FR2758821A1 - Use of peptide(s) for facilitating or modulating attachment of adenovirus to cells - Google Patents

Abstract

Use of a peptide (I) homologous or identical with \- 6 consecutive amino acids of one of the sequences (S1)-(S5) to allow, or facilitate, attachment of an adenovirus (Ad) to a host cell and/or entry of Ad into the cell is new: LRAYLEGTCVEWLRRYLQNGKETLQ (S1) NSHPIQWNAPQPSHISKTILRWRPKN (S2) VKVTIMWTPPESAVTGYRVDVIPVN (S3) STVLVRWTPPRAQITGYRLTVGLTR (S4) NSLLVSWQPPRARITGYIIKYEKPS (S5) Also new are: (1) cells that express (I); (2) ligands (II) homologous or identical with \- 6 consecutive amino acids of the sequences (S6) or (S7): RAIVGFRVQWLRRYFVNGSR (S6) RHILWTPANTPAMGYLARVS (S7).

Description

 The subject of the present invention is the use of all or part of an antigen of the major histocompatibility complex of class I and / or of a type III fibronectin module to allow or facilitate the attachment of an adenovirus. on a host cell and / or its entry therein. The invention also relates to the use of a ligand capable of modulating the infectivity of an adenovirus with respect to a host cell, mediated by one or the other of the polypeptides mentioned above. Finally, the invention relates to a method of bio-blasting to identify or select a cellular receptor for an adenovirus or one of these ligands, in particular of viral origin.

Adenoviruses are DNA viruses with a broad host spectrum. They have been found in many animal species and can infect various cell types. Many serotypes have been characterized within each species which have a comparable genomic organization and infectious cycle. In general, the adenoviral genome consists of a linear, double-stranded DNA molecule of approximately 36kb containing the genes coding for the viral proteins and at its ends two inverted repeats (designated
ITR) involved in replication and the packaging region.

 Adenoviruses replicate in the nuclei of infected cells. The infectious cycle takes place in 2 stages. The early phase precedes the initiation of replication and makes it possible to produce the early proteins regulating the replication and transcription of viral DNA. These stages are followed by the late phase during which the structural proteins which constitute the viral particles are synthesized. The assembly of new virions takes place in the nucleus.

First, the viral proteins assemble so as to form empty capsids of icosahedral structure, in which the adenoviral DNA is packaged. Viral particles are released and can infect other permissive cells. In this respect, the fiber and the base penton present on the surface of the capsids play a critical role in the cellular attachment of virions and their internalization.

 The adenovirus binds to the surface of permissive cells via the trimeric fiber and a hitherto unidentified cellular receptor.

Then, the particle is internalized by endocytosis by binding of the penton base to the cellular integrins avss3 and avss5 (Belin and Boulanger, 1993, J. Gen. Virol. 74, 1485-1497; Mathias et al., 1994, J. Virol. 68 , 6811-6814; Nemerow et al., 1994,
Trends Cell. Biol. 4, 52-55; Wickham et al., 1993, Cell 73, 309-319; Wickham et al., 1994, J. Cell Biol. 127, 257-264). The fiber of Ad2 contains 580 amino acids (aa) whose sequence is disclosed in Herissé et al. (1981, Nucleic Acid
Res. 9, 4023-4042). That of Ad 5 has 582 amino acids (Chroboczek and
Jacrot, 1987, Virology 161, 549-554). Its molecular mass is 62 kDa, but the native fiber behaves like a molecule of 160-180 kDa confirming its assembly in the form of a tnmère.

 The fiber is made up of 3 domains (Chroboczek et al., 1995, Current Top.

Microbiol. Immunol. 199, 165-200): (1) In N-terminal, the very conserved "tail" from one serotype to another, interacts
with the base penton and anchors the molecule in the capsid.

(2) The "rod" is a rod structure of variable length according to the
serotypes. For example, the stem of the Ad5 fiber contains 22 repeats of a pattern of 15 residues which could adopt a sheet-like conformation ss P.

The number of these repetitions differs from one serotype to another, which explains
variations in length.

(3) Finally, at the distal end of the stem, the "head" or terminal sphericle is a
globular structure containing the trimerization signals (Hong and Engler,
1996, J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991, J. Biol. Chem.

266, 9299-9303; Novelli and Boulanger, 1991, Virology 185, 365-376). The
most experimental data shows that this is the domain of the head
which is responsible for binding to permissive cells.

 The complexity of adenoviral attachment suggests that it is serotype dependent and that several cellular proteins could participate.

With regard to Ad2, Hong and Boulanger (1995, EMBO J. 14,4714-4727) have identified a number of peptide motifs found in several surface cell proteins capable of interacting with the capsid proteins (penton base and fiber), in particular the type III modules 5 and 14 of human fibronectin. The authors operated by immobilization on an inert support of the penton base or of the fiber (ligand) on which they reacted a library of phages expressing random hexapeptides (designated phagotopes). The adsorbed phages, which in theory express phagotopes interacting with a motif carried by the adenoviral protein, are then eluted either conventionally at acid pH or by competition with the other non-immobilized capsid partner (eluent).

However, the cellular adenovirus receptor and the region of the head specifically involved in binding to the receptor have not yet been clearly identified.

 We have now carried out a new technique of "bio-mulching" (for biopanning in English) in which the immobilized ligand is constituted by the domain of the head of the AdS fiber and the eluent by a neutralizing antibody directed against the latter and isolated two classes of phagotopes according to the antibody used. The first corresponds to a sequence conserved within the a -2 domain of the antigens of the major histocompatibility complex of class I (a-2 MHC-I) and the second to a sequence found in modules III of human fibronectin (FNIII ). The data reported in the following examples support the hypothesis that cc-2 MHC-I constitutes the primary receptor for serotype C adenoviruses and confirm the participation of FNIII as co-receptor or co-factor. The regions of these two receptors and of the fiber interacting with each other have also been highlighted. In addition, an antagonist peptide reproducing the motif of the α-2 MHC-I domain which neutralizes the attachment of adenoviruses was generated and an agonist peptide reproducing the FNIII motifs which stimulates attachment.

This is why the subject of the present invention is the use of a polypeptide comprising an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown (a) in SEQ ID NO: 1 beginning with the leucine residue in position 1 and
ending with the glutamine residue in position 25, (b) in SEQ ID NO: 2 starting with the asparagine residue in position 1
and ending with the asparagine residue in position 26, (c) in SEQ ID NO: 3 starting with the valine residue in position I and
ending with the asparagine residue in position 25, (d) in SEQ ID NO: 4 starting with the serine residue in position 1 and
ending with the arginine residue in position 25, and / or (e) in SEQ ID NO: 5 starting with the asparagine residue in position 1
and ending with the serine residue in position 25 to allow or facilitate the attachment of an adenovirus to a host cell and / or the entry of said adenovirus into said host cell.

 For the purposes of the present invention, the term "polypeptide" means any molecule constituted by a chain of at least 6 and, preferably at least 8, amino acids. The term polypeptide includes both peptide molecules of short length (from 6 to a few tens of residues) as molecules of greater length (up to several hundred residues), on the condition, however, of allowing the intended use. It is specified that a polypeptide in use in the context of the present invention can be derived from a native polypeptide as found in nature, in particular in humans, or from a part thereof. It can also be chimeric and include additional residues of any origin fused in N and / or C-terminal and / or inserted so as to form an open reading frame. It is also possible to use a mutant obtained by mutation, deletion, insertion and / or substitution of one or more amino acids with respect to the sequences disclosed in the sequence identifiers (SEQ ID).

 A preferred polypeptide in the context of the present invention further comprises elements suitable for ensuring its anchoring in a cell membrane or its presentation on the surface of a cell. Such elements are known to those skilled in the art. As an indication, the presence of a signal peptide generally associated in the N-terminal position and of a transmembrane region having a high degree of hydrophobicity is mentioned. However, other techniques, for example chemical techniques, can also be used to anchor or bind a polypeptide to a membrane or a cell surface.

 By "homologous amino acid sequence" is meant a sequence having a degree of homology of at least 70%, advantageously of at least 80%, preferably of at least 90% with at least minus 6 continuous amino acids from one of the sequences listed. The identical term refers to 100% homology. Those skilled in the art know the general rules which make it possible to calculate the degree of homology between two sequences. This is generally done by aligning the sequences, possibly using specialized computer programs. It may be necessary to artificially introduce vacant locations. Once the optimal alignment is achieved, the degree of homology is established by counting all the positions in which the amino acids of the two sequences are found identically, compared to the total number of positions.

 By "attachment of an adenovirus to a host cell" is meant the binding of the viral particle to the cell. By "entry of an adenovirus into a host cell" is meant the penetration of the virus into the host cell.

The attachment and / or entry are preferably mediated at least in part by the polypeptide (s) in use in the context of the present invention by interaction with the adenoviral capsid. Of course, other polypeptide molecules or not can also participate in these processes recognized in the art field as complex and multifactorial. They can be evaluated by any technique of the art, such as those described below using a permissive cell line and radioactively labeled particles or expressing a reporter gene, for example the luciferase gene. At 0 C, only attachment can take place, viral penetration requiring a temperature of 37 "C.

For the purposes of the present invention, an adenovirus can be of human or animal origin (canine, avian, bovine, etc.) or a hybrid comprising genome fragments. These viruses and their genome are described in the literature (see for example
Graham and Prevec, Methods in Molecular Biology, Vol 7; Gene Transfer and
Expression Protocols; Ed: EJ Murray, 1991, The Human Press Inc., Clinton,
NJ). It is preferred to use a recombinant adenovirus defective for replication and expressing in particular a gene of therapeutic interest.

Advantageously, the adenoviral genome is modified by deletion or mutation of sequences essential for replication and, in particular, included in the regions E1, E2, E4 and / or L1-L5 (see for example the international application
WO 94/28152).

 According to a first variant, the subject of the present invention is the use of a polypeptide comprising an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 1 starting with the alanine residue in position 1 and ending with the isoleucine residue in position 24.

 Advantageously, a polypeptide in use in the context of the present invention comprises an amino acid sequence homologous or identical to all or part of an antigen of the major histocompatibility complex of class I (MHC-I) and, preferably , of the latter's heavy chain.

 All the cells of an organism present on their membrane molecules called histocompatibility antigens which define each individual.

The corresponding genes, more than a dozen, are located on chromosome 6 in humans and exhibit significant polymorphism, which makes it possible to ensure great variability of these identity markers. There are two different categories of these histocompatibility antigens, respectively Class I and II, whose structure and functions are distinct. Class I molecules, called HLA (for Human Leukocyte Antigen in English) intervene to present the antigenic peptides on the cell surface and play an essential role in the antiviral immune responses exerted by cytotoxic T lymphocytes.

 The MHC-I molecules are heterodimers composed of a non-MHC light chain designated p2-microglobulin (P2m) and a heavy chain coded by the MHC genes, linked noncovalently. The heavy chain is a membrane protein whose N-terminal part is oriented outside the cell while the C-terminal part is cytoplasmic. The first comprises 3 domains designated a 1, a2 and a3 of approximately 90 amino acids each. It is followed by a transmembrane region of about 25 amino acids and then by the C-terminal region of around thirty amino acids. Most of the variations between the products of the different alleles are localized in the domains ai and a2, the domain a3 being relatively conserved and the ss2m invariable (for a review and the comparison of sequence between the members of MHC-I, see Bjorkman and Parham , 1990, Annu.

Rev. Biochem. 59, 253-288).

 Among the polypeptides suitable for the purposes of the present invention, there may be mentioned more particularly the HLA antigens A, B, C, D, E and F or polypeptides derived therefrom.

In a particularly advantageous manner, the polypeptide used in the context of the present invention comprises a sequence homologous or identical to all or part of the C-terminal region of the a2 domain of the heavy chain of
MHC-I and, more particularly, to the part centered on the tryptophan residue in position 167, in particular that extending from residues 156 to 180 (SEQ ID NO: 1).

The numbering referred to is consistent with that used for example in Bjorkman and Parham (1990, supra).

According to another variant, a polypeptide in use in the context of the present invention comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown - in SEQ ID NO: 2 starting with the residue asparagine in position 1
and ending with the asparagine residue in position 26, - in SEQ ID NO: 3 starting with the valine residue in position 1 and
ending with the asparagine residue in position 25, - in SEQ ID NO: 4 starting with the serine residue in position 1 and
ending with the arginine residue in position 25, and / or - in SEQ ID NO: 5 starting with the asparagine residue in position 1
and ending with the serine residue in position 25.

 A preferred polypeptide comprises an amino acid sequence homologous or identical to fibronectin and, in particular, to at least one of its type III modules and, in particular, to modules FNIII 1, 4, 5 and / or 14. Good heard, he can understand several. One can also consider the use of human fibronectin or a peptide derived therefrom, for example by mutation or fragmentation. For information, fibronectin encoded by a single gene is a molecule involved in the phenomena of cell adhesion and contact.

Its sequence and characteristics are described in the literature accessible to those skilled in the art (see in particular Bork and Doolittle, 1992, Proc. Natl. Acad.

Sci. USA 89, 8990-8994 and Dickinson et al., 1994, 236, 1079-1092). It is made up of 14 so-called type III modules (numbered from 1 to 14), the primary sequence of which may vary, but whose conformation in sheet ss is preserved.

 According to a particularly advantageous embodiment, a polypeptide as defined above is more particularly intended to allow or facilitate the attachment of an adenovirus of serotype C to a host cell and / or its entry therein. Among the possible adenoviruses, mention may be made more particularly of serotypes 2 and 5.

 The present invention also relates to a host cell capable of expressing a polypeptide in use in the context of the present invention and its use to allow or facilitate the attachment of an adenovirus to its surface and / or the entry of said adenovirus. Various types of host cells can be considered. They can be cells of any origin, for example microorganisms, yeasts, insects, plants, animals. We prefer in particular a mammalian cell and, in particular, a human cell of primary type, tumor or from a line cultivable in vitro. It can have a hematopoietic origin (totipotent stem cell, leukocyte, lymphocyte, monocyte, macrophage ...), hepatic, renal, central nervous system, fibroblast, epithelial, pulmonary or muscular (myocyte, myoblast, satellite cell, cardiomyocyte .. .). A particularly preferred cell is or is derived from line 293 established from embryonic kidney cells by integration of the El adenoviral region (Graham et al., 1977, J. Gen. Virol. 36, 59-72). It is indicated that the expression of one or more polypeptides in use in the context of the present invention on the surface of a host cell not usually expressing MHC-I and / or fibronectin should allow its infectivity by an adenovirus . It could be used as a new cell producing adenoviral vectors.

One can also consider the case of an overexpression in a cell naturally expressing said polypeptide. An overexpression line derived from line 293 should make it possible to improve the production yields of an adenovirus of interest. Of course, the polypeptide used in the context of the present invention can be associated with the cell by chemical means or by means of a ligand recognizing a cell surface protein. However, one can also envisage expression by recombinant DNA techniques. Such an embodiment is within the reach of those skilled in the art. As an indication, the nucleotide sequence coding for the polypeptide in question can be isolated (by standard PCR or cloning techniques) or chemically synthesized before being inserted into a conventional expression vector under the control of appropriate regulatory elements , the vector being introduced into the host cell by any technique of the art. The host cell used in the context of the present invention can also be modified so as to complement a defective adenovirus by transfection of appropriate fragment (s) of adenoviral genome.

 The present invention also relates to the use of a ligand capable of influencing the attachment of an adenovirus to a host cell and / or its entry within the latter mediated by a polypeptide as defined above. The ligand used in the invention can be of any nature. Mention may be made, for example, of peptides, hormones, antibodies or their derivatives and, in particular, single chain antibodies of scFv type (for variable monkey chain fragment in English) and soluble receptors lacking their transmembrane region. In particular, such a ligand can be derived from a polypeptide used in the present invention. In accordance with the aims pursued by the present invention, the ligand can have a negative (antagonist) or positive (agonist) influence. Preferably, a preferred ligand has a dissociation constant with respect to the adenovirus of between 0.01 and 100 nM, advantageously between 0.1 and 50 nM and, most preferably, between 0.5 and 10 nM.

 In the case of an antagonist, the interaction of the ligand with the fiber will make it possible to reduce or inhibit the processes of attachment and / or entry of an adenovirus.

In this context, a particularly preferred ligand is based on a polypeptide as defined in SEQ ID NO: 1. By way of example, mention may be made of a polypeptide comprising an amino acid sequence homologous or identical to at least 6 amino acids Continues included in the sequence as shown in SEQ ID NO: 6 starting with the arginine residue in position 1 and ending with the arginine residue in position 20. It will be preferred to use the peptide designated MH20 in the examples which follow.

 In the case of a positive influence, the ligand used in the context of the present invention is used to allow or stimulate the attachment and / or entry of adenoviruses. A ligand suitable for the purposes of the invention comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 7 starting with the arginine residue in position 1 and ending with the serine residue at position 20. A preferred example consists of the peptide designated below FN20.

 The present invention also relates to a ligand comprising an amino acid sequence which is homogeneous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 6 or 7.

 However, it can also be a ligand of adenoviral origin. According to this embodiment, a preferred ligand is derived from the fiber of an adenovirus, in particular, from the part of the head interacting with the abovementioned polypeptides. A peptide motif chosen in this region should therefore influence the infectivity of adenoviruses with respect to a host cell expressing the polypeptide.

Advantageously, use is made of a ligand covering residues 438 to 486 of the fiber of an adenovirus. More particularly, a ligand of a polypeptide as defined by SEQ ID NO: I preferably derives from an Ad5 and comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 8, starting with the amino acid leucine in position 1 and ending with the amino acid aspartic acid in position 18. A ligand also conceivable can derive from the fiber of a serotype 2 adenovirus and include a amino acid sequence homologous or identical to at least 6 amino acids of the sequence as shown in SEQ ID NO: 9 starting at the threonine residue in position 1 and ending at the valine residue in position 16.

The ligand of a polypeptide as defined by SEQ ID NO: 2 to 5 is more particularly characterized by an amino acid sequence homologous or identical to at least 6 amino acids of the sequence as shown in the
SEQ ID NO: 10 starting at the leucine residue in position 1 and ending at the threonine residue in position 14 (Ad5) or in SEQ ID NO: 11 starting at the asparagine residue in position 1 and ending at the asparagine residue in position 13 (Ad2 ).

 The present invention also relates to the use of a ligand according to the invention, for the preparation of a medicament intended to inhibit or reduce an infection by an adenovirus. In this context, the use of an antagonist ligand will be preferred for a therapeutic or prophylactic purpose. The use of a ligand according to the invention, preferably an agonist, is suitable for the preparation of a medicament intended to promote or facilitate infection by an adenovirus, and, in particular, of a recombinant adenovirus carrying a gene therapeutic for gene therapy (curative) or anti-viral (AIDS) or anti-cancer. Such a drug finds its utility for example in combination with gene therapy treatments in order to improve viral infection in a patient treated with a recombinant adenovirus. One can consider a parenteral, oral or aerosol route of administration. The administration can take place in single dose or repeated one or more times after a certain interval of interval. The appropriate dosage and formulation vary according to different parameters, for example the individual, the disease to be treated, the desired effect, the route of administration or the adenovirus in question.

The present invention also relates to a method for selecting or identifying a cellular receptor for a virus in an appropriate sample, comprising (a) immobilization on an inert support of a reagent of viral origin
comprising all or part of a surface protein of said determining virus
its attachment to the cellular receptor, (b) incubation for a determined time with the sample, (c) elution of the sample retained in step (b) with all or part of an antibody
directed against said reagent of viral origin, and (d) analysis of the sample eluted in step (c).

The inert support can be without limitation in any form (cone, tube, well, beads or the like) and of any material (natural, synthetic such as polymers, chemically modified or not ...). The fixing of the reagent on the inert support can be carried out directly or indirectly. Directly, the procedure is preferably by adsorption, that is to say in a non-covalent manner although the establishment of covalent bonds can also be considered. Indirectly, an anti-reactive compound capable of interacting with the reagent can be fixed beforehand so as to immobilize the assembly on the inert support. According to an advantageous embodiment, the sample consists of a library called random and, in particular of expression (genomic fragments, cDNA) or peptide or, preferably, phages expressing peptide motifs (phagotopes). Such libraries are described in the literature or commercially accessible. In order to select or identify a cellular receptor for an adenovirus, all or part of the fiber and, in particular, of the head of an adenovirus and, preferably, as reagent of viral origin. as eluent, a neutralizing anti-fiber antibody (inhibitor of the attachment of the virus to the surface of the host cell). The fiber or its fragments can be produced recombinantly and the antibodies by the hybridoma technique or by genetic engineering (production of single chain antibodies scFv,
Fab ...). Most anti-fiber antibodies are said to be neutralizing.

The analysis is carried out by comparison of the sequence of the eluted sample with the databases. Such an analysis is within the reach of those skilled in the art.

Finally, the present invention also relates to a method for selecting or identifying the part of a viral protein determining the attachment of a virus to a cellular receptor in an appropriate sample, comprising (a) immobilization on an inert support of any or part of a directed antibody
against said viral protein, (b) incubation for a determined time with said sample, (c) elution of the sample retained in step (b) with a reagent of viral origin
comprising all or part of said viral protein, and (d) analyzing the sample eluted in step (c).

 The specific embodiments mentioned above can also be applied in this context.

 Figure 1 shows the phagotopes obtained after bio-mulching using the antibody 1D6.3 (a) or 7A2.7 (b) as a ligand. The peptide motifs of the phagotopes are aligned with respect to the sequence of the Ad5 head (the methionine residue initiating the fiber representing +1. The regions forming P-sheet structures (Xia et al., 1994, supra) are underlined and indicated by (D), (E) and (F). Identical or conserved residues in the sequences are indicated in bold.

Figure 2 shows the phagotopes obtained after bio-blasting using (a)
The antibody 7A2.7 or (c) 1D6.3 as eluent, (b) and (d) the consensus sequences determined from phagotopes (a) and (c) respectively as well as the homologous sequences found by analysis of the SWISS database
PROT. Residues stored at similar positions are shown in bold.

 Figure 3 illustrates the expression of the lucifer growing gene of peptide (a) FN20 (0 to 500, 1M) or (b) MH20 (0 to 50 ptM). The controls correspond to the incubation of the peptides after the attachment of Ad5Luc3 () or after endocytosis (o).

Figure 4 illustrates the expression of the luciferase gene in Daudi cells
HLA- () or Daudi-HLA + (O) infected with increasing concentrations of Ad5Luc3 (0.3 to 150 fia / 105 cells). The AdSLuc3 is brought into contact with the cells precooled at 0 C for 1 h to allow viral attachment but not entry. The luciferase activity is evaluated after 18 h of culture at 37 "C. The values
RLU represent the average of three separate experiments.

EXAMPLES
HeLa cells (ATCC CCL2) are cultured in monolayers according to the techniques of the art. Preferably a DMEM medium (Dulbecco's
Modified Eagle's Medium; Gibco) containing 10% heat-inactivated fetal calf serum (FCS), L-glutamine and usual antibiotics. Cells
Daudi HLA- (ATCC CCL213) and HLA + (Quillet et al., 1988, J. Immunol. 141, 17-20) are maintained in RPMI 1640 medium (Gibco) supplemented with 15% FCS.

Wild-type Ad5 and recombinant Ad5Luc3 are propagated in cells
HeLa by standard techniques. As an indication, Ad5Luc3 is an adenovirus competent for replication which contains the luciferase gene placed under the control of the early promoter of the SV40 virus (Simian virus 40) inserted in the E3 region of the adenoviral genome (Mittal et al., 1993, Virus Research 28, 67-90).

EXAMPLE 1: Productlon of monoclonal antlcoms (mAb canable to inhlber
attachment of Ad5 to permissive cells
The murine mAbs 1D6.3 and 7A2.7 were generated by conventional techniques by injecting the head of the Ad5 fiber produced in the bacteria by the recombinant route (Henry et al., 1994, J. Virol. 68, 5239- 5246) to Balb / C mice. The fusion and obtaining of hybridoma clones are conventional techniques within the reach of those skilled in the art. The secreting clones are selected by their recognition of the antigen which served for immunization in ELISA. They are said to have neutralizing activity against virions (Michael et al., In preparation).

Sero-reactivity of monoclonal antibodies 1D6.3 and 7A2. 7.

 The reactivity of the antibodies is tested with regard to the fiber head domain of 3 different serotypes (Ad2, Ad5 and Ad3) prepared by recombinant route.

The corresponding sequences are isolated by PCR (Polymerase Chain Reaction) from viral genomic DNA and then introduced into the AcNPV virus (Autographa californica Nuclear Polyhedrosis Virus) under the control of the polyhedrine promoter (Luckow and Summer, 1989, Virology 170, 31- 39). Recombinant proteins are expressed in Sf9 insect cells (Spodoptera frugiperda). The general technology is detailed in Karayan et al.

(1994, Virology 202, 782-796) and Novelli and Boulanger (1991, Virology 185, 365376). More specifically, the Ad5 sequences carrying the last repeated motif of the stem followed by the head of the fiber, are cloned using the primers represented in SEQ
ID NO: 12 and 13. The sense primer corresponds to nucleotides 32164 to 32205 of the Ad5 genome (Chroboczek and Jacrot, 1987, Virology 161, 549-554), includes 4 mismatch so as to create a BamHI site and to replace threonine at position 388 of the native fiber by an initiating ATG codon. The antisense primer corresponds to nucleotides 32919 to 32883 of the Ad5 genome and makes it possible to create a KpnI site to facilitate the subsequent cloning steps. The recombinant protein harvested from the supernatants of Sf9 cells is designated F5-AT386. The Ad2 head is produced from the baculovirus vector described in Louis et al. (1994, J. Virol. 68, 4104-4106). The expression product designated F2-AT388 begins at position 388 (by replacing IAla of the native sequence with a Met) and carries, in addition to the domain of the head, the last repeated motif of the stem. Finally, for the corresponding Ad3 sequences, a sense primer (SEQ ID NO: 14) is used which is designed to introduce an NcoI cloning site and to replace the Asn and Ser codons at position 124 and 125 with Met and Ala codons respectively. . The antisense primer (SEQ ID NO: 15) introduces a KpnI site. The expression product is designated F3
AT124.

 The wells of an ELISA plate are covered with the recombinant protein F5-AT386, F2-AT388 or F3-AT124 on which the mAb 1D6.3 or 7A2.7 is reacted and then a labeled anti-mouse antibody (for example with phosphatase or peroxidase). A positive reaction is observed with regard to the recombinant protein F5-AT386 native. No reaction is detected in the wells containing the F5-AT386 protein denatured in SDS or those containing the native or denatured products F2-AT388 and F3-AT124. These data suggest that these antibodies recognize a conformational epitope specific for serotype C.

Inhibitory effect of monoclonal antibodies in6.3 and 7A2.7 on the cellular attachment of Ad5.

A microlocation test on HeLa cells in culture is carried out using Ad5 virions labeled with valine [14C] (specific activity from 2200 to 2500 cpm / 108 virions) followed by in situ autoradiography (Silver and Anderson, 1988, Virology 165, 377-387). To do this, the cells in the semi-confluent state are placed in the presence of a constant quantity of radioactive virions (103 cpm for 5 × cells) at a multiplicity of infection (MOI) of 1000 virions per cell for 1 h at 0 ° C. presence of mAb 1D6.3 or 7A2.7 (dilutions 1:10, 1: 8, 1: 4 and 1: 2 of the supernatants of respective hybridomas whose mAb concentration is estimated at 0.1-0.2 llg / ml, which corresponds to an excess in mAb compared to the virions present in the inoculum of 100, 250, 500 and 1000 respectively). The cells are then washed in the presence of PBS, fixed with 0.1% of paraformaldehyde in PBS, dried and covered with the K4 emulsion in the form of a gel (Ilford
Nuclear Research). After a week-long exposure and development (development agent D19B, Kodak), the samples are stained briefly with 0.5% toluidine blue taken up in H2O and examined under a microscope. The density of the reduced silver grains around the contour of the cells is representative of the number of [14C] virions bound to the cell surface.

 In the absence of anti-head mAb or at a low concentration (1:10 dilution), a dark halo of reduced silver grains is visible around the cells indicating adsorption of the virions on their surface. A reduction in the halo depending on the mAb concentration is observed for the 1: 8, 1: 4 and 1: 2 dilutions. These results reflect a blockage of the binding of the adenoviral head to the primary cellular receptor due to mAb 1D6.3 and 7A2.7 directed against this part of the fiber. A comparison of the surface of the halos for the same dilutions shows that the antibody 1D6.3 is more inhibitor of the attachment of Ad5 to the cellular HeLa receptor than mAb 7A2.7.

 EXAMPLE 2. Identification of the epitopes of mAbs 1D6 and 7A2.7.

Since the fiber epitopes are presumed to be conformational (Fender et al., 1995, Virology 214, 110-117), the conventional method of identifying epitopes by scanning peptides is not appropriate. We proceed according to a biofeeding technique derived from those described by Smith and Scott (1993, Methods
Enzymol. 217, 228-257) and Hong and Boulanger (1995, EMBO J. 14, 4714-4727).

In this case, mAb 1D6.3 or 7A2.7 is adsorbed overnight at 40C on a microtiter plate (Nunc Immunomodule MaxiSorp F8) at a concentration of I ugluits in 0.1 M sodium carbonate buffer pH9.6. The immobilized antibodies are brought into contact with a phage library expressing hexapeptides (phages fUSE5; Scott and Smith, 1990, Science 249, 386-390). In a second step, the phages retained are eluted either by a conventional acid elution buffer or, more selectively, by competition in the presence of an excess of recombinant protein F5-AT386. The hexapeptide motifs (phagotopes) carried by the eluted phages are determined by sequencing of the protein plil fUSES by the method of Sanger et al. (1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467).

Sequence homologies with phagotopes are sought in the Swiss Prot database and the FASTA 1.6 program (Pearson and Lipman, 1988,
Proc. Natl. Acad. Sci. USA 85, 2444-2448) and the sequence alignments carried out using the W (1; 4) version of the Clustal program (Higgins and Sharp, 1988, Gene 73, 237-244).

 As shown in Figure 1, mAb 1D6.3 retains different phagotopes whose sequences overlap, and are homologous to the head of AdS extending between the residues Val at position 438 and Asp at position 462. Despite a certain degree of degeneration and dispersion, it was possible to determine a central motif of sequence LAPISGTVQSAHLIIRFD corresponding to the amino acids at positions 445 to 462 (SEQ ID NO: 8). This motif is centered on the His residue at position 456, which corroborates the presence of histidine in several independent phagotopes.

In addition, three phagotopes contain histidine close to a serine (GISHTG and GASHTV) and a homologous sequence is found in the head (453QSAHLI458).

On the N-terminal side, the LAPIS sequence is represented in several phagotopes in the form L-P, VAP-S and LIPFNS.

 Sequence analyzes of the 15 phagotopes retained by mAb 7A2.7, demonstrated the presence of a proline 4 times, tryptophan and histidine 8 times and the association in the same phagotope of two aromatic residues is found 5 time. It therefore seems that the epitope of mAb 7A2.7 contains a proline residue, such as that at position 475 of the fiber, near a group of aromatic residues, such as 477YWNF480. In addition, two phagotopes FWLAVR and WALFRS are homologous to the motif 477YWNFR48 '. On the basis of these data, the epitope of mAb 7A2.7 was mapped between residues 473 and 486 of the Ad5 fiber (SEQ ID NO: 10).

In summary, mAbs 1D6.3 and 7A2.7 recognize adjacent segments of 15-20 aa in the linear sequence of the AdS head, the residues extending from positions 445 to 462 and 473 to 486 respectively. According to the three-dimensional model of the head proposed by Xia et al. (1994, Curr. Biol. Structure 2, 1259-1270), the two epitopes occupy continuous regions from a spatial point of view. The mAb epitope 1D6.3 covers part of the CD loop and the sheet p D, while
The epitope of mAb 7A2.7 is located at the level of the adjacent segment DE and of the two sheets p E and F. The epitope 1D6.3 is located inside a sheet R while the epitope 7A2.7 is oriented more laterally with respect to sheet R.

EXAMPLE F.3: Identification of cellular Ad5 receptors by the
reverse bio-paillage technique.

 This technique is said to be reverse compared to the previous one since fiber is used as a ligand and mAb as an eluent. We therefore proceed by immobilization of the head of the Ad5 fiber on which we react the phage library expressing hexapeptide phagotopes. The adsorbed phages can be eluted either by a conventional acid buffer or by mAb 1D6.3 or 7A2.7 and the recombinant pIII protein carrying their respective phagotopes is sequenced. The search in the databases is carried out as above. The hexapeptide patterns identified are shown in Figure 2.

 The phagotopes produced by competition with mAb 7A2.7 are shown in Figure 2a. Their analysis makes it possible to derive a consensus sequence (FIG. 2b) which exhibits homology with motifs 1, 3, 5 and 14 of the type III module of human fibronectin (SEQ ID NO: 2 to 5) (Main et al., 1992, Cell 71, 671-678).

The latter are situated at the level of the sheet p B and of the adjacent loop BC of the module FNIII (Dickinson et al., 1994, J. Mol. Biol. 236, 1079-1092).

 The phagotopes eluted after the action of mAb 1D6.3 are described in Figure 2c.

All the sequences overlap and also make it possible to determine a consensus sequence (Figure 2d). The search for homology with the sequences listed in the databases reveals a homology with the Cterminal region of the a-2 domain of the heavy chain of MHC class I molecules (MHC-Ia-2) (positions 156 to 180) ( SEQ ID NO: 1).

FXFMPI: Interactions of the adenoviral fiber with the FNTTT module and
the MHC-Ia-2 domain
The interaction is studied in vitro using a chimeric protein resulting from the C-terminal fusion of the protein GST (Glutathione S-transferase) with a pentadecapeptide RHILWTPANTPAMGY reproducing the consensus sequence homologous to the sheet p B and the loop BC of FNIII (see previous example and
Figure 2b). To do this, the oligonucleotides presented to the sequence identifiers 16 and 17 are hybridized and introduced into the XhoI site of the plasmid pGEX-KG (Guan and Dixon, 1991, Anal. Biochem. 192, 262-267). It should be noted that the oligonucleotides once rehybridized generate an XhoI site at the 5 ′ end if the insert is cloned in the correct orientation. This makes it possible to integrate a single copy or multiple copies in tandem at the level of the reconstructed XhoI site. The sequence of the fusion product comprising a copy of the pentadecapeptide (designated GST FNxl) can be schematized as follows: GST- (thrombin cleavage site-PGIS-GGGGG-ILDSMGRLE-RHILWTPANTPAMGY (V) -ELKLNS stop. GST-FNx2 and GST-FNx3 have 2 and 3 repeats of the pentadecapeptide respectively between the LE and EL residues of the cloning cassette.

The sense and antisense oligonucleotides (SEQ ID NO: 18 and 19) coding for the consensus sequence obtained by competition with mAb 1D6.3 (Figure 2d) are inserted according to the same strategy as above at the C-terminal end of the
GST to give the constructions GST-MHCx 1, GST-MHCx2 and GST-MHCx3 according to the number of motifs present. The chimeric proteins GST-FN and GST-MHC are produced in E. coli, extracted and affinity-purified on agarose-glutathione beads (Sigma) according to conventional methods (Smith and
Johnson, 1988, Gene 67, 31-40).

 In parallel, the complete fibers of serotypes 2, 3 and 5 are produced recombinantly according to the baculovirus / Sf9 cell technology already used.

The F2-FL582 construction carrying the Ad2 fiber gene is described in Novelli and Boulanger (1991, Virology 185, 365-376). Fiber coding sequences
Ad5 and Ad3 are isolated by PCR using viral DNA as a template and appropriate sense and antisense primers such as those reported in SEQ ID
NO: 20 and 13 and 21 and 15. The amplified segment is introduced into a baculovirus vector under the control of the polyhedrin promoter and the expression product recovered in the culture supernatants. F5-FL581 and F3-FL320 are obtained corresponding to the fibers Ad5 and Ad3 respectively.

The capacity of the FNIII module and of the recombinant α-2 MHC-I domain to bind the adenoviral fiber is evaluated by immunoblotting after in vitro incubation of the fusion proteins GST-FN and GST-MHC and of the recombinant fibers F2-FL582,
F5-FL581 and F3-FL320. The complexes formed are isolated on glutathione agarose beads under the conditions described by Johnson et al. (1995, J. Biol. Chem.

270, 24352-24360) then analyzed on 12.5% polyacrylamide gel (SDS
PAGE) using a discontinuous buffer system (Laemmli, 1970, Nature 227, 680-685). The proteins are transferred onto a nitrocellulose membrane (0.8 mA / cm 2; Cambridge Electrophoresis system, UK) in a 25 mM Tris-HCl buffer, 192 mM glycine, pH 8.3 containing 20% methanol. After treatment with a blocking solution (skim milk 5%, calf serum 1% in buffer
TBS-T: 20 mM Tris-HCI pH 7.8, 0.15 M NaCl, 0.05% Tween 20), the membrane is brought into contact with the antibody 4D2.5 (Hong and Engler, 1991, Virology 185, 758-767) then an anti IgG conjugate labeled with horseradish peroxidase. Revelation by the chemiluminescent substrate peroxidase (SuperSignal, Pierce
Chemicals) is carried out according to Carrière et al. (1995, J. Virol. 69, 2366-2377) and the luminograms (Hyperfilm P-max, Amersham) are analyzed at 610 nm using an automatic densitometer (REP-EDC, Helena Laboratories, Beaumont, TX) .

As an indication, the antibody 4D2.5 recognizes the FNPVYP epitope of the tail domain conserved in most mammalian adenoviruses.

GST-FNxl, GST-FNx2 and GST-FNx3 bind to F5-AT386 and F2-AT388 with great efficiency while F3-AT124 is retained at a lower level. Similar behavior is observed with the chimeric proteins GST-MHCxl,
GST-MHCx2 and GST-MHCx3 which retain F5-AT386 and F2-AT388 with an efficiency greater than F3-AT124.

The Ad5 fiber binds to the GST-FN and GST-MHC fusion proteins and this with an affinity 2 to 3 times greater compared to the Ad2 fiber and 10 to 15 times greater compared to the Ad3 fiber. Furthermore, the binding efficiency is not dependent on the number of motifs present in the fusion protein, the intensity being comparable and even sometimes lower between GST-FNxl and GST-FNx3 and
GST-MHCxl and GST-MHCx3. This can be explained by the fact that the tandem patterns can adopt a conformation which harms the bond.

EXEMPLUTED. influence of synthetic peptides derived from FNIII and
MHC-1a2 on attachment of viruses to the cell surface.

Two synthetic peptides reproducing the motifs FNIII and MHC-Ia2 were chemically synthesized and purified according to the techniques of the art. FN20 (SEQ
ID NO: 7) reproduces the consensus sequence of phagotopes eluted by the antibody 7A2.7 and MH20 (SEQ ID NO: 6) corresponds to that of phagotopes eluted by mAb 1D6.3.

The FN20 and MH20 peptides are tested for the attachment of the reporter adenovirus AdSLuc3 to HeLa cells cultured in vitro. The test is carried out in part at 0 C, a temperature which allows the attachment of viruses to the surface of permissive cells but, on the other hand, blocks the entry of viruses and the recycling of receptors. The AdSLuc3 (ME 0.16 pfu / 105 cells) is previously incubated with increasing amounts of peptides (0.01 to 500 nM) at room temperature for 2 hours then the mixture is added to a cell culture placed on ice. After 1 h at 0 C, the non-adsorbed viruses and the peptides are eliminated by washing and the culture is continued for 18 h at 37 "C after addition of a preheated medium. The cell lysates are prepared in a conventional manner and the activity luciferase expressed in RLU (for Relative Light
Units in English) is determined (Promega, Madison, Wl substrate; luminometer
Lumat LB-9501, Brethold Bioanalytical, Wildbad, Germany).

 The results of competition with the FN20 peptide are presented in Figure 3a. No significant effect is obtained up to a molarity of 10 MIC, then a gradual increase in luciferase activity appears beyond 25 MIC. In particular, the activity increases by a factor of 100 between 25 and 100 μM. The FN20 peptide therefore has a stimulating effect on viral attachment. It does not confer any apparent cytotoxicity and has no negative effect on the expression of the luciferase gene once the virus is pre-attached (peptide added to cell culture after the viral attachment step at 0 C) or pre-endocytosed (peptide added to cell culture after the virus attachment and penetration step).

 In the case of the MH20 peptide (FIG. 3b), a slight increase in the expression of the luciferase gene is observed for the molarities of between 0.05 and 2.5 μM (activity 5 to 6 times higher at 2.5; M). This phenomenon is followed by a rapid decrease in luciferase levels when the molarities used are greater than S 1M, with a decrease of a factor of 100 compared to the control at 25 pM and by almost four orders of magnitude at 50 I1M, showing that linked to the virus, it blocks attachment to the cellular receptor. As before, the peptide MH20 at concentrations of 50 M does not exhibit any cytotoxic effect and does not influence the expression of the reporter gene after the pre-attachment or pre-endocytosis of Ad5Luc3. The almost total inhibition of luciferase activity in the presence of 50 HM of MH20 is the reflection of a complete neutralization of the virus.

 EXAMPLE 6. Sero-specificity of viral neutralization by synthetic peptides.

 The wild adenoviruses Ad5, Ad2 Ad3 are preincubated for 2 h at room temperature with the inhibiting peptide MH20 at a constant molarity (25 pM), the MOI varying from 0.2 to 2 pfu / cell. The mixture is placed in the presence of the HeLa cells for 1 h at 0 ° C. and the culture continued at 370 ° C. after removal of the non-adsorbed viruses under the same conditions as those described above. The level of synthesis of the hexon, of the 100k protein, of the penton base and of the fiber is estimated by immunotitration (Wohlfart, 1988, J. Virol. 62, 2321 2328) on the cell extracts collected 48 h after infection. .

 Infection of HeLa cells with AdS or Ad2 at an MOI of 0.2 to 2 pfu / cell in the presence of 25 .muM MH 2 O during the virus attachment phase, is followed by inhibition of protein synthesis of the hexon viral capsid, protein 100 k, penton base and fiber 48 hours after infection (factor 15 to 30).

On the other hand, when the infection is carried out under the same conditions with wild-type Ad3, the synthesis of structural proteins is reduced only by a factor 1.5 to 2, which suggests that the adenoviral neutralization by MH20 is serotype dependent.

 FXEMPIF7. Affinity of the Ad5 fiber for the peptides FN20 and MH20.

5, 10 and 25 μM of synthetic peptides FN20 or MH20 are immobilized on the polystyrene surface of a 96-well microtiter plate (Nunc,
Maxisorb) overnight at 40C. After washing and then blocking with a solution of bovine serum albumin (BSA) 3% in PBS buffer, increasing concentrations of F5-FL581 fibers taken up in PBS buffer and reacted with radioactivity are reacted (labeling with [35S] methionine and [35S] cysteine specific activity of 50,000 to 65,000 cpm / g of protein). The fiber adsorbed on either peptide is eluted with an adequate solution (1M urea, 1 M NaOH and 1% SDS) and then precipitated in the presence of trichloroacetic acid. The radioactivity contained in the precipitate recovered on a GF / C filter is counted using a liquid scintillation spectrometer (Beckman LS-6500) and the dissociation constants (Kd) determined according to Scatchard (1949, Annls NY
Acad. Sci. 51, 660-672).

 The Kd of the AdS fiber labeled with respect to the peptide MH20 is evaluated at 3.0+ 0.6 nM and that found for the peptide FN20 is 8.0+ 1.9 nM (n = 3 in both cases ).

EXAMPLE 8 The Ad5 infection is dependent on the expression of
MHC-I on the surface of permissive cells
The Daudi line of B lymphoblastoids, established from a lymphoma of
Burkitt, is naturally deficient in the expression of p-2 microglobulin and, therefore, does not have on its surface the HLA class I molecules (Daudi HLA). The Daudi-derived E8.1 cell line was generated by transfection of a gene coding for p-2 microglobulin in order to restore the expression of molecules
HLA class I on their surface (Daudi-HLA +; Quillet et al., 1988, J. Immunol.

141, 17-20).

The attachment experiments of Ad5Luc3 at 0 C were carried out on Daudi HLA- and Daudi-HLA + cells with an MOI of three orders of magnitude higher than that used in the case of HeLa cells (0.3 to 150 pfu / 105 cells). The luciferase activity measured in the cell lysates 18 h postinfection is shown in Figure 4. When the infection concerns the cells
Daudi-HLA +, the luciferase activity increases regularly in a dependent MOI manner until it reaches a plateau beyond 5 pfu / 105 cells. Regarding Daudi HLA- cells, the luminescent signal is very weak (3 to 4 orders of magnitude) compared to that observed with cells provided with molecules.
HLA functional on their surface. These experiments show that the expression of
MHC-I on the cell surface is necessary for AdS infection.

LIST OF SEQUENCES (1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: National Center for Scientific Research
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(ii) TITLE OF THE INVENTION: Cellular receptors for adenoviruses.

(iii) NUMBER OF SEQUENCES: 21
(iv) COMPUTER-READABLE FORM:
(A) TYPE OF SUPPORT: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release # 1.0, Version # 1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: peptide
(iii) HYPOTHETIC: NO
(v) TYPE OF FRAGMENT: internal
(vi) ORIGIN:
(A) ORGANIZATION: Homo sapiens
(C) INDIVIDUAL ISOLATED: alpha 2 domain of MHC-I antigens
(positions 156 to 180)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1:
Leu Arg Ala Tyr Leu Glu Gly Thr Cys Val Glu Trp Leu Arg Arg Tyr
1 5 10 15
Leu Glu Asn Gly Lys Glu Thr Leu Gln
20 25 (2) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 26 amino acids
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(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: peptide
(iii) HYPOTHETIC: NO
(v) TYPE OF FRAGMENT: internal
(vi) ORIGIN:
(A) ORGANISM: homo sapiens
(C) INDIVIDUAL ISOLATED: module type III 1 of the
human fibronectin
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:
Asn Ser His Pro Ile Gln Trp Asn Ala Pro Gln Pro Ser His Ile Ser
1 5 10 15
Lys Tyr Ile Leu Arg Trp Arg Pro Lys Asn
20 25 (2) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(D) CONFIGURATION: linear
(ii) TY (2) INFORMATION FOR SEQ ID NO: 4:
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(A) LENGTH: 25 amino acids
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(ii) TYPE OF MOLECULE: peptide
(iii) HYPOTHETIC: NO
(v) TYPE OF FRAGMENT: internal
(vi) ORIGIN:
(A) ORGANISM: homo sapiens
(C) INDIVIDUAL ISOLATED: type III module 5 of the
human fibronectin
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4:
Ser Thr Val Leu Val Arg Trp Thr Pro Pro Arg Ala Gln Ile Thr Gly
l 5 10 15
Tyr Arg Leu Thr Val Gly Leu Thr Arg
20 25 (2) INFORMATION FOR SEQ ID NO: 5:
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(v) TYPE OF FRAGMENT: internal
(vi) ORIGIN:
(A) ORGANISM: homo sapiens
(C) INDIVIDUAL ISOLATED: type III module 14 of the
human fibronectin
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5:
Asn Ser Leu Leu Val Ser Trp Gln Pro Pro Arg Ala Arg Ile Thr Gly
l 5 10 15
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20 25 (2) INFORMATION FOR SEQ ID NO: 6:
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(vi) ORIGIN:
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6:
Arg Ala Ile Val Gly Phe Arg Val Gln Trp Leu Arg Arg Tyr Phe Val
l 5 10 15
Asn Gly Ser Arg
20 (2) INFORMATION FOR SEQ ID NO: 7:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(vi) ORIGIN:
(A) ORGANISM: synthetic peptide FN20
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
Arg His Ile Leu Trp Thr Pro Ala Asn Thr Pro Ala Met Gly Tyr Leu
1 5 10 15
Ala Arg Val Ser
20 (2) INFORMATION FOR SEQ ID NO: 8:
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(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(C) INDIVIDUAL ISOLATED: serotype 5
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
Leu Ala Pro Ile Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg
1 5 10 15
Phe Asp (2) INFORMATION FOR SEQ ID NO: 9:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(ii) TYPE OF MOLECULE: peptide
(iii) HYPOTHETIC: NO
(v) TYPE OF FRAGMENT: internal
(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(C) INDIVIDUAL ISOLATED: serotype 2
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
Thr Val Ala Ser Val Ser Ile Phe Leu Arg Phe Asp Gln Asn Gly Val
1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 10:
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(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(C) INDIVIDUAL ISOLATED: serotype 5
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10:
Leu Asp Pro Glu Tyr Trp Asn Phe Arg Asn Gly Asp Leu Thr
1 5 10 (2) INFORMATION FOR SEQ ID NO: 11:
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(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(B) STRAIN: serotype 2
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11:
Asn Ser Ser Leu Lys Lys His Tyr Trp Asn Phe Arg Asn
1 5 10 (2) INFORMATION FOR SEQ ID NO: 12:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(B) STRAIN: serotype 5
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide (nt32164
32205 Ad5)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12:
CCTAAACTAG GATCCGGCCT TAGTTTTGAC AGCATGGGTG CC 42 (2) INFORMATION FOR SEQ ID NO: 13:
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(iii) ANTI-SENSE: YES
(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(B) STRAIN: serotype 5
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide (nt
32919 32883 Ad5)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13:
CTGTGAGTTT GATTAAGGTA CCGTGATCTG TATAAGC 37 (2) INFORMATION FOR SEQ ID NO: 14:
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(A) ORGANISM: Mastadenovirus
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(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14:
GGTCTTACAT TTGACTCTTC CATGGCTATT GCACTG 36 (2) INFORMATION FOR SEQ ID NO: 15:
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(iii) ANTI-SENSE: YES
(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(B) STRAIN: serotype 3
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide (Ad3)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15:
CAATAAAAAA TGTGGTACCT TATTTTTGTT GTCAG 35 (2) INFORMATION FOR SEQ ID NO: 16:
(i) CHARACTERISTICS OF THE SEQUENCE:
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(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomics)
(iii) HYPOTHETIC: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: homo sapiens
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide
(FNIII consensus sequence)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16:
TCGAGAGGCA TATACTTTGG ACTCCTGCTA ATACACCGGC AATGGGGTAT G 51 (2) INFORMATION FOR SEQ ID NO: 17:
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(iii) HYPOTHETIC: NO
(iii) ANTI-SENSE: YES
(vi) ORIGIN:
(A) ORGANISM: homo sapiens
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide
(FNIII consensus sequence)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17:
TCGACATACC CCATTGCCGG TGTATTAGCA GGAGTCCAAA GTATATGCCT C 51
(2) INFORMATION FOR SEQ ID NO: 18:
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(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: homo sapiens
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide encoding
for the consensus sequence MHC-I
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: TCGAGAGGGC TATAGTTGGG TTTAGGGTGC AATGGCTTAG GCGGTATTTT GTGAATGGGT 60
CGAGG 65
(2) INFORMATION FOR SEQ ID NO: 19:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 65 base pairs
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(D) CONFIGURATION: linear
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(iii) ANTI-SENSE: YES
(vi) ORIGIN:
(A) ORGANISM: homo sapiens
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide encoding
for aa 157-176 of MHC-I
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19:
TCGACCTCGA CCCATTCACA AAATACCGCC TAAGCCATTG CACCCTAAAC CCAACTATAG 60
CCCTC 65
(2) INFORMATION FOR SEQ ID NO: 20:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomics)
(iii) HYPOTHETIC: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(B) STRAIN: serotype 5
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide (nt
31021-31050 Ad5)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20:
CCATCCGCAC CCACTATGAT CACGTTGTTG 30
(2) INFORMATION FOR SEQ ID NO: 21:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: DNA (genomics)
(iii) HYPOTHETIC: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: Mastadenovirus
(B) STRAIN: serotype 3
(C) INDIVIDUAL ISOLATED: synthetic oligonucleotide (Ad3)
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21:
CTTCATTTCT TTATCCCCCC ATGGCCA 27

Claims (7)

Claims 1. Use of a polypeptide comprising an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown (a) in SEQ ID NO: 1 starting with the leucine residue in position I and ending with the glutamine residue in position 25, (b) in SEQ ID NO: 2 starting with the asparagine residue in position I and ending with the asparagine residue in position 26, (c) in SEQ ID NO: 3 starting with the residue valine in position 1 and ending with the asparagine residue in position 25, (d) in SEQ ID NO: 4 starting with the serine residue in position 1 and ending with the arginine residue in position 25, and / or (e) in the SEQ ID NO:  5 starting with the asparagine residue in position  1 and ending with the serine residue in position 25  to allow or facilitate the attachment of an adenovirus to a host cell  and / or the entry of said adenovirus into said host cell. 2. Use according to claim 1, characterized in that the polypeptide  includes an amino acid sequence homologous or identical to at least  6 continuous amino acids of the sequence as shown in SEQ ID  NO: 1 starting with the leucine residue in position 1 and ending with the  glutamine residue in position 25. 3. Use according to claim 2, characterized in that the polypeptide  comprises an amino acid sequence homologous or identical to all or  part of a major class I histocompatibility complex antigen  (MHC-I) and, preferably, the heavy chain of said MHC-I. 4. Use according to claim 3, characterized in that the polypeptide  comprises an amino acid sequence homologous or identical to all or  part of the a2 domain of said heavy chain.  5. Use according to claim 3 or 4, characterized in that the polypeptide  comprises an amino acid sequence homologous or identical to all or  part of an HLA antigen A, B, C, D, E or F. 6. Use according to claim 1, characterized in that the polypeptide  includes an amino acid sequence homologous or identical to at least  6 continuous amino acids of the sequence as shown  - in SEQ ID NO: 2 starting with the asparagine residue in position  1 and ending with the asparagine residue in position 26,  - in SEQ ID NO: 3 starting with the valine residue in position 1  and ending with the asparagine residue in position 25,  - in SEQ ID NO: 4 starting with the serine residue in position I and  ending with the arginine residue in position 25, and / or  - in SEQ ID NO: 5 starting with the asparagine residue in position  1 and ending with the serine residue in position 25. 7. Use according to claim 6, characterized in that said polypeptide  comprises an amino acid sequence homologous or identical to all or  part of fibronectin. 8. Use according to claim 7, characterized in that said polypeptide  comprises an amino acid sequence homologous or identical to all or  part of at least one type III fibronectin module. 9. Use according to one of claims 1 to 8, characterized in that said  adenovirus is serotype C. 10. Use according to claim 9, characterized in that said adenovirus is  serotype 2 or 5. 11. Cell capable of expressing a polypeptide as defined according to one of  claims 1 to 10. 12. Cell according to claim 11, characterized in that said cell  overexpressed said polypeptide. 13. Use of a cell according to claim 11 or 12, to allow or  facilitate the attachment of an adenovirus to said cell and / or the entry of said  adenovirus within said cell. 14. Use of a ligand to influence the attachment of an adenovirus to a  host cell and / or the entry of said adenovirus into said mediated host cell  by a polypeptide as defined in one of claims 1 to 10. 15. Use of a ligand according to claim 14, to influence so  negative the attachment of an adenovirus to said host cell and / or the entry of said  adenovirus within said host cell mediated by a polypeptide such as  defined according to one of claims 1 to 5 and 9 and 10. 16. Use of a ligand according to claim 15, characterized in that said  ligand comprises an amino acid sequence homologous or identical to at  minus 6 continuous amino acids included in the sequence as shown  in SEQ ID NO: 6 starting with the arginine residue in position I and  ending with the arginine residue in position 20. 17. Use of a ligand according to claim 14, to influence so  positive the attachment of an adenovirus to said host cell and / or the entry of said  adenovirus within said host cell mediated by a polypeptide such as  defined according to one of claims 1 and 6 to 10. 18. Use of a ligand according to claim 17, characterized in that said  ligand comprises an amino acid sequence homologous or identical to at  minus 6 continuous amino acids included in the sequence as shown  in SEQ ID NO: 7 starting with the arginine residue in position 1 and  ending with the serine residue in position 20. 19. Use of a ligand according to one of claims 14 to 18, characterized in that  that said ligand has a dissociation constant (kd) with respect to said adenovirus  from 0.01 to 100 nM, and in particular from 0.1 to 50 nM. 20. Ligand as defined in claim 16, 18 or 19. 21. Use of a ligand according to claim 14, characterized in that said  ligand is of adenoviral origin. 22. Use according to claim 21, characterized in that said ligand derives  of the fiber of an adenovirus and, in particular, of the head domain. 23. Use according to claim 22 for interacting with a polypeptide such as  defined according to one of claims 1 to 5 and 9 and 10, characterized in that said  ligand is derived from the fiber of a serotype 5 adenovirus and includes a  amino acid sequence homologous or identical to at least 6 amino acids  of the sequence as shown in SEQ ID NO: 8 beginning  with the leucine residue in position I and ending with the aspartic acid residue  in position 18. 24. Use according to claim 22 for interacting with a polypeptide such as  defined according to one of claims 1 to 5 and 9 and 10, characterized in that said  ligand is derived from the fiber of a serotype 2 adenovirus and includes a  amino acid sequence homologous or identical to at least 6 amino acids  of the sequence as shown in SEQ ID NO: 9, starting  with the threonine residue in position 1 and ending with the valine residue in  position 16.. 25. Use according to claim 22 for interacting with a polypeptide such as  defined according to one of claims 1 and 6 to 10, characterized in that said  ligand is derived from the fiber of a serotype 5 adenovirus and includes a  amino acid sequence homologous or identical to at least 6 amino acids  of the sequence as shown in SEQ ID NO: 10 beginning  with the leucine residue in position 1 and ending with the threonine residue in  position 14. 26. Use according to claim 22 for interacting with a polypeptide such as  defined according to one of claims 1 and 6 to 10, characterized in that said  ligand is derived from the fiber of a serotype 2 adenovirus and includes a  amino acid sequence homologous or identical to at least 6 amino acids  of the sequence as shown in SEQ ID NO: 11 beginning  with the asparagine residue in position 1 and ending with the asparagine residue in  position 13. 27. Use of a ligand according to one of claims 14 to 26, for the  preparation of a drug intended to inhibit or reduce infection by a  adenovirus. 28 Use of a ligand according to one of claims 14 to 26, for the  preparation of a drug intended to promote or facilitate infection by  an adenovirus and, in particular, a recombinant adenovirus.