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J Virol, July 1998, p. 6138-6145, Vol. 72, No. 7
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Role of E4 in Eliciting CD4 T-Cell and B-Cell
Responses to Adenovirus Vectors Delivered to Murine and Nonhuman
Primate Lungs
Narendra
Chirmule,
Joseph V.
Hughes,
Guang-Ping
Gao,
Steven E.
Raper, and
James M.
Wilson*
Institute for Human Gene Therapy and
Department of Molecular and Cellular Engineering, University of
Pennsylvania, and the Wistar Institute, Philadelphia, Pennsylvania
19104
Received 12 January 1998/Accepted 27 February 1998
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ABSTRACT |
Adenovirus vectors delivered to lung are being considered in the
treatment of cystic fibrosis (CF). Vectors from which E1 has been
deleted elicit T- and B-cell responses which confound their use in the
treatment of chronic diseases such as CF. In this study, we directly
compare the biology of an adenovirus vector from which E1 has been
deleted to that of one from which E1 and E4 have been deleted,
following intratracheal instillation into mouse and nonhuman primate
lung. Evaluation of the E1 deletion vector in C57BL/6 mice demonstrated
dose-dependent activation of both CD4 T cells (i.e., TH1 and TH2
subsets) and neutralizing antibodies to viral capsid proteins. Deletion
of E4 and E1 had little impact on the CD4 T-cell proliferative response
and cytolytic activity of CD8 T cells against target cells expressing
viral antigens. Analysis of T-cell subsets from mice exposed to the vector from which E1 and E4 had been deleted demonstrated preservation of TH1 responses with markedly diminished TH2 responses compared to the
vector with the deletion of E1. This effect was associated with reduced
TH2-dependent immunoglobulin isotypes and markedly diminished
neutralizing antibodies. Similar results were obtained in nonhuman
primates. These studies indicate that the vector genotype can modify
B-cell responses by differential activation of TH1 subsets. Diminished
humoral immunity, as was observed with the E1 and E4 deletion vectors
in lung, is indeed desired in applications of gene therapy where
readministration of the vector is necessary.
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INTRODUCTION |
Adenovirus vectors have been used
widely in preclinical and clinical applications of gene therapy
(21). First-generation constructs with deletions of E1
efficiently transduce a variety of cells in vivo. Therapeutic doses of
vector are often associated with inflammation, transient gene
expression, and problems with vector readministration. Early
experiments in immune-deficient or immune-suppressed animals suggested
that these problems may be related to host immune responses (4,
22, 25). Initially, we proposed that cytotoxic T lymphocytes
(CTLs) in response to the vector-transduced cells contribute to the
loss of transgene expression whereas B-cell responses to the input
viral capsid proteins elicit neutralizing antibodies which block
repeated attempts at gene transfer (24).
The concept of cellular immunity to vector-encoded viral antigens led
to the development of a number of advanced-generation adenovirus
vectors further disabled by the inactivation of other essential genes.
The most extensive experimentation has been in applications of
liver-directed gene transfer in murine models, regarding which the
literature has been conflicting and somewhat difficult to reconcile.
Several themes have emerged, however. It appears that vector-encoded
viral proteins as well as the transgene product can serve as targets
for CTLs in a major histocompatibility complex (MHC) class I-restricted
manner (9, 19, 23). Improvements in transgene stability were
modest at best with vectors in which E1 and E2a were defective,
although inflammation was substantially diminished (6).
Results with constructs from which E1 and E4 were deleted have been
more encouraging. Three independent groups have demonstrated marked
prolongation of transgene expression in mouse liver with vectors from
which E1 and E4 have been deleted, although this advantage was not
demonstrated in two other experimental models (2, 5, 9, 14,
20). Studies with vectors with deletions of all viral open
reading frames have yielded impressive results in mouse liver, where
they are associated with substantially diminished toxicity and
extremely stable transgene expression (18). Less impressive
results were obtained with an adenovirus vector with deletions of all
genes except E4 (15). Modifications in the vector genome
described above do not significantly impact the development of
neutralizing antibodies, which presumably are elicited by the input
viral capsid proteins.
The application to the lung of E1 deletion vectors has confirmed the
role of humoral and cellular immunity. Transgene expression is stable
and vector readministration is possible in lungs of mice that are
genetically immunodeficient or transiently immunosuppressed (24,
25). Further disabling the vector through the incorporation of a
temperature-sensitive mutation in E2a resulted in a modest increase in
transgene stability in both mice and nonhuman primates (7,
10). Analysis of vectors with deletions of E1 and E4 has been
complicated by problems of transcriptional extinction (2).
Apparently, ongoing expression of the transgene in mouse lung from a
viral promoter such as cytomegalovirus (CMV) requires the presence of
E4 viral open reading frames (2). The impact of vector
genotype on humoral immune responses is less well characterized in the
lung.
In this study, we performed a direct comparison of host immune
responses to adenovirus vectors expressing the cystic fibrosis gene
with deletions of E1 or of E1 and E4. Comparisons were performed in
both C57BL/6 mice and nonhuman primates.
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MATERIALS AND METHODS |
Animals.
C57BL/6 (H-2b) mice were
purchased from Taconic Laboratory Animals and Services, Germantown,
N.Y., and housed in a specific-pathogen-free environment. Mice were
prepared for intratracheal instillation by dissecting the tracheas and
directly instilling various concentrations of the vector through small
incisions. Animals were sacrificed on days 11 and 29, and spleen,
serum, and bronchoalveolar lavage (BAL) fluid were obtained for
immunological analyses.
Rhesus monkeys were acclimatized for a 4-week period before initiation
of experiments. Vector administration (5 × 1012
particles) was performed by bronchoscopy as described previously (10). Monkeys were anesthetized with ketamine and atropine. A physical exam was performed on and a 22-gauge intravenous needle was
inserted into each monkey. In the operating room suite, a pulse
oximeter was applied and each monkey was placed supine with its head in
the "sniffing position." The vocal cords were visualized with a
laryngoscope and sprayed with Cetacaine, and the bronchoscope was
passed through the vocal cords and the membranous trachea. The left
main stem bronchus was identified and entered under direct vision.
Sterile saline (10 ml) was injected into a peripheral branch and
aspirated into a mucus trap. Vector (1.75 ml containing 5 × 1012 particles) was instilled into the left main stem
bronchus through the biopsy port of the bronchoscope. The bronchoscope
was withdrawn under direct vision and the monkey was allowed to emerge
from anesthesia. Two animals received the E1 deletion vector and two received an equivalent dose of vector with deletions of E1 and E4.
Peripheral blood and BAL fluids were drawn for immunological analyses.
One animal from each group was sacrificed on day 11 and day 29. Extensive toxicological analyses of these experiments will be published
elsewhere. All animal studies were approved by the University of
Pennsylvania, Institutional Animal Care and Use Committee and
Institutional Biosafety Committee.
Recombinant adenoviruses.
The structure and production of
the vector with the deletion of E1, H5.020CBCFTR, has been
described. Basically, the vector expresses a human cystic fibrosis
transmembrane conductance regulator (CFTR) cDNA from a chicken
-actin promoter enhanced by sequences of the immediate early gene of
CMV. Sequences spanning E1 and E3 are deleted. The plasmid containing
the minigene and 5' adenovirus type 5 sequences were used to construct
H5.001CBCFTR, from which E1 and virtually all of E4 (except
orf1) are deleted. To construct H5.001CBCFTR
virus, the E1 and E4 double-complementing cells (27-18) were seeded in
60-mm plates and cotransfected with ClaI-digested dl1004 (mutant from which E4 was deleted) viral DNA and
NheI-digested pAdCBCFTR plasmid DNA (2 µg of
viral DNA and 10 µg of plasmid DNA per plate) by the calcium
phosphate precipitation method (9). Twenty hours
posttransfection, the cells were overlaid with top agar containing 20 mM dexamethasone to induce expression of E4. Well-isolated plaques were
picked 10 days posttransfection following neutral red staining,
amplified in 27-18 cells, and screened for recombinant viruses by viral
DNA analysis (PCR and restriction endonuclease digestion). Positive
plaques were confirmed by infecting HeLa cells with the viral lysates
and detecting CFTR protein by immunofluorescent staining. After three
rounds of plaque purification, the recombinant viruses were amplified
in cells expressing E1 and E4 and purified by standard protocols
(9). The CFTR viruses used in these studies were derived
from the production lots used in phase I clinical trials. Recombinant
adenovirus-expressing -galactosidase (H5.010CMVlacZ) used
for in vitro assays has been described previously (7).
Selected in vivo experiments were performed with a CMV-driven
lacZ vector with deletions of E1 (H5.000CMVlacZ) or of E1 and E4 (H5.001CMVlacZ) (9).
Lymphoproliferative assays.
Splenocytes from mice or
peripheral blood samples from monkeys were obtained on day 11 of each
study. Mouse splenocytes were prepared as a single-cell suspension made
on a wire mesh following passage through a nylon filter. Rhesus monkey
lymphocytes were isolated by standard Ficoll-Hypaque density gradients.
Triplicate cultures of lymphocytes (105 cells) were
cultured with either inactivated lacZ virus (multiplicity of
infection [MOI] based on particles equal to 10) or medium alone. Antigen-stimulated cultures were harvested on day 6. Proliferation was
measured by a 16-h [3H]thymidine (1-µCi/well) pulse.
Cytokine release assays.
Lymphocytes were cultured with or
without antigen (i.e., inactivated lacZ virus at a particle
MOI equal to 10) for 72 h in a 24-well plate. Cell-free
supernatants were collected and analyzed for presence of interleukin-2
(IL-2), IL-4, gamma interferon (IFN-
), and IL-10 by enzyme-linked
immunosorbent assay (ELISA). For the mouse cytokine ELISA, 96-well
flat-bottomed, high-binding Immulon-IV plates were coated with 200 µl
of rat anti-mouse IL-2, IL-4, IFN-, and IL-10 in phosphate-buffered
saline (PBS) overnight at 4°C, washed four times in PBS-0.05%
Tween, and blocked in PBS-1% bovine serum albumin for 2 h at
4°C. Culture supernatants were added to antibody-coated plates and
incubated overnight at 4°C. Plates were washed four times in
PBS-0.05% Tween and incubated with biotin-conjugated rat anti-mouse
IL-2, IL-4, IFN-, and IL-10 (1:1,000 dilution; Pharmingen, San
Diego, Calif.) for 2 h at 4°C. Plates were washed as described
above, and peroxidase-conjugated streptavidin was added to the plates
for 2 h at 4°C. After another washing, ABTS [2,2'-azinobis(3-ethylbenzthiazolesulfonic acid)] substrate
(Kirkegaard and Perry, Gaithersburg, Md.) was added. Optical densities
were read at 405 nm on a MRX Dynatech Microplate reader. Cytokine
secretion in the mouse BAL fluid and culture supernatants of rhesus
monkey and mouse lymphocytes was analyzed with commercial ELISA kits for IL-2, IL-4, IFN-, IL-10 (BioSource), and IL-8 (R&D Systems).
Cytotoxicity assay.
The CTL assay was performed as described
previously (24). In brief, mice were sacrificed on day 11 and a single-cell suspension of spleen cells from groups of three to
six mice was cultured for 5 days at a concentration of 5 × 106 cells/well in a 24-well plate. Purified lacZ
virus was added at an MOI of 0.8. After secondary in vitro stimulation,
nonadherent spleen cells were harvested and assayed on MHC-compatible
target cells (M57SV) in different ratios of effectors to target cells. Target cells (2 × 106) were infected overnight with
adenovirus at a particle MOI of 100. Cells (106) were
labeled with 100 µCi of 51Cr
(Na251CrO4; NEN Research Products)
for 1 h, washed three times with 10 ml of Dulbecco's modified
Eagle medium, and resuspended in assay medium at 5 × 104/ml. Aliquots of target cells (100 µl) were plated
with spleen cells (100 µl) at various effector/target cell ratios in
V-bottom microtiter plates. The plates were spun down for 3 min at
1,100 rpm and incubated for 6 h at 37°C in 10% CO2.
A 100-µl sample of the supernatant was removed from each well and
counted in a Wallach gamma counter. The percentage of specific
51Cr release was calculated as the following: [(cpm of
sample 
cpm of spontaneous release)/(cpm of maximal release cpm of spontaneous release)] × 100, where cpm stands for counts
per minute. All sample values represent the averages of quadruplicate
wells; maximum (i.e., target cells incubated with 5% sodium dodecyl
sulfate) and spontaneous (i.e., target cells incubated with medium
only) releases were averaged from eight wells.
Neutralizing-antibody assays.
Neutralizing-antibody titers
were evaluated by measuring the ability of serum antibody to inhibit
transduction of reporter lacZ virus into HeLa cells. Various
dilutions of antibodies were preincubated with reporter virus for
1 h at 37°C and added to 90% confluent HeLa cell cultures.
Cells were incubated for 16 h and expression of lacZ
was measured by X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) staining. The neutralizing titer of antibody was calculated as the
highest titer that inhibited transduction to 
50% of the cells.
Adenovirus-specific Ig.
Serum (diluted 1:200) and BAL fluid
(diluted 1:20) from animals were analyzed for adenovirus-specific,
isotype-specific immunoglobulins (Ig) (IgM, IgG, IgA, and IgE) by
ELISA. For the ELISA, 96-well flat-bottomed, high-binding Immulon-IV
plates were coated with lacZ virus (109
particles) in PBS overnight at 4°C, washed four times in PBS-0.05% Tween, and blocked in PBS-1% bovine serum albumin for 2 h at
4°C. Appropriately diluted samples were added to antigen-coated
plates and incubated overnight at 4°C. Plates were washed four times in PBS-0.05% Tween and incubated with peroxidase- and
biotin-conjugated rat anti-mouse IgM, IgG1, IgG2a, IgG2b, IgG3, or IgA
(1:2,000 dilutions; Pharmingen) for 2 h at 4°C. Plates were
washed as described above and ABTS substrate (Kirkegaard and Perry) was
added. Optical densities were read at 405 nm on an MRX Dynatech
Microplate reader.
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RESULTS |
Adenovirus vectors.
The first-generation adenovirus vector
H5.020CBCFTR has been evaluated in mice, nonhuman primates,
and humans. It has a deletion of E1 and expresses the human CFTR cDNA
from a CMV-enhanced, chicken -actin promoter. The nonessential gene
E3 is also deleted to make room for the CFTR minigene, which is
inserted in place of E1. The next generation vector, called
H5.001CBCFTR, has deletions of the two essential genes E1
and E4. This is grown in a cell line stably expressing E1 and E4 from
an inducible promoter. H5.001CBCFTR is currently being
evaluated in a phase I clinical trial.
Clinical-grade production lots of both vectors were made in a pilot
manufacturing facility at the University of Pennsylvania
that operates
under good manufacturing practices. The yields and
ratios of PFU to
particles were consistently twofold lower for
the vector from which E1
and E4 were deleted than for the vector
with a deletion of E1. The
protein compositions of the resulting
virions were indistinguishable in
Western blot analyses (data
not shown).
Dose-dependent activation of T cells by adenovirus vectors in the
mouse.
C57BL/6 mice were intratracheally given various
concentrations (i.e., 1.5 × 108 to 5 × 1010 particles) of vectors with deletions of either E1 or
E1 and E4. Six animals were given each vector at each concentration.
Extensive analyses of cell-mediated immune responses were performed on
splenocytes obtained on day 11.
Activation of spleen-derived T cells by vector was characterized in a
lymphoproliferation (LPR) assay. This assay represents
CD4 T-cell
responses, since in vivo administration of a depleting
anti-CD4
antibody completely abrogates LPR (data not shown). Figure
1 shows that the administration of at
least 5 × 10
8 particles of the adenovirus vector was
required to induce in
vitro LPR which was indistinguishable from those
of the vectors
with deletions of E1 or E1 and E4. Further analyses of
activated
CD4 and CD8 T cells by three-color flow cytometry showed that
animals that were given either vector had equivalent CD69 expression
(data not shown). These results indicate that both vectors that
were
tested are capable of activating CD4 T-cell responses in
mice.
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FIG. 1.
Threshold of in vivo adenovirus instillation vector
required for inducing measurable T-cell proliferative responses.
C57BL/6 mice were given vehicle alone or various concentrations of
either H5.020CBCFTR or H5.001CBCFTR
intratracheally. Splenocytes harvested on day 11 were cultured in the
presence or absence of inactivated adenovirus (Adeno) for 7 days. LPR
responses were measured by [3H]thymidine incorporation.
The results are means of triplicate cultures from spleens of six
animals in each group and one of three separate experiments.
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Analyses of the cytokine secretion profiles of activated CD4 T cells
showed concentrations of released cytokines that increased
in
proportion to the concentrations of the administered vectors
(Fig.
2). Significant IL-2 and IFN-
responses were observed with
both vectors, although they were
attenuated with the construct
with deletions of E1 and E4. There was a
marked difference in
the patterns of TH2 cytokine secretion in animals
given the vector
with deletions of E1 and E4, which failed to mount
appreciable
IL-4 and IL-10 responses. This contrasts with the patterns
in
animals given the E1 deletion vector, which consistently developed
IL-4 and IL-10 responses in a dose-dependent manner.
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FIG. 2.
Dose-dependent induction of cytokine secretion profile.
C57BL/6 mice were given vehicle alone or various concentrations of
either H5.020CBCFTR or H5.001CBCFTR
intratracheally. Splenocytes harvested on day 11 were cultured in the
presence or absence of inactivated adenovirus (Adeno) for 48 h.
Culture supernatants were analyzed for IL-2, IFN-, IL-4, and IL-10
by ELISA. Panels a to d show cytokines for H5.001CBCFTR
vector and panels e to h show cytokines for the H5.020CBCFTR
vector. Values are for duplicate culture supernatants from spleens of
six animals in each group and one of three separate experiments.
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CTL activity against viral proteins has been shown previously to
correlate with the elimination of transgene expression (
23,
24). Cytotoxic T-cell responses were assessed by the ability
of
splenocytes to kill adenovirus-infected autologous
H-2b target cells. Figure
3 shows that animals given either vector
that was tested in this study generated CTL against the vector.
Note
that this is an assay of bulk-cultured in vitro-stimulated
lymphocytes
and, therefore, is not necessarily quantitative.
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FIG. 3.
CTL responses in mice infected with
H5.020CBCFTR and H5.001CBCFTR. C57BL/6 mice were
given either H5.020CBCFTR or H5.001CBCFTR
intratracheally. Splenocytes harvested on day 11 were cultured in the
presence of adenovirus for 5 days and tested for specific lysis on
mock-infected or adenovirus-lacZ-infected C57SV target cells
in a 51Cr release assay. The percentage of specific lysis
is expressed as a function of different ratios of effector cells to
target cells. Dashed lines with diamonds indicate values from
mock-infected target cells while solid lines with squares indicate
values from target cells infected with the first-generation
lacZ virus. The results show CTL responses of six spleens in
each group and one of three separate experiments.
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Deletion of E4 in an adenovirus vector with a deletion of E1
substantially diminishes humoral immune responses in mouse lung.
Humoral immune responses following intratracheal administration of
vector were analyzed in serum and BAL fluid on day 29. Figure
4 shows that mice instilled with the
vector from which E1 was deleted developed neutralizing antibodies in
serum in a dose-dependent manner that was substantially blunted in
animals given the vector with deletions of E1 and E4. Analysis of BAL fluid on day 29 following the instillation of 5 × 1010 particles revealed reciprocal dilutions of
neutralizing antibodies of 340 ± 30 (mean ± standard
deviation) with the vector with one deletion and 30 ± 14 with the
vector with two deletions. Vehicle control yielded a
neutralizing-antibody reciprocal dilution equal to 20.
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FIG. 4.
Dose-dependent induction of adenovirus-specific
neutralizing antibodies. Day 29 sera obtained from mice given either
vehicle or various concentrations of H5.020CBCFTR or
H5.001CBCFTR were analyzed for the presence of neutralizing
antibody by the ability of sera to block infection of HeLa cells with
lacZ virus. The reciprocal dilution is plotted against
vector dose. The results represent means and standard deviations of six
animals in each group and one of five separate experiments.
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Analyses of the adenovirus-specific Ig isotypes in Fig.
5 showed that both vectors generated
levels of IgM and IgG3 equivalent
to those generated by adenoviral
antigens, both of which are T-cell
independent. Substantially more
adenovirus-specific TH2-dependent
isotype, IgG1, was obtained following
exposure to the E1 deletion
vector than following exposure to the
vector from which E1 and
E4 were deleted. The TH1-dependent isotypes
IgG2a and IgG2b were
generated in essentially equivalent quantities by
the two vectors.
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FIG. 5.
Anti-adenovirus vector-specific Ig isotypes. Day 29 sera
obtained from animals given either vehicle, H5.020CBCFTR, or
H5.001CBCFTR were analyzed for the presence of
adenovirus-specific IgM, IgG2a, IgG2b, IgG3, IgG1, and IgA by ELISA.
The optical densities determined by the ELISAs are presented. The
results represent means and standard deviations of six animals in each
group and one of three separate experiments.
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Western blot analyses of adenoviral capsid proteins were performed to
determine whether there was preferential induction of
antibody
responses directed against hexon, penton, or fiber (Fig.
6). Animals given the vector with a
deletion of E1 generated antibodies
against all three capsid proteins.
The vector from which E1 and
E4 had been deleted induced primarily
antihexon antibodies, weak
antipenton antibodies, and no antifiber
antibodies.
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FIG. 6.
Western blot analyses of sera of mice receiving
adenovirus vectors. Day 29 sera obtained from five animals given either
vehicle (lanes 5 through 8), H5.020CBCFTR (lanes 10 through
14), or H5.001CBCFTR (lanes 15 through 19) were analyzed for
the presence of adenovirus-specific antibodies to hexon, penton, and
fiber by Western blot analyses. Control lanes show control polyclonal
sera to complete adenovirus (Ad) (lane 1) and polyclonal antibodies to
hexon (H) (lane 2), penton (P) (lane 3), and fiber (F) (lane 4). The
results represent one of two separate experiments.
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Inflammatory responses following vector administration were analyzed by
measuring IL-6 and IL-8 levels in BAL fluid. Figure
7 shows that mice given the vector with a
deletion of E1 had significantly
higher levels of IL-6 and IL-8
cytokines than animals given the
vector with deletions of E1 and E4. No
measurable cytokines were
detected in BAL fluid from monkeys that had
been given vehicle
(data not shown).
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FIG. 7.
Induction of inflammatory cytokines in BAL fluid of
mice. BAL fluid obtained from animals given either vehicle,
H5.020CBCFTR, or H5.001CBCFTR intratracheally was
analyzed for the presence of IL-8 and IL-6 by commercial ELISA kits as
recommended by the manufacturer. The results are means and standard
deviations of six animals in each group.
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Humoral and TH2 responses are substantially diminished in nonhuman
primate lungs when E4 is deleted from the first-generation adenovirus
vector.
Antigen-specific immune responses to the CFTR adenovirus
vectors were analyzed in lungs of rhesus monkeys. A total of four monkeys were studied. Two received the vector with a deletion of E1 and
two received the vector with deletions of E1 and E4. Equivalent
concentrations of vector were directly instilled into the airways of
monkeys with a bronchoscope. Animals from each group were necropsied at
days 11 and 29. A report of the clinical and pathological analyses of
these animals will be provided elsewhere.
CD4 T-cell responses to adenovirus vector were studied from peripheral
blood harvested at day 11. LPR was identical in animals
that received
either vector (Fig.
8). The vector with
one deletion
resulted in secretion of TH1 (i.e., IL-2 and IFN-
) and
TH2 (i.e.,
IL-10 and IL-4) cytokines. The TH1-specific cytokines were
retained
with the vector with two deletions, although the TH2-specific
cytokines (IL-4 and IL-10) were diminished (Fig.
9).
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FIG. 8.
Lymphoproliferative responses in rhesus monkeys.
Peripheral blood mononuclear cells obtained on day 11 from animals
given either H5.020CBCFTR (animal AA6X) or
H5.001CBCFTR (animal AA3B) intratracheally were cultured in
medium alone or in the presence of inactivated adenovirus for 7 days.
LPR responses were measured by [3H]thymidine
incorporation and are presented as means of triplicate cultures.
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FIG. 9.
Cytokine secretion profiles in rhesus monkeys. Rhesus
monkeys were given either H5.020CBCFTR (animal AA6X) or
H5.001CBCFTR (animal AA3B) intratracheally. Peripheral blood
mononuclear cells isolated from heparinized blood on day 11 were
cultured in medium alone (med) or in the presence of inactivated
adenovirus (Adeno) for 48 h. Culture supernatants were analyzed
for the presence of IL-2, IFN-, IL-4, and IL-10 by commercial ELISA
kits, and results are presented as means of duplicate cultures.
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Humoral immune responses following vector administration were analyzed
in the serum and BAL fluid obtained on day 29. Animals
that were given
the vector with a deletion of E1 developed a strong
neutralizing-antibody responses both in serum and BAL fluid, whereas
those given vector with deletions of E1 and E4 showed a weaker
neutralizing-antibody response in the serum and failed to generate
neutralizing antibody in BAL fluid, as observed in the studies
in mice
(Fig.
10). IgM responses to adenovirus
were equivalent
with both vectors while IgG and IgA responses were
weaker in the
animal that received the vector with deletions of E1 and
E4 (Fig.
11).
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FIG. 10.
Neutralizing antibodies in serum and BAL fluid of
rhesus monkeys. Day 29 sera and BAL fluid obtained from animals given
either H5.020CBCFTR (animal AA6R) or H5.001CBCFTR
(animal AA7B) intratracheally was analyzed for the presence of
neutralizing antibody by the ability of sera to block infection of HeLa
cells with lacZ virus. The reciprocal dilution is presented.
The results are means of duplicate wells.
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FIG. 11.
Adenovirus-specific Ig in rhesus monkeys. Day 29 sera
obtained from animals given either H5.020CBCFTR (animal
AA6R) or H5.001CBCFTR (animal AA7B) intratracheally were
analyzed for the presence of adenovirus-specific IgM, IgG, and IgA by
ELISA. The results denote means of duplicate wells. OD, optical
density.
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DISCUSSION |
Immunologic responses to recombinant adenoviruses have emerged as
a major issue in the success of in vivo gene therapy (21). The initial paradigm suggested that the activation of CTLs in response
to proteins expressed from the vector genome (i.e., in first-generation
constructs this includes products from the transgene and viral genes)
and the formation of neutralizing antibodies to the input viral capsid
proteins were critical for the transient transgene expression and
problems with readministration. The role of CTLs in vector performance
for transgene persistence and inflammation has been confirmed in
multiple settings. The relative importance of different vector-derived
antigens in cellular immunity as well as the contribution of other
factors, such as transcriptional inactivation or vector genome
instability, is more controversial and is likely to be vector and model
specific.
The formation of neutralizing antibodies to viral capsid proteins is a
reproducible finding that was present in virtually all models studied.
The emergence of neutralizing antibodies to inactivated adenovirus
vectors at levels similar to that obtained with an identical dose of
infectious virus from which E1 had been deleted suggested that this
B-cell response is primarily mediated by MHC class II presentation of
capsid proteins from the input vector (24). Our studies
implicate factors other than the capsid antigens in modulating the
resulting humoral response. Specifically, identical particle quantities
of viruses with the deletions described above administered to lung
yielded identical LPR and TH1 responses but very different TH2 and
B-cell responses. The net effect was substantially diminished
production of neutralizing antibody to the vectors with deletions of E1
and E4.
Our findings are consistent with the concept that both the antigen and
the context in which it is presented influence immune responses.
Activation of naïve T cells requires presentation of antigen by
a professional antigen-presenting cell (APC), such as a dendritic cell.
Environmental factors, such as the inflammation associated with an
infection, trigger key steps in the activation and maturation of the
APC to modulate the T-cell response (3, 16). The innate
immune system, which is capable of recognizing and responding to
infection, may provide a key link to the acquired immune response via
the regulation of APCs (8, 11).
How can vector genotype affect the innate immune response and impact
antigen presentation? A vector with deletions of E1 and E4 is more
attenuated than one with a deletion of E1, which leads to diminished
inflammation prior to the development of antigen-specific processes.
Relevant to our studies is the observation that the vectors from which
E1 and E4 have been deleted induced lower IL-6 and IL-8 levels in BAL
fluid than did the vectors with deletions of E1. Previous experiments
in mice, in the setting of autoimmune disease and parasitic infections,
have demonstrated a critical role of IL-6 in the expression of B7 and
in the induction of TH2 responses (12, 17). The E4 gene
products are toxic and interfere with a number of cellular proteins
involved in the cell cycle and apoptosis (13). One can
envision models in which direct transduction of the APC or a bystander
effect from a nearby transduced cell could impact antigen presentation.
The adenovirus vectors characterized in this study are being or have
been evaluated in clinical trials. The vectors differ at the E4 and E3
loci. Introduction of the CFTR minigene into our first-generation
construct required the deletion of the nonessential E3 genes to
maintain an overall genome length of <105% of wild type. For a
variety of reasons, however, we do not believe that E3 genes play a
role in the observed effect. In the absence of E1, there is no
detectable transcription of E3 from its endogenous promoter. We have in
fact evaluated cellular and humoral immune responses to vectors with
deletions of E1 which express the smaller transgene -galactosidase
and which differ by the presence or absence of E4. The lacZ
vector with a deletion of E1 that contained an intact E3 region
generated far less neutralizing antibody after intratracheal
administration than did the version from which E1 and E4 had been
deleted (Fig. 12). Another question
concerns the impact of the host on the differential CD4 T-cell and
humoral responses noted in our studies. In fact, qualitatively
different T-helper subset responses to pathogens such as
Leishmania in different strains of mice have been observed
(1). The fact that the E4 effects were observed in multiple
strains of mice (H-2b, C57BL/6;
H-2d, BALB/c; and H-2k,
C3H; data not shown) and in two species (mice and nonhuman primates) suggests that it may have broader implications in gene therapy directed
to the lung.
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|
FIG. 12.
Neutralizing-antibody response to lacZ
vectors. Day 29 sera obtained from animals given either vehicle
H5.000CMVlacZ or H5.001CMVlacZ intratracheally
were analyzed for the presence of neutralizing antibody by the ability
of sera to block infection of HeLa cells with lacZ virus.
The dose of vector was 5 × 1010 particles. The figure
shows means for three animals in each group plus standard deviation.
|
|
Our studies point out another advantage of using advanced-generation
adenovirus vectors with deletions of multiple essential genes. Problems
of neutralizing antibody are substantial in applications of gene
therapy that require repeated administrations of vectors. It remains to
be seen if the advantage afforded by the deletion of E4 is sufficient
to enable effective repeated administrations of vector. Administration
of a pharmacologic inhibitor of T cells with the vector from which E1
and E4 have been deleted may provide sufficient additive and/or
synergistic inhibition of B-cell activation necessary for their
repeated use in chronic diseases such as cystic fibrosis.
| |
ACKNOWLEDGMENTS |
These studies were performed in the context of the Translational
Research Program at the Institute for Human Gene Therapy (study numbers
96-20, 96-27, 96-32, 96-39, and 97-11). Support from the Vector and
Cell Morphology Cores and the excellent technical help of George Qian
and Ruth Qian in the Immunology Core are appreciated.
This work was supported by grants from the Cystic Fibrosis Foundation,
the NIDDK of the NIH (P30 DK47757-05), CFSCOR (P50 DK49136-04), and
Genovo, Inc.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The Wistar
Institute, 3600 Spruce St., Philadelphia, PA 19104. Phone: (215)
898-1979. Fax: (215) 573-7414. E-mail:
jurmu{at}wista.wistar.upenn.edu.
| |
REFERENCES |
| 1.
|
Abbas, A. K.,
M. M. Murphy, and A. Sher.
1996.
Functional diversity of helper T lymphocytes.
Nature
383:787-793[Medline].
|
| 2.
|
Armentano, D.,
J. Zabner,
C. Sacks,
C. C. Sookdeo,
M. P. Smith,
J. A. St. George,
S. C. Wadsworth,
A. E. Smith, and R. J. Gregory.
1997.
Effect of the E4 region on the persistence of transgene expression from adenovirus vectors.
J. Virol.
71:2408-2416[Abstract].
|
| 3.
|
Cella, M.,
A. Engering,
V. Pinet,
J. Pieters, and A. Lanzavecchia.
1997.
Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells.
Nature
388:782-787[Medline].
|
| 4.
|
Dai, Y.,
E. M. Schwarz,
D. Gu,
W. W. Zhang,
N. Sarvetnick, and I. M. Verma.
1995.
Cellular and humoral immune responses to adenoviral vectors containing factor IX gene: tolerization of factor IX and vector antigens allows for long-term expression.
Proc. Natl. Acad. Sci. USA
92:1401-1405[Abstract/Free Full Text].
|
| 5.
|
Dedieu, J.-F.,
E. Vigne,
C. Torrent,
C. Jullien,
I. Mahfouz,
J.-M. Caillaud,
N. Aubailly,
C. Orsini,
J.-M. Guillaume,
P. Opolon,
P. Delaère,
M. Perricaudet, and P. Yeh.
1997.
Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses.
J. Virol.
71:4626-4637[Abstract].
|
| 6.
|
Engelhardt, J. F.,
L. Litzky, and J. M. Wilson.
1994.
Prolonged transgene expression in cotton rat lung with recombinant adenoviruses defective in E2a.
Hum. Gene Ther.
5:1217-1229[Medline].
|
| 7.
|
Engelhardt, J. F.,
X. Ye,
B. Doranz, and J. M. Wilson.
1994.
Ablation of E2A in recombinant adenoviruses improves transgene persistence and decreases inflammatory response in mouse liver.
Proc. Natl. Acad. Sci. USA
91:6196-6200[Abstract/Free Full Text].
|
| 8.
|
Fearon, D. T., and R. M. Locksley.
1996.
The instructive role of innate immunity in the acquired immune response.
Science
272:50-54[Abstract].
|
| 9.
|
Gao, G.-P.,
Y. Yang, and J. M. Wilson.
1996.
Biology of adenovirus vectors with E1 and E4 deletions for liver-directed gene therapy.
J. Virol.
70:8934-8943[Abstract].
|
| 10.
|
Goldman, M. J.,
L. A. Litzky,
J. F. Engelhardt, and J. M. Wilson.
1995.
Transfer of the CFTR gene to the lung of nonhuman primates with E1-deleted, E2a-defective recombinant adenoviruses: a preclinical toxicology study.
Hum. Gene Ther.
6:839-851[Medline].
|
| 11.
|
Janeway, C. A.
1989.
In
Approaching the asymptote? Evolution and revolution in immunology, p. 1-13.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 12.
|
Lenschow, D. J.,
K. C. Herold,
L. Rhee,
B. Patel,
A. Kooks,
H.-Y. Qin,
E. Fuchs,
B. Singh,
C. B. Thompson, and J. A. Bluestone.
1996.
CD28/B7 regulation of TH1 and TH2 subsets in the development of autoimmune diabetes.
Immunity
5:285-293[Medline].
|
| 13.
|
Leppard, K. N.
1997.
E4 gene function in adenovirus, adenovirus vector and adeno-associated virus infections.
J. Gen. Virol.
78:2131-2138[Medline].
|
| 14.
|
Lieber, A.,
C.-Y. He, and M. A. Kay.
1997.
Adenoviral preterminal protein stabilizes mini-adenoviral genes in vitro and in vivo.
Nat. Biotechnol.
15:1383.
[Medline] |
| 15.
|
Lieber, A.,
C.-Y. He,
I. Kirillova, and M. A. Kay.
1996.
Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo.
J. Virol.
70:8944-8960[Abstract].
|
| 16.
|
Pierre, P.,
S. J. Turley,
E. Gatti,
M. Hull,
J. Meltzer,
A. Mirza,
K. Inaba,
R. M. Steinman, and I. Mellman.
1997.
Developmental regulation of MHC class II transport in mouse dendritic cells.
Nature
388:787-792[Medline].
|
| 17.
|
Rincon, M.,
T. Anguita,
T. Nakamura,
E. Fikrig, and R. A. Flavell.
1997.
Interleukin 6 directs the differentiation of IL-4 producing CD4 T cells.
J. Exp. Med.
185:461-469[Abstract/Free Full Text].
|
| 18.
|
Schiedner, G.,
N. Morral,
R. J. Parks,
Y. Wu,
S. C. Koopmans,
C. Langston,
F. L. Graham,
A. L. Beaudet, and S. Kochanek.
1998.
Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity.
Nat. Genet.
18:180-183[Medline].
|
| 19.
|
Tripathy, S. K.,
H. B. Black,
E. Goldwasser, and J. M. Leiden.
1996.
Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors.
Nat. Med.
2:545-550[Medline].
|
| 20.
|
Wang, Q.,
G. Greenburg,
D. Bunch,
D. Farson, and M. H. Finer.
1997.
Persistent transgene expression in mouse liver following in vivo gene transfer with a delta E1/delta E4 adenovirus vector.
Gene Ther.
4:393-400[Medline].
|
| 21.
|
Wilson, J. M.
1996.
Adenoviruses as gene-delivery vehicles.
N. Engl. J. Med.
334:1185-1187[Free Full Text].
|
| 22.
|
Yang, Y.,
F. A. Nunes,
K. Berencsi,
E. E. Furth,
E. Gonczol, and J. M. Wilson.
1994.
Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy.
Proc. Natl. Acad. Sci. USA
91:4407-4411[Abstract/Free Full Text].
|
| 23.
|
Yang, Y.,
K. U. Jooss,
Q. Su,
H. C. Ertl, and J. M. Wilson.
1996.
Immune responses to viral antigens versus transgene product in the elimination of recombinant adenovirus-infected hepatocytes in vivo.
Gene Ther.
3:137-144[Medline].
|
| 24.
|
Yang, Y.,
Q. Li,
H. C. J. Ertl, and J. M. Wilson.
1995.
Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses.
J. Virol.
69:2004-2015[Abstract].
|
| 25.
|
Zsengeller, Z. K.,
S. E. Wert,
W. M. Hull,
X. Hu,
S. Yei,
B. C. Trapnell, and J. A. Whitsett.
1995.
Persistence of replication-deficient adenovirus-mediated gene transfer in lungs of immune-deficient (nu/nu) mice.
Hum. Gene Ther.
6:457-467[Medline].
|
J Virol, July 1998, p. 6138-6145, Vol. 72, No. 7
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
