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Prepublished online as a Blood First Edition Paper on November 21, 2002; DOI 10.1182/blood-2002-07-2146.
GENE THERAPY
From the Center for Molecular and Vascular Biology,
Department for Molecular and Cardiovascular Research, Leuven,
Belgium.
Adenoviral transfer of human apo A-I in Balb/c mice induces a
strong humoral immune response against the transgene product when
expression is driven from the ubiquitously active CMV
promoter but induces no immune response when driven by the
hepatocyte-specific 256-base pair apo A-I promoter.
Here the hypothesis was tested, which is that the humoral immune
response against the circulating transgene product correlates
with its expression in antigen-presenting cells. No humoral immune
response was observed after adenoviral transfer of vectors with human
apo A-I expression driven by the hepatocyte-specific apo
C-II or 1.5-kilobase (kb) human
Humoral immune responses are initiated with the
cellular uptake and processing of foreign antigens by professional
antigen-presenting cells. After migration to secondary lymphoid organs,
interaction of antigen-presenting cells with naive T cells provides the
T cell with all signals for their activation.1,2
Activation and clonal B-cell expansion occur after primed
antigen-specific CD4+ T cells engage with the
antigen-specific B cells through T-cell receptor/major
histocompatibility complex (MHC) II
interaction.3-5
Previously, we observed a strong humoral immune response after
adenoviral gene transfer of human apo A-I in Balb/c and Fvb mice when
expression was driven by the ubiquitously active
cytomegalovirus (CMV) promoter-driven construct,
but not after transfer of a hepatocyte-specific minimal 256-base pair
(bp) apolipoprotein (apo) A-I promoter-driven construct.6 Pastore et al7 showed that
expression of human Generation of E1-deleted recombinant adenoviral
vectors
The MHCII E Animal experiments
Human apo A-I ELISA Human apo A-I levels were determined by sandwich enzyme-linked immunosorbent assay (ELISA) as described previously.6Detection of anti-human apo A-I antibodies by ELISA Human apo A-I antibodies were determined as described previously.11 Briefly, polystyrene microtiter plates (Elscolab, Kruibeke, Belgium) were coated with 0.14 µM human apo A-I (Biovalley, Paris, France) or 0.14 µM of human tissue-type plasminogen activator (t-PA), which was used as control antigen. Dilutions of plasma samples up to 1:256 000 were added to the wells for 2 hours. After washing of the plates, rabbit anti-mouse polyclonal antibodies conjugated with peroxidase (DAKO, Denmark) were diluted 1:10 000 and added to the wells for 2 hours. Peroxidase reaction was performed with H2O2 and orthophenylenediamine, and absorbance was measured at 492 nm. The titer of antibodies was defined as the plasma dilution for which the optical density of the antibody assay was higher than 0.100, which is significantly above background.Detection of antiadenoviral antibodies by ELISA Serial dilutions of plasma samples up to 1:640 000 were added overnight to 96-well polystyrene microtiter plates (Elscolab) coated with 100 µL control vector AdRR5 at a concentration of 2 × 107 plaque-forming units (pfu)/mL. After washing of the plates, rabbit anti-mouse polyclonal antibodies (DAKO) were diluted 1:10 000 and added to the wells for 2 hours. Peroxidase reaction was performed with H2O2 and orthophenylenediamine, and absorbance was measured at 492 nm. The titer of IgG antibodies against adenovirus serotype 5 was defined as the plasma dilution for which the optical density of the antibody assay was higher than 0.100, which is significantly above background.Immunization with human apo A-I protein Immunization was performed by injecting 25 µg human apo A-I formulated in complete Freund adjuvant in the footpad of each hindlimb.Quantification of human apo A-I transgene DNA in the liver Quantification of human apo A-I DNA was performed as described previously by real-time polymerase chain reaction (PCR).6 The human apo A-I DNA copy number was normalized to the copy number of the Prion Protein (PrP) gene.Quantification of human apo A-I mRNA Human apo A-I mRNA was quantified by real-time PCR after cDNA synthesis as described previously.6 The human apo A-I cDNA copy number was normalized to the copy number of the glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) housekeeping gene.Isolation of splenic dendritic cells, macrophages, B and T cells
after transfer with AdE  . Because the absolute
number of dendritic cells isolated from one spleen is too low to obtain
sufficient mRNA, splenocytes isolated from 4 Balb/c were pooled, and
human apo A-I mRNA of each cell type was quantified on 4 independent samples, each consisting of a pool of 4 mice. Ficoll Paque
(Lymphoprep; Axis-Shield POC AS, Oslo, Norway) was used according to
the instructions of the manufacturer to obtain mononuclear cells from
the spleen. Subsequently, positive selection with CD11c+,
CD11b+, and CD45R MACS Micro-beads (Miltenyi Biotec,
Auburn, CA) was performed to isolate dendritic cells, macrophages, and
B cells, respectively. All selections were performed on MiniMacs
(MS) separation columns as instructed by the manufacturer
(Miltenyi Biotec). The remaining eluent, containing T cells, was
purified by negative Pan T-cell selection (Miltenyi Biotec). The purity
of the enriched splenic dendritic cells, macrophages, and B and T cells
was confirmed by flow cytometry using phycoerythrin-conjugated
antibodies against CD11c+, CD11b+, CD19, and
the Pan T cell Marker CD3 (BD Pharmingen, Heidelberg, Germany),
respectively. To isolate mRNA, the Quickprep micro mRNA purification
kit (Amersham Pharmacia, Uppsala, Sweden) was used.
Statistical analysis Human apo A-I data are expressed as mean ± standard error of the mean (SEM). Antibody titers are expressed as geometric mean ± standard error of the geometric mean. Comparison of antibody titers was performed on logarithmically transformed data by nonparametric Mann-Whitney test using the INSTAT V2.05a statistical program (Graph Pad Software, San Diego, CA). A 2-sided P value less than .05 was considered statistically significant.
Humoral immune response against human apo A-I after transfer with CMV promoter and U1b promoter-driven constructs but not after transfer with apo C-II and hAAT promoter-driven constructs We have previously demonstrated a strong humoral immune response against human apo A-I in Balb/c mice after transfer with a CMV promoter-driven construct (AdCMV), but follow-up was limited to 35 days.6 Human apo A-I expression after transfer with AdCMV was 6.2 ± 2.0 mg/dL at day 6 and below detection limit (1 mg/dL) within 14 days.6 To investigate whether high-titer antibodies against human apo A-I persisted after transfer with AdCMV, the titer of antibodies against human apo A-I was determined up to 12 months after transfer with 5 × 108 pfu AdCMV. Figure 1B illustrates that the geometric mean of the inverse of the titer of antibodies 12 months after transfer was 90 000 ± 16 000, and antibody titers had not declined at this time point.
We have previously shown that after transfer with a construct driven by the hepatocyte-specific 256-bp minimal apo A-I promoter (AdA4), no humoral immune response against human apo A-I occurred in Balb/c mice. To corroborate a potential relationship between hepatocyte-specificity of promoters and absence of a humoral immune response against human apo A-I, human apo A-I expression (Figure 1A), and antibodies against human apo A-I (Figure 1B) were compared after transfer with constructs driven by the hepatocyte-specific apo C-II and 1.5-kb hAAT promoters (AdC4 and AdAT4, respectively) and after transfer with the ubiquitously active U1b small nuclear RNA promoter (AdU). Human apo A-I expression was sustained for the duration of the experiment (4 months) after transfer with AdC4 and AdAT4, and no antibodies against human apo A-I were observed. The decrease of expression levels after day 35 correlated with a 3.9-fold (P < .01) and a 3.3-fold (P < .01) decrease of the human apo A-I transgene DNA copy number between day 35 and 4 months after transfer with AdC4 and AdAT4, respectively. After transfer with AdU, human apo A-I expression was below detection limit (1 mg/dL) within 10 days (Figure 1A), and antibodies against human apo A-I were observed. However, compared with AdCMV, the geometric mean of the inverse of the titer of antibodies after transfer with AdU was more than 300-fold lower. Humoral immune response against human apo A-I after transfer with a
murine MHCII E
promoter-driven construct (AdE). Human apo A-I was below
detection limit (1 mg/dL) at all time points after transfer with
AdE. Figure 1B illustrates the inverse of the titer of
antibodies against human apo A-I after transfer with
5 × 108 pfu and 108 pfu AdE.
After transfer with 5 × 108 pfu, the geometric mean of
the inverse of the titer of antibodies reached a peak of 180 000 ± 11 000 at 3 months after transfer, and high antibody titers
persisted for 10 months. After transfer with 108 pfu, the
geometric mean of the inverse of the titer of antibodies reached a peak
of 37 000 ± at 4 months after transfer. No humoral immune
response against human apo A-I was observed after transfer with
2 × 107 pfu of AdE (n = 5) nor in Balb/c
mice treated with 5 × 108 pfu 10 days after splenectomy
(n = 5) (data not shown).
To investigate whether humoral immune responses against human apo A-I
correlate with human apo A-I expression in the spleen, human apo
A-I mRNA in the spleen was quantified by real-time PCR after
reverse transcription. Figure 2A
illustrates that human apo A-I mRNA was detected in the
spleen 7 days after transfer with 5 × 108 pfu of
AdCMV or AdE
In transgenic mice, the E Time-course of human apo A-I expression may be a determinant of the humoral immune response against human apo A-I after adenoviral gene transfer To investigate the mechanism of absence of a humoral immune response against human apo A-I, 5 × 108 pfu AdE and 5 × 108 pfu AdAT4 were
coinjected in Balb/c mice. Human apo A-I expression after coinjection
was very similar compared with transfer with AdAT4 (Figure
3A). Figure 3B illustrates that high
titers of antibodies against human apo A-I were observed at day 10 and
day 14 after coinjection, but human apo A-I antibodies decreased
rapidly and were undetectable at day 56 after transfer. Coinjection of
AdE and of AdAT4 induces human apo A-I
expression in the spleen and the liver. Because the CMV
promoter is a ubiquitously active promoter and human apo A-I expression
is induced both in the liver and the spleen (Figure 2B), human apo A-I
expression (Figure 3A) and titers of antibodies against human apo A-I
(Figure 3B) were determined after gene transfer with
2 × 109 pfu AdCMV. Human apo A-I levels at
day 3 and day 6 were similar to those after coinjection of
AdE and AdAT4, but declined to 3.2 ± 1.3
mg/dL at day 14 and were below detection limit at day 21 and later. The
titer of antibodies against human apo A-I at day 14 after transfer was
not significantly different than after coinjection of AdE
and AdAT4, but in contrast to the coinjection experiment,
titers persisted for several months after transfer. In aggregate, these
data suggest that the time course of human apo A-I antigen levels may
determine whether a sustained humoral immune response against human apo
A-I or tolerance for human apo A-I develops. Expression of human apo
A-I in the liver per se is insufficient for the induction of tolerance
against the transgene product after adenoviral gene transfer. The
presence of antibodies against human apo A-I in the first weeks after
gene transfer in the coinjection experiment indicates that the initial
absence of a humoral immune response against human apo A-I after
transfer with AdAT4 may be due to immunologic ignorance and
cannot be explained by anergy or deletion of immunoreactive cells at
these early time points.
To evaluate whether the development of tolerance in the coinjection experiment was specific for human apo A-I, antibodies against adenovirus serotype 5 were determined. The inverse of the antibody titer was 200 000 ± 19 000 at 21 days and 190 000 ± 17 000 at 3 months, indicating the specificity of tolerance induction. Gene transfer with AdAT4 induces tolerance for human apo A-I To investigate directly tolerance for human apo A-I at later time points after transfer with AdAT4, a subcutaneous immunization with human apo A-I protein was performed at 3 weeks (n = 8) after transfer with 5 × 108 pfu AdAT4. No immune response was observed in 7 of 8 mice injected at 3 weeks after transfer. A weak immune response was seen in one mouse (1-1000). A rechallenge subcutaneous immunization with human apo A-I protein was performed 6 weeks after gene transfer. No immune response was observed in the 7 mice without antibodies after the first immunization, whereas a slight increase of antibody titer occurred in the eighth mouse (1-4000). Human apo A-I level is shown in Figure 4A and was not significantly different compared with mice without subcutaneous immunization (Figure 1A). The geometric mean of the inverse of the titer is shown in Figure 4B. As a positive control, 4 Balb/c mice not injected previously with an adenoviral vector were immunized by subcutaneous injection of human apo A-I and a strong humoral immune response was observed (Figure 4B).
Antibodies against human apo A-I induced by subcutaneous immunization persist after subsequent gene transfer with AdAT4 To investigate human apo A-I expression and titers of antibodies against human apo A-I in mice preimmunized with human apo A-I protein, subcutaneous immunization with human apo A-I protein was performed 4 months before gene transfer. Figure 5A illustrates that high human apo A-I protein levels were observed in preimmunized mice, notwithstanding persisting high titers of antibodies against human apo A-I (Figure 5B). Because the simultaneous presence of high human apo A-I antigen levels and anti-human apo A-I antibodies represents a paradox, 2 µg/mL of human apo A-I either as free protein or as human apo A-I present on high density lipoprotein (HDL) particles from mice injected with AdAT4 was added to 1:2000 dilutions of plasma samples obtained at day 56, 3 months, and 4 months after gene transfer with AdAT4 in preimmunized mice. The optical density in the antibody titer assay decreased 1.8-fold (P = .0005) after addition of human apo A-I protein, but decreased not at all (ratio = 1.0) after addition of human apo A-I containing HDL particles nor after addition of 2 µg/mL bovine serum albumin. This observation may be explained by the tertiary structure of human apo A-I in HDL particles15-17 leading to the shielding of epitopes.
Subcutaneous gene transfer with AdE promoter-driven vectors may be used for
vaccination purposes. For safety reasons, subcutaneous gene transfer is
likely to be more suitable than intravenous gene transfer. Subcutaneous gene transfer performed by injecting 2.5 × 108 pfu
AdE into the footpath of each hindlimb induced similar
antibody titers than those induced after intravenous gene transfer with the same viral dose (data not shown).
The main findings of the present study are that (1) gene transfer with hepatocyte-specific promoters is associated with absence of a humoral immune response against human apo A-I in Balb/c mice; (2) notwithstanding the fact that human apo A-I is a circulating antigen, a strict correlation was observed between antigen expression in antigen-presenting cells and a humoral immune response against human apo A-I after adenoviral gene transfer; and (3) the time course of human apo A-I expression may be a determinant of the development of tolerance for human apo A-I after adenoviral gene transfer. Because the adenoviral backbone and the adenoviral capsid are
identical in the different vectors, the presence or absence of an
immune response against human apo A-I cannot be explained by
differences in adjuvant effect related to cytokine release induced by
adenoviral gene transfer.18 The absence of a humoral immune response against human apo A-I after adenoviral gene transfer with 1.5-kb human Because the induction of IgG antibodies requires CD4-cell
help19 and activation of CD4+ T cells is
dependent on MHC II presentation of processed antigen by
antigen-presenting cells, the question why endogenous expression of the
antigen leads to more efficient MHC II presentation arises. MHC class
II molecules are classically involved in the presentation of exogenous
antigens but can also present endogenous antigens to CD4 T cells.
MHC class II presentation of endogenous secreted hen egg lysozyme was
much more efficient when compared with that of exogenous hen egg
lysozyme and was not explained by reuptake of secreted
antigen.20 Rowell et al21 demonstrated that
class II presentation of the HIV-1 envelope protein by infected
antigen-presenting cells may be mediated by rapid endocytosis from the
cell surface, but also that class II MHC presentation occurred under
conditions that prevented reuptake by endocytosis, indicating that an
internal pathway for class II presentation exists. The in vivo data in the present investigation indicate that expression by
antigen-presenting cells of secreted and circulating antigens
potentiates humoral immune responses and is likely to be useful for
efficient vaccination against such antigens. The potential of MHC
class II promoters for vaccination purposes is underscored by the
induction of high titers of antibodies against human apo A-I after
subcutaneous gene transfer with AdE Interestingly, the lower human apo A-I mRNA signal in the
spleen after transfer with AdU also resulted in
significantly lower antibody titers. Lowering the dose of
AdE The route of administration may play a role in the induction of a
humoral immune response against a xenogenic transgene
product.22 An antibody response against factor VIII was
observed after intramuscular transfer of an AAV vector in C57BL/6 mice
but not after intraportal vein administration, indicating that
liver-directed gene transfer may result in mitigation of immune
responses against the transgene product.22 Liver-derived
dendritic cells have been shown to induce donor-specific
hyporesponsiveness in transplantation studies23 and may
also play a role in the induction of tolerance after adenoviral gene
transfer, since adenoviral vectors in mice demonstrate a predominant
hepatotropism and the production of antigen after transfer with
hepatocyte-specific promoters is restricted to the liver. However, the
present result suggests that the time course of human apo A-I
expression and not human apo A-I expression in the liver per se
determines development of tolerance for human apo A-I after adenoviral
gene transfer. Coinjection of 5 × 108 pfu
AdE As expected, a pre-existing humoral immune response against human apo A-I induced by subcutaneous immunization with human apo A-I protein formulated in complete Freund adjuvant could not be abrogated by subsequent gene transfer with AdAT4. Notwithstanding persisting antibodies, high human apo A-I antigen levels were detected. We suggest that epitopes recognized by many of the anti-human apo A-I antibodies may be shielded in the specific tertiary structure of apo A-I on discoidal or spherical HDL particles.16,17 Higher reactivity of anti-apolipoprotein A-I antibodies against apo A-I and HDL particles than against denatured apo A-I also has been described in patients with systemic lupus erythematosus.15 In conclusion, a strict correlation was observed between antigen expression in professional antigen-presenting cells and a humoral immune response against human apo A-I after adenoviral gene transfer. This may be relevant both for inducing efficient humoral immune responses in vaccination studies and for avoiding such responses in gene transfer applications for correction of protein deficiencies.
Bart De Geest is a postdoctoral fellow of the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen. Sophie Van Linthout is a research assistant at the Instituut voor Wetenschappelijk en Technisch Onderzoek-Vlaanderen. We thank Jessy Hendrix and Zhiyong Zhang for superb technical assistance.
Submitted July 18, 2002; accepted November 7, 2002.
Prepublished online as Blood First Edition Paper, November 21, 2002; DOI 10.1182/blood- 2002-07-2146.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Bart De Geest, Center for Molecular and Vascular Biology, Campus Gasthuisberg, Herestraat 49, 3000 Leuven, Belgium; e-mail: bart.degeest{at}med.kuleuven.ac.be.
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© 2003 by The American Society of Hematology.
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  B. D. Brown, A. Cantore, A. Annoni, L. S. Sergi, A. Lombardo, P. Della Valle, A. D'Angelo, and L. Naldini A microRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice Blood, December 15, 2007; 110(13): 4144 - 4152. [Abstract] [Full Text] [PDF]
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A. Annoni, M. Battaglia, A. Follenzi, A. Lombardo, L. Sergi-Sergi, L. Naldini, and M.-G. Roncarolo The immune response to lentiviral-delivered transgene is modulated in vivo by transgene-expressing antigen-presenting cells but not by CD4+CD25+ regulatory T cells Blood, September 15, 2007; 110(6): 1788 - 1796. [Abstract] [Full Text] [PDF] |
Top
Abstract
Introduction
1-antitrypsin promoter, but antibodies were
induced after transfer with vectors driven by the ubiquitously active
U1b promoter and the murine MHCII E
), AdC4 (
), AdU
(
), and AdCMV (
) in Balb/c mice. Human apo A-I levels
were below detection limit after transfer with any dose of
AdE
) and after transfer
with 108 pfu AdE
) in
Balb/c mice.
) and after
gene transfer with 5 × 108 pfu AdAT4 in
Balb/c mice, which were subcutaneously immunized with human apo A-I 3 weeks after gene transfer and rechallenged with subcutaneous human apo
A-I immunization 6 weeks after gene transfer (
the central role of immunoglobulin receptor engagement and T-cell help.
Int Rev Immunol.
1997;15:33-52