0.05; ns, not significant).
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MICROBIOLOGY
compelling evidence that Ad5-T3D
1 displaying Fibtail-T3D
1
retains both the JAM1- and sialic acid-binding functions of the T3D
1 protein.
We envision at least four applications for chimeric Ad vectors in
which the CAR-binding functions of fiber have been replaced with
the JAM1- and sialic acid-binding functions of
1. First, Ad vectors
based on fiber-
1 chimeras may serve to efficiently target mucosal
sites for enhanced induction of immune responses at mucosal
surfaces. Second, because JAM1 and sialic acid are expressed on a
variety of cells, Ad5-T3D
1 and its derivatives may have utility for
transducing cells deficient in CAR (e.g., DCs and certain types of
cancer cells). Third, because
1 incorporates its own trimerization
motifs, fiber-
1 fusions may provide a trimeric scaffold for the
display of other cell-targeting ligands in a manner analogous to
fiber-fibritin chimeras (46). In support of this approach, we recently
appended single-chain antibodies onto truncated forms of Fibtail-
T3D
1 (unpublished data). Fourth, Ad vectors based on Ad5-
T3D
1 can be used as a simple genetic platform for directed
mutagenesis of
1 for studies of reovirus tropism and receptor-
linked signaling.
The opportunity to use Ad vectors encoding fiber-
1 chimeras
for mucosal targeting is especially appealing. Increased delivery of
antigens to intestinal epithelial cells and Peyer’s patch lymphocytes
by such vectors might result in more potent and less toxic gene-
based vaccines. Reovirus binds to murine microfold cells (15, 16,
18), and the
1 protein plays an important role in conferring this
tropism (18, 47). Interactions of Ad5-
1 vectors with microfold cells
may facilitate efficient delivery to underlying Peyer’s patches for
induction of immune responses in the gut. Alternatively,
1-bearing
Ad vectors may directly infect DCs at the luminal surface, which are
known to shuttle bacteria across epithelial monolayers by opening
tight junctions and sampling the intestinal lumen (48). DCs express
tight junction proteins, including JAM1 (27), which are hypothe-
sized to facilitate epithelial barrier penetration. Our finding that
Ad5-T3D
1 transduces primary DCs more efficiently than wild-
type Ad5 suggests that Ad5-
1 vectors may be useful for antigen
gene delivery to DCs in the intestine and other sites.
Findings described in this report indicate that Ad vectors can be
efficiently retargeted to cells expressing JAM1 and sialic acid by the
reovirus attachment protein
1. By virtue of the capacity to infect
both intestinal epithelial cells and DCs, Ad5-
1 vectors may have
utility in the induction of immune responses at mucosal surfaces
and thus prevention of infection at the site of pathogen entry. These
vectors also will allow a precise determination of the contribution
of the JAM1- and sialic acid-binding properties of
1 to interactions
of
1 with cells in vivo. This approach should lead to improved Ad
vectors for gene delivery and enhance an understanding of
1
biology.
We thank Mary E. Barry and Jared Abramian for excellent technical
assistance; members of the Barry and Dermody laboratories for many useful
discussions; Chuck Parkos for providing hJAM1-specific mAb J10.4; and
Dan Von Seggern for providing the 633 cells. This work was supported by
National Science Foundation IGERT Award DGE-0114264 (to G.T.M.),
Public Health Service Awards T32 CA09385 (to J.A.C.), T32 HL07751 (to
J.D.C.), R01 GM67853 (to T.S. and T.S.D.), R01 AI38296 (to T.S.D.), and
R01 AI42588 (to M.A.B.), and the Elizabeth B. Lamb Center for Pediatric
Research. Additional support was provided by Public Health Service
Awards AI36211 for the Center for AIDS Research at Baylor College of
Medicine, DK056338 for the Texas Gulf Coast Digestive Diseases Center
(Baylor College of Medicine), CA68485 for the Vanderbilt Cancer Center,
and DK20593 for the Vanderbilt Diabetes Research and Training Center.
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