Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-09-30
2002-07-16
Clark, Deborah J. R. (Department: 1633)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S320100, C435S325000
Reexamination Certificate
active
06420120
ABSTRACT:
The subject of the present invention is the use of all or part of an antigen of the class I major histocompatibility complex and/or of a type III module of fibronectin to allow or facilitate the attachment of an adenovirus onto a host cell and/or its entry into the latter. The invention also relates to the use of a ligand capable of modulating the infectivity of an adenovirus toward a host cell, mediated by either of the polypeptides mentioned above. Finally, the invention relates to a biopanning method for identifying or selecting 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 detected in numerous animal species and can infect various cell types. Numerous serotypes have been characterized within each species which exhibit a genomic organization and an infectious cycle which are comparable. In general, the adenoviral genome consists of a double-stranded linear DNA molecule of about 36 kb containing the genes encoding the viral proteins and, at its ends, two inverted repeats (designated ITRs) which are involved in replication and the encapsidation region.
Adenoviruses replicate in the nuclei of the cells infected. The infectious cycle occurs in two stages. The early phase precedes the initiation of replication and makes it possible to produce the early proteins regulating the replication and the transcription of the viral DNA. These stages are followed by the late phase during which the structural proteins which constitute the viral particles are synthesized. The assembling of the new virions takes place in the nucleus. In the first instance, the viral proteins assemble so as to form empty capsids having an icosahedral structure, into which the adenoviral DNA is encapsidated. The viral particles are released and are capable of infecting other permissive cells. In this regard, the fiber and the penton base which are present at the surface of the capsids play a critical role in the cellular attachment of the virions and their internalization.
The adenovirus binds to the surface of permissive cells through the intermediacy of the trimeric fiber and a cellular receptor which has so far not been identified. Next, the particle is internalized by endocytosis through the binding of the penton base to the cellular integrins &agr;
v
&bgr;
3
and &agr;
v
&bgr;
5
(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 Ad2 fiber comprises 580 amino acids (aa) whose sequence is disclosed in Herissé et al. (1981, Nucleic Acid Res. 9, 4023-4042). That of Ad5 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 160-180 kDa molecule, confirming its assembly in the form of a trimer.
The fiber is composed of 3 domains (Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199, 165-200):
(1) At the N-terminus, the “tail”, which is highly conserved from one serotype to another, interacts with the penton base and ensures the anchorage of the molecule in the capsid.
(2) The “stem” is a structure in the form of a rod of variable length depending on the serotypes. For example, the stem of the Ad5 fiber contains 22 repeats of a motif of 15 residues which could adopt a &bgr; sheet conformation. The number of these repeats differs from one serotype to another, which explains the variations in length.
(3) Finally, at the distal end of the stem, the “head” or terminal sphericle is a globular structure containing 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). Most of the experimental data show that it is the head domain which is responsible for the binding to permissive cells (Krasnykh et al., 1996, J. Virol. 70, 6839-6846).
(4) The complexity of the adenoviral attachment suggests that it could be serotype-dependent and that several cellular proteins could participate in it. As regards Ad2, Hong and Boulanger (1995, EMBO J. 14, 4714-4727) have identified a number of peptide motifs found in several cellular surface proteins which are capable of interacting with the capsid proteins (penton base and fiber), in particular the type III 5 and 14 modules of human fibronectin. The authers proceeded by immobilizing, on an inert support, penton base or fiber (ligand) with which they reacted a library of phages expressing random hexapeptides (designated phagotopes). The phages adsorbed, which in theory express phagotopes interacting with a motif carried by the adenoviral protein, are then eluted either conventionally at acidic pH or by competition with the other nonimmobilized capsid partner (eluent). However, the cellular receptor for adenoviruses and the region of the head precisely involved in the binding to the receptor have so far not yet been clearly identified.
A new technique of “biopanning” has now been carried out in which the immobilized ligand consists of the head domain of the Ad5 fiber and the eluent consists of a neutralizing antibody directed against the latter and two classes of phagotopes isolated depending on the antibody used. The first corresponds to a conserved sequence within the &agr;-2 domain of the antigens of the class I major histocompatibility complex (&agr;-2 MHC-I) and the second to a sequence found in the III modules of human fibronectin (FNIII). The data presented in the examples which follow support the hypothesis that the &agr;-2 MHC-I constitutes the primary receptor for the serotype C adenoviruses and confirm the participation of the FNIIIs as coreceptor or cofactor. The regions of these two receptors and of the fiber which interact with each other have also been identified. In addition, an antagonist peptide has been generated which reproduces the motif of the &agr;-2 MHC-I domain which neutralizes the attachment of adenoviruses and an agonist peptide reproducing the FNIII motifs which stimulates attachment.
Accordingly, 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 starting with the leucine residue at position 1 and ending with the glutamine residue at position 25,
(b) in SEQ ID NO: 2 starting with the asparagine residue at position 1 and ending with the asparagine residue at position 26,
(c) in SEQ ID NO: 3 starting with the valine residue at position 1 and ending with the asparagine residue at position 25,
(d) in SEQ ID NO: 4 starting with the serine residue at position 1 and ending with the arginine residue at position 25, and/or
(e) in SEQ ID NO: 5 starting with the asparagine residue at position 1 and ending with the serine residue at position 25; to allow or facilitate the attachment of an adenovirus to a host cell and/or the entry of the said adenovirus into the said host cell.
For the purposes of the present invention, “polypeptide” is understood to mean any molecule consisting of a succession of at least 6, and preferably of at least 8, amino acids. The term polypeptide comprises both peptide molecules of short length (from 6 to a few tens of residues) and molecules which are longer (up to several hundreds of residues), provided, however, the envisaged use is allowed. It is specified that a polypeptide in use within the framework of the present invention may be derived from a native polypeptide as found in nature, in particular in humans, or a portion thereof. It may also be a chimera and comprise additional residues of any origin fused at the N- and/or C-terminus 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 relative to the sequences disclosed in the s
Boulanger Pierre
Hong Saw See
Karayan Lucie
Burns Doane Swecker & Mathis L.L.P.
Centre National de la Recherche Scientifique "CNRS"
Clark Deborah J. R.
Sorbello Eleanor
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