Cytotoxic T Lymphocyte Responses to Transgene Product, Not Adeno-Associated Viral Capsid Protein, Limit Transgene Expression in Mice

Article (PDF Available)inHuman gene therapy 20(1):11-20 · November 2008with22 Reads
DOI: 10.1089/hum.2008.055 · Source: PubMed
Abstract
The use of adeno-associated viral (AAV) vectors for gene replacement therapy is currently being explored in several clinical indications. However, reports have suggested that input capsid proteins from AAV-2 vector particles may result in the stimulation of cytotoxic T lymphocyte (CTL) responses that can result in a loss of transduced cells. To explore the impact of anti-AAV CTLs on AAV-mediated transgene expression, both immunocompetent C57BL=6 mice and B cell-deficient muMT mice were immunized against the AAV2 capsid protein (Cap) and were injected intravenously with an AAV-2 vector encoding alpha-galactosidase (alpha-Gal). C57BL=6 mice, which developed both CTL and neutralizing antibody responses against Cap, failed to show any detectable alpha-Gal expression. In contrast, serum alpha-Gal levels comparable to those of naive mice were observed in muMT mice despite the presence of robust CTL activity against Cap, indicating that preexisting Cap-specific CTLs did not have any effect on the magnitude and duration of transgene expression. The same strategy was used to assess the impact of CTLs against the alpha-Gal transgene product on AAV-mediated gene delivery and persistence of transgene expression. Preimmunization of muMT mice with an Ad=alpha-Gal vector induced a robust CTL response to alpha-Gal. When these mice were injected with AAV2=alpha-Gal vector, initial levels of alpha-Gal expression were reduced by more than 1 log and became undetectable by 2 weeks postinjection. Overall, our results indicate that CTLs against the transgene product as opposed to AAV capsid protein are more likely to interfere with AAV transgene expression.
Cytotoxic T Lymphocyte Responses to Transgene
Product, Not Adeno-Associated Viral Capsid
Protein, Limit Transgene Expression in Mice
William M. Siders,
1
Jacqueline Shields,
1
Johanne Kaplan,
1
Michael Lukason,
2
Lisa Woodworth,
1
Sam Wadsworth,
2
and Abraham Scaria
2
Abstract
The use of adeno-associated viral (AAV) vectors for gene replacement therapy is currently being explored in
several clinical indications. However, reports have suggested that input capsid proteins from AAV-2 vector
particles may result in the stimulation of cytotoxic T lymphocyte (CTL) responses that can result in a loss of
transduced cells. To explore the impact of anti-AAV CTLs on AAV-mediated transgene expression, both im-
munocompetent C57BL=6 mice and B cell-deficient mMT mice were immunized against the AAV2 capsid protein
(Cap) and were injected intravenously with an AAV-2 vector encoding a-galactosidase (a-Gal). C57BL=6 mice,
which developed both CTL and neutralizing antibody responses against Cap, failed to show any detectable
a-Gal expression. In contrast, serum a-Gal levels comparable to those of naive mice were observed in mMT mice
despite the presence of robust CTL activity against Cap, indicating that preexisting Cap-specific CTLs did not
have any effect on the magnitude and duration of transgene expression. The same strategy was used to assess
the impact of CTLs against the a-Gal transgene product on AAV-mediated gene delivery and persistence of
transgene expression. Preimmunization of mMT mice with an Ad=a-Gal vector induced a robust CTL response to
a-Gal. When these mice were injected with AAV2=a-Gal vector, initial levels of a-Gal expression were reduced
by more than 1 log and became undetectable by 2 weeks postinjection. Overall, our results indicate that CTLs
against the transgene product as opposed to AAV capsid protein are more lik ely to interfere with AAV trans-
gene expression.
Introduction
T
he diversity and design of gene therapy vectors have
advanced greatly in their design and utility since their
initial introduction for clinical use (Meyer and Wagner, 2006;
Alton et al., 2007a,b). Nevertheless, for monogenetic diseases
such as hemophilia or lysosomal storage diseases, the goal of
gene therapy remains the same: to deliver a therapeutic pro-
tein to patients who either do not correctly express the wild-
type protein or express it at subtherapeutic levels. Research
has focused on the use of adeno-associated viral (AAV) vec-
tors for several reasons including their ability to transduce
nondividing cells and potential to mediate long-term trans-
gene expression (Chao and Walsh, 2004; Wu et al., 2006). Be-
cause AAV vectors do not contain any viral genes, they are
generally thought to be less immunogenic and less toxic than
other gene therapy vectors such as adenoviral vectors, which
retain some viral gene expression and can induce immune
responses to virus-associated proteins (Yang et al., 1994; Jooss
and Chirmule, 2003) as well as induce the production of
several proinflammatory cytokines (Bessis et al., 2004). The
lack of viral gene expression by AAV vectors suggests that
any immune response generated after AAV administration
would be directed against the transgene product or the input
capsid protein from the initial injection of vector particles.
It has been demonstrated by several groups that neutral-
izing antibodies (NAbs) to AAV can significantly affect viral
transduction (Murphy et al., 2008; Scallan et al., 2008). Because
NAbs are serotype specific and more than 100 serotypes or
variants of AAV have been described to date, it is possible that
by using AAV vectors of different serotypes, the issue of
NAbs can be circumvented (Halbert et al., 2000). In addition to
anti-viral antibody responses, humoral responses against the
transgene product have also been observed after intramus-
cular administration of AAV expressing foreign proteins such
as ovalbumin (OVA) (Brockstedt et al., 1999) or therapeutic
proteins such as factor IX (FIX) (Ge et al., 2001). By altering
the route of administration, it was possible to circumvent
1
Department of Immunotherapy Research and
2
Department of Molecular Biology, Genzyme, Framingham, MA 01701.
HUMAN GENE THERAPY 20:11–20 (January 2009)
ª Mary Ann Liebert, Inc.
DOI: 10.1089=hum.2008.055
11
antibody responses to FIX in mice; OVA-specific antibodies
were still generated irrespective of the route of injection. It is
clear that not only the nature of the transgene itself, but also
the route of administration, will affect the induction of an
immune response to the transgene product. Although the
impact of humoral responses on AAV transduction and sub-
sequent gene expression has been well characterized, the role
of T cell responses to AAV and the transgene product is less
well understood.
In a hemophilia clinical trial, administration of an AAV
vector encoding the FIX protein resulted in an initial increase
in circulating levels of FIX that declined over a short period of
time (Manno et al., 2006). The decline was accompanied by an
increase in the level of serum transaminase levels and the
appearance of a T cell response to the AAV capsid protein in
one patient. Manno and colleagues (2006) suggested that the
host immune response to AAV capsid protein may signifi-
cantly affect long-term transgene expression resulting in de-
struction of the transduced hepatocytes by capsid-specific
cytolytic T cell responses. However, reports suggest that AAV
capsid-specific T cell responses may have little effect on
transgene expression in the mouse (H. Li et al., 2007; C. Li et al.,
2007; Wang et al., 2007). To further investigate this issue, we
have compared the impact of cytotoxic T lymphocytes (CTLs)
against AAV capsid protein versus the encoded transgene
product on AAV-mediated transgene expression in immu-
nocompetent C57BL=6 mice and mMT mice, which lack the
ability to mount a humoral response. The transgene used in
these studies encodes the a-galactosidase (a-Gal) protein,
which is the therapeutic protein currently used to treat pa-
tients with Fabry disease. Fabry disease is a rare lysosomal
storage disorder resulting in the accumulation of the glyco-
sphingolipid globotriaosylceramide in tissues because of a
deficiency in the a-Gal enzyme as a result of mutations, gene
rearrangement, and deletions in the a-Gal gene (Ashley et al.,
2001). Enzyme replacement therapy has proven to be a suc-
cessful treatment for this disease, making it a possible candi-
date disease for gene therapy (Desnick, 2004). However,
similar to factor IX therapy, immunogenicity may be a po-
tential problem as some patients may not be tolerized against
the protein.
Using a plasmid or adenoviral vector expression system to
generate strong CTL responses to the capsid protein or the a-
Gal transgene product, we demonstrate that, in the absence of
NAb responses in mMT mice, preexisting capsid-specific CTL
responses have no effect on the strength or duration of a-Gal
expression. In contrast, preexisting a-Gal-specific CTL re-
sponses significantly affected long-term transgene expression,
resulting in a 2-log decrease in initial expression that dropped
to undetectable levels by 2 weeks after AAV administration.
The drop in expression was also accompanied by an increase
in serum transaminase levels. These results suggest that CTL
responses against the transgene product, as opposed to AAV
capsid protein, are the major factor limiting transgene ex-
pression from AAV vectors.
Materials and Methods
Cell lines
The C57BL=6 histocompatible (H-2
b
) SVB6 fibroblast cell
line was used in CTL assays and was a gift from L. Gooding
(Emory University, Atlanta, GA). HeLa cells were purchased
from the American Type Culture Collection (ATCC, Manassas,
VA). Cells were grown in Dulbecco’s modified Eagle’s me-
dium (DMEM) supplemented with 10% fetal calf serum (Lonza
Walkersville, Walkersville, MD), penicillin (100 units=ml),
streptomycin (100 mg=ml), and 2 mM glutamine. Cells were
maintained at 378C in a 5% CO
2
atmosphere and confirmed to
be free of mycoplasma by routine testing.
Generation of viral vectors
AAV2=a-Gal contained serotype 2 inverted terminal re-
peats and the human a-Gal cDNA under the control of the
DC190 liver-restricted promoter (Ziegler et al., 2004; Barbon
et al., 2005; McEachern et al., 2006). Recombinant vectors were
produced by triple-plasmid transfection of 293 cells and were
column purified as reported (O’Riordan et al., 2000). The final
titer of AAV2=a-Gal was determined by TaqMan polymerase
chain reaction (PCR) of the bovine growth hormone poly-
adenylation signal sequence. Quality control of each lot of
recombinant included analysis for bioburden (presence of
gram-positive bacteria) and endotoxin testing (<0.6 EU=ml).
The Ad5=Cap virus expressing the capsid protein under the
control of the cytomegalovirus (CMV) promoter was a kind
gift from N. Muzyczka (University of Florida, Gainesville, FL).
Immunization of mice to generate Cap-specific
cytotoxic T cell responses
Groups of three to five C57BL=6 or BALB=c mice were
immunized with peptide, plasmid, or recombinant adenoviral
vectors to induce capsid-specific CTL responses. AAV2 capsid
peptides were identified by the BIMAS (BioInformatics and
Molecular Analysis Section, Center for Information Technol-
ogy, National Institutes of Health, Bethesda, MD) peptide
prediction algorithm. For peptide analysis, mice were im-
munized with 100 mg of peptide emulsified in Freund’s in-
complete adjuvant and injected intradermally into mice. To
generate T cell-specific responses using plasmid or recombi-
nant adenoviral vectors, mice were immunized intramuscu-
larly with either 100 mg of pCAP-plasmid (plasmid expressing
AAV2 Cap driven by the CMV promoter) or 110
10
particles
of Ad5=Cap virus. Two weeks postimmunization, mice were
killed and spleens were collected for analysis as detailed
subsequently.
Long-term transgene expression experiments
Six- to 8-week-old female C57BL=6 or B cell-deficient mMT
mice were purchased from Charles River Laboratories (Wil-
mington, MA). To generate antigen-specific T cell responses,
mice were immunized intradermally with 110
10
particles of
an adenoviral vector expressing either the a-Gal or AAV2
capsid protein or with plasmid as previously described. Two
weeks after immunization, a subset of mice was killed and
spleens were collected to confirm the induction of a CTL re-
sponse. The remaining animals were injected intravenously
with 110
11
particles of a recombinant AAV2=a-Gal vector
under the control of a liver-specific promoter. At various
time points postinjection, serum was collected and analyzed
for the presence of circulating a-Gal protein. In some studies,
long-term transgene expression was examined in the pres-
ence and absence (no preimmunization) of specific cytolytic
T cell responses. All animal experiments were conducted in
12 SIDERS ET AL.
accordance with the guidelines established by the Institutional
Animal Care and Use Committee at Genzyme (Framingham,
MA).
Cytotoxic T lymphocyte assay
Evaluation of CTL activity was performed 2 weeks after
immunization of mice with plasmid or adenoviral vectors.
Naive mice were used as a negative control. Spleen cells from
naive or immunized mice were stimulated with mitomycin
C-inactivated syngeneic SVB6 cells infected with Ad vector
encoding either a-Gal or the AAV2 capsid protein. Cells were
cultured in 24-well plates containing 510
6
spleen cells and
610
4
stimulator fibroblasts in 2 ml of RPMI 1640 medium
with 10% fetal calf serum. Cytolytic activity was assayed after
6 days of culture. Target cells consisted of SVB6 fibroblasts
infected for 48 hr with an Ad vector expressing either the
a-Gal protein or the capsid protein, or with an Ad vector
lacking a transgene (Ad=empty vector [EV]) as a control.
Target cells were treated with recombinant mouse interferon
(IFN)-g (100 U=ml; R&D Systems, Madison, WI) for the last
24 hr to enhance MHC class I presentation, labeled with
chromium-51 (PerkinElmer Life and Analytical Sciences,
Boston, MA) overnight (25 mCi=110
5
cells), and plated in
round-bottom 96-well plates at 510
3
cells per well. Effector
cells were added at various effector-to-target (E:T) cell ratios
in triplicate in a total volume of 200 ml. After a 5-hr incubation,
25 ml of cell-free supernatant was collected from each well and
counted in a Wallac MicroBeta TriLux scintillation counter
(PerkinElmer Life and Analytical Sciences). The amount of
51
Cr spontaneously released was determined by incubating
target cells in medium alone. Spontaneous release from target
cells was typically less than 20%. The total amount of
51
Cr
incorporated was determined by adding 1% Triton X-100 in
distilled water, and the percentage lysis was calculated as
follows: [(sample cpm spontaneous cpm)=(total cpm
spontaneous cpm)]100.
Analysis of circulating a-Gal protein
Levels of circulating a-Gal protein in individual mice were
determined using a sandwich enzyme-linked immunosorbent
assay (ELISA). For this assay, 96-well plates were coated with
a rabbit polyclonal anti-a-Gal antibody for 2 hr at 378C. After
washing with phosphate-buffered saline (PBS)–Tween,
blocking buffer (Tris-buffered saline with Tween [TBS-T] and
5% milk) was added and plates were incubated overnight at
48C. Serum samples were serially diluted 2-fold, plated in
duplicate, and incubated at 378C for 1 hr. Washed plates were
incubated with a biotinylated goat anti-a-Gal monoclonal
antibody for 1 hr at 378C followed by the addition of strep-
tavidin–horseradish peroxidase (HRP) for 90 min. SIGMA-
FAST substrate (Sigma-Aldrich, St. Louis, MO) was added
and assay plates were analyzed with a VMax plate reader
(Molecular Devices, Sunnyvale, CA) at 490 nm. The amount
of a-Gal protein contained in the serum was derived by
comparison with a standard curve with known concentra-
tions of recombinant a-Gal protein.
Assessment of AAV2-neutralizing antibodies
For the AAV2 neutralization assay, mouse serum was tes-
ted for its ability to inhibit infection of HeLa cells by an AAV2
vector encoding b-galactosidase (b-Gal), resulting in de-
creased b-Gal transgene expression. Briefly, HeLa cells were
plated into 96-well tissue culture plates at a density of 210
4
cells per well and allowed to adhere for 2 hr. The adhered cells
were incubated with an Ad2 wild-type helper virus for 4 hr at
378C, 5% CO
2
. During this incubation period, mouse serum
samples were serially diluted 2-fold in a separate 96-well
plate. AAV2=b-Gal vector was added to each well containing
serum, and incubated for 1 hr at 378C, 5% CO
2
. At the end of
both incubation periods, the neutralizing sera–AAV samples
were added to the wild-type Ad2-infected HeLa cells, and the
plates were incubated for approximately 3 days at 378C, 5%
CO
2
. The medium was removed from all wells of the assay
plate, the cells were lysed, and the supernatants were tested
with a Tropix Galacto-Star kit for b-Gal expression according
to the manufacturer’s instructions (Applied Biosystems, Fos-
ter City, CA). The neutralizing serum titer was defined as the
dilution of serum that reduced b-Gal expression of the posi-
tive control (naive serum sample) by: 50%.
Statistical analysis
Statistical analysis of transgene expression data was per-
formed with GraphPad Prism version 3.0a for the Macintosh
(GraphPad Software, San Diego CA). Differences were con-
sidered to be statistically significant when p < 0.05.
Results
Generation of AAV2 capsid-specific cytolytic
T cell responses in C57BL=6 mice
To study the impact of preexisting CTLs on AAV transgene
expression, we first sought to induce AAV-specific CTLs
using peptides for immunization. Previously, other investi-
gators have used several methods to identify potentially im-
munogenic peptides from the AAV2 capsid protein sequence
including capsid peptide libraries or computer prediction al-
gorithms (Sabatino et al., 2005; Chen et al., 2006). Using these
computer prediction algorithms, we were able to identify
potential CD8
þ
T cell epitopes contained within the capsid
protein sequence predicted to bind to MHC class I molecules
with high affinity for both the C57BL=6 (H-2K
b
) and BALB=c
(H-2K
d
) MHC class I molecules. In some cases, immuniza-
tion with these peptides generated a CD8
þ
T cell response
that resulted in IFN-g production on restimulation of sple-
nocytes with the immunizing peptide (Table 1). However,
no direct cytolytic activity was observed from these T cells
(Table 1).
As an alternative method for immunization, recombinant
adenovirus and plasmid expression vectors were generated
that expressed high levels of the full-length AAV2 capsid
protein (Cap) under the control of a CMV promoter. To gen-
erate capsid-specific cytolytic T cell responses, mice were
immunized either intramuscularly with the pCap plasmid or
intradermally with the recombinant Ad5=Cap vector. Ten
days after immunization, spleens were collected and analyzed
for the presence of a capsid-specific cytolytic T cell response.
As shown in Fig. 1, immunization with a single dose of
the pCap plasmid resulted in a strong capsid-specific CTL
response that was present in all mice as evidenced by the
ability of effector cells to lyse syngeneic targets expressing the
AAV2 capsid protein. The strength of this response was not
CTL RESPONSES TO TRANSGENE PRODUCT 13
Table 1. Assessment of Immunogenicity of Computer-Predicted AAV2 Capsid Peptides
Immunogenic
c
Recognized as target
Peptide
a
H2 locus Start position
b
Strain ELISPOT CTL ELISPOT
d
CTL
e
KYLGPFNGL H2-K
d
(9-mer) 50 BALB=c Yes No Not tested Not tested
QYGSVSTNL H2-K
d
(9-mer) 574 BALB=c No No Not tested Not tested
VFQAKKRVL H2-K
d
(9-mer) 117 BALB=c No No Not tested Not tested
VFTDSEYQL H2-K
d
(9-mer) 341 BALB=c No No Not tested Not tested
KFFPQSGVL H2-K
d
(9-mer) 531 BALB=c No No Not tested Not tested
FFPQSGVLI H2-K
d
(9-mer) 532 BALB=c No No Not tested Not tested
QYSTGQVSV H2-K
d
(9-mer) 671 BALB=c No No Not tested Not tested
VPQYGYLTL H2-K
b
(9-mer) 371 C57BL=6 No No Yes No
LVLPGYKYL H2-K
b
(9-mer) 44 C57BL=6 No No Yes No
FMVPQYGYL H2-K
b
(9-mer) 369 C57BL=6 No No Yes No
DSLVNPARA H2-D
b
(9-mer) 513 C57BL=6 No No Yes Yes
GEPVNEADA H2-D
b
(9-mer) 61 C57BL=6 No No Yes No
LTLNNGSQA H2-D
b
(9-mer) 377 C57BL=6 No No Yes No
a
Peptide prediction was done with the BIMAS program.
b
Sequence start position relative to VP1.
c
Following direct peptide immunization.
d
For ELISPOT Ad5=CAP-generated CTLs were incubated with the indicated peptide and IFN-g release was analyzed.
e
Peptide was pulsed onto target cells and incubated with Ad5=CAP-generated CTLs.
FIG. 1. Generation of AAV2 capsid-specific CTL responses. To generate capsid-specific CTL responses, mice were immu-
nized either intramuscularly with a plasmid or intradermally with an adenoviral vector expressing the AAV2 capsid protein
under the control of a CMV promoter. Two weeks later, spleens were collected to analyze the ability of T cells to lyse targets
in a chromium release assay. Targets consisted of uninfected SVB6 cells (squares), SVB6 cells infected with the Ad5=Cap virus
(diamonds), and SVB6 cells infected with Ad5=EV control virus (circles). Representative graphs are shown from three inde-
pendent experiments and data are expressed as means standard deviation at each effector-to-target ratio.
14 SIDERS ET AL.
enhanced after a second dose of plasmid. Similarly, injection
of the Ad5=Cap vector also induced a potent Cap-specific CTL
response that resulted in a significant degree of target lysis.
However, a slight CTL response was also observed against the
adenoviral proteins themselves (Ad5=EV-transduced targets),
demonstrating one of the drawbacks to Ad-based gene ther-
apy, that is, the ability to induce T cell responses to both the
transgene product and Ad proteins. Interestingly, immuni-
zation with the Ad5=Cap virus generated a response that re-
sulted in IFN-g secretion after restimulation with each of the
predicted H-2K
b
-binding peptides, but T cells from Ad5=Cap-
immunized mice lysed targets pulsed with only one of the
predicted peptides (Table 1). Taken together, these data sug-
gest that the strength of the stimulus and the context in which
it is delivered (peptide vs. viral vector) play a role in gener-
ating cytolytic T cell responses. In addition, these data confirm
published reports indicating that discordant results can be
obtained using different immunoassays (Whiteside et al.,
2003), implying that not every CD8
þ
T cell that secretes IFN-g
in response to peptide stimulation has the ability to lyse cells
expressing the target antigen.
Neutralizing antibodies, but not preexisting
CTLs against AAV2 capsid protein, inhibit
transgene expression
It has been previously demonstrated that infection of cells
with AAV vector can be severely inhibited by the presence of
neutralizing antibodies (Sun et al., 2002, 2003). In addition, it
has been suggested that uncoating of the vector within the cell
may result in the presentation of capsid peptides on the cell
surface in the context of MHC class I molecules (Manno et al.,
2006), rendering infected cells susceptible to lysis by capsid-
specific CTLs. Therefore, both T and B cell responses could
potentially affect the overall strength and duration of trans-
gene expression after the administration of AAV vector. To
explore the contribution of humoral and CTL responses, ex-
periments were conducted in immunocompetent C57BL=6
mice and mMT mice, which do not possess functional B cells.
The mMT mouse harbors a disruption of the mM locus result-
ing in an arrest during B cell maturation (Kitamura et al.,
1991). These mice can mount normal T cell responses but do
not have the ability to generate antibodies (Perricone et al.,
2004). The mMT mouse allows for assessment of the impact of
preexisting CTLs on AAV transduction and subsequent
transgene expression separate from any contribution from
neutralizing antibodies. Administration of the pCap plasmid
to either C57BL=6 mice (Fig. 2A) or mMT mice (Fig. 2B) re-
sulted in the generation of capsid-specific CTLs that lysed
target cells expressing the capsid antigen to a similar extent. In
addition, immunized C57BL=6 mice also generated capsid-
specific neutralizing antibodies (Fig. 2C); this response was
not present in mMT mice. After induction of these anti-Cap
immune responses, mice were injected intravenously with an
AAV2 vector expressing the a-galactosidase (a-Gal) protein to
determine the strength and duration of transgene expression.
Control mice primed with an empty plasmid followed by the
AAV2=a-Gal vector, or mice injected with the AAV2=a-Gal
vector alone, expressed similarly high, sustained levels of
a-Gal protein during the course of the experiment (Fig. 3A). In
contrast, C57BL=6 mice that were treated with the pCap
plasmid (and developed a capsid-specific CTL response as
well as neutralizing antibodies) did not demonstrate mea-
surable levels of a-Gal in the serum at any of the time points
FIG. 2. Immune responses in mice after administration of pCap
plasmid. C57BL=6 and mMT mice were injected intramuscularly with
100 mg of the pCap plasmid. Two weeks later both T and B cell
responses were analyzed. CTL responses were evaluated from in-
dividual C57BL=6(A)ormMT (B) mice in a chromium release assay.
Targets consisted of uninfected SVB6 cells or SVB6 cells infected
with the Ad5=Cap virus. Data are expressed as means standard
deviation at an 80:1 effector-to-target cell ratio. Serum samples were
analyzed for the presence of anti-AAV2 neutralizing antibodies
(C) by ELISA as described in Materials and Methods.
CTL RESPONSES TO TRANSGENE PRODUCT 15
analyzed, indicating that either the neutralizing anti-Cap
antibody response, the anti-Cap CTL response, or a combi-
nation of the two limited the transduction efficiency of the
vector or transgene expression. Similar expression studies
were carried out in the mMT mouse. As shown in Fig. 3B, after
the injection of AAV2=a-Gal vector, there was no difference in
the level of a-Gal expression in mice injected with
AAV2=a-Gal vector alone, mice primed with empty plasmid,
or mice primed with pCap plasmid. Although mMT mice
primed with the pCap plasmid developed a strong capsid-
specific CTL response, high levels of transgene expression
were still observed, comparable to those seen in mice that did
not possess capsid-specific cytolytic T cells. These data sug-
gest that AAV2-specific CTL responses do not limit the level
of transgene expression, a finding consistent with data from
C. Li and colleagues (2007) and Wang and colleagues (2007).
The results also confirm that neutralizing antibodies can play
a significant role in inhibiting transduction and subsequent
transgene expression by AAV2 vector in C57BL=6 mice.
Presence of preexisting CTLs to a-Gal,
but not capsid protein, severely limits
a-Gal expression in mMT mice
Because capsid-specific cytolytic T cells did not appear to
affect transgene expression, we examined whether preexist-
ing CTLs to the a-Gal transgene product would have an im-
pact on the strength and duration of expression after an IV
delivery of AAV-2=a-Gal vector. C57BL=6 mice were pre-
immunized with an Ad vector expressing the a-Gal protein
driven by a CMV promoter to generate both antibody and
CTL responses to the a-Gal transgene. Two weeks later, mice
were shown to have both a CTL response (90% specific lysis at
an E:T ratio of 80:1), as well as antibodies to the a-Gal trans-
gene (average titer of 6400) and were injected with the
AAV=a-Gal vector. Mice that received no priming or received
the Ad2=EV control vector as a priming agent displayed
similarly high levels of a-Gal expression (Fig. 4A). In contrast,
no circulating a-Gal protein could be detected in the group of
preimmunized C57BL=6 mice that developed both antibody
and CTL responses to a-Gal, suggesting that either the CTLs
played a role in limiting transgene expression or the presence
of an antibody response to a-Gal limited our ability to quan-
titatively measure it in serum. To circumvent any involve-
ment of neutralizing antibodies to the transgene product, a
similar experiment was performed in the mMT mice. mMT
mice were immunized with either the Ad5=Cap vector or the
Ad2=a-Gal vector to determine the individual effects of cy-
tolytic T cell responses to Cap and a-Gal on transgene ex-
pression. Mice immunized with the Ad5=Cap vector
generated a capsid-specific CTL response (54% specific lysis at
an
E:T cell
ratio of 80:1), whereas mice immunized with the
Ad2=a-Gal
vector generated an a-Gal-specific CTL response
(47% specific lysis at an E:T cell ratio of 80:1). As shown in
Fig. 4B, the presence of capsid-specific CTLs alone did not
impair the level of transgene expression compared with the
expression levels observed in unprimed mice, confirming
the results shown in Fig. 3. However, in the group of mMT
mice with CTLs but no antibodies to a-Gal, there was a sig-
nificant decrease in the level of transgene expression; a 2-log
difference at the onset of expression that quickly declined to
background levels. These data indicate that CTLs to the a-Gal
FIG. 3. a-Gal expression in mice with
preexisting Cap-specific immune re-
sponses. C57BL=6 mice (A) and mMT
mice (B) were unprimed (squares),
primed with a control pNull plasmid
(diamonds), or primed with pCap plas-
mid (circles) to generate capsid-specific
immune responses. Two weeks later
mice were injected intravenously with
110
11
particles of AAV2=a-Gal vec-
tor. At various time points throughout
the course of the experiment serum
was collected to analyze the circulating
levels of a-Gal protein by ELISA, as
described in Materials and Methods.
16 SIDERS ET AL.
transgene product, and not the AAV capsid protein, severely
limit transgene expression.
Decrease in a-Gal transgene expression
is accompanied by increase in serum
transaminase levels
Results from a clinical trial using AAV as a delivery vehicle
for factor IX therapy implicate immune responses to the AAV
capsid protein as a complicating issue (Manno et al., 2006). A
decrease in circulating factor IX levels was observed in some
patients and was accompanied by elevations in serum trans-
aminase levels, suggesting liver toxicity. Although the au-
thors demonstrated that CD8
þ
T cell responses could be
detected against the capsid protein, T cell responses against
the factor IX protein were not examined. In light of the finding
that preexisting CTLs against a-Gal and not the capsid protein
play a significant role in limiting transgene expression, serum
from mMT mice with preexisting CTL responses (Fig. 4B) was
analyzed to determine whether the decrease in expression
was accompanied by increases in serum transaminase levels.
As shown in Table 2, a direct correlation exists between the
decrease in transgene expression in the Ad=a-Gal-primed
group and the increase in serum transaminase levels. Mice
that were not primed with antigen or primed with the
Ad=Cap vector to induce a capsid-specific CTL response did
not display an increase in either alanine aminotransferase
(ALT) or aspartate aminotransferase (AST) level. In contrast,
mice that were primed with Ad=a-Gal displayed significantly
higher levels of both ALT and AST in serum at the time when
transgene expression was reduced (days 7 to 14). Transami-
nase levels returned to vehicle control levels by day 28, at
which point circulating a-Gal could no longer be detected.
Discussion
AAV vectors have long been regarded as having little po-
tential to induce a CTL response because they do not contain
any viral genes. However, it has been suggested that injection
of AAV2 vectors may actually result in the generation of
FIG. 4. a-Gal expression is inhibited
in the presence of preexisting a-Gal-
specific immune responses. C57BL=6
mice (A) were unprimed (squares),
primed with a control Ad2=EV virus
(diamonds), or primed with Ad2=a-Gal
vector (circles) to generate a-Gal-
specific immune responses. mMT mice
(B) were unprimed (squares), primed
with a control Ad5=Cap virus (dia-
monds), or primed with Ad2=a-Gal
vector (circles) to generate capsid or
a-Gal-specific immune responses. Ten
days later both groups of mice were
injected intravenously with 110
11
particles of AAV2=a-Gal vector. At
various time points throughout the
course of the experiment serum was
collected to analyze the circulating
levels of a-Gal protein by ELISA, as
described in Materials and Methods.
Table 2. Serum Transaminase Levels in Experimental Groups
Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)
Priming agent Day 7 Day 14 Day 21 Day 28 Day 7 Day 14 Day 21 Day 28
Vehicle 21.2 9.6 24.0 7.1 12.0 2.4 19.8 1.8 24.4 6.1 34.2 2.8 18.8 6.5 28.2 11.1
Ad=CAP 17.2 2.2 25.8 4.7 12.2 1.6 21.2 3.8 24.8 5.5 34.0 9.4 19.0 5.8 37.6 7.6
Ad=agal 101.8 21.5
a
88.6 16.9
a
28.8 6.4
a
25.8 6.3 170.0 41.9
a
143.4 34.8
a
30.4 14.5 44.8 15.0
a
p # 0.001 compared with vehicle group or Ad=CAP-primed group.
CTL RESPONSES TO TRANSGENE PRODUCT 17
de novo CTL responses elicited by the input particles or in the
activation of a memory T cell response due to prior AAV
exposure (Manno et al., 2006). In a clinical trial, patients
treated with an AAV-factor IX vector were found to express
factor IX only transiently and the decline in expression cor-
related with a rise in serum transaminase levels and the
appearance of capsid-specific T cells. On the basis of these
observations, it was hypothesized that the loss of expression
was due to the induction of an AAV-specific CTL response
leading to the destruction of transduced liver cells. In view
of these observations, it becomes important to better un-
derstand the role that the host immune response plays in
affecting transgene expression after the administration of
AAV vectors. Although much attention has been focused on
the possible involvement of AAV-specific CTLs, it is im-
portant to consider that host immune responses can be di-
rected against not only the viral vector itself but also the
transgene being expressed and can consist of both T and B
cell components.
In this study, we have explored the relative contribution of
B and T cell responses as well as the impact of responses
directed against the AAV capsid and the transgene product
on AAV-mediated expression. Our results confirm that neu-
tralizing antibodies against AAV2 can prevent transduction
and subsequent transgene expression by an AAV2=a-Gal
vector. Analysis of CTL responses indicated that a CTL re-
sponse against the a-Gal transgene product is capable of
limiting transgene expression whereas CTLs against AAV
capsid have little impact on the magnitude or duration of
expression.
Adenoviral and plasmid expression systems were used to
create immune responses against the Cap protein of AAV2. In
immune-competent C57BL=6 mice primed with plasmid en-
coding Cap, both antibody and CTL responses developed
against the AAV2 capsid protein and no transgene expression
was detected, making it difficult to determine whether T cells,
B cells, or a combination of the two was responsible for af-
fecting AAV transduction and subsequent transgene expres-
sion. The presence of neutralizing antibodies to AAV has been
previously reported to negatively influence AAV transduc-
tion, resulting in low levels of transgene expression (Scallan et
al., 2008). To assess the role of Cap-specific CTLs in the ab-
sence of neutralizing antibodies, the experiment was repeated
in B cell-deficient mMT mice, which developed a strong CTL
response to the AAV capsid protein but no antibodies. Under
these conditions, the Cap-specific CTL response in mMT mice
failed to affect the strength or duration of transgene expres-
sion. This would suggest that in immunocompetent mice,
Cap-specific CTLs did not affect transgene expression but that
neutralizing antibodies effectively prevented transduction
with AAV vector. These results are consistent with the reports
of C. Li and colleagues and Wang and colleagues, who
demonstrated that factor IX expression remains constant in
the presence of capsid-specific CTLs. Therefore, although
CTLs can be raised against the AAV Cap protein and may be
detected in the host, they are unlikely to be the main con-
tributing factor in the loss of transgene expression.
Comparatively little attention has been paid to the potential
role of CTLs against the transgene product in limiting ex-
pression from AAV vectors. The use of AAV for gene therapy
delivery of a protein that is either truncated or not expressed
in a patient may result in the generation of a therapy-specific
immune response. In these cases, T cell responses induced by
the transgene may significantly impact the overall therapeutic
benefit of the gene therapy being used. For proof-of-concept
experiments, we have chosen to use the a-Gal transgene as a
way to mimic the delivery of a therapeutic protein against
which the patient is not immunologically tolerized. Our re-
sults demonstrate that transgene-specific CTLs have a direct
effect on the level and duration of expression from an AAV
vector. This was clearly demonstrated by experiments in
which B cell-deficient mMT mice were preimmunized with
Ad2=a-Gal vector to generate CTLs, but no antibodies, against
a-Gal. When subsequently injected with AAV2=a-Gal vector,
these mice exhibited a more than 2-log decrease in initial
levels of transgene expression compared with unprimed mice
or mice that were preimmunized with Ad5=Cap to generate
CTLs to the capsid protein. Within 2 weeks, circulating levels
of a-Gal protein were undetectable, indicating that transgene
expression was severely compromised by the presence of a
CTL response against the transgene product whereas Cap-
specific CTLs had no impact. Similar to results observed in the
hemophilia clinical trial, the decrease in circulating levels of
therapeutic protein was accompanied by an elevation in se-
rum transaminase levels.
It remains unclear why preformed CTLs against the
transgene product are capable of terminating transgene ex-
pression whereas CTLs against Cap, when present at equiv-
alent levels of lytic activity (as measured in vitro), have no
measurable impact on transgene expression. One possible
explanation
may
reside in the degree of antigen presentation
required for sensitization of target cells to the lytic activity of
specific CTLs. After AAV vector administration, there is
abundant transgene expression that should result in robust
presentation on the cell surface in association with MHC class
I. However, the Cap protein is not expressed by the vector and
transduced hepatocytes must process and present capsid
protein from the initial input number of viral particles by a
nonclassical method of MHC class I presentation. It is con-
ceivable that the amount of processed capsid peptide pre-
sented on the cell surface is not sufficient to render the cells
susceptible to lysis by capsid-specific CTLs. It has also been
hypothesized that murine hepatocytes may process and
present antigen less efficiently than human hepatocytes, ex-
plaining observed differences between mouse experiments
and the hemophilia clinical trial described previously. On the
basis of our results, a-Gal-specific CTLs appear capable of
directly affecting transduced cells and abolish transgene ex-
pression, suggesting that the processing and presentation
pathway is fully functional in transduced murine hepato-
cytes.
Results from our studies have potential implications for the
clinical application of AAV-based gene therapy. Although
preexisting CTLs against AAV appear unlikely to represent a
significant impediment to transgene expression, the induction
of CTLs against the transgene product potentially represents a
hurdle in the clinic. For many genetic diseases such as he-
mophilia, the goal of gene therapy is to introduce a functional
protein into patients who either do not express the protein,
express a truncated version, or express nontherapeutic levels
of the protein. During the course of T cell development, these
patients may not become tolerized to the wild-type protein
because of this alteration in or lack of expression. Therefore,
the likelihood exists that T cell responses to these proteins
18 SIDERS ET AL.
may affect long-term expression from a gene therapy vector.
In such instances, the therapeutic protein expressed by the
vector may be recognized as a nonself antigen, resulting in
the induction of a destructive CTL response. To circumvent
the potential effect of CTL responses against the transgene
product, it may be necessary to develop strategies to tolerize
patients against the therapeutic protein before AAV vector
administration or to establish transient immunosuppression
regimens that can be delivered at the time of vector admin-
istration to minimize the generation of CTL responses.
Author Disclosure Statement
The authors are employees of Genzyme Corporation and
own stock in the company.
References
Alton, E., Ferrari, S., and Griesenbach, U. (2007a). Progress and
prospects: Gene therapy clinical trials (part 1). Gene Ther. 14,
1439–1447.
Alton, E., Ferrari, S., and Griesenbach, U. (2007b). Progress and
prospects: Gene therapy clinical trials (part 2). Gene Ther. 14,
1555–1563.
Ashley, G.A., Shabbeer, J., Yasuda, M., Eng, C.M., and Desnick,
R.J. (2001). Fabry disease: Twenty novel a-galactosidase A
mutations causing the classical phenotype. J. Hum. Genet. 46,
192–196.
Barbon, C.M., Ziegler, R.J., Li, C., Armentano, D. Cherry, M.,
Desnick, R.J., Schuchman, E.H., and Cheng, S.H. (2005). AAV8
mediated hepatic expression of acid sphingomeylinase cor-
rects the metabolic defect in the visceral organs of a mouse
model of Neimann-Pick disease. Mol. Ther. 12, 431–440.
Bessis, N., Garcia Cozar, F.J., and Biossier, M.-C. (2004). Immune
responses to gene therapy vectors: Influence on vector func-
tion and effector mechanisms. Gene Ther. 11, S10–S17.
Brockstedt, D.G., Podsakoff, G.M., Fong, L., Kurtzman, G.,
Mueller-Ruchholtz, W., and Engleman, E.G. (1999). Induction
of immunity to antigens expressed by recombinant adeno-
associated virus depends on the route of administration. Clin.
Immun. 92, 67–75.
Chao, H., and Walsh, C.E. (2004). AAV vectors for hemophilia B
gene therapy. Mt. Sinai J. Med. 71, 305–313.
Chen, J., Wu, Q., Yang, PA., Hsu, H.-C., and Mountz, J.D. (2006).
Determination of specific CD4 and CD8 T cell epitopes after
AAV2 and AAV8-hF.IX gene therapy. Mol. Ther. 13, 260–269.
Desnick, R.J. (2004). Enzyme replacement and enhancement
therapies for lysosomal diseases. J. Inherit. Metab. Dis. 27,
385–410.
Ge, Y., Powell, S., van Roey, M., and McArthur, J.G. (2001).
Factors influencing the development of an anti-factor IX (FIX)
immune response following administration of adeno-associ-
ated virus-FIX. Blood 97, 3733–3737.
Halbert, C.L., Rutledge, E.A., Allen, J.M., Russell, D.W., and
Miller, D. (2000). Repeat transduction in the mouse lung by
using adeno-associated virus vectors with different serotypes.
J. Virol. 74, 1524–1532.
Jooss, K., and Chirmule, N. (2003). Immunity to adenovirus and
adeno-associated viral vectors: Implications for gene therapy.
Gene Ther. 10, 955–963.
Kitamura, D., Roes, J., Kuhn, R., and Rajewsky, K. (1991). A B-
cell deficient mouse by targeted disruption of the membrane
exon of the immunoglobulin m chain gene. Nature 350, 423–
426.
Li, C., Hirsch, M., Asokan, A., Zeithaml, B., Ma, H., Kafri, T., and
Samulski, J. (2007). Adeno-associated virus type 2 (AAV2)
Capsid-specific cytotoxic T lymphocytes eliminate only vec-
tor-transduced cells coexpressing the AAV2 capsid in vivo.
J. Virol. 81, 7540–7547.
Li, H., Murphy, S.L., Giles-Davis, W., Edmonson, S., Xiang, Z.,
Li, Y., Lasaro, M.O., High, K.A., and Ertl, H.C. (2007). Pre-
existing AAV capsid-specific CD8
þ
T cells are unable to
eliminate AAV-transduced hepatocytes. Mol. Ther. 4, 792–800.
Manno, C.S., Arruda, V.R., Pierce, G.F., Glader, B., Ragni, M.,
Rasko, J., Ozelo, M.C., Hoots, K., Blatt, P., Konkele, B., Dake,
M., Kaye, R., Razavi, M., Zajko, A., Zehnder, J., Nakai, H.,
Chew, A., Leonard, D., Wright, J.F., Lessard, R.R., Sommer,
J.M., Tigges, M., Sabatino, D., Luk, A., Jiang, H., Mingozzi, F.,
Couto, L., Ertl, H.C., High, K.A., and Kay, M.A. (2006). Suc-
cessful transduction of liver in hemophilia by AAV-factor IX
and limitations imposed by the host immune response. Nat.
Med. 12, 342–347.
McEachern, K.A., Nietupski, J.B., Chuang, W.L., Armentano, D.,
Johnson, J., Hutto, E., Grabowski, G.A., Cheng, S.H., and
Marshall, J. (2006). AAV8-mediated expression of glucocer-
ebrosidase ameliorates the storage pathology in the visceral
organs of a mouse model of Gaucher disease. J. Gene Med. 8,
719–729.
Meyer, M., and Wagner, E. (2006). Recent developments in the
application of plasmid DNA-based vectors and small inter-
fering RNA therapeutics for cancer. Hum. Gene Ther. 17,
1062–1076.
Murphy, S.L., Li, H., Zhou, S., Schlachterman, A., and High, K.
(2008). Prolonged susceptibility to antibody-mediated neu-
tralization for adeno-associated vectors targeted to the liver.
Mol. Ther. 16, 138–145.
O’Riordan, C.R., Lachapelle, A.L., Vincent, K.A., and Wads-
worth, S.C. (2000), Scaleable chromatographic purification
process for recombinant adeno-associated virus (rAAV).
J. Gene Med. 2, 444–454.
Perricone, M.A., Smith, K.A., Claussen, K.A., Plog, M.S., Hem-
pel, D.M., Roberts, B.L., St. George, J.A., and Kaplan, J.K.
(2004). Enhanced efficacy of melanoma vaccines in the absence
of B lymphocytes. J. Immunother. 4, 273–281.
Sabatino, D.E., Mingozzi, F., Hui, D.J., Chen, H., Colosi, P., Ertl,
H.C.J., and High, K.A. (2005). Identification of mouse AAV
capsid-specific CD8
þ
T cell epitopes. Mol. Ther. 6, 1023–1033.
Scallan, C.D., Jiang, H., Liu, T., Patarroyo-White, S., Sommer,
J.M., Zhou, S., Couto, L.B., and Pierce, G.F. (2006). Human
immunoglobulin inhibits liver transduction by AAV vectors at
low AAV2 neutralizing titers in SCID mice. Blood 107, 1810–
1817.
Sun, J.Y., Chatterjee, S., and Wong, K.K. (2002). Immunogenic
issues concerning recombinant adeno-associated virus vectors
for gene therapy. Curr. Gene Ther. 2, 485–500.
Sun, J.Y., Anand-Jawa, V., Chatterjee, S., and Wong, K.K. (2003).
Immune responses to adeno-associated virus and its recom-
binant vectors. Gene Ther. 10, 964–976.
Wang, L., Figueredo, J., Calcedo, R., Lin, J., and Wilson, J.M.
(2007). Cross-presentation of adeno-associated virus serotype
2 capsids activates cytotoxic T cells but does not render he-
patocytes effective cytolytic targets. Hum. Gene Ther. 18, 185–
194.
Whiteside, T.L., Zhao, Y., Tsukishiro, T., Elder, E.M., Gooding,
W., and Baar, J. (2003). Enzyme-linked immunospot, cytokine
flow cytometry, and tetramers in the detection of T-cell re-
sponses to a dendritic cell-based multipeptide vaccine in pa-
tients with melanoma. Clin. Cancer Res. 9, 641–649.
CTL RESPONSES TO TRANSGENE PRODUCT 19
Wu, Z., Asokan, A., and Samulski, R.J. (2006). Adeno-associated
virus serotypes: Vector toolkit for human gene therapy. Mol.
Ther. 14, 316–327.
Yang, Y., Nunes, F.A., Berencsi, K., Furth, E.E., Go
¨
nczo
¨
l, E., and
Wilson, J.M. (1994). Cellular immunity to viral antigens limits
E1-deleted adenoviruses for gene therapy. Proc. Natl. Acad.
Sci. U.S.A. 91, 4407–4441.
Ziegler, R.J., Lonning, S.M., Armentano, D., Li, C., Souza, D.W.,
Cherry, M., Ford, C., Barbon, C.M., Desnick, R.J., Gao, G.,
Wilson, J.M., Peluso, R., Godwin, S., Carter, B.J., Gregory, R.J.,
Wadsworth, S.C., and Cheng, S.H. (2004). AAV2 vector har-
boring a liver-restricted promoter facilitates sustained expres-
sion of therapeutic levels of a-galactosidase A and the induction
of immune tolerance in Fabry mice. Mol. Ther. 9, 231–240.
Address reprint requests to:
Dr. Abraham Scaria
Department of Molecular Biology
49 New York Avenue
Framingham MA 01701
E-mail: abraham.scaria@genzyme.com
Received for publication May 9, 2008;
accepted after revision September 30, 2008.
Published online: January 14, 2009.
20 SIDERS ET AL.
    • "Transduction efficiency can be also be increased by mutating surface exposed tyrosine residues on the capsid, which is thought to reduce proteasomal degradation, increasing trafficking to the nucleus (Zhong et al., 2008; Markusic et al., 2010). Though a variety of mechanisms are involved in these studies, they, along with other studies in animals, are united by a common theme: in current murine models, functional CD8+ T cell infiltrates in AAV transduced tissues are primary directed against the transgene product rather than the capsid, while an antibody response is often observed to both potential immunogens (Siders et al., 2009). "
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