Papillomavirus cellular receptor

Details Papillomavirus cellular receptor Papillomavirus cellular receptor

1999-07-07

2001-06-05

Salimi, Ali R. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S007100, C435S007240, C435S345000, C435S343000, C435S343200, C435S343100, C436S548000

Reexamination Certificate

active

06242176

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to methods and reagents for treating or preventing papillomavirus infection.
BACKGROUND OF THE INVENTION
Papillomavirus are causative agents for several types of epithelial and mucosal diseases. Of particular concern is that certain strains of papillomavirus are associated with cervical and penile cancers (see, e.g., Iwasawa et al., J.
Urol
., 149, 59-63 (1993); Koutsky et al.,
N. Engl. J. Med.,
327, 1272-78 (1992)). Considerable efforts, therefore, are underway to prevent the spread of this virus by developing a prophylactic vaccine and novel treatments for papillomavirus-induced lesions (see, e.g., Cason et al.,
Vaccine,
11, 603-11 (1993); Crawford,
Cancer Surv.,
16, 215-29 (1993)). Such efforts, however, have been hampered by the particular difficulties of working with papillomavirus in vitro. To complete its life cycle, papillomavirus requires its host cell to undergo a differentiation event. Currently, no in vitro culture system duplicates this state adequately to permit efficient papillomavirus growth in vitro.
Papillomaviruses are nonenveloped double-stranded DNA viruses about 55 nm in diameter with an approximately 8-kb genome in a nucleohistone core (Baker et al.,
Biophys J.
60, 1445-56 (1991)). The capsids include two viral proteins (L1 and L2) of about 55 kDa and 75 kDa, respectively (Larson et al.,
J. Virol.,
61, 3596-3601 (1987)). L1 is the major capsid protein, and it is arranged in 72 pentameres within the capsid. In fact, L1 has the ability to self-assemble into virus-like particles (VLPs) upon production of the L1 protein in eukaryotic cells (see, e.g., Hagensee et al.,
J. Virol.,
67, 315-22 (1993); Kirnbauer et al.,
J. Virol.,
67, 6929-36 (1993)). The function and position of L2 within the virion are not clear, although the protein is assembled with L1 into VLPs when coexpressed in cells.
Because of the lack of suitable papillomavirus culture conditions, VLPs typically are used for in vitro studies of papillomavirus infection, as opposed to intact papillomavirus (see, e.g., Roden et al.,
J. Virol.,
68, 7260-66 (1994); Volpers et al.,
J. Virol.,
69, 3258-64 (1995)). Using VLPs, it is now thought that a putative cell receptor for papillomavirus is expressed across a wide range of cell types and is highly conserved between a diverse group of organisms (see, e.g., Müller et al.,
J Virol.,
69, 948-54 (1995); Volpers et al., supra; Roden et al., supra). Currently, little is understood about papillomavirus infection, particularly the identity of a cell surface receptor recognizing papillomavirus.
In view of the foregoing problems, there exists a need for methods and reagents for treating or preventing papillomavirus infection.
BRIEF SUMMARY OF THE INVENTION
The present invention is predicated, at least in part, on the discovery that papillomavirus selectively binds the CD16 protein present in a variety of cells. Using this discovery, the present invention provides methods and reagents for treating or preventing papillomavirus infection. In one aspect, the present invention provides reagents and methods for attenuating the ability of papillomavirus to bind to cells by blocking access of papillomavirus to its cellular receptor. In another aspect, the present invention provides a method of attenuating the ability of papillomavirus to infect cells by reducing the free titer of papillomavirus. In another aspect, the present invention provides a complex comprising a biologically active substance and a ligand that recognizes CD16 and a method of delivering a biologically active substance to an HVP-infected cell using the complex. These and other advantages of our invention, as well as additional inventive features, will be apparent from the following detailed description.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods and reagents for treating or preventing papillomavirus infection. In one aspect, the method attenuates the ability of papillomavirus to bind cells. One method of attenuating the ability of papillomavirus to bind cells is to expose the cells to a ligand that recognizes CD16 under conditions sufficient for the ligand to bind CD16. The bound ligand interferes with subsequent interaction between papillomavirus and the CD16 molecules; thus, by such treatment, the ability of papillomavirus to subsequently bind cells is attenuated.
In this context, the ligand is present on any molecule suitable for blocking the interaction between papillomavirus and the bound CD16 protein. While many types of molecules can provide the ligand, generally the ligand is present as part of a protein. For example, the ligand can be an antibody recognizing an epitope on CD16. In other embodiments, the ligand is on a protein including an external domain of an papillomavirus capsid protein (e.g., L1, L2, or a soluble derivative of L1 or L2 retaining an extracellular domain). The residues that comprise the ligand in a protein need not necessarily be contiguous in the chain of amino acids that make up the protein. In other words, the ligand can be generated by the particular conformation of the protein, e.g., through folding of the protein in such a way as to bring contiguous and/or noncontiguous sequences into mutual proximity. Such proteinatious species can be synthesized using standard direct peptide synthesizing techniques (see, e.g., Bodanszky,
Principles of Peptide Synthesis
(Springer-Verlag, Heidelberg: 1984)), such as via solid-phase synthesis (see, e.g., Merrifield,
J Am. Chem. Soc.,
85, 2149-54 (1963); Barany et al., Int.
J Peptide Protein Res.,
30, 705-739 (1987); and U.S. Pat. No. 5,424,398). Alternatively, such modified proteins can be chemically crosslinked, and a variety of cross-linking agents are known in the art and widely available (e.g., succinimidyl or maleimidyl cross-linkers). Methods for conjugating peptides and polyamines are also well-known in the art (see, e.g., Staros,
Biochem.,
21, 3990 (1982)). Alternatively, a DNA fragment encoding the protein can be subcloned into an appropriate vector using well known molecular genetic techniques. The fragment is then transcribed and the peptide subsequently translated in vitro within a host cell. Any appropriate expression vector (see, e.g., Pouwels et al.,
Cloning Vectors: A Laboratory Manual
(Elsevior, NY: 1985)) and corresponding suitable host cells can be employed for production of recombinant peptides. Expression hosts include, but are not limited to, bacterial species, mammalian or insect host cell systems including baculovirus systems (e.g., as described by Luckow et al.,
Bio/Technology,
6, 47 (1988)), and established cell lines such 293, COS-7, C 127, 3T3, CHO, HeLa, BHK, ctc. From such cells, the modified chimeric proteins can be harvested by standard techniques.
The ligand is exposed to the cells, e.g., under conditions suitable for the ligand to bind the CD16 protein. For example, for cells in vitro, the ligand-bearing molecules can be added to the cell culture for a time sufficient for them to bind the CD16 molecules on the cells. For cells in vivo, the ligand-bearing molecules can be supplied in a pharmacologically acceptable carrier (e.g., a solution, gel, magma, or salve). In this regard, the ligand can be delivered topically to cells within a discrete organ or tissue or systemically to attenuate papillomavirus infection on a more wide-spread scale.
The ligand recognizing CD16 is exposed to the cells under conditions sufficient for the ligand to bind the cells. The concentration of ligand, as well as the conditions required for efficient binding, will depend on the type of ligand employed and location of delivery. However, it is within the routine skill in the art to investigate the kinetic profile of such ligands in advance of an application.
In accordance with the present invention, another method of attenuating the ability of papillomavirus to infect cells is to reduce the free titer of papillomavirus. In the context of the invention, this can be achieved by exposing papillomavirus to

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