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Adenovirus serotype 5 infects human dendritic cells
via a coxsackievirus–adenovirus receptor-
independent receptor pathway mediated by
lactoferrin and DC-SIGN
William C. Adams,1,2,3Emily Bond,1,2Menzo J. E. Havenga,4
Lennart Holterman,5Jaap Goudsmit,5Gunilla B. Karlsson Hedestam,2,6
Richard A. Koup3and Karin Lore ´1,2
Karin Lore ´
1Center for Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden
2The Swedish Institute for Infectious Disease Control, Stockholm, Sweden
3Immunology Laboratory, Vaccine Research Center, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD, USA
4TNO Biosciences, Leiden, The Netherlands
5Crucell Holland, Leiden, The Netherlands
6Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
Received 29 October 2008
Accepted 10 March 2009
The coxsackievirus–adenovirus receptor (CAR) is the described primary receptor for adenovirus
serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene
therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as
freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are
susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression.
Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly
decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of
these cells. Lactoferrin (Lf), an abundant protein in many bodily fluids known for its antiviral and
antibacterial properties, promoted rAd5 infection in all cell populations except plasmacytoid DCs
using a CAR-independent process. Lf caused phenotypic differentiation of the DCs, but cell
activation played only a minor role in the increase in infection frequencies. The C-type lectin
receptor DC-SIGN facilitated viral entry of rAd5–Lf complexes and this was dependent on high-
mannose-type N-linked glycans on Lf. These results suggest that Lf present at high levels at
mucosal sites can facilitate rAd5 attachment and enhance infection of DCs. A better
understanding of the tropism and receptor mechanisms of Ad5 may help explain Ad5
pathogenesis and guide the engineering of improved rAd vectors.
Adenoviruses are frequent causes of acute upper respir-
atory tract infections and can be responsible for ocular and
gastrointestinal illnesses in humans. They have also been
exploited for the development of replication-incompetent
viral vectors because their genomes allow large inserts of
expression cassettes, thereby offering efficient gene deliv-
ery. Recombinant adenoviruses (rAds), serotype 5 (rAd5)
in particular, are commonly used vectors for gene therapy
and vaccination trials (Barouch & Nabel, 2005; McConnell
& Imperiale, 2004; Tatsis & Ertl, 2004). The primary
receptor described for infection of most Ad species (A, C,
D, E and F) is the coxsackievirus and adenovirus receptor
(CAR) (Bergelson et al., 1997; Roelvink et al., 1998; Tomko
et al., 1997). As currently understood, infection of Ad5
(species C) involves binding of the viral fiber knob to CAR
on the target cells (Roelvink et al., 1996), followed by an
interaction between the viral penton base with avintegrins
on the cell surface (Wickham et al., 1993). This allows
virus–receptor complexes to enter clathrin-coated pits via
endocytosis (Li et al., 1998; Wang et al., 1998; Wickham et
al., 1993). Efficient infection of Ad5 via CAR has mainly
been demonstrated in vitro using immortalized cell lines
with well-exposed CAR. However, CAR is naturally
expressed in tight junctions between cells and therefore
A supplementary figure showing that Lf can mediate rAd5 infection via
langerin is available with the online version of this paper.
Journal of General Virology (2009), 90, 1600–1610
1600 008342Printed in Great Britain
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has limited accessibility on the apical cell surface (Cohen et
al., 2001). As a likely consequence of this, CAR has been
shown to play a minor role in Ad5 infection of epithelial
cells (Johansson et al., 2007), hepatocytes (Smith et al.,
2003), fibroblasts (Hidaka et al., 1999; Leopold et al., 2006)
and antigen-presenting cells (APCs) such as dendritic cells
(DCs) (Cheng et al., 2007; Lore et al., 2007; Rozis et al.,
2005; Tillman et al., 1999). Thus, CAR-independent
infection pathways used by Ad5 have been proposed. For
example, heparan sulfate glycosaminoglycans have been
demonstrated to mediate Ad5 infection (Cheng et al., 2007;
Dechecchi et al., 2001, 2000; Xie et al., 2006). As opposed
to the fiber knob, the hexon of Ad5 was shown to be able to
directly bind the coagulation factor X and mediate Ad5
infection of the liver (Waddington et al., 2008). Moreover,
lactoferrin (Lf), an iron-binding protein present at mucosal
sites and in many bodily fluids, was shown to facilitate
binding of Ad5 to human epithelial cells and enhance
infection via a CAR-independent manner using a yet
undefined receptor (Johansson et al., 2007). Interestingly,
Lf is mainly known for its immunomodulatory properties,
which enhance anti-microbial and anti-inflammatory
responses (Legrand et al., 2005). High levels of Lf present
during inflammation were recently shown to directly
recruit and activate DCs (de la Rosa et al., 2008; Spadaro
et al., 2008).
The receptor pathways used for Ad5 entry into DCs are
poorly understood. We have previously demonstrated that
primary human CD11c+myeloid DCs (MDCs) and
CD123+plasmacytoid DCs (PDCs) are susceptible to
rAd5 and are able to present rAd5-encoded antigen to
activate specific T-cell responses (Lore et al., 2007). In this
study, we investigated the involvement of CAR and the
influence of Lf in rAd5 infection in relevant primary
human APC populations from both blood and skin.
Isolation of blood DCs. This study was approved by the ethical
committee at the Karolinska Institutet, Stockholm, Sweden, and the
NIH, Bethesda, MD, USA. Our sorting procedures for direct isolation
of DCs from blood have been described previously (Lore et al., 2003,
2005, 2007; Smed-Sorensen et al., 2005). DCs were cultured in
complete medium composed of RPMI 1640 and 10% fetal calf serum
(FCS; Sigma-Aldrich). To maintain viability, the medium for PDCs
and MDCs was supplemented with interleukin (IL)-3 (1 ng ml21;
R&D Systems) or granulocyte–monocyte colony-stimulating factor
(GM-CSF, 2 ng ml21; PeproTech), respectively.
In vitro differentiation of DCs from monocytes. Monocyte-
derived dendritic cells (MDDCs) were prepared as described
previously (Lore et al., 1998) with some modifications. Adherent
monocytes were cultured for 6 days in complete medium supple-
mented with IL-4 (150 ng ml21; R&D Systems) and GM-CSF
(300 ng ml21) to obtain .90% CD1a+CD142immature DCs.
Human monocyte enrichment. Adherent monocytes were collected
by cell scraper and cultured in complete medium to obtain enriched
monocyte cultures (.90% CD14+).
Isolation of skin DC subsets. Skin was collected from patients
undergoing breast-reconstruction surgery (Karolinska University
Hospital, Stockholm, Sweden). Skin DCs were isolated as described
previously with some modifications (Lore et al., 1998). Following
surgery, skin was processed with a skin graft mesher (Zimmer).
Meshed skin was incubated with dispase (3.4 U ml21in PBS; Gibco)
for 90 min at 37 uC before the dermis and epidermis were separated
with forceps. The epidermis and dermis were incubated separately in
complete medium (with 40 ng GM-CSF ml21) for 48 h at 37 uC to
induce the migration of epidermal Langerhans cells (LCs; CD1a+,
HLA-DR+) and dermal DCs (dDCs; CD1a+/2, HLA-DR+),
respectively. All experiments were carried out on freshly harvested
Phenotypic characterization of DCs. Cells were harvested, washed
in PBS supplemented with 2% FCS and surface stained with different
combinations of anti-CD1a, anti-CD11c, anti-CD123, anti-CD14,
anti-CD80, anti-CD86, anti-HLA-DR (BD Biosciences), anti-CAR
(Beckman-Coulter) monoclonal antibodies (mAbs) for 15 min at
4 uC and then washed. The cells were collected on a FACSCalibur
flow cytometer (BD Biosciences) and all data were analysed using
FlowJo software (version 8.5; Treestar).
Production of Ad construct. E1/E3-deleted, replication-incompet-
ent rAd5 or Ad35 vectors were generated in PER.C6/55K cells as
described previously (Barouch et al., 2004; Havenga et al., 2001). Viral
titres were determined by high performance liquid chromatography
and infectivity was assessed by plaque assays using PER.C6/55K cells.
rAd5 and rAd35 infection of cells. Cell populations were cultured
at 16106cells ml21(with ¢36105cells per tube) at 37 uC in
polystyrene round-bottomed tubes (BD Labware) and exposed to
either rAd5 or rAd35 encoding green fluorescent protein (rAd35–
GFP) at the indicated inoculums of infectious particles (i.p.) per cell
for 24 h. Infections were evaluated using flow cytometry.
Lf and transferrin treatment of rAd. rAd5 or rAd35 was incubated
with human Lf from milk or recombinant human Lf produced in rice
(HLf), bovine Lf from milk (BLf) or human transferrin (HTf) (Sigma-
Aldrich) at the indicated concentrations for 3 h at 25 uC before
exposure to cells. In the indicated experiments, the various Lfs or HTf
were added simultaneously with rAd to the cells.
Stimulation of MDDCs. MDDCs were stimulated with Lf or
lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma-
Aldrich) for 24 h and evaluated for CD86 expression using flow
Endotoxin removal of Lf stocks. Endotoxin contents were reduced
in all protein stocks using 1% Triton X-114 extraction (Calbiochem).
HLf and BLf stocks at 1 mg ml21contained ¡1.25 and ¡0.125 ng
endotoxin ml21, respectively, levels that were reduced 10-fold by this
procedure. The final concentrations of endotoxin in the Lf/rAd5
mixes added to DC cultures were 23-fold more diluted. Endotoxin
was measured using a Limulus amoebocyte lysate assay.
rAd blocking assays. Cells were pre-treated with anti-CAR (clone
RmcB, 20 mg ml21; Upstate), anti-CD46 (clone 13/42, 5 mg ml21;
BMA Biomedicals), anti-DC-SIGN (clone 120507, 10 mg ml21; R&D
Systems) or anti-langerin (clone DCMG4, 20 mg ml21, Beckman-
Coulter) mAbs, mannan (200 mg ml21; Sigma-Aldrich) or heparin
sodium salt (200 mg ml21; Sigma-Aldrich) for 1 h at 37 uC before
exposure to rAd for 24 h.
denatured HLf or BLf (5 mg) was incubated with 15 mU endoglyco-
digestionofN-linkedcarbohydrates. Native or
Lactoferrin mediates rAd5 infection of human DCs
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