1 UMR INRA-AFSSA-ENVA 1161, Virologie, Ecole Nationale Vétérinaire d'Alfort, 94704 Maisons-Alfort Cedex, France
2 Crucell Holland BV, PO Box 2048, 2301 CA Leiden, The Netherlands
Correspondence
M. Eloit
meloit{at}vet-alfort.fr
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ABSTRACT |
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INTRODUCTION |
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Attachment and entry of numerous human Ads, except those belonging to subgroup B, depend on the binding of the viral fibre protein to a high-affinity Ad receptor, i.e. the coxsackieadenovirus receptor (CAR) (Bergelson et al., 1997; Tomko et al., 1997
). After fibre binding, interaction between the Arg-Gly-Asp (RGD) sequence present in the viral penton base and
v integrins present on the host cell triggers the internalization of the virus in clathrin coated pits (Wickham et al., 1993
).
Several studies have demonstrated that the fibre protein carried by rAds can be modified so as to alter tropism in vitro (Barnett et al., 2002). Since different Ads display different tropisms (Horwitz, 1996
), one approach to modify the tropism of rAds is to replace the entire fibre protein of one serotype with that of another, thus generating pseudotyped rAds (Wickham et al., 1997
). Such replacement, however, may give rise to unstable recombinant vectors. Therefore, replacing only a portion of the fibre is preferred. In this manner, Rea et al. (2001)
have shown that pseudotyped rAds carrying fibres from human subgroup B Ads were able to transduce human DCs more efficiently than rAd5 (Rea et al., 2001
). Very few studies, however, have compared the ability of these pseudotyped rAds to induce an immune response against the transgene product in vivo.
In the present study, we have used a library of pseudotyped, fibre-chimeric rAds with the aim (i) to identify vectors with distinct capacities to express transgene product in murine cell types naturally present in the muscle and (ii) to identify whether such distinct transgene product expression patterns could influence the ability of such viruses to induce potent immune responses against the transgene in vivo. Hereto, pseudotyped vectors selected on the basis of their differential ability to express transgene in murine myoblasts, endothelial cells and DCs were injected i.m. into C57BL/6 mice.
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METHODS |
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Cell lines and cell culture.
Primary mouse myoblasts were kindly provided by C. Pinset and D. Montarras (Pasteur Institute, Paris, France). Cells were cultivated in medium composed of high-glucose DMEM and MCDB 202 medium (1 : 1, v/v) (both from BICEF, l'Aigle, France) supplemented with 20 % FCS (Biomedia) and 2 % (v/v) Ultroser (Biosepra). Endothelial cells derived from mouse heart and immortalized with SV40 T antigen were provided by D. Paulin and C. Delouis (Vicart et al., 1994). These cells were grown in DMEM containing 10 % FCS (Invitrogen). RMA-S cells are lymphoid cells that were cultivated in RPMI (Invitrogen) with 10 % FCS. Primary murine DCs were generated from bone marrow of C57BL/6 mice as described previously (Lutz et al., 1999
). Briefly, total bone marrow leukocytes were plated in bacteriological Petri dishes in RPMI medium containing 10 % FCS and recombinant murine granulocyte-colony-stimulating factor (20 ng ml1) at 2x106 cells per plate. The medium was partially replaced every 2 to 3 days. On day 8 of in vitro culture, non-adherent cells were used for transduction. Chinese hamster ovary (CHO) cells stably transfected with either cDNA constructs encoding human coxsackie and adenovirus receptor (CHO-CAR cells) or with vector alone (CHO-pcDNA) were cultured in MEM (Invitrogen) with 10 % FCS (Bergelson et al., 1997
). All media were supplemented with L-glutamine (2 mM), penicillin (50 IU ml1), streptomycin (50 µg ml1) and sodium pyruvate (1 mM). Cells were cultured at 37 °C in a 5 % CO2 incubator, except endothelial cells, which were cultured in an 8 % CO2 incubator.
Attachment experiments with fibre-chimeric rAds.
Binding of fibre-chimeric rAds to CHO-pcDNA and CHO-CAR was assayed by flow cytometry. Hereto, 5x105 cells were incubated for 1 h at 4 °C at different ratios of VP per cell in 50 µl of PBS. After washing twice with PBS, cells were incubated with a monoclonal antibody, 1D2, specific for the Ad penton base protein (Hong et al., 2000) for 30 min on ice and then with a 1 : 15 dilution of goat anti-mouse immunoglobulin coupled to phycoerythrin (DAKO). Following additional washes in PBS, cells were fixed in 400 µl of 1 % paraformaldehyde (PFA) and analysed by flow cytometry.
Transduction and analysis of gene transfer.
Transduction experiments using rAds were performed in triplicate in 96-well plates with each well containing approximately 5x104 cells in serum-free medium. Transduction experiments on myoblasts, endothelial cells and DCs were performed at 1000, 5000 and 8000 VP per cell, respectively. CHO-pcDNA and CHO-CAR cells were incubated with increasing ratios of VP per cell (ranging from 20 to 2500 VP per cell). Cells were exposed to virus for 2 h at 37 °C. The inoculum was then replaced by fresh complete medium. Gene transfer efficiency was assessed 24 (for CHO-CAR and CHO-pcDNA) or 48 h later (for DCs, myoblasts and endothelial cells) by measurement of Luc activity with commercially available reagents (luciferase reporter gene assay; Roche Diagnostics) using a Berthold luminometer (Lumat LB 9507) for DCs, myoblasts and endothelial cells and a Thermo Labsystems luminometer (Fluoroskan ascent FL) for CHO-CAR and CHO-pcDNA.
Blocking experiments.
For blocking experiments, transduction assays on myoblast and endothelial cells were performed with 1000 or 5000 VP per cell, respectively, in the presence of 10 % rabbit polyclonal antiserum against recombinant soluble CAR (Spiller et al., 2002) or of a control rabbit polyclonal serum in a final volume of 50 µl. Cells were exposed to the virus for 2 h at 37 °C and then fresh complete medium was added. Gene transfer efficiency was assessed 24 h later using a Thermo Labsystems luminometer (Fluoroskan ascent FL).
Immunization with rAds or rAd-transduced DCs.
Female C57BL/6 mice, 6 weeks of age, were purchased from Iffa Credo (L'Arbresle, France). Animals were handled in accordance with the institutional guidelines of the National Veterinary School of Alfort. Groups of seven mice were injected by the i.m. route at one site in the quadriceps with 50 µl of inoculum. The inoculum was either 108·5 or 1010 VP of rAd5 or fibre-chimeric rAd vectors, or 2x104 Gal-expressing DCs (transduced either with rAd5
Gal or with rAdF35
Gal at 25 000 VP per cell) or control DCs. Viral DNA contained in each rAd inoculum was quantified by real-time PCR. Peripheral blood was collected at the indicated time points. For all analyses, blood samples were pooled from animals in the same group.
Anti-Gal antibody assay.
Anti-Gal plasma antibody titres were determined by ELISA. A 96-well plate (Maxisorb; Nunc) was coated overnight at 4 °C with 500 ng of
Gal (Roche Diagnostic) in 100 µl of 0·1 M Tris, 2 mM EDTA, 0·15 M NaCl (pH 9·6) buffer. A solution containing 0·1 % (v/v) Tween 20 and 0·5 % (w/v) gelatin in PBS was used as a blocking buffer. The plate was washed with 0·05 % Tween 20 in water, and dilutions of plasma were incubated in the coated wells for 30 min at 37 °C. Secondary antibodies were as follows: goat anti-mouse IgG (Amersham Biosciences), anti-mouse IgG1 (Caltag Laboratories) and anti-mouse IgG2a (Caltag Laboratories). The peroxidase (POD) activity was analysed by using BM-Blue POD as a substrate. Absorbance was measured at 405 nm. Preimmune sera were used to determine the background level.
Anti-IFN- ELISPOT assay.
The interferon gamma (IFN-) ELISPOT assay was performed as described previously (Mercier et al., 2002
). Briefly, 96-well nitrocellulose plates (Multiscreen HA; Millipore) were incubated overnight at 4 °C with 40 ng of anti-mouse IFN-
monoclonal antibody (MAb) R4-6A2 (PharMingen) per well. Wells were washed repeatedly with culture medium and saturated with complete medium for 30 min at 37 °C. MHC I compatible target cells, RMA-S cells, were pulsed with a synthetic peptide (40 µg ml1) representing the CD8+ epitope [ICPMYARV; I8V (Isochem)] of
Gal in the H-2b haplotype (Oukka et al., 1996
). Serially diluted PBMC (1x1061·25x105) were co-cultured with 105 pulsed RMA-S in the ELISPOT wells, in medium supplemented with recombinant human interleukin 2 (30 IU ml1) (Roche Molecular Biochemicals), for 24 h at 37 °C under 5 % CO2. After extensive washing with PBS/0·05 % Tween 20, plates were incubated overnight at 4 °C with 50 ng per well of biotinylated anti-mouse IFN-
Mab XMG1.2 (PharMingen) and then with ExtrAvidin alkaline phosphatase conjugate (Sigma) for 1 h at room temperature. Spots were developed by an alkaline phosphatase conjugate substrate (Bio-Rad Laboratories). The number of spots, corresponding to IFN-
secreting cells, was determined with a KS-ELISPOT (Zeiss-Kontron).
Statistical analysis.
Statistical analysis was carried out using single factor ANOVA. Comparisons of means were performed at P<0·05. Computations were done using the SAS statistical software package.
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RESULTS |
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rAds binding to and transduction of CHO-pcDNA and CHO-CAR
To compare selected pseudotyped viruses with rAd5 as regards to their mechanisms of attachment and internalization, binding and transduction assays were performed using two different CHO cell lines, i.e. CHO-CAR, stably transfected with a vector expressing CAR and CHO-pcDNA, transfected with the empty vector. The binding of rAd5, rAdF40L, rAdF35 and rAdF50 was evaluated by flow cytometry at seven different ratios of VP per cell (156, 312, 625, 1250, 2500, 5000 and 10 000 VP per cell). Both rAd5 and rAdF40L bound efficiently to the CHO-CAR cell line (Fig. 2A). In contrast, rAdF50 bound very weakly to these cells, and no attachment of rAdF35 to CHO-CAR could be detected, even at the highest ratio tested. Of the four rAds, only rAdF50 bound to CHO-pcDNA to a similar extent as to CHO-CAR cells, indicating that the serotype 50 fibre bound to a receptor other than CAR expressed by both CHO-pcDNA and CHO-CAR cells.
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By using blocking experiments, we determined that an anti-CAR polyclonal serum could significantly inhibit rAd5 internalization in myoblasts and endothelial cells (P<0·05), although part of the transduction efficiency might be mediated by a CAR independent pathway in endothelial cells (Fig. 2C)
Antibody responses against Gal after i.m. inoculation of fibre-chimeric rAds
C57BL/6 mice were inoculated by the i.m. route with rAdF40L, rAdF50, rAdF35 and rAd5, all carrying the lacZ transgene encoding Gal. Blood samples were collected from immunized mice at days 10, 18, 35 and 42 after inoculation. Serum samples from seven mice of each group were pooled and assayed for anti-
Gal antibodies by ELISA (Fig. 3
). Mice inoculated with 1010 VP of each of the four viruses developed similar and high anti-
Gal IgG titres at day 42 (Fig. 3A
). In animals inoculated with rAdF50
Gal a slower antibody response was seen, which was even more pronounced when mice were inoculated with a lower dose of the different fibre-chimeric rAds (108·5 VP) (Fig. 3B
). The slower kinetics of the IgG response may be explained by the lower level of
Gal protein expression by this virus in mouse cells, which should reflect lower transduction efficiency in vivo, compared with the other viruses. To determine whether differences between the vectors could be observed at the level of Th1 or Th2 induction, the production of IgG2a and IgG1 was investigated, which is characteristic for Th1- and Th2-dominant conditions, respectively. All four rAds elicited both IgG1 and IgG2a after i.m. injection (Fig. 3C
), indicating that both Th1 and Th2 subsets were activated by all four viruses.
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DISCUSSION |
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We first used a classical approach that involves studying the ability of viruses to express a transgene product in vitro in targeted cell types, and notably in DCs, before testing them in vivo in animal experiments (Fontana et al., 2003; Brandao et al., 2003
; Wang et al., 2003
). In such an approach, each virus of a fibre-chimeric rAds library was first tested for its ability to express the transgene product after in vitro transduction of cell types present in skeletal muscle (endothelial cells, myoblasts and DCs). These experiments led to the selection of four viruses that were used to study the influence of distinct in vitro expression patterns on the ability of these viruses to induce potent immune responses against the transgene product in vivo. We selected rAd5 and rAdF40L, which were highly efficient at expressing the transgene product in myoblasts and endothelial cells in vitro, rAdF35, which was able to express transgene product as efficiently as rAd5 only in DCs, and rAdF50, which was very inefficient at expressing the transgene product in any of the three cell types. It was notable that none of the fibre-chimeric viruses tested in this study appeared to express the transgene product in murine DCs more efficiently than rAd5, although Rea et al. (2001)
observed that human DCs were transduced more readily with rAd35 than with rAd5 (Rea et al., 2001
). It is well known that human monocyte-derived DCs do not express CAR, which serves as a high-affinity receptor for the Ad fibre protein and mediates attachment of subgroup C Ads, such as Ad2 and Ad5, to the majority of permissive human cell types (Bergelson et al., 1997
). Moreover, the results obtained in our study indicate (based on transgene expression levels) that murine DCs do not express a high affinity receptor for any of the fibre-chimeric viruses tested. It is noteworthy that the three rAds tested carrying a subgroup B fibre (rAdF11, rAdF35 and rAdF50) expressed the transgene product in murine DCs with markedly different efficiencies. In particular, rAdF11 and rAdF35 transduced these cells 15 to 20 times more efficiently than rAdF50 (P<0·05). It is well-documented that human subgroup B Ads use a fibre receptor distinct from CAR. This subgroup has been divided into B1 (Ad3, Ad7, Ad50) and B2 (Ad11 and Ad35) species, which present different tropisms in vivo (De Jong et al., 1999
). It has recently been demonstrated that on human cells there are two different receptors for subgroup B Ads, one common to B1 and B2 species and the other exclusively used by the B2 species (Segerman et al., 2003a
). Also, CD46 has been identified as a receptor for Ad11 (Segerman et al., 2003b
). Since rAdF11 and rAdF35 transduce DCs more efficiently than rAdF50, it is possible that murine DCs express a B2 receptor.
The immunization of C57BL/6 mice by the i.m. route with all of the selected fibre-chimeric rAds induced strong antibody responses, but with different kinetics of IgG production. Resting B cells can be stimulated to proliferate and differentiate into antibody-producing cells by a combination of cell contact and soluble signals provided by activated primed helper T (Th) cells. In this study, we observed that the inoculation of rAd5Gal, rAdF40L
Gal, rAdF50
Gal and rAdF35
Gal induced both IgG1 and IgG2a at similar levels. As the isotype profile is guided mostly by cell-secreted cytokines, we postulate that the four viruses induced a similar cytokine profile in vivo. Thus, the replacement of the Ad5 fibre with one derived from either subgroup B or F did not prevent activation of both Th1 and Th2 subsets, classically obtained also after rAd5 inoculation in C57BL/6 mice. In our experiments, the
Gal antigen has a nuclear targeting signal and thus is a cell-associated protein. In order to stimulate the B cell response, the newly synthesized
Gal protein, after rAd-mediated transduction, needs to be released into the extracellular environment, for instance by the induction of cytotoxic NK and T cell responses (Yang et al., 1996
). The different kinetics of the IgG response observed after inoculation of 108·5 VP per mice may be explained by a difference in the level of
Gal expression after i.m. inoculation of the different fibre-chimeric rAds, which should reflect different transduction efficiencies in vivo (Huard et al., 1995
). For example, a recent study has described that the rAdF35 induces lower transgene expression than rAd5 in muscle after i.m. inoculation in C57BL/6 mice (Sakurai et al., 2003
). Here we show that rAd5 and rAdF40L, which efficiently express transgene product in myoblasts and endothelial cells in vitro, induced a predominantly faster antibody response than rAdF35 and rAdF50. Many studies have shown that myoblasts are efficiently transduced after i.m. inoculation of rAd and may serve as a reservoir of transgene in vivo. In fact, rAd5 and rAdF40L were similarly efficient in inducing an anti-
Gal antibody response. Interestingly, these two fibre-chimeric rAds were able to transduce cells via a CAR-dependant pathway, as shown by CHO-CAR/CHO-pcDNA transduction experiments. Moreover, CAR is involved in myoblast and endothelial cell transduction by rAd5, as shown by using blocking experiments with an anti-CAR polyclonal serum. Together, these data suggest that the induction of the anti-
Gal antibody response, after i.m. inoculation in C57BL/6 mice, might be increased following gene delivery by rAds able to induce high levels of transgene product expression, notably by transducing the cells via a CAR dependent mechanism.
It has been previously shown that Ad5 is able to elicit a CD8+ T cell response through both the direct transduction and cross-priming of DCs (Jooss et al., 1998). A key question that remains is whether one pathway is dominant over the other. In our hands, some viruses (rAd5 and rAdF40L) were much more efficient than others (rAdF35 and rAdF50) in expressing transgene product in murine heart endothelial cells and myoblasts in vitro, but were not more efficient in eliciting a CD8+ T cell response against the transgene product than rAdF35 and rAdF50. Thus, our results suggest that cross-priming from dead or living cells expressing high level of transgene product (Ramirez & Sigal, 2002
) is not a key event in eliciting a CD8+ T cell response, in contrast with the major role of cross-priming for the induction of CD8+ immune responses against vaccinia virus following i.m. injection (Shen et al., 2002
). Another possibility is that low levels of expression of foreign proteins in non-APCs are sufficient to establish cross-priming. The latter hypothesis is unlikely however, since it has been demonstrated that only high level of Ag expression allows efficient cross-presentation (Kurts et al., 1998
). Of the four viruses selected for the in vivo experiments, the efficiency of transgene product expression was much less divergent in mouse bone marrow-derived DCs (between 1·15 and 20) than in endothelial cells (between 1·05 and 1800) and myoblasts (between 1·4 and 350), even though the DCs were transduced at a higher m.o.i. This limited divergence did not substantially modify the induction of a CD8+ T cell response in vivo. It is therefore probable that only a small number of transgene product-expressing DCs or a low level of transgene product expression is required for induction of such a response after inoculation of rAds. Timares et al., (1998)
have shown that transfection of 500 DCs is sufficient to initiate a wide variety of immune responses in vivo. Nevertheless, it is still not known whether these responses represent an upper limit, or whether more efficient transduction of DCs could lead to a higher CD8+ T cell response against the transgene product. Immunization of C57BL/6 mice with
Gal-expressing DCs after ex-vivo transduction with either rAd5 or rAdF35 induced the same anti-
Gal CD8+ T cell response. These results indicated that the fibre replacement itself does not alter the ability of rAd-transduced DCs to present foreign Ags and that these two viruses could probably induce the maturation of murine DCs to allow an efficient stimulation of CD8+ T cells. Previously, fibre- and thus receptor-independent transduction of Ad5 in the muscle has been described (Einfeld et al., 2001
; van Deutekom et al., 1999
). Receptor-independent uptake of adenoviruses in muscle could also explain the similar T cell responses with the four fibre-chimeric rAds, despite their distinct cellular tropisms observed in vitro. To discriminate between those possibilities, further studies on which cell types are transduced in vivo are required.
The results of the experiments described do not show a direct correlation between the in vitro tropism and the level of the immune response induced after i.m. administration of different fibre-chimeric rAds. Furthermore, we did not find any evidence for significant participation of cross-priming in the development of the CD8+ T cell response against the transgene product. Further studies, including in vivo tropism of these fibre-chimeric adenoviruses, should help in further unravelling the underlying mechanisms for immune induction after i.m. administration.
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ACKNOWLEDGEMENTS |
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Received 25 November 2003;
accepted 22 January 2004.