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Journal of Virology, April 2000, p. 3345-3352, Vol. 74, No. 7
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Readministration of Adenovirus Vector in Nonhuman
Primate Lungs by Blockade of CD40-CD40 Ligand Interactions
Narendra
Chirmule,1
Steven E.
Raper,1
Linda
Burkly,2
David
Thomas,2
John
Tazelaar,1
Joseph V.
Hughes,1 and
James M.
Wilson1,*
Institute for Human Gene Therapy, Department
of Molecular and Cellular Engineering, University of Pennsylvania, and
The Wistar Institute, Philadelphia,
Pennsylvania,1 and Biogen, Inc.,
Cambridge, Massachusetts2
Received 26 August 1999/Accepted 5 January 2000
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ABSTRACT |
The interaction between CD40 on B cells and CD40 ligand (CD40L) on
activated T cells is important for B-cell differentiation in
T-cell-dependent humoral responses. We have extended our previous murine studies of CD40-CD40L in adenoviral vector-mediated immune responses to rhesus monkeys. Primary immune responses to adenoviral vectors and the ability to readminister vector were studied in rhesus
monkeys in the presence or absence of a transient treatment with a
humanized anti-CD40 ligand antibody (hu5C8). Adult animals were treated
with hu5C8 at the time vector was instilled into the lung.
Immunological analyses demonstrated suppression of adenovirus-induced lymphoproliferation and cytokine responses (interleukin-2 [IL-2], gamma interferon, IL-4, and IL-10) in hu5C8-treated animals. Animals treated with hu5C8 secreted adenovirus-specific immunoglobulin M (IgM)
levels comparable to control animals, but did not secrete IgA or
develop neutralizing antibodies; consequently, the animals could be
readministered with adenovirus vector expressing alkaline phosphatase.
A second study was designed to examine the long-term effects on immune
functions of a short course of hu5C8. Acute hu5C8 treatment resulted in
significant and prolonged inhibition of the adenovirus-specific humoral
response well beyond the time hu5C8 effects were no longer significant.
These studies demonstrate the potential of hu5C8 as an immunomodulatory
regimen to enable administration of adenoviral vectors, and they
advocate testing this model in humans.
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INTRODUCTION |
Adenoviral vectors are attractive
tools for effectively transducing a wide range of cells (7,
26). The major limitation of adenovirus vectors in gene therapy
has been the ensuing immune response to viral proteins and transgene
product (8, 24, 29, 30). Extensive studies in mice have
demonstrated that a vigorous cell-mediated immune response generated
against the late gene products and transgene products eliminate the
vector-transduced cells through activation of CD4+
T-cell-dependent, gamma interferon (IFN-
)-activated, cytotoxic T
cells (reviewed in reference 4). Activation of
humoral immunity results in the induction of neutralizing antibodies,
which prevents readministration. Several studies have now demonstrated
that blocking of both T- and B-cell responses results in prolonged
transgene expression and effective readministration of adenovirus
vector in mice (4, 8, 16, 28). We have established in murine models that blocking antibodies to CD40L abolish
adenovirus-vector-specific B-cell functions and severely compromise
T-cell responses, allowing for efficient readministration of the vector
(32, 33).
The crucial role of the CD40 molecule, expressed on B cells,
professional and nonprofessional antigen-presenting cells (APC), endothelial cells, and some epithelial cells for effector cell function, has been clearly established (13, 25). The
regulation of T-cell-dependent B-cell functions by CD40-CD40L
interactions involves signals transduced through the CD40 molecule. The
CD40-mediated signals have been involved in multiple functional
responses, e.g., immunoglobulin (Ig) class switching and induction of
anti-apoptotic protein BclxL in B cells, upregulation of B7
family proteins on macrophages and dendritic cells, induction of
regulatory cytokines and inflammatory cytokines (interleukin-12
[IL-12], IL-1
, IL-6, lymphotoxin-tumor necrosis factor alpha, and
IL-8) (15, 23). Thus, the wide distribution of CD40 has
implicated its signaling pathway at multiple levels in the regulation
of the effector functions of the immune system.
The T-cell counter-receptor for CD40 is the CD40 ligand (CD40L)
(gp39, T-BAM, CD154), a type-II integral membrane glycoprotein, transiently expressed on antigen-activated CD4+ T
cells. Experiments performed in mice, with in vivo infusion of
blocking CD40L monoclonal antibody (MAb) or genetic mutations in its
gene, have shown marked dysfunction of humoral immunity as indicated by
decreased B-cell proliferation, Ig secretion, and class switching,
maintenance of germinal centers, and memory B cells (1). The
importance of the CD40-CD40L interactions in the regulation of T cells
was implicated in observations of opportunistic infections with
Pneumoncystis carinii, Cryptosporidium, and
cytomegalovirus in patients with Hyper-IgM syndrome, who have a
mutational defect in the CD40L gene (9). Indeed, it is now widely recognized that CD40L-CD40 interactions play pivotal roles in
the development of CD4+ T-cell-dependent immune responses.
Several mechanisms by which these molecules regulate T-cell functions
have been identified, including involvement of interacting
costimulatory molecules on APC (e.g., B7-1) and T cells (e.g., CD28,
adhesion molecules ICAM-1, CD44, and cytokines) or IL-12 (10,
13).
The central role of the CD40-CD40L interactions in the regulation of
immune responses has been exploited in strategies of transplantation
immunology of graft acceptance or tolerance (3, 20, 22). The
present study was undertaken with the hypothesis that in vivo
administration of adenoviral vectors elicits cell-mediated and humoral
immune responses, both of which require functional CD4+ T
helper cells (28, 31). The in vivo model predicts that inhibition of CD4+ T-cell responses at the time of
administration of the vector would interfere with the induction of
cellular and humoral responses to adenoviral proteins, resulting in
prolonged transgene expression and efficient readministration. In this
study, we have analyzed the use of a short course of anti-CD40 ligand
antibody (hu5C8) in lung-directed gene therapy in rhesus monkeys.
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MATERIALS AND METHODS |
Animals and specimen collection.
Wild, caught juvenile
rhesus monkeys were purchased from Southwest Foundation for Biomedical
Research (San Antonio, Tex.) and underwent full quarantine. The monkeys
weighed approximately 3 to 4 kg, and were serologically negative for
simian immunodeficiency virus, simian T-cell leukemia virus, other
simian retroviruses, and human adenovirus. The protocol was approved by
the Infection Control Committee of The Hospital of the University of
Pennsylvania, the Environmental Health and Safety Office, the
Institutional Biosafety Committee, and The Institutional Animal Care
and Use Committee of The University of Pennsylvania. The monkeys are
identified in the two protocols as denoted in Fig.
1.
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FIG. 1.
Schematic representation of the study designs.
(Top) Experimental protocol 1 comprised six animals (in three groups:
1a, 1b, and 1c), four of which received hu5C8 antibody (T3A, T6A, T8P,
and T7X) in a six-injection regimen of 5 mg/kg/dose intravenously on
days  2, 1, 1, 3, 5, and 7 while two served as controls (T8J and
T7N). All the animals were intratracheally instilled with
Ad-LacZ in the left lung on day 1. Ad-ALP vector was
instilled in the right lung in animals T7N, T3A, and T6A on day 43, and
in animals T8J, T7X, and T8P on day 85. Peripheral blood and BAL were
taken at various time points. Animals were necropsied for analyses of
transgene expression in the lung on either day 46 (T7N, T3A, and T6A)
or day 88 (T8J, T7X, and T8P). (Bottom) Experimental protocol 2 comprised eight animals in four groups (2a, 2b, 2c, and 2d). hu5C8 was
administered intravenously in a six-injection regimen of 5 mg/kg/dose
on days 2, 1, 1, 3, 5, and 7 (in group 2b and 2c) and again in
group 2b on days 178, 179, 180, 183, 185, and 187. Animals were
intratracheally instilled with Ad-CFTR on days 1 and 180. Group 2a,
animals received Ad-CFTR on day 1 and readministered Ad-CFTR on day
180; Group 2b, animals were administered hu5C8, both during first and
second vector instillation; Group 2c, animals were administered hu5C8
during the first, but not the second vector instillation; Group 2d,
animals served as controls and received only Ad-CFTR on day 180. Peripheral blood and BAL were drawn at various time points. All animals
were necropsied on day 210 for analysis of toxicity and immune
responses.
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Antibody.
The anti-CD154 MAb hu5C8 was prepared as
previously described (18) and humanized (Biogen, Inc.,
Cambridge, Mass.). hu5C8 dosing is as specified in the legend to Fig.
1.
Vectors.
The construction and production of
H5.010CBLacZ (henceforth called Ad-LacZ) and
H5.100CBALP (henceforth called Ad-ALP), the E1-deleted
recombinant adenovirus vectors expressing LacZ and alkaline
phosphatase (ALP), respectively, have been described previously
(11, 12). Virus was titered at a particle-to-PFU ratio of
100. For the study protocol 2, H5.020CBCFTR (henceforth called Ad-CFTR) was used, which expresses the human cystic fibrosis transmembrane receptor gene. This vector has been used in a Phase I
clinical trial and has been previously described (5).
Vector administration.
Monkeys were anesthetized with
ketamine-atropine. Physical examination was performed, and a 22-gauge
intravenous needle was inserted for emergency medications. After chest
X ray and blood draws, the monkey was brought to the operating room
suite. Pulse oximetry was applied, and the monkey was placed supine
with the head in the sniffing position. Using the laryngoscope, the
vocal cords were visualized and sprayed with Cetacaine. The
bronchoscope was passed through the vocal cords, and the membranous
trachea and carina were identified. By using these landmarks, we
identified the right mainstem bronchus and entered under direct vision.
Sterile saline (10 ml) was injected into a peripheral branch and
aspirated into a mucus trap (for bronchoalveolar lavage [BAL]). For
administration, 1 ml of the virus (5 × 1012
particles) was instilled into the mainstem bronchus through the biopsy
port of the bronchoscope. The bronchoscope was withdrawn under direct
vision, and the monkeys were allowed to emerge from anesthesia and were
returned to the colony in stable condition.
ALP histochemistry for ALP expression.
Cryostat sections (6 µm) of lung tissue were fixed in 0.5% glutaraldehyde and were rinsed
with 1 mM MgCl2 in phosphate-buffered saline (PBS) (all
reagents were obtained from Sigma Chemical Co., St. Louis, Mo.). Slides
were incubated in ALP substrate consisting of
5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium (NBT) in PBS for 30 min in the dark at 37°C to detect the presence of
ALP enzyme activity. Sections were counterstained with neutral red and mounted.
Lymphoproliferative (LPR) responses.
Peripheral blood
mononuclear cells (PBMC) from the various time points of the study were
separated by Ficoll-Hypaque density gradient centrifugation. In some
cases, cells were cryopreserved in liquid nitrogen. At the time of the
assay, PBMC were thawed by standard protocols. Triplicate cultures of
106 PBMC per ml (100 µl) were cultured with either 10 multiplicity of infection (MOI) of inactivated Ad-LacZ, 10 µg of phytohemagglutinin (PHA) per ml (Difco, Franklin Lakes, N.J.),
100 ng of Staphylococcus enterotoxin B (SEB) per ml (Toxin
Technologies, Sarasota, Fla.) or medium alone. PHA-stimulated cultures
were harvested on day 3, and SEB- and antigen-stimulated cultures were
harvested on day 6. In some cases, anti-CD28 (1 µg/ml) (Coulter
Immunotech, Miami, Fla.) was added to the cultures. Proliferation was
measured by a 16-h [3H]thymidine (1 µCi/well) pulse.
Results were presented as a stimulation index, which represents a ratio
of counts per minute of adenovirus-stimulated cultures to counts per
minute of cultures with medium alone.
Cytokine release assays.
PBMC were cultured with or without
antigen (i.e., inactivated Ad-LacZ at a particle-to-cell
ratio of 10) for 48 h in a 24-well plate. Cell-free supernatants
were collected and analyzed for the presence of IL-2, IL-4, IFN-,
and IL-10 by commercial enzyme-linked immunosorbent assay (ELISA) kits
(BioSource International, Camarrillo, Calif.) by following
manufacturers' protocols.
Adenovirus-specific Igs.
Serum (diluted 1:200) and BAL
(diluted 1:20) samples from animals were analyzed for
adenovirus-specific isotype-specific Igs (IgM, IgG, and IgA) by ELISA.
For the ELISA, 96-well flat-bottomed, high-binding Immulon-IV plates
were coated with 100 µl of Ad-LacZ antigen (5 × 109 particles/ml) in PBS overnight at 4°C, were washed
four times in PBS-0.05% Tween, and were blocked in PBS-1% bovine
serum albumin for 1 h at 37°C. Appropriately diluted samples
were added to antigen-coated wells and were incubated for 4 h at
37°C. Plates were washed four times in PBS-0.05% Tween and were
incubated with peroxidase-conjugated goat anti-human IgM, IgG, or IgA
(1:2,000 dilution) (Sigma Chemical Co.) for 2 h at 37°C. Plates
were washed as described above, and ABTS substrate (Kirkegaard and
Perry, Gaithersburg, Md.) was added. Optical densities were read at 405 nm on a microplate reader (Dynatech Laboratories, Chantilly, Va.).
Anti-adenovirus neutralizing antibodies.
Neutralizing
antibody (NAB) titers were analyzed by determining the ability of serum
or BAL antibody to inhibit transduction of reporter virus,
Ad-LacZ, into HeLa cells. Various dilutions of antibodies
(twofold dilutions, 1:20 to 1:2560) preincubated with the reporter
virus (100 MOI) for 1 h at 37°C were added to 90% confluent
HeLa cell cultures (104 cells/well in a 96-well plate).
Cells were incubated for 16 h at 37°C in a humidified
CO2 incubator, and expression of -galactosidase was
analyzed by
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal)
staining. The neutralizing titer of antibody was calculated by the
highest dilution with which 50% of the cells turned blue.
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RESULTS AND DISCUSSION |
In vivo treatment of rhesus monkeys with hu5C8 markedly
inhibits initiation of the adenovirus-specific immune responses.
Experimental protocol 1 (Fig. 1) comprised six animals. Group 1a
animals (T3A and TGA) received Ad-LacZ with hu5C8 on day 1 and were subsequently administered Ad-ALP on day 43. Group 1b animals
(T8P and T7X) were treated the same as those of group 1a except that
the Ad-ALP was administered on day 85. Group 1c was a control with both
animals receiving Ad-LacZ without antibody on day 1 and
Ad-ALP on either day 43 (animal T7N) or day 85 (animal T8J). In
experimental protocol 1, cell-mediated and humoral immune responses
were analyzed in animals administered Ad-LacZ (Fig. 2). Intratracheal administration of
Ad-LacZ in the absence of hu5C8 (group 1c) resulted in
generation of a brisk LPR response, measured on day 14. Analyses of
humoral immune responses showed that these animals generated strong
neutralizing antibodies and adenovirus-specific IgA in BAL. The
presence of a neutralizing antibody response was confirmed by the
inability to readminister Ad-ALP in bronchoepithelial cells of these
animals (T7N and T8J) either on day 43 or 85 (Fig.
3 and Table
1).
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FIG. 2.
In vivo treatment of rhesus monkeys with hu5C8 antibody
inhibits adenovirus-vector-mediated, cell-mediated, and humoral immune
responses. Rhesus monkeys were treated with Ad-LacZ with
(groups 1a and 1b) or without (group 1c) hu5C8 as described in the
legend of Fig. 1 top. (A and B) PBMC were isolated from heparinized
blood, drawn on day 14 post-vector administration from experimental
protocol 1. Cells were cultured with inactivated adenovirus antigen or
PHA. Lymphoproliferative responses were measured by
[3H]thymidine incorporation as described in Materials and
Methods. Results are denoted as a stimulation index (counts per minute
of adenovirus stimulated cells/counts per minute of cells in medium
alone) or counts per minute of [3H]thymidine
incorporation. All the 5C8-treated animals had a significant decrease
in induction of adenovirus-induced LPR responses (P < 0.05, Student's t test); PHA responses were not
significantly different from controls. (C and D) BAL samples taken on
day 28 were analyzed for the presence of adenovirus-specific
neutralizing antibodies and IgA. The NAB titer was calculated from the
highest dilution of the sera required to block 50% of the cells
turning blue and was denoted as reciprocal dilution. IgA in BAL was
measured by ELISA.
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FIG. 3.
Micrographs of readministration of ALP staining in the
broncholes of rhesus monkeys. Rhesus monkeys were administered Ad-ALP
in the right lower lobe as depicted in Fig. 1 top. Animals were
necropsied, and the right lower lobe was sectioned for ALP expression,
as described in Materials and Methods. Alkaline phosphatase expression
in the broncholes of animals T7N (A) and T8P (B) were analyzed by
staining with buffer containing BCIP and NBT, as described in Materials
and Methods. There was significant alveolar endogenous ALP staining in
all the tissue. However, extensive analyses of lungs of rhesus monkeys
not administered ALP-expressing adenovirus showed that none of the
broncholar epithelial cells had endogenous ALP staining. Panel A,
animal T7N; panel B, animal T8P; magnification, ×186.
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TABLE 1.
Morphometric analyses of recipient lungs for efficiency
of recombinant gene expression in experimental
protocol 1a
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Treatment of animals with hu5C8 resulted in marked inhibition of
adenovirus-specific LPR responses. PHA-induced responses
were not
significantly affected, indicating that hu5C8 did not
result in the
depletion of T-cell populations (Fig.
2B). Animals
treated with hu5C8
had markedly suppressed adenovirus-specific
neutralizing
antibodies and IgA responses, as compared to untreated
animals (Fig.
2C
and D). Animals could be efficiently readministered
Ad-ALP on day
43 (animals T3A and T6A) (Table
1) and day 85 (animals
T8P and T7X)
(Table
1 and Fig.
3), demonstrating that the transient
antibody
treatment resulted in prolonged inhibition of the vector-specific
B-cell responses, sufficient to readminister Ad-ALP. In the
hu5C8-treated
animals, readministration of Ad-ALP on day 43 (T3A
and T6A) induced
a strong NAB response (data not shown), demonstrating
that the
hu5C8 treatment does not induce tolerance to adenovirus
vector,
and only affects initiation of a primary immune
response.
Effect of treatment of rhesus monkeys with hu5C8 MAb results in
long-term inhibition of adenovirus-specific cell-mediated and humoral
immune responses.
Experimental protocol 2 (Fig. 1 bottom) was
designed to analyze the impact of transient treatment with hu5C8
treatment on suppression of adenovirus vector-specific cell-mediated
and humoral immune responses up to 180 days. This study also addressed
whether treatment of animals with hu5C8, along with readministration of vector 2, suppressed subsequent immune responses.
Experimental protocol 2 comprised eight animals in four groups (2a, 2b,
2c, and 2d) as follows: Group 2a, Ad vector on days
1 and 180; Group
2b, Ad vector on days 1 and 180 with hu5C8 administered
with each
vector; Group 2c, Ad vector on days 1 and 180 with hu5C8
administered
with the first vector; and Group 2d, only vector
on day
180.
Intratracheal administration of Ad-CFTR into the lungs of rhesus
monkeys resulted in a strong inflammatory response, as measured
by IL-8
secretion (Fig.
4). Antigen-specific LPR
responses to
adenoviral antigens were determined at various time points
during
the study (Fig.
5). Intratracheal
instillation of Ad-CFTR to rhesus
monkeys in the absence of hu5C8
treatment (animals AA8J and AA7V)
induced an LPR response which peaked
on day 90 and diminished
by day 136. Antigen specificity was confirmed
by the lack of LPR
responses in control animals, which did not receive
Ad-CFTR on
day 1 (Group 2d). The qualitative nature of the response was
determined
by analyses of cytokine secretion profiles on day 36. PBMC
from
animals treated with vector alone secreted IL-2, IFN-
, IL-4,
and IL-10 in response to in vitro adenovirus stimulation (Fig.
6). Humoral immune responses were
measured by analyzing NABs in
serum and BAL fluids. Table
2 shows that none of the animals
had
preexisting NABs to adenovirus on day
2. NAB was observed
in serum
samples of animals treated with vector alone by day 14,
which peaked at
day 29 and diminished through day 180. The mucosal
humoral immune
response was analyzed by measuring NAB in BAL fluid.
BAL from control
animal Group 2a (i.e., vector but no hu5C8) developed
NAB which peaked
on day 29. Interestingly, the NAB in BAL in these
animals (AA8J and
AA7V) diminished to baseline titers by day 90.
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FIG. 4.
hu5C8 treatment inhibits generation on inflammatory
cytokine IL-8 in the lung. Rhesus monkeys were instilled with Ad-CFTR
on day 1 with or without hu5C8 as described in the legend to Fig. 1
bottom. BAL obtained on day 2 (prior to vector instillation) and 29 were analyzed for the presence of IL-8 by ELISA. Treatments of the
animals in the various groups are described in the legend to Fig. 1
bottom. All of the 5C8-treated animals had a significant decrease in
induction of IL-8 (P < 0.05, Wilcoxon's test) as
compared to those who received vector but were not administered 5C8.
Since the rhesus monkeys were caught in the wild, the prevector
inflammatory status of the lung could not be controlled. All the
monkeys underwent a 3-month quarantine period, when they were tested
for a variety of infectious agents; all tests were negative.
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FIG. 5.
Adenovirus-vector-mediated LPR responses in rhesus
monkeys in experimental protocol 2. Rhesus monkeys were instilled with
Ad-CFTR on days 1 and 180 with or without hu5C8 as described in the
legend to Fig. 1 bottom. PBMC isolated from heparinized blood were
cultured with inactivated adenovirus vector. Lymphoproliferative
responses, measured by [3H]thymidine incorporation, are
presented as a stimulation index, which is the ratio of the counts per
minute of PBMC cultured with adenovirus vector and counts per minute of
PBMC cultured with medium alone. Adenovirus-specific responses induced
following the first intratracheal vector instillation were analyzed on
various days of the experimental protocol. Blue lines represent animals
in Group 2a, red lines represent animals treated with hu5C8 (Groups 2b
and 2c), and green lines represent animals in Group 2d.
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FIG. 6.
Cytokine secretion profiles of animals in experimental
protocol 2. Rhesus monkeys were instilled with Ad-CFTR on days 1 and
180 with or without hu5C8 as described in the legend to Fig. 1 bottom.
IFN-, IL-2, IL-4, and IL-10 cytokines secreted by PBMC were measured
on day 36 after vector-1 instillation and on day 210 (i.e., 30 days
after vector-2 instillation). PBMC were cultured in the presence or
absence of inactivated Ad-LacZ for 48 h, and culture
supernatants were analyzed for the indicated cytokines by commercial
ELISA kits. None of the culture supernatants from PBMC cultured in the
absence of adenovirus vector antigen induced cytokines; only
adenovirus-vector-induced cytokines are shown in the figure.
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CD40-CD40L interactions have been recently implicated in induction of
inflammatory responses in the lung. In this study, animals
administered
vector alone generated a brisk local inflammatory
response, as
measured by secretion of IL-8. hu5C8-treated animals
elicited
a markedly blunted IL-8 response (Fig.
4). Since IL-8
is predominantly
secreted by inflammatory cells, our results suggest
that hu5C8
treatment inhibits recruitment of inflammatory cells
into the lung or
activation of those recruited cells. These observations
are
consistent with several studies, which have implicated CD40-CD40L
interactions in inflammatory responses (
13).
The effect of hu5C8 treatment on adenovirus-specific LPR responses at
various time points of the study is shown in Fig.
5.
None of the
four hu5C8-treated animals elicited an adenovirus
LPR response through
at least day 36. By day 90, animals administered
hu5C8 elicited modest
LPR responses, which were less than those
of control animals, and which
remained relatively low through
day 136. During the course of the
study, hu5C8 treatment did not
affect SEB-induced LPR responses (data
not
shown).
Additional experiments were performed to determine whether lack of
T-cell priming in the absence of CD40L was due to inhibition
of T-cell
activation, anergy induction, or deletion of antigen-specific
T cells.
PBMC were treated in vitro with anti-CD28 MAb and were
stimulated with
vector-derived antigens. Costimulation of PBMC
from hu5C8-treated
animals in vitro with adenovirus antigen and
anti-CD28 MAb completely
restored lymphoproliferative responses
and IL-2 secretion
(Table
3), suggesting that hu5C8
treatment
inhibited antigen-specific T-cell functions, and did not
delete
them. Concanavalin-A-induced LPR responses were within normal
ranges in all animals. Furthermore, extensive phenotype analyses
showed
no significant changes in CD4, CD8, and CD19 cells (data
not shown).
These studies demonstrate that inhibition of CD40L-CD40
interactions
lead to a marked inhibition of induction of T-cell
responses without
cell depletion.
Treatment of animals with hu5C8 antibody resulted in diminished serum
NAB, compared to untreated controls, up to day 36 of
the study, after
which a modest increase in NAB titer was observed.
These NAB responses
were maintained at low titer throughout the
180-day period and in some
cases returned to baseline. Analyses
of the mucosal response in BAL
showed that hu5C8-treated animals
did not elicit a NAB response on day
29 and remained below the
level of detection through day
180.
Ad-CFTR was readministered to all the animals in the study on day 180 (Fig.
1 bottom). LPR responses following vector administration
were
analyzed on days 184 and 210 (4 and 30 days after vector
readministration). Figure
5 shows that all the animals generated
LPR
responses, irrespective of hu5C8 treatment status, indicating
that
acute hu5C8 treatment did not induce tolerance to the adenovirus
vector.
Cytokine secretion profile was analyzed 30 days post-second-vector
instillation (day 210 of the study) (Fig.
6). Animals which
received
vector alone (i.e., no hu5C8) on days 1 and 180 (Group
2a) or only on
day 180 (Group 2d) induced secretion of all cytokines
tested.
Interestingly, animals in Group 2c (which received vector
2 without
hu5C8 treatment) generated a modest IL-2, IFN-
, and
IL-10 response,
while animals in Group 2b (which received vector
2 along with a second
course of hu5C8) were suppressed in their
ability to secrete IL-2,
IFN-
, and IL-4, but not IL-10.
The development of NAB to adenovirus was not inhibited after
second-vector instillation, irrespective of whether the animals
were
retreated with hu5C8. Thus, following readministration of
the vector on
day 180, all animals (either untreated or treated
with hu5C8) developed
high titers of NAB both in serum and BAL
by day
210.
Interaction of CD40L on activated T cells with CD40 on APC and B cells
plays a central role in activation of B- and T-cell
responses (
13,
25). As we have demonstrated in mice (
19,
32),
intratracheal administration of adenoviral vectors in rhesus
monkeys
generated strong cell-mediated immune responses, comprised
of both
TH
1-type and TH
2-type responses, as evidenced
by secretion
of IFN-
and IL-4. Blocking of CD40L-CD40 interaction
with hu5C8
treatment in vivo abrogated induction of lymphoproliferative
responses
and cytokine secretion, through day 36. Longitudinal analyses
of lymphoproliferative responses showed that LPR responses emerged
at
low levels over time as the circulating levels of hu5C8 were
diminished
(unpublished observations). The immunomodulatory effects
of hu5C8 are
consistent with various models of in vivo primary
immune responses, and
confirm in a large animal model that CD40-CD40L
interactions are
critical for induction of immune
responses.
Several studies have demonstrated that administration of adenovirus
vectors induce NABs (for a review, see reference
4).
CD4 cell depletion studies in mice have established that the humoral
response is CD4
+ T-cell-dependent. Consistent with the
observations in mice (
32,
33), treatment of monkeys with
hu5C8 antibody markedly impaired
development of
anti-adenovirus-specific IgG antibodies in serum
(data not shown) and
anti-IgA antibodies in BAL. The lack of isotype
switching correlated
with the inability of these antibodies to
neutralize Ad-
LacZ
infection of HeLa cells in vitro. The absence
of neutralizing
antibodies in BAL was confirmed in vivo when animals
instilled with
Ad-ALP on day 85 showed impressive transgene expression
in the
epithelial cells of the bronchoalveolar cells, compared
to untreated
animals. In the second experimental protocol, hu5C8-treated
animals
elicited markedly decreased NABs, compared to untreated
animals, until
day 36 post-vector administration. Following this
time point, there was
an emergence of modest anti-adenovirus-specific
LPR and NAB responses,
correlating with a decrease in circulating
hu5C8 concentrations. These
decreased hu5C8 levels correlated
with the appearance of low titers of
antibodies to hu5C8 (unpublished
results). These studies further
demonstrate that although acute
treatment of rhesus monkeys with a
humanized anti-CD40L MAb abrogates
development of a primary
adenovirus-neutralizing response and
limits the response to itself,
hu5C8 does not prevent the elicitation
of a virus-specific antibody
response upon secondary challenge
with vector. The diminished
effectiveness of the second sequential
immune blockade could be due to
a number of mechanisms such as
insufficient initial CD40L-CD40
inhibition, persistence of antigen
beyond the duration of CD40L-CD40
inhibition, the requirement
of inhibiting an alternate pathway, or
incomplete blockade of
CD40 signaling due to antibodies that interfere
with hu5C8
function.
The duration of systemic and mucosal humoral immune responses (in serum
and BAL, respectively) clearly diverged. Animals administered
adenovirus vector in the absence of hu5C8 in experimental protocol
2 elicited strong NABs in serum and BAL on day 29. While the NAB
in serum
persisted until at least day 180, antibodies in BAL reverted
to
baseline levels by day 90. It has been postulated that most
of the
antibody present in the serum comes from plasma cells in
the bone
marrow, whereas mucosal antibody levels are induced by
plasma cells in
the mucosal sites (
21). It is likely that possible
differences in the life spans of plasma cells in the two anatomical
locations may contribute to the short-lived antibody levels in
the BAL.
These observations may have important implications for
the
readministration of viral vectors in the
lung.
Induction of TH
1 or TH
2 responses was evaluated
following vector 2 administration by analyzing cytokine secretion
profiles
on day 210. Animals treated with repeat doses of hu5C8 (Group
2b) were inhibited in their ability to secrete IL-2, IFN-
, or
IL-4,
but secreted moderate levels of IL-10. It is possible that
monocyte/macrophages contributed to the IL-10 response. Alternatively,
hu5C8 treatment affected TH
1-type responses more
effectively than
IL-10 responses during secondary immune responses. To
this effect,
a previous report has shown that CD40L blockade results in
inhibition
of TH
1, but not TH
2 responses
(
20). Further studies need to
be done to clarify the role of
CD40-CD40L interactions in regulation
of TH
1 and
TH
2 responses in this animal
model.
In summary, these studies have investigated the effect of blocking
CD40L-CD40 interactions in adenovirus vector-mediated somatic
gene
transfer. Administration of hu5C8 MAb resulted in a marked
inhibition
of induction of antigen-specific T- and B-cell responses.
Analyses of
secondary immune responses showed that generation
of secondary B-cell
responses was not markedly affected. Another
unexpected observation was
that during secondary responses, TH
1 cytokine responses
were more affected than TH
2 responses. These
studies
demonstrate the potential of hu5C8 to prolong transgene
expression in
immune-suppressive regimens and permit vector readministration.
Further
studies using a combination of blocking agents (e.g.,
TRANCE, CTLA4-Ig,
and anti-CD4 MAb) (
2,
6,
14,
17,
27)
along with anti-CD40L
MAbs may provide a more complete abrogation
of T- and B-cell immune
responses.
| |
ACKNOWLEDGMENTS |
We thank the members of the Wilson laboratory for helpful
discussions. We thank Eric Wheeldon for performing the experiments on
the rhesus monkeys. Support from Cell Morphology Core, Immunology Core
(Ruth Qian, George Qian, and Parag Dhagat), and Animal Services Unit
(Ernest Glover and Lisa Stephens) of the Institute for Human Gene
Therapy is greatly appreciated.
This work was funded by grants from the NIH (P30 DK47757-05 and R01
HL49040-08), Cystic Fibrosis Foundation, and Genovo, Inc., a
biotechnology company J. M. Wilson founded and has equity in.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 204 Wistar
Institute, 3601 Spruce St., Philadelphia, PA 19104. Phone: (215)
898-0226. Fax: (215) 898-6588. E-mail:
wilsonjm{at}mail.med.upenn.edu.
| |
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Top
Abstract
Introduction
