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*
Department of Immunohematology and Blood Bank and
Department of Cell Biology, Leiden University Medical Center, Leiden, The Netherlands; and
Crucell, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC loaded ex vivo with tumor Ags and administered in vivo to tumor-bearing hosts induce T cell responses responsible for the regression or eradication of established tumors both in mice (12, 13, 14, 15) and in human beings (16, 17, 18). In addition, DC are the best APC for ex vivo expansion of tumor-specific CTL in T cell-based immunotherapy (19). Multiple approaches have been designed to deliver Ags to DC such as loading with peptides, full-length proteins, and tumor cell lysates, transfection with DNA and RNA, fusion between DC and tumor cells, and gene transfer via recombinant viral vectors (20, 21). Genetic modification of DC with recombinant viruses offers major advantages including persistent Ag presentation over time and exposure to potentially immune-activating viral components. We have previously shown that subgroup C serotype 5 recombinant adenoviruses (rAd5) are attractive vectors for gene transfer into human DC because of their synergistic action with maturation signals for the generation of highly immunostimulatory APC (22).
To date, 51 serotypes of human Ad have been identified and divided into
six subgroups from A to F (23). Their entry into
susceptible cells is a two-step process consisting of virus attachment
to the membrane via the Ad fiber knob (24), followed by
internalization upon binding of the penton base RGD motifs to cellular
integrins (25). Although the penton-integrin interactions
may represent a common pathway for internalization (26),
attachment strategies vary among Ad subgroups and play an important
role in dictating Ad tropism. In this regard, the fiber is a crucial
mediator for high-efficiency binding to target cells. Subgroup C fibers
use the coxsackie-adenovirus receptor (CAR) as high affinity cellular
ligand (27). In CAR-deficient cells, Ad5 attachment occurs
at much lower efficiency through alternative pathways involving
interactions between the fiber and the MHC class I heavy chain 
2
domain or between the penton and cellular integrins
(28, 29, 30). Subgroup A, D, E, and F fibers bind to soluble
recombinant CAR, but whether CAR is their physiologically relevant
receptor is not clear (31). Finally, subgroup B fibers do
not bind CAR and bind poorly to MHC class I molecules (28, 31).
Because human DC lack CAR, prolonged exposure to high amounts of subgroup C rAd are required to achieve significant gene transfer (22, 32, 33). Although high virus doses allow the generation of transgene-specific T cell responses (33, 34, 35), they prevent the broad application of rAd5-infected DC as therapeutic vaccines in the clinic. Therefore, creating new rAd vectors with increased DC tropism is essential. Toward this goal, we constructed a library of rAd5 vectors modified to express the tail of the Ad5 fiber (F5) fused with the shaft and knob of fibers from other Ad serotypes. We show that compared with parental rAd5, rAd5 with chimeric subgroup B fibers allow an up to 100-fold increase in gene transfer and expression in immature and mature DC, while not impairing the immune-activating properties of rAd5. Consequently, DC infected with these novel vectors are highly potent in presenting transgene-encoded epitopes to CTL.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Monocytes were purified from human PBMC by positive selection
with CD14 microbeads (Miltenyi Biotec, Auburn, CA). Monocytes (>95%
pure) were cultured in RPMI 1640 (Life Technologies, Grand Island, NY)
with 2 mM glutamine, 10% FCS, 800 U/ml GM-CSF (Leukomax; Novartis
Pharmaceuticals, East Hanover, NJ), and 500 U/ml IL-4 (PeproTech, Rocky
Hill, NJ). After 6 days, cells developed into typical immature DC being
CD14-, CD1a+,
CD11c+, CD80+,
CD86+, HLA-DR+, HLA class
I+, and CD83- (data not
shown). In some experiments, immature DC were generated from monocytes
cultured in medium supplemented with 2% autologous plasma instead of
10% FCS. For maturation, DC were incubated with 200 ng/ml of LPS from
Escherichia coli (Sigma, St. Louis, MO) for 48 h. In
some experiments, 50 µg/ml poly(I:C) (Sigma), 30%
monocyte-conditioned medium (MCM), 100 ng/ml TNF- (PeproTech), 250
ng/ml agonistic anti-CD40 Ab (B-B20) (Cymbus Bioscience
Laboratories, Chandlers Ford, U.K.), or 100 IU/ml IFN- (PeproTech)
were used.
Plasmid and cosmid constructs
The plasmid pBr/Ad.Bam-rITR contains the Ad5 sequence from the
BamHI site (nt 21562) until the 3' end. This plasmid was
used to delete the fiber sequence from nt 31042 to nt 32787. For this
purpose, a PCR was performed with the oligonucleotides NY-up
5'-CGACATATGTAGATGCATTAGTTTGTGTTATGTTTCAACGTG-3',
which contains a NdeI and a NsiI site (in bold),
and NY-down 5'-GGAGACCACTGCCATGTTG-3'. The 2200-bp PCR fragment was
digested with SbfI (just upstream NY-down) and
NdeI, and cloned into a SbfI and
NdeI-digested pBr/Ad.Bam-rITR. Thus the resulting plasmid
(pBr/Ad.BamR
FIB) lacks part of the fiber starting from the
NdeI site until the stop codon, but instead contains a
unique NsiI site directly after the fiber stop codon. The
restriction sites NdeI and NsiI were introduced
into the tail of degenerate oligonucleotides for amplification of fiber
sequences from other Ad serotypes and for subsequent cloning into
pBr/Ad.BamRFIB. All human wild-type Ad (serotype 1–50) except for
types 8, 40, and 41 were propagated on PER.C6 (36), after
which virus DNA was isolated from crude lysates as previously described
(37). The plasmid library generated was named
pBr/Ad.BamRFIBXX where XX represents the serotype number from which
the fiber was amplified. The amplified sequences inserted into
pBr/Ad.BamRFIBXX constructs were sequenced to confirm the existence
of an open reading frame. Protein sequences were aligned, and putative
fiber domains such as the trimerization domain (tlwt) were localized.
To maximize the chances of generating fiber chimeric rAd, an additional
cloning step was performed. All pBr/Ad.BamRFIBXX constructs and
pBr/Ad.AflIII-BamHI were digested with
BamHI and PacI. The isolated fragments were
cloned into a PacI-digested pWE.pac cosmid using a packaging
kit (Stratagene, La Jolla, CA). The cosmid pWE.pac was generated from
pWE15 (Clontech Laboratories, Palo Alto, CA) by inserting into the
EcoRI sites a synthetic DNA fragment containing a
PacI restriction site. Cosmids obtained were verified by
restriction enzyme digestion. The cosmid library generated was named
pWE/Ad.AflIII-rITR.pac/FIBXX.
Generation of rAd5 chimeric for the fiber protein
To generate rAd, two DNA molecules were cotransfected in PER.C6 cells: pWE/Ad.AflIII-rITR.pac/FIBXX digested with PacI and a plasmid encoding Ad5 sequences 1–454 and 3511–6095 in which the E1 region is replaced by a marker gene. This plasmid named either pCLIP or pADAPT contains Ad5 sequence from nt 1 to 454 (left ITR and packaging signal), a cassette for transgene expression containing the CMV promoter (nt -601 to -14 for pCLIP, nt -735 to +95 for pADAPT), a polylinker, the SV40 intron/poly(A) sequence from pcDNAI (HhaI-AvrII fragment, Invitrogen, San Diego, CA), and a second Ad5 sequence ranging from nt 3511 to nt 6095. pADAPT lacks the SV40 intron sequences. The Ad5 sequence nt 3511–6095 enables the generation of rAd through homologous recombination with pWE/Ad.AflIII-rITR.pac/FIBXX. The recombinant vectors were purified by double cesium chloride density centrifugation, aliquoted, and stored at -80°C until further use. The number of virus particles (VP)/ml was determined by HPLC as described (38). The production yields in VP/ml for green-fluorescent protein (GFP)-encoding vectors were: rAd5GFP: 8.4 x 1011 and 5.1 x 1011 VP/ml (two batches), rAd5F16GFP: 4.8 x 1011 and 5.1 x 1011 VP/ml (two batches), rAd5F35GFP: 7.8 x 1011 and 4.9 x 1011 VP/ml (two batches), rAd5F40LGFP: 8.6 x 1011 VP/ml (one batch), rAd5F50GFP: 1.1 x 1012 VP/ml (one batch), rAd5 human (h)gp100: 5.1 x 1011 VP/ml (one batch), and rAd5F35hgp100: 7.1 x 1011 VP/ml (one batch).
Infection of DC with rAds
DC (105) were incubated with viral doses ranging from 103 to 105 VP/DC on ice in serum-free medium, unless otherwise indicated. After 1 h, the cells were extensively washed and shifted at 37°C. Gene transfer efficiency was assessed 24 h later by measurement of luciferase (Luc) activity with a Promega (Madison, WI) luciferase assay kit on a luminometer or by measurement of GFP fluorescence by flow cytometry (FACSCalibur; Becton Dickinson, Mountain View, CA). In the former case, the results were normalized for the number of cells present during infection. Cell viability was determined by trypan blue exclusion or by propidium iodide staining. In coculture experiments, DC (105) were mixed with primary skin-derived fibroblasts (105). The cells were incubated with 109 VP total for 15 min or 2 h at 37°C. After 24 h, CD11c staining was performed to discriminate between CD11c+ DC and CD11c- fibroblasts, and GFP expression was measured by flow cytometry.
Quantification of Ad genomes by real-time PCR
The principle of real-time PCR has been described by Klein et
al. (39). Briefly, genomic and Ad DNA were extracted
simultaneously from cells using a DNAeasy Tissue Kit (Qiagen,
Chatsworth, CA). The DNA concentrations were determined with a
Picogreen dsDNA quantification kit (Molecular Probes, Eugene, OR). To
test for the amount of Ad genomes present, the following primers were
used: Ad5Clip-F: 5'-CGACGGATGTGGCAAAAGT-3', Ad5Clip-R:
5'-CCTAAAACCGCGCGAAAA-3'. To detect amplification, a probe (Ad5Clip-Pr:
5'-CACCGGCGCACACCAAAAACG-3') carrying a fluorescent group
(6-carboxyfluorescein, FAM) and a quencher dye
(6-carboxytetramethylrhodamine, TAMRA) was used. Both the primers and
the probe were obtained from Perkin-Elmer (Norwalk, CT). To quantify
the amount of Ad genomes per cell, a second pair of oligonucleotides
and a probe recognizing 18SrDNA was added to the reaction
(40). The amplification mixture contained 1x buffer A, 3
mM MgCl2, 200 µM dATP, dCTP, dGTP, and dTTP, 90
nM concentrations of each Ad primer, 100 nM of each 18S rDNA primer,
200 nM concentrations of both probes, 0.6 U Amplitaq Gold polymerase
(all obtained from Perkin-Elmer), and 5 µl of DNA. The reaction was
initiated by a 10-min 95°C predenaturation step. Amplification
occurred during 45 cycles, each cycle consisting of 15 s at 95°C
and 1 min at 60°C. As a standard for the estimation of the amount of
Ad genomes present per sample, a plasmid was used that contains 
5000
bp of the left part of the Ad serotype 5 genome (pADAPT). As a standard
for cellular DNA, genomic DNA extracted from human 293 cells was
used.
Analysis of DC phenotype by flow cytometry
DC were stained on ice with mouse mAb for 30 min in PBS-1% FCS
and analyzed by flow cytometry. The following mAbs were used:
PE-anti-CD1a (HI149), FITC-anti-CD80 (BB1), PE-anti-CD86
(FUN-1), FITC-anti HLA-A,B,C (G46–2.6) (all obtained from PharMingen,
San Diego, CA), PE anti CD11c (Leu-M5), PE anti-CD14 (L243),
FITC-anti-HLA-DR (m
P9), PE- and FITC-conjugated isotype controls
(all obtained from Becton Dickinson), and PE-anti-CD83 (Immunotech,
Westbrook, ME).
Cytokine detection by ELISA
Culture supernatants were analyzed in serial 2-fold
dilutions in duplicate. Human IL-12p70 was detected with by solid phase
sandwich ELISA (Diaclone Research, Besançon, France)
(sensitivity, 3 pg/ml). Human IL-10 was detected with a Pelikine
compact ELISA kit (CLB, Amsterdam, The Netherlands) (sensitivity, 3
pg/ml). IFN-
was detected using a capture rat anti-mouse IFN-
mAb (R4-6A2) and a detection biotinylated rat anti-mouse mAb
(XMG1.2) (both obtained from PharMingen).
HLA-A2-restricted hgp100-specific CTL and Ag presentation assays
HLA-A2*0201/Kb
(A2Kb) transgenic mice (provided by L. Sherman,
Scripps Laboratories, San Diego, CA) were immunized three times by i.v.
injection of 107 PFU of a recombinant ALVAC
canary pox virus encoding for hgp100 (Virogenetics, Troy, NY). Ten days
after the last injection, spleen cells were cultured with LPS blasts
loaded with the hgp100154–162 HLA-A2-restricted
peptide. After three rounds of stimulation, hgp100 recognition by T
cells was assessed by standard 51Cr release
assay, and the T cells were cloned by limiting dilution. The 8J CTL
clone specific for the hgp100154–162
HLA-A2-restricted epitope recognizes human
HLA-A2+ and murine A2Kb+
cell lines expressing hgp100. For Ag presentation assays,
HLA-A2+ DC infected with rAd5hgp100 or
rAd5F35hgp100 were incubated with LPS for 48 h or left untreated.
Uninfected DC and DC infected with rAd5GFP and rAd5F35GFP were taken
along as negative controls. hgp100-specific CTL
(104) were cultured with different amounts of DC
in triplicate in 96-well U-bottom plates, and supernatants were
collected 24 h later to measure T cell-derived IFN-
production.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the search for rAd with increased tropism for human DC, we
constructed a library of 19 rAd5 vectors encoding the Luc reporter gene
and expressing fibers from subgroup A (F12), B (F16, F35, F50), D (F8,
9, 10, 17, 24, 27, 30, 32, 33, 38, 45, 47, 49), and F (both the
long (F40L) and the short (F40S) fiber) viruses. The N-terminal part of
the Ad5 tail was conserved to ensure proper interaction with the Ad5
penton, whereas the shaft, whose length is thought to interfere with
the binding of the penton to v integrins (41), and the
head, which carries receptor specificity, were exchanged (Fig. 1
). Immature DC were incubated with rAd5
or with fiber-modified rAd5 vectors at 105 viral
particles (VP)/DC and were analyzed for Luc activity 24 h later.
Unlike vectors carrying fibers from subgroups A, D, and F Ad, rAd5 with
fibers from subgroup B viruses type 16, 35, and 50 mediated a level of
Luc activity 4- to 9-fold superior to that obtained with rAd5 (Fig. 2).
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The differential gene transduction properties of rAd5 and
rAd5 carrying types 16, 35, and 50 fibers were characterized at the
single cell level using GFP-encoding vectors. rAdF40L was taken along
as a control for the fiber-exchange strategy. Immature DC were
incubated with viral doses ranging from 103 to
105 VP/DC at 0°C. At low temperatures, virus
attachment occurs but not internalization. After 1 h, the cells
were extensively washed to remove unbound viruses and shifted at 37°C
to allow virus entry. DC were analyzed for GFP expression 24 h
later. Viable cell recovery was similar in uninfected and infected
cultures. Overall, the transduction patterns of GFP-encoding viruses
and Luc-encoding vectors were comparable. The percentage of
GFP+ DC detected after infection with rAd5F16,
rAd5F35, or rAd5F50 was markedly higher than that obtained with rAd5 at
all virus doses tested, and rAd5F40L failed to induce GFP expression
(Fig. 3A). At
105 VP/DC of rAd5, only 20% of DC expressed GFP,
whereas at a lower virus dose the percentage of
GFP+ DC was decreased to <5%. In sharp
contrast, 105 VP/DC of rAd5F16, rAd5F35, or
rAd5F50 conferred GFP expression in up to 90% of the cells and at
103 VP/DC, 20% of GFP+ DC
were still present. Especially rAd5F35 was the most efficient of the
three chimeric vectors. This was not only apparent from the percentage
of GFP+ cells but also from the GFP expression
levels achieved (Fig. 3B). Similar results were obtained
with 
-galactosidase-encoding vectors (data not shown). Notably, DC
that are to be administered to patients for vaccination purposes cannot
be grown in medium containing FCS, but instead have to be cultured in
serum-free medium or medium supplemented with autologous human plasma.
We found that immature DC generated in medium containing FCS vs
autologous plasma were equally susceptible to rAd5F35, an important
feature with respect to the clinical applicability of DC engineered by
means of subgroup B fiber-modified adenoviral vectors (data not shown).
To conclude, rAd5 carrying the subgroup B fibers type 16, 35, and 50
show an increased tropism for immature DC, and transgene expression
requires 10- to 100-fold less virus than with unmodified rAd5.
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Mature DC have developed resistance to infection by certain
viruses (6). Thus we analyzed the susceptibility of mature
DC to subgroup B fiber-modified rAd5. LPS-treated DC were incubated
with the different GFP-encoding vectors as described for immature
cells. No significant cell death was observed with either vector. The
number of GFP+ LPS-treated DC (Fig. 4A) and the levels of GFP
expression (Fig. 4B) with rAd5F16, rAd5F35, and rAd5F50
largely exceeded that obtained with rAd5 at all virus doses tested. No
GFP was found in cells incubated with rAd5F40L. At
105 VP/DC, only 34% of rAd5-infected LPS-treated
cells expressed GFP, whereas with rAd5F16, rAd5F35, and rAd5F50, GFP
was detected in >90% of the cells. Upon a 10- to 100-fold decrease of
the virus dose, the number of rAd5-infected GFP+
DC went down to <5%. In contrast, at 103 VP/DC,
GFP was present in over 50% of DC infected with rAd5F16, rAd5F35, and
rAd5F50. Therefore, rAd5 carrying subgroup B fibers type 16, 35, and 50
equally transduce mature DC, and transgene expression requires at least
100-fold less viral particles than with unmodified rAd5.
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The results obtained with immature and mature DC suggest that the
transduction levels achieved by the three subgroup B fiber-modified
vectors depend on the DC maturation stage. Therefore, we compared
immature DC and DC incubated with a panel of maturation agents for
their susceptibility to subgroup B fiber-modified rAd5-mediated gene
expression. The number of GFP+ DC was increased
when the cells were exposed to LPS, TNF-, MCM, poly(I:C), or
agonistic anti-CD40 mAb before infection with subgroup B
fiber-carrying rAd5 (Table I). At
104 VP/DC, GFP was detected in 22, 66, and 33%
of immature DC infected with rAd5F16, rAd5F35, and rAd5F50,
respectively, whereas it was expressed in >85% of mature DC with all
three vectors. As a control, IFN-, which does not trigger DC
maturation but acts in synergy with maturation factors (7, 42), did not enhance GFP expression.
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Several mechanisms may explain the rise in GFP expression in
mature DC infected with subgroup B fiber-carrying rAd5. These include
an increased uptake of viral particles, due for instance to the
up-regulation of the subgroup B fiber receptor, or a better activation
state of the CMV promoter driving the expression of the transgene. To
get insight into these mechanisms, we compared GFP expression in three
different settings: rAd-infected immature DC, rAd-infected LPS-matured
DC, and rAd-infected DC matured with LPS after infection. Exposure of
DC to LPS after infection increased the number of
GFP+ cells (Fig. 5A) and the levels of GFP
expression (Fig. 5B) to the same extent as rAd infection of
already LPS-activated cells. Similar findings were obtained with other
DC maturation agents such as MCM and agonistic anti-CD40 mAb (data
not shown). We also quantified the amount of rAd DNA present in DC.
Subgroup B fiber-modified rAd5-infected immature and mature DC
contained on average 15- and 100-fold more Ad genomes, respectively,
than rAd5-infected counterparts (Fig. 5C). As expected, LPS
treatment did not influence the amount of Ad genomes detected in DC
infected in an immature state. Comparative analysis among the three
subgroup B fiber-modified rAd5 showed that the number of genomes
incorporated by LPS-activated DC averaged 3-fold higher than that by
immature cells (Fig. 5C). In conclusion, the superiority of
mature DC over immature DC for gene expression driven by subgroup B
fiber-modified rAd5 is due to at least two distinct events: an enhanced
virus entry and a better expression of the transgene.
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An important feature of rAd5 vectors for their use in DC-based
immunization strategies resides in their ability to increase DC
costimulatory and Ag-presenting functions and to act in synergy with DC
maturation signals (22, 43, 44). Because the viral
determinants responsible for this phenomenon are not fully identified
(44), it was crucial to search for a potential impact of
the exchange between fiber 5 and fibers 16, 35, and 50 on DC
activation. Immature DC infected with rAd5, rAd5F16, rAd5F35, and
rAd5F50, and further incubated in the presence or absence of LPS, were
analyzed for their surface phenotype and for their capacity to produce
the bioactive IL-12p70 cytokine. In the absence of LPS, the three
subgroup B fiber-carrying vectors were able to up-regulate the
costimulatory molecule CD86 and MHC class I and II molecules (Table II). As previously described for rAd5
(22, 32), the expression of the maturation marker CD83 was
only weakly enhanced, and none of the viruses triggered IL-12
production. However, all vectors strongly cooperated with LPS for the
induction of high CD83 levels and IL-12p70 secretion (Table II),
whereas IL-10 could not be detected (data not shown). Finally, the
strongest synergistic effects were exerted by rAd5F35, which correlates
with its capacity to infect the highest proportion of DC.
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We also compared DC infected by rAd5 with DC infected by subgroup
B fiber-modified rAd5 for their capacity to present transgene-encoded
Ag to specific T cells. For this purpose, we constructed vectors
encoding the human gp100 (hgp100), a melanocyte lineage-restricted Ag
expressed in a vast majority of malignant melanoma and a major target
for CTL-mediated tumor rejection in vivo (45). We chose to
focus on rAd5F35, which displays the greatest gene transduction
efficacy among the fiber-modified vectors tested in our study.
HLA-A2+ DC infected with rAd5hgp100 or
rAd5F35hgp100 at 105–103
VP/DC and further incubated in the presence or absence of LPS were
cultured with hgp100154–162-specific
HLA-A2-restricted CTL, which recognize human targets in a
costimulation-independent fashion. With rAd5hgp100, T cell-derived
IFN- secretion was triggered by DC only when
105 VP/DC were used in combination with LPS
stimulation at high DC to T cell ratios (Fig. 6A). At lower virus doses,
IFN- production was similar to that obtained with control DC. In
contrast, rAd5F35hgp100-infected immature DC and their LPS-treated
counterparts stimulated the secretion of high levels of IFN- when
105 and 104 VP/DC were used
(Fig. 6, A and B). As expected, mature DC were
the most potent APC. At 103 VP/DC, considerable
amounts of IFN- were still detected with LPS-matured cells (Fig. 6C). Therefore, increased rAd5F35-mediated transduction of
DC correlates with enhanced transgene-encoded Ag presentation.
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Although most immunotherapeutic strategies rely on ex vivo
manipulation of DC, in vivo Ag delivery to DC, especially those present
in the skin, is also being explored (46). We investigated
how rapidly rAd5F35 could infect DC. Immature cells exposed to rAd5F35
or to rAd5 at 104 VP/DC for time periods varying
from 15 min to 2 h at 37°C were analyzed for GFP expression
24 h later. The kinetics of rAd5F35 infection strikingly differed
from those of rAd5. Maximum GFP expression in 70% of DC was already
achieved from rAd5F35 particles that had entered DC within the first 15
min of incubation and remained stable thereafter (Fig. 7A). Only 10% of DC that had
been exposed to rAd5 for 15 min expressed GFP, and expression slowly
increased with time without reaching that obtained with rAd5F35.
Finally, the DC infection pattern of rAd5F35 was fully maintained upon
addition of primary skin-derived fibroblasts (Fig. 7B),
despite the fact that these neighboring cells were also susceptible to
the vector. These results suggest that unlike rAd5, rAd5F35 can be
exploited for genetic modification of DC in vivo and warrant studies in
experimental animals to test its potency.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our approach reveals that fibers from different Ad subgroups interact differently with DC. Among all chimeric vectors tested here, only rAd5F16, rAd5F35, and rAd5F50 emerged as vectors with high DC tropism. They all possess fibers with shaft and head domains from Ads belonging to subgroup B, which is the only subgroup that cannot use the CAR (31). Recent reports indicate that subgroup B Ads exhibit tropism toward other cell subsets of the hemopoietic system. rAd5 equipped with a subgroup B Ad3 fiber was shown to successfully target EBV-transformed B cells (47). Subgroup B Ad11 and Ad35 were found to infect lymphocytic and myelomonocytic tumor cell lines (48). Ad35 and rAd5 with Ad35 fibers appeared to efficiently enter CAR-deficient CD34+ stem cells (49). Moreover, the data presented in this latter report suggest that Ad3 and Ad35 target different cell surface receptors. Interestingly, we found that rAd5F35 was more potent than rAd5F16 and rAd5F50 for transducing immature DC. Thus, the possibility exists that these subgroup B fiber-modified vectors use different strategies for entry into DC. Alternatively, their fibers may differentially modulate intracellular trafficking and transport of the rAd genome into the nucleus (50). Irrespective of these considerations, our data and previous studies by others suggest that cells of hemopoietic origin express one or more receptors for subgroup B viruses.
Enhanced gene expression mediated by rAd5F16, rAd5F35, and rAd5F50 does
not only occur in immature DC but also in mature cells. In fact, mature
DC express transgenes at even higher levels than immature cells. This
most likely relates to at least two different events. The first factor
is an increased uptake of viral particles, probably through the
up-regulation of the subgroup B fiber receptor(s) on mature DC. Which
receptor accounts for this phenomenon is currently unknown but is
unlikely to involve cellular integrins because these are not
up-regulated upon DC maturation (22). The second factor
concerns the activation of transcriptionally silent transgenes by
maturation-induced signaling events. Efficient transcription from the
CMV promoter controlling the transgene relies in part on NF-
B
activation, and maturation signals function at least through activation
of NF-B (51, 52). Recently, Tillman et al. have
constructed bispecific mAb directed against the Ad5 fiber and CD40 to
retarget Ad5 to human DC via their CD40 receptor (32).
Because DC mature upon CD40 cross-linking (10, 11), these
bispecific mAb may not only increase virus uptake by DC, but may also
improve transgene expression in a similar fashion as observed in our
study.
In addition to targeting DC more efficiently, rAd5 with subgroup B fibers conserve the immune-activating properties of the rAd5 parental virus. These novel vectors increase the expression of costimulatory and MHC molecules on DC and act in synergy with LPS to promote the maturation of DC into fully immunostimulatory APC. In fact, due to more efficient infection, subgroup B fiber-modified rAd5 exert an enhanced effect on DC maturation. Altogether, these characteristics lead to a better presentation of the transgene-encoded Ag to specific T cells, a key issue for rAd-infected DC-based immunization strategies.
In conclusion, our findings demonstrate major progress toward the clinical applicability of rAd vectors as vaccines to battle cancer and pathogens and support the use of subgroup B fiber-modified viruses, especially rAd5F35, for ex vivo Ag delivery into DC. Moreover, the favorable kinetics of DC infection by rAd5F35 even in the presence of other susceptible cells may enable this vector to reach the DC network in vivo and suggest that this vector is an attractive new genetic vaccine. Whether rAd5F35 can truly serve for in vivo targeted DC-based immunotherapy is currently under investigation in primate models.
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Acknowledgments |
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Footnotes |
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2 Current address: Service d’Hématologie Adulte, Hôpital Saint Louis, 1 avenue Claude Vellefaux, 75010 Paris, France.
3 Address correspondence and reprint requests to Dr. Rienk Offringa, Department of Immunohematology and Blood Bank, Leiden University Medical Center, Albinusdreef 2, Postbus 9600, 2300 RC Leiden, The Netherlands.
4 Abbreviations used in this paper: DC, dendritic cells; A2Kb, HLA-A*0201/Kb; CAR, coxsackie-adenovirus receptor; F, fiber; hgp100, human gp100; GFP, green- fluorescent protein; Luc, luciferase; rAd, recombinant adenovirus; VP, virus particle(s); MCM, monocyte-conditioned medium; MFI, mean fluorescence intensity.
Received for publication October 6, 2000. Accepted for publication January 24, 2001.
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References |
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  W. C. Adams, E. Bond, M. J. E. Havenga, L. Holterman, J. Goudsmit, G. B. Karlsson Hedestam, R. A. Koup, and K. Lore Adenovirus serotype 5 infects human dendritic cells via a coxsackievirus-adenovirus receptor-independent receptor pathway mediated by lactoferrin and DC-SIGN J. Gen. Virol., July 1, 2009; 90(7): 1600 - 1610. [Abstract] [Full Text] [PDF]
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 K. Lore, W. C. Adams, M. Havenga, M. L. Precopio, L. Holterman, J. Goudsmit, and R. A. Koup Myeloid and Plasmacytoid Dendritic Cells Are Susceptible to Recombinant Adenovirus Vectors and Stimulate Polyfunctional Memory T Cell Responses J. Immunol., August 1, 2007; 179(3): 1721 - 1729. [Abstract] [Full Text] [PDF]
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 S. Tuve, H. Wang, C. Ware, Y. Liu, A. Gaggar, K. Bernt, D. Shayakhmetov, Z. Li, R. Strauss, D. Stone, et al. A New Group B Adenovirus Receptor Is Expressed at High Levels on Human Stem and Tumor Cells J. Virol., December 15, 2006; 80(24): 12109 - 12120. [Abstract] [Full Text] [PDF]
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H. Wang and A. Lieber A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette J. Virol., December 1, 2006; 80(23): 11699 - 11709. [Abstract] [Full Text] [PDF]
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T. D. de Gruijl, O. J. A. E. Ophorst, J. Goudsmit, S. Verhaagh, S. M. Lougheed, K. Radosevic, M. J. E. Havenga, and R. J. Scheper Intradermal Delivery of Adenoviral Type-35 Vectors Leads to High Efficiency Transduction of Mature, CD8+ T Cell-Stimulating Skin-Emigrated Dendritic Cells J. Immunol., August 15, 2006; 177(4): 2208 - 2215. [Abstract] [Full Text] [PDF]
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 F. Pichiorri, F. Trapasso, T. Palumbo, R. I. Aqeilan, A. Drusco, B. W. Blaser, D. Iliopoulos, M. A. Caligiuri, K. Huebner, and C. M. Croce Preclinical Assessment of FHIT Gene Replacement Therapy in Human Leukemia Using a Chimeric Adenovirus, Ad5/F35. Clin. Cancer Res., June 1, 2006; 12(11): 3494 - 3501. [Abstract] [Full Text] [PDF]
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S. Lecollinet, F. Gavard, M. J. E. Havenga, O. B. Spiller, A. Lemckert, J. Goudsmit, M. Eloit, and J. Richardson Improved Gene Delivery to Intestinal Mucosa by Adenoviral Vectors Bearing Subgroup B and D Fibers J. Virol., March 15, 2006; 80(6): 2747 - 2759. [Abstract] [Full Text] [PDF]
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S. Verhaagh, E. de Jong, J. Goudsmit, S. Lecollinet, G. Gillissen, M. de Vries, K. van Leuven, I. Que, K. Ouwehand, R. Mintardjo, et al. Human CD46-transgenic mice in studies involving replication-incompetent adenoviral type 35 vectors J. Gen. Virol., February 1, 2006; 87(2): 255 - 265. [Abstract] [Full Text] [PDF]
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C. Fleischli, S. Verhaagh, M. Havenga, D. Sirena, W. Schaffner, R. Cattaneo, U. F. Greber, and S. Hemmi The Distal Short Consensus Repeats 1 and 2 of the Membrane Cofactor Protein CD46 and Their Distance from the Cell Membrane Determine Productive Entry of Species B Adenovirus Serotype 35 J. Virol., August 1, 2005; 79(15): 10013 - 10022. [Abstract] [Full Text] [PDF]
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S. M. Sumida, D. M. Truitt, A. A. C. Lemckert, R. Vogels, J. H. H. V. Custers, M. M. Addo, S. Lockman, T. Peter, F. W. Peyerl, M. G. Kishko, et al. Neutralizing Antibodies to Adenovirus Serotype 5 Vaccine Vectors Are Directed Primarily against the Adenovirus Hexon Protein J. Immunol., June 1, 2005; 174(11): 7179 - 7185. [Abstract] [Full Text] [PDF]
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D. Stone, S. Ni, Z.-Y. Li, A. Gaggar, N. DiPaolo, Q. Feng, V. Sandig, and A. Lieber Development and Assessment of Human Adenovirus Type 11 as a Gene Transfer Vector J. Virol., April 15, 2005; 79(8): 5090 - 5104. [Abstract] [Full Text] [PDF]
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P. Krebs, E. Scandella, B. Odermatt, and B. Ludewig Rapid Functional Exhaustion and Deletion of CTL following Immunization with Recombinant Adenovirus J. Immunol., April 15, 2005; 174(8): 4559 - 4566. [Abstract] [Full Text] [PDF]
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D. H. Barouch, M. G. Pau, J. H. H. V. Custers, W. Koudstaal, S. Kostense, M. J. E. Havenga, D. M. Truitt, S. M. Sumida, M. G. Kishko, J. C. Arthur, et al. Immunogenicity of Recombinant Adenovirus Serotype 35 Vaccine in the Presence of Pre-Existing Anti-Ad5 Immunity J. Immunol., May 15, 2004; 172(10): 6290 - 6297. [Abstract] [Full Text] [PDF]
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D. Sirena, B. Lilienfeld, M. Eisenhut, S. Kalin, K. Boucke, R. R. Beerli, L. Vogt, C. Ruedl, M. F. Bachmann, U. F. Greber, et al. The Human Membrane Cofactor CD46 Is a Receptor for Species B Adenovirus Serotype 3 J. Virol., May 1, 2004; 78(9): 4454 - 4462. [Abstract] [Full Text] [PDF]
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S. Mercier, S. Verhaagh, J. Goudsmit, A. Lemckert, M. Monteil, M. Havenga, and M. Eloit Adenovirus fibre exchange alters cell tropism in vitro but not transgene-specific T CD8+ immune responses in vivo J. Gen. Virol., May 1, 2004; 85(5): 1227 - 1236. [Abstract] [Full Text] [PDF]
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L. Fontana, M. Nuzzo, L. Urbanelli, and P. Monaci General Strategy for Broadening Adenovirus Tropism J. Virol., October 15, 2003; 77(20): 11094 - 11104. [Abstract] [Full Text] [PDF]
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 V. Molinier-Frenkel, A. Prevost-Blondel, S.-S. Hong, R. Lengagne, S. Boudaly, M. K. Magnusson, P. Boulanger, and J.-G. Guillet The Maturation of Murine Dendritic Cells Induced by Human Adenovirus Is Mediated by the Fiber Knob Domain J. Biol. Chem., September 26, 2003; 278(39): 37175 - 37182. [Abstract] [Full Text] [PDF]
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V. Thiel, N. Karl, B. Schelle, P. Disterer, I. Klagge, and S. G. Siddell Multigene RNA Vector Based on Coronavirus Transcription J. Virol., September 15, 2003; 77(18): 9790 - 9798. [Abstract] [Full Text] [PDF]
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A. Segerman, J. P. Atkinson, M. Marttila, V. Dennerquist, G. Wadell, and N. Arnberg Adenovirus Type 11 Uses CD46 as a Cellular Receptor J. Virol., September 1, 2003; 77(17): 9183 - 9191. [Abstract] [Full Text] [PDF]
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R. Vogels, D. Zuijdgeest, R. van Rijnsoever, E. Hartkoorn, I. Damen, M.-P. de Bethune, S. Kostense, G. Penders, N. Helmus, W. Koudstaal, et al. Replication-Deficient Human Adenovirus Type 35 Vectors for Gene Transfer and Vaccination: Efficient Human Cell Infection and Bypass of Preexisting Adenovirus Immunity J. Virol., August 1, 2003; 77(15): 8263 - 8271. [Abstract] [Full Text] [PDF]
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X. Wang, M. Messerle, R. Sapinoro, K. Santos, P. K. Hocknell, X. Jin, and S. Dewhurst Murine Cytomegalovirus Abortively Infects Human Dendritic Cells, Leading to Expression and Presentation of Virally Vectored Genes J. Virol., July 1, 2003; 77(13): 7182 - 7192. [Abstract] [Full Text] [PDF]
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 Y. Kawakami, H. Li, J. T. Lam, V. Krasnykh, D. T. Curiel, and J. L. Blackwell Substitution of the Adenovirus Serotype 5 Knob with a Serotype 3 Knob Enhances Multiple Steps in Virus Replication Cancer Res., March 15, 2003; 63(6): 1262 - 1269. [Abstract] [Full Text] [PDF]
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 S. Gottschalk, O. L. Edwards, U. Sili, M. H. Huls, T. Goltsova, A. R. Davis, H. E. Heslop, and C. M. Rooney Generating CTLs against the subdominant Epstein-Barr virus LMP1 antigen for the adoptive immunotherapy of EBV-associated malignancies Blood, March 1, 2003; 101(5): 1905 - 1912. [Abstract] [Full Text] [PDF]
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E. H. Slager, M. Borghi, C. E. van der Minne, C. A. Aarnoudse, M. J. E. Havenga, P. I. Schrier, S. Osanto, and M. Griffioen CD4+ Th2 Cell Recognition of HLA-DR-Restricted Epitopes Derived from CAMEL: A Tumor Antigen Translated in an Alternative Open Reading Frame J. Immunol., February 1, 2003; 170(3): 1490 - 1497. [Abstract] [Full Text] [PDF]
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J. W. Schoggins, J. G. D. Gall, and E. Falck-Pedersen Subgroup B and F Fiber Chimeras Eliminate Normal Adenovirus Type 5 Vector Transduction In Vitro and In Vivo J. Virol., December 20, 2002; 77(2): 1039 - 1048. [Abstract] [Full Text] [PDF]
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M. J. E. Havenga, A. A. C. Lemckert, O. J. A. E. Ophorst, M. van Meijer, W. T. V. Germeraad, J. Grimbergen, M. A. van den Doel, R. Vogels, J. van Deutekom, A. A. M. Janson, et al. Exploiting the Natural Diversity in Adenovirus Tropism for Therapy and Prevention of Disease J. Virol., March 27, 2002; 76(9): 4612 - 4620. [Abstract] [Full Text] [PDF]
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D. M. Shayakhmetov, Z.-Y. Li, S. Ni, and A. Lieber Targeting of Adenovirus Vectors to Tumor Cells Does Not Enable Efficient Transduction of Breast Cancer Metastases Cancer Res., February 1, 2002; 62(4): 1063 - 1068. [Abstract] [Full Text] [PDF]
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