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Journal of Virology, June 1999, p. 5156-5161, Vol. 73, No. 6
CNRS-IGR-Rhône Poulenc Rorer UMR1582,
Institut Gustave Roussy, 94805 Villejuif Cedex, France
Received 19 November 1998/Accepted 1 February 1999
Hypervariable region 5 (HVR5) is a hydrophilic, serotypically
nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. We have replaced the HVR5 sequence of Ad5 with
that of heterologous peptides and studied their effects on virus
viability and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine "isogenic"
viruses that differed in their flanking spacers. Whereas virus
productivity was not profoundly altered by any of these modifications,
immunoprecipitation experiments under nondenaturing conditions
demonstrated that epitope recognition by its cognate monoclonal
antibody (C3 MAb) was strongly linker dependent and
correlated perfectly with the ability of C3 MAb to inhibit
transgene delivery and expression. An Subgroup C-based adenovirus (Ad)
vectors are being used in a growing number of clinical trials for
acquired (e.g., cancer) and monogenic hereditary disorders.
Ideally, these vectors should deliver and express their transgene
only in the targeted cells in vivo. However, barriers exist that limit
their efficacy: (i) widespread distribution of the virus receptors
makes it difficult to restrict infection to particular cells; (ii)
therapeutic targets expressing small amounts of receptors require high
vector doses, thereby favoring spreading; and (iii) neutralizing
antibodies specific to the capsid proteins can preclude efficient
readministration in vivo (7). For example, intratumoral
administration of recombinant Ad in murine models has been associated
with vector dissemination to distant organs (e.g., lungs, liver, and
spleen). Clinical data have also established the presence of the
recombinant virus in bodily fluids of cancer patients intratumorally
injected with the highest doses (3, 21). Taken together,
these data emphasize the need to adapt the vector tropism to particular
target cells in order to enhance infection while reducing vector
dissemination and shedding.
Virus cell attachment, the initial step that dictates viral tropism,
proceeds through high-affinity binding of the distal domain (i.e., the
knob) of the capsid fibers to cell surface receptors (2, 10,
19). Accordingly, targeting strategies have focused primarily on
the knob-mediated attachment step. For example, incubation of
recombinant virus with an anti-fiber neutralizing antibody chemically
conjugated to a cell-specific ligand (e.g., folate or fibroblast growth
factor) has been shown to enhance the transduction of cells displaying
the appropriate receptors (6, 16). A conceptually different
strategy relies on the incorporation of chimeric fibers within the
capsid to switch the tropism of different serotypes (8,
18), especially that of subgroup B (15). Additional strategies are being investigated, including peptidic insertion and C-terminal extension of the fiber knob
(11-13, 23, 24). Although these approaches are not examples
of a true retargeting strategy, since the native tropism is
maintained, they should reduce viral dissemination by allowing the use
of lower doses to reach clinical efficacy. Indeed, a polylysine
extension of the fiber C terminus increases transduction both in vitro
and in vivo, most probably because virus internalization occurred after
virus attachment to heparan sulfate receptors (23, 24). However, genetic engineering of the fiber C terminus is tricky: deletions as small as 2 residues can prevent fiber trimerization in
vitro, whereas addition of larger peptides often impairs virus growth
(9, 13). Peptide insertion within the knob domain constitutes a more permissive approach, as recently reported for HI
loop insertion between residues 546 and 547 (11) and the successful recovery of 15 of 15 HI-modified viruses with a foreign peptide ranging in size from 11 to 19 residues (5a).
Genetic engineering of the capsid hexon monomer is an attractive
approach because this polypeptide is by far the most abundant capsid
component (for a review, see reference 17). If the
approach is successful, hexon-modified vectors should exhibit profound modifications of their physical and biological properties: target avidity for a particular ligand may reduce vector dissemination following local delivery and/or may alter tropism (see below). The
primary sequence of the hexon monomer is well conserved among serotypes; the main differences are clustered within seven stretches (the so-called hypervariable regions [HVRs]) that are localized mostly within the extruding domains of the molecule (1).
Some of these HVRs, including HVR5, must be quite flexible, as
suggested by the absence of assignable structure in the
crystallographic study of the Ad2 hexon monomer (1).
Additional arguments prompted us to select HVR5 for peptide insertion.
First, in contrast to HVR3, HVR4, and HVR7, HVR5 apparently does not
include residues involved in intramolecular interactions within the
molecule (1). Second, HVR5 is quite variable in length,
ranging from 9 residues (e.g., in Ad30 and Ad37) to 20 residues (e.g.,
in Ad10, Ad19, and Ad45). Finally, HVR5 displays linear epitopes
capable of eliciting anti-Ad2 neutralizing antibodies in vivo
(20), and it could be replaced with a vaccinating poliovirus
epitope (4). Based on these observations, we performed the
present study by substituting HVR5 residues 269 to 281 (TTEAAAGNGDNLT), keeping intact its most highly conserved
residues on either side (Ser268 and Pro282).
To facilitate construction of the HVR5-modified viruses by
recombinational cloning in Escherichia coli (5),
we first introduced the xylE marker gene from
Pseudomonas putida in place of HVR5, since its phenotypic
expression in E. coli is readily detectable in the presence
of catechol (26). Shuttle plasmid IE27 was therefore engineered to contain the flanking regions of HVR5 (residues 135 to 268 and 282 to 414) flanking the xylE marker gene (Fig.
1A). This ColE1-based episome was
introduced into E. coli G4977, a polymerase A
(polA)-deficient strain that cannot replicate ColE1-derived plasmids, and recombined with plasmid AE18c (an RK2-based plasmid which
does not require the polA gene product for replication). Following a two-step recombinational gene replacement in E. coli (5), plasmid IE27c was recovered, which contains a
PacI-excisable
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
RGD Inclusion in the Hexon Monomer Provides
Adenovirus Type 5-Based Vectors with a Fiber Knob-Independent
Pathway for Infection
ABSTRACT
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Abstract
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v-specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-modified capsids would
enhance the transduction of cells displaying limiting amounts
of the virus attachment receptors. Interestingly, although hexon
has never been implicated in Ad entry, the modified virus
significantly increased the transduction of human vascular smooth
muscle cells in vitro. Competition experiments with 293 cells
saturated with recombinant knob further indicated that the
hexon-modified virus could use an additional, knob-independent pathway
for entry. We concluded that genetic engineering of the Ad5
hexon monomer constitutes a novel and feasible approach to equip the
virus with additional targeting ligands.
TEXT
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Abstract
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References
E1E3 viral backbone with a
cytomegalovirus-lacZ expression cassette in place of the E1
genes (the deletion extends from Ad5 nucleotides 382 to 3512), together
with the xylE phenotypic marker inserted within HVR5 (Fig.
1C).
View larger version (26K):
[in a new window]
FIG. 1.
Modification of the Ad5 hexon monomer by recombinational
cloning in E. coli. (A) Shuttle plasmid IE27 (the detailed
construction strategy is available upon request) is a kanamycin
(Km)-selectable plasmid in which the xylE phenotypic marker
has been inserted in place of HVR5 of Ad5 (residues 268 to 282). Its
replication requires the polA gene product of E. coli. SacB is a suicide gene for E. coli in the
presence of sucrose as the carbon source. a and b refer to
HVR5-flanking sequences from Ad5 (numbers in parentheses refer to hexon
amino acid numbering). (B) The xylE phenotypic marker of
shuttle plasmid IE27 is bordered by unique NruI and
Bsu36I sites to facilitate subsequent HVR5 modifications.
(C) Principle of HVR5 modification by homologous recombination in
E. coli (adapted from reference 5).
Homologous recombination (HR) between identical sequences (black bars a
and b) from a suicide ColE1 shuttle plasmid (e.g., IE27) and an
RK2-derived (replication is polA independent) plasmid (e.g.,
AE18c) in a polA mutant of E. coli generates a
kanamycin (Km)- and tetracycline (Tet)-selectable cointegrate.
Resolution of the cointegrate by HR leads to the loss of the
sacB suicide gene, which is selected by using sucrose as a
carbon source. Depending on the recombination pathway, resolution of
the cointegrate generates either the starting backbone AE18c or the
xylE-containing IE27c backbone, which can be differentiated
upon catechol addition (26). IE27c has been used as a
recipient backbone to generate all HVR5-modified backbones and viruses
used in this study (see the text).
The DNPASTTNKDK model peptide, a well-studied neutralizing epitope from
poliovirus type 1 (22), was then inserted in HVR5: shuttle
plasmids IE35, IE37, IE39, IE40, IE43, IE44, IE45, IE46, and IE47,
which are identical to IE27 except that the xylE marker had
been replaced with the poliovirus epitope bordered by connecting linkers of different sizes and/or sequences, were thus constructed (Table 1). These plasmids were recombined
with backbone IE27c in E. coli G4977 by using the
xylE screening to generate a serie of
PacI-excisable E1E3 lacZ-expressing viral
backbones (Table 1). Plasmid IE31c
contains a control viral backbone in which peptide
NLDSLEQPTTRAQKPRLD was inserted in place of HVR5 residues 269 to
285 as reported previously (4). IE30c, IE32c, and IE33c are
additional controls in which the HVR5 Ad5 sequence was replaced with that from Ad2, Ad30, and Ad19, respectively (Table 1). In all
cases, the substitutions within HVR5 were checked by nucleotide sequencing.
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All viral backbones were excised by PacI restriction
and transfected in 293 cells as described previously (5).
With the exception of IE31c, they all induced a cytopathic effect that could be progressively amplified. Laboratory-scale batches
(3 × 108 infected cells; one assay per virus) were
prepared 48 h after infection at a multiplicity of infection (MOI)
ranging from 5 to 10 transducing units, and viral productivity was
assessed by counting the number of virus particles produced
(VP/cell). The Ad5/Ad2, Ad5/Ad19, or Ad5/Ad30 chimeric viruses
exhibited a correct productivity (i.e., 2,000 to 6,600 VP/cell),
as did all nine HVR5-modified viruses containing the poliovirus epitope
(i.e., 8,500 ± 4,700 VP/cell). HVR5 is thus quite permissive for
insertion, since peptides ranging in size from 6 to 17 residues can be
included with no obvious deleterious effect. The finding that virus
AdIE31 could not be recovered despite repeated attempts was not
expected. Possibly, the original virus of Crompton et al., which was
constructed by homologous recombination between an Ad5-based chromosome
and a recombinant Ad2-based HVR5 mutant plasmid, did exhibit extragenic compensatory mutations (4). In fact, the sole deletion
of residues 269 to 285 in our construct most probably affected
viability, since residues 281 to 285 have been assigned to a
-strand located immediately downstream of HVR5 (1).
That the physical properties of the virus capsid were modified after HVR5 insertion was first evidenced by its different elution profile during anion-exchange chromatography (2a). The accessibility of the inserted epitope was first assessed by immunoprecipitating nondenatured capsids with C3 MAb, a monoclonal antibody (MAb) specific to the poliovirus epitope (22). Briefly, 1010 particles of CsCl-purified virus were resuspended in 400 µl of 50 mM Tris-HCl (pH 7.5)-150 mM NaCl-0.05% Nonidet P-40 prior to C3 MAb incubation for 1 h at 4°C. A 400-µl volume of protein A-Sepharose complex equilibrated in the same buffer was then added, and the mixture was incubated for another 1 h at 4°C. The complex was then collected by centrifugation, washed twice, and further incubated at 4°C for 20 min in 1 ml of 10 mM Tris (pH 7.5)-0.1% NP40. It was then resuspended in 50 µl of Laemmli buffer, boiled for 2 min, and subjected to Western analysis (enhanced chemiluminescence [Amersham]) with an anti-Ad5 rabbit serum. The presence of a spacer larger than 2 residues downstream of the poliovirus epitope drastically affected the outcome of this assay: whereas AdIE43, AdIE44, AdIE45, AdIE46, and AdIE47 were efficiently immunoprecipitated by C3 MAb, apparently with a similar efficacy, this was not the case for the other viruses (Fig. 2A).
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Virus opsonization by C3 MAb also inhibited lacZ
transduction of W162 reporter cells (Fig. 2B). Briefly, C3 MAb was
incubated in phosphate-buffered saline for 1 h at 37°C with
105 CsCl-purified infectious particles (i.e.,
105 lacZ-transducing units [TDU])
(25), at which time subconfluent W162 cells were infected at
37°C for 1 h at an MOI of 0.1 TDU/cell. After the cells
were washed, fresh culture medium was added, and the number of
lacZ-expressing cells was quantified 2 days later by X-Gal (5-bromo-4-chloro-3-indolyl--D-galactopyranoside) staining. Again, the flanking spacers had a major influence in this assay: whereas lacZ transduction was inhibited between 97 and
99% upon incubation of AdIE43, AdIE44, AdIE45, AdIE46, and
AdIE47 with a 10
3 dilution of C3 MAb, that of the other
HVR5-modified viruses remained unchanged and paralleled that of virus
AE18, a nonmodified "isogenic" E1E3 virus expressing
lacZ from the cytomegalovirus promoter (Fig. 1C). C3 MAb
binding to the HVR5-modified capsids therefore correlated perfectly
with the inclusion of linker sequences on both sides of the inserted
epitope (Table 1).
An RGD-containing peptide specific to v integrins
(14) was then flanked by GS linkers (GSDCRGDCFGS) and
inserted into HVR5 by recombinational cloning in E. coli
(virus AE57 [Table 1]). Again, CsCl-purified batches were prepared
with a correct productivity (6,000 VP/cell). We then asked
whether capsid inclusion of this targeting ligand would
facilitate the infection of human cells naturally
refractory to Ad5 infection. Human vascular smooth muscle cells
were selected for this assay because they display v
integrins at the cellular surface but have only small numbers of knob
primary attachment receptors (24). Interestingly, 30% of
the smooth muscle cells stained positive for X-Gal following
infection with 10-fold less of the RGD-containing virus (AE57)
(1,000 VP/cell) compared to its unmodified counterpart (AE18)
(Fig. 3A). In this assay,
X-Gal-positive cells were scored in five fields for each condition, i.e., AE18 (MOI 1,000; 14 ± 2 per field), AE18
(MOI 10,000; 113 ± 17), AE57 (MOI 1,000; 90 ± 14), and
AE57 (MOI 10,000; 337 ± 107), and correlated with
detectable levels of intracellular vector DNA following infection
with virus AE57 but not AE18 (results not shown). Transgene expression
was also significantly increased when the RGD virus was used in place
of virus AE18 (Fig. 3B). We next assessed the behavior of virus
AE57 toward cells readily infectable by Ad5. AE57 infection of 293 cells incubated with saturating amounts of competitor knob (i.e.,
amounts almost completely abolishing lacZ transduction when
virus AE18 was used) led to the transduction of a significant
proportion of cells (Fig. 4A). In this
assay, quantitation of X-Gal-positive cells in the presence of knob was
associated with 128 ± 9 positive cells per field with AE57 versus
32 ± 9 for the AE18 control virus (n = 5) and
correlated with a significant increase in -galactosidase specific
activity (Fig. 4B). That 293 cells remained amenable to transduction
following inhibition of the normal attachment pathway supports a direct recruitment of the inserted RGD peptide during the initial
step of infection. Possibly, the inserted RGD peptide also
contributed to the endocytic internalization process.
|
) or AE57 (
) at the indicated MOI. Extracts
were prepared 48 h postinfection, at which time total protein and
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Taken together, our data demonstrate that the Ad5 hexon monomer can accommodate a foreign peptide within HVR5 with no dramatic effect on virus viability and growth. Conditions can also be found that allow the inserted peptide to recognize its cognate receptor either in a soluble phase or at the cellular surface. In particular, the observations that hexon-modified capsids can be efficiently internalized by cells lacking the virus primary receptors or when the initial attachment step has been artificially abolished should open up new avenues to adapt the vector tropism to particular cellular subsets. Genetic engineering of the hexon monomer may also constitute a useful prerequisite to design truly retargeted vectors following ablation of the native entry pathway.
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ACKNOWLEDGMENTS |
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We thank F. Blanche, J. Crouzet, J.-D. Guitton, M. Latta, and J. F. Mayaux (CRVA, Vitry-Sur-Seine) for their support and expertise. J. Douglas, V. Krasnykh, and D. Curiel (Gene Therapy Program, University of Alabama at Birmingham) are acknowledged for sharing unpublished data and material, and R. Crainic (Institut Pasteur, Paris, France) is thanked for providing C3 MAb.
This work initiated under the BioAvenir program was supported by Rhône-Poulenc, the French Ministry of Research, and the French Ministry of Industry.
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FOOTNOTES |
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* Corresponding author. Mailing address: CNRS-IGR-Rhône Poulenc Rorer UMR1582, Institut Gustave Roussy, Rue Camille Desmoulins, 94805 Villejuif Cedex, France. Phone: (33-1) 42 11 50 89. Fax: (33-1) 42 11 52 46. E-mail: pyeh{at}igr.fr.
Present address: Vector Development, Rhône-Poulenc Rorer
Gencell, CRVA, 94403 Vitry-Sur-Seine, France.
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Journal of Virology, June 1999, p. 5156-5161, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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