WO2002087609A1

WO2002087609A1 - Identification of a cell surface receptor for papillomaviruses

Info

Publication number: WO2002087609A1
Application number: PCT/US2002/013579
Authority: WO
Inventors: Douglas R. Lowy; Patricia Day; John T. Schiller
Original assignee: The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2001-05-01
Filing date: 2002-04-30
Publication date: 2002-11-07

Abstract

The invention relates to a pharmaceutical composition for preventing papillomavirus infection in a mammal comprising, in an amount sufficient to prevent papillomavirus infection, a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached. Small molecules that inhibit or prevent binding of HPV to syndecans can also be used for preventing of papillomavirus infection.

Description

IDENTIFICATION OF A CELL SURFACE RECEPTOR FOR PAPILLOMAVIRUSES

Field of the Invention The invention relates to a pharmaceutical composition for preventing papillomavirus infection in a mammal comprising, in an amount sufficient to prevent papillomavirus infection, a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached. Small molecules that inhibit or prevent binding of HPV to syndecans can also be used for preventing of papillomavirus infection.

Background of the Invention Identification of the cell surface receptor(s) by which papillomaviruses enter cells has remained conjectural. Studies in our laboratory and elsewhere have suggested that human and animal papillomaviruses use a common receptor(s) and that this receptor is a protein that is widely expressed and evolutionarily conserved (Roden et al. J Virol 68:7260; Muller et al. 1995 J Virol 69:948). The alpha-6/beta-4 integrin has been proposed as a candidate for this receptor, based upon biochemical results (McMillan et al. 1999 Virology 261:271). However, other investigators have found that bovine papillomavirus type 4 (BPV-4) can infect cells that lack this integrin (Sibbet et al. 2000 J Gen Virol 81:327). Thus, alpha-6/beta-4 integrin may not be the primary papillomavirus receptor.

In some virus systems, such as herpesviruses, the presence of heparan sulfate, which is a constituent of proteoglycans, has been shown to act as a co-receptor. Proteoglycans, which contain complex glycosaminoglycans, function in cell adhesion and signaling. They are cell surface molecules composed of multiple copies of glycosaminoglycans, such as heparan sulfate, linked to a protein core. The pharmacologic agent heparin is essentially the isolated glycosaminoglycan devoid of the protein moiety. There are two main families of proteoglycans, the glypicans and the syndecans. In the case of herpesviruses, it is believed that the specific sequence of the protein core of the proteoglycan is not critical to the ability of the proteoglycan to act as a co-receptor. Rather, it is the heparan sulfate on the proteoglycan that serves this function. The initial binding of the viral glycoprotein to the heparan sulfate is believed to enable other cell surface proteins to undergo high affinity binding with the virus particle (reviewed in Spear et al. 2000 Virology 275:1).

Based on the herpesvirus model, researchers at Merck carried out a series of biochemical studies with HPV-11 particles (Joyce et al. 1999 J Biol Chem 274:5810). They found that HPN-11 virus-like particles (VLPs) are able to bind heparin in vitro, to bind cells that contain heparan sulfated proteoglycans, and to bind less efficiently to cells treated with heparanase (also called heparitinase, an enzyme that cleaves heparan sulfate from the surface of the cells). Martin Sapp's laboratory in Germany has extended these observations by reporting that treatment of cultured cells with heparanase can convert cells from being permissive for infection by HPV-33 pseudovirions to being non-permissive for this infection (Giroglou et al. 2001 J Virol 75:1565). The Merck authors (Joyce et al. 1999 J Biol Chem 274:5810) pointed out that their "study establishes the ability of recombinant HPN VLPs to interact with heparin and cellular glycosaminoglycans in vitro. It does not identify a defined class of glycosaminoglycans as high specificity receptors for papillomaviruses, but it does demonstrate that these cell-surface molecules could be involved in initial recognition and association of virus in vivo."

Summary of the Invention The invention relates to a pharmaceutical composition for preventing papillomavirus infection in a mammal comprising, in an amount sufficient to prevent papillomavirus infection, a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDΝA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, in admixture with conventional excipients for topical or other application.

Brief Description of the Drawings Figure 1 shows the major cell surface heparan sulfate proteoglycans (HSPGs).

Detailed Description of the Preferred Embodiment The invention is based on a new understanding of how papillomaviruses enter cells, which has direct implications for preventing papillomavirus infection. We have identified the receptors by which these viruses bind to cells. This finding is envisioned as leading to the use of a soluble form of the receptor to prevent papillomavirus infection by serving as a molecular decoy. This finding is also envisioned as leading to identifying small molecules that prevent the interaction of the papillomavirus virion with its cellular receptor.

Papillomaviruses cause benign epithelial tumors (warts) in humans and animals. In humans, the vast majority of genital infections with human papillomavirus (HPN) are sexually transmitted. HPV infection of the internal and external genitalia is extremely common, and sub-clinical (inapparent) HPV infection is even more common than clinical infection. It is estimated there are around 10 million new cases of cervical HPV infection per year in the U.S. Some papillomaviruses are carcinogenic. In humans, HPV are etiologically involved in many forms of genital malignancy, especially cancer of the cervix, which is the third most common cancer of women throughout the world. Several different HPV genotypes (types) have been found in cervical cancer, with HPV- 16 (less commonly HPV- 18) being the one most frequently isolated. Otherwise, HPV-1 causes skin warts, and HPV-6 and HPV-11 produce genital warts. The invention is envisioned as preventing these infections and the diseases they induce.

We have determined that papillomaviruses enter cells via syndecans. Our results have been obtained with lymphoid cells, which ordinarily are not permissive for papillomavirus infection. To determine whether a particular cell surface proteoglycan might be implicated in papillomavirus infection, we have tested a lymphoid cell line (ARH- 77) and its derivatives that had been stably transfected with one of three different proteoglycans: syndecan-1, syndecan-4, or glypican-1. The transfectants and the parental cells were obtained from Dr. Ralph Sanderson, University of Arkansas for Medical Sciences, and the transfectants were described in Liu et al. 1998 J Biol Chem 273:22825. We have found that the syndecan-1 transfectants are permissive for infection by BPV-1 or HPV- 16 pseudovirions, while the parental cells and the glypican-1 transfectants are non- permissive for BPV-1 or HPV- 16 infection. The syndecan-4 transfectants were non- permissive for BPV-1 but had a weak infectious signal for the HPV- 16 pseudovirions. Our results confirm the data published previously by Dr. Sanderson's laboratory indicating that each line should express its transfected proteoglycan, with the syndecan-1 and -4 expressing lines containing similar levels of heparan sulfate (although our studies measured somewhat less heparan sulfate in the glypican-1 expressing line). We envision that one or more syndecans represent a high affinity receptor for papillomaviruses, while glypicans do not serve this function. The invention relates to the prevention of papillomavirus infection by use of a version of a syndecan that binds virus particles at relatively high affinity, with a Kd as low as 1-5 nM. Since genital HPVs are transmitted primarily via sexual intercourse, the inhibitor might be formulated for topical application for use prior to intercourse, and other formulations to prevent this and other sexually transmitted diseases (STDs) are also contemplated. This approach would be expected to be equally effective against all HPV types, in contrast to current candidate papillomavirus vaccines, whose effectiveness is predicted to be limited to only those HPV types included in the vaccine.

Syndecans have several desirable properties for use as an inhibitor. Soluble forms of the extracellular forms of syndecan-1 and syndecan-4 are made physiologically by cells, these soluble forms are stable, and they are able to bind heparin-binding proteins. Moreover, active soluble forms can be produced by recombinant DNA techniques. Since syndecans are endogenous molecules, they are unlikely to be immunogenic. Intermittent topical (or other) application of soluble syndecan would unlikely be associated with serious side effects. 1. Functioning of Syndecans

Referring to Figure 1, all syndecan core proteins, which are type 1 membrane proteins, possess unique ectodomains, which bear homology particularly at the sites to which three heparan sulfate glycosaminoglycan chains are normally attached. Additionally, a protease cleavage site adjacent to the transmembrane domain is highly conserved. Each core protein has a highly conserved transmembrane domain. The short cytoplasmic domains contain two highly conserved regions, between which the sequence is unique. Comparisons between species of derived protein sequences for the syndecans show an evolutionary relationship in which the cytoplasmic and transmembrane domains do not evolve among species, but the extracellular domain is evolving more rapidly, except for highly conserved sequences surrounding the glycosaminoglycan (GAG) attachment sites and at the protease cleavage site. Syndecan-1 was the first transmembrane heparan sulfate proteoglycan to be sequenced, followed by syndecan-2 (fibroglycan), syndecan-3 (N- syndecan) and most recently syndecan-4 (amphiglycan, ryudocan). A diagram of syndecan structure is shown in Figure 1. Syndecan proteins range in size from ~45 kDa (syndecan-3) to -20 kDa (syndecan-4) as deduced from cDNA cloning. The apparent molecular mass on SDS-PAGE is, however, much larger owing to core protein association and glycosaminoglycan substitution. Most of the core protein is extracellular: syndecans have a single membrane-spanning region and a short cytoplasmic domain. All four syndecans normally bear three covalently attached heparan sulfate glycosaminoglycan chains, and chondroitin sulfate chains can sometimes be present as well. The glycosaminoglycan chains are known to bind to heparin-binding moieties of matrix molecules, but roles for the core proteins are only beginning to be elucidated. The four mammalian syndecans are expressed in a development-, cell-type- and tissue-specific manner. In adult tissues, syndecan-1, -2, and -3 are the major syndecans in epithelial cells, fibroblasts and neuronal cells, respectively. During development, however, there are fluxes of syndecan expression. For example, there is a transient expression of syndecan-1 in presumptive skin appendage mesenchyme. Syndecan-4, by contrast, is usually present in lower amounts than the major syndecan species but is more widespread in distribution.

2. Analysis of the Papillomavirus Infectivity Pathway

It is presently unclear how papillomaviruses (PV) enter the host cell to initiate their infective cycle. Previous analyses of PV entry pathways have been in disaccord as to both the kinetics of virion penetration and the endocytic pathway utilized. The current emphasis on identification of PV receptors accentuates the need for a systematic consideration of these questions. To assess these issues we have assayed both bovine papillomavirus (BPV) virus-like particle (VLP) trafficking and authentic virus mRNA expression as a measure of infection. An early spliced mRNA was easily detectable by RT-PCR at 10 hours postinfection. Disruption of both processes occurred when cells were exposed to biochemical inhibitors that affect receptor-mediated endocytosis (i.e. azide, latrunculin) and those that affect vesicular trafficking (i.e. bafilomycin, nocodozole). No effect was observed when caveolae-mediated uptake was prevented with filipin or nystatin. This would indicate a subcellular trafficking pathway more similar to that utilized by the human polyomavirus type BK than by SV40 (simian virus 40). However, the kinetics of entry, measured by loss of antibody-reactive cell surface VLPs, are substantially delayed by comparison to those typically observed for endocytosis via clathrin-coated pits. We estimated a t_1/2 of 90 minutes versus 5 to 15 minutes for internalization of a typical ligand via this pathway. Additionally, we have used these techniques to examine potential PV receptor proteins. In summary, our results show: (1) bovine papillomavirus type 1 (BPV-1) is internalized via an atypical endocytic pathway; (2) BPV-1 is not internalized via caveolae; (3) expression of syndecan-1 allows BPV-1 infection of ARH-77 cells; and (4) expression of syndecan-1 and -4 allows human papillomavirus type 16 (HPV- 16) pseudoinfection of ARH-77 cells.

3. The Syndecan Gene

The syndecan nucleotide sequences of the invention include: (a) the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number

#A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number

#075056), and syndecan-4 (accession number #CAA16520), which is known in the art; (b) a nucleotide sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), and syndecan-4 (accession number #CAA16520); (c) any nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number

#P34741), syndecan-3 (accession number #075056), and syndecan-4 (accession number

#CAA16520) under highly stringent conditions, e.g., hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C, and washing in

0.1X SSC/0.1% SDS at 68°C. (Ausubel F.M. et al. eds., 1989 Current Protocols in

Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes a functionally equivalent gene product; and (d) any nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), and syndecan-4 (accession number #CAA16520) under moderately stringent conditions, e.g., washing in 0.2X SSC/0.1% SDS at 42°C (Ausubel et al. 1989 Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.), yet which still encodes a functionally equivalent syndecan gene product. Functional equivalents of the syndecan include naturally occurring syndecan present in the same or other species, and mutant syndecans whether naturally occurring or engineered. The invention also includes degenerate variants of sequences (a) through (d).

In addition to the syndecan nucleotide sequences described above, full length syndecan cDNA or gene sequences present in the same species and/or homologs of the syndecan gene present in the same or other species can be identified and readily isolated, without undue experimentation, by molecular biological techniques well known in the art. Genes at other genetic loci within the genome that encode proteins which have extensive homology to one or more domains of the syndecan gene product can be identified via these techniques. For example, expression libraries of cDNAs synthesized from epithelium mRNA derived from the organism of interest can be screened using labeled syndecan derived from that species. Alternatively, such cDNA libraries, or genomic DNA libraries derived from the organism of interest can be screened by hybridization using the nucleotides described herein as hybridization or amplification probes. In the case of cDNA libraries, such screening techniques can identify clones derived from alternatively spliced transcripts in the same or different species. Screening can be by filter hybridization, using duplicate filters. The labeled probe can contain at least 15-30 base pairs of the syndecan cDNA sequence. The hybridization washing conditions used should be of a lower stringency when the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived. With respect to the cloning of a human syndecan homo log, using murine syndecan probes, for example, hybridization can, for example, be performed at 65°C overnight in Church's buffer (7% SDS, 250 mM NaHPO₄, 2 μM EDTA, 1% BSA). Washes can be done with 2X SSC, 0.1% SDS at 65°C and then at 0.1 X SSC, 0.1% SDS at 65°C. Low stringency conditions are well known to those of skill in the art, and will vary predictably depending on the specific organisms from which the library and the labeled sequences are derived. For guidance regarding such conditions see, for example, Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al. 1989 Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.

Further, a syndecan gene homolog may be isolated from nucleic acid of the organism of interest by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the syndecan gene product disclosed herein. The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or tissue, such as epithelium, known or suspected to express a syndecan gene allele.

The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a syndecan gene. The PCR fragment may then be used to isolate a full-length cDNA clone by a variety of methods. For example, the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library. Alternatively, the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.

PCR technology may also be utilized to isolate full-length cDNA sequences. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source (i.e., one known, or suspected, to express the syndecan gene, such as, for example, epithelium). A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis. The resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer. Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of cloning strategies which may be used, see e.g., Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y. The invention also encompasses nucleotide sequences that encode peptide fragments of the syndecans, truncated syndecans, and syndecan fusion proteins. These include, but are not limited to nucleotide sequences encoding polypeptides or peptides corresponding to the extracellular domain (ECD) of the syndecans or portions of these domains, advantageously the evolutionarily conserved domains, conveniently the GAG attachment sites and the protease cleavage sites; and truncated syndecans in which one or two of the domains is deleted, e.g., a soluble syndecan lacking the transmembrane domain (TM) or both the TM and cytoplasmic domain (CD). Nucleotides encoding fusion proteins may include by are not limited to full length syndecan, truncated syndecan or peptide fragments of syndecan fused to an unrelated protein or peptide, such as for example, a transmembrane sequence, which anchors the syndecan ECD to the cell membrane; an Ig Fc domain which increases the stability and half life of the resulting fusion protein (e.g., syndecan-Ig) in the bloodstream; or an enzyme, fluorescent protein, luminescent protein which can be used as a marker.

The invention also encompasses (a) DNA vectors that contain any of the foregoing syndecan coding sequences; (b) DNA expression vectors that contain any of the foregoing syndecan coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing syndecan coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell. As used herein, regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.

4. Syndecan Proteins and Polypeptides

Syndecan protein, polypeptides and peptide fragments, truncated or deleted forms of the syndecan and/or syndecan fusion proteins can be prepared for a variety of uses, including but not limited to, as reagents in diagnostic assays, reagents in purifying VLPs, and reagents useful in the prevention of papillomavirus infection and horizontal spread of existing papillomavirus infection.

The syndecan amino acid sequences of the invention include the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), and syndecan-4 (accession number #CAA16520). Further, syndecans encoded by genes at other genetic loci within the genome are encompassed by the invention. In fact, any syndecan protein encoded by the syndecan nucleotide sequences described above are within the scope of the invention. The invention also encompasses proteins that are functionally equivalent to the syndecan encoded by the nucleotide sequences described above, as judged by any of a number of criteria, including but not limited to the ability to bind papillomavirus particles with high affinity, with a Kd as low as 1-5 nM. Such functionally equivalent syndecan proteins include but are not limited to additions or substitutions of amino acid residues within the amino acid sequence encoded by the syndecan nucleotide sequences described but which result in a silent change, thus producing a functionally equivalent gene product. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. While random mutations can be made to syndecan DNA (using random mutagenesis techniques well known to those skilled in the art) and the resulting mutant syndecans tested for activity, site-directed mutations of the syndecan coding sequence can be engineered (using site-directed mutagenesis techniques well known to those skilled in the art) to generate mutant syndecans with increased function, e.g., higher binding affinity for papillomavirus particles.

For example, the alignment of mouse, rat, hamster, and human syndecans indicates the position of the amino acids that are identical. Mutant syndecans can be engineered so that regions of identity are maintained, whereas the variable residues are altered, e.g., by deletion or insertion of an amino acid residue(s) or by substitution of one or more different amino acid residues. Conservative alterations at the variable positions can be engineered in order to produce a mutant syndecan that retains function; e.g., papillomavirus binding affinity. Non-conservative changes can be engineered at these variable positions to alter function, e.g., papillomavirus binding affinity. Alternatively, where alteration of function is desired, deletion or non-conservative alterations of the conserved regions (i.e., identical amino acids) can be engineered. Non-conservative alterations to the residues in the ECD can be engineered to produce mutant syndecans with altered binding affinity for papillomaviruses. Other mutations to the syndecan coding sequence can be made to generate syndecans that are better suited for expression, scale up, etc. in the host cells chosen. For example, cysteine residues can be deleted or substituted with another amino acid in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites. To this end, a variety of amino acid substitutions at one or both of the first or third amino acid positions of any one or more of the glycosylation recognition sequences which occur in the ECD (N-X-S or N-X-T), and/or an amino acid deletion at the second position of any one or more such recognition sequences in the ECD will prevent glycosylation of the syndecan at the modified tripeptide sequence (see, e.g., Miyajima et al. 1986 EMBOJ5:1193-1197).

Peptides corresponding to one or more domains of the syndecan (ΕCD), truncated or deleted syndecans (e.g., syndecan in which the TM and/or CD is deleted) as well as fusion proteins in which the full length syndecan, or syndecan peptide or truncated syndecan is fused to an unrelated protein are also within the scope of the invention and can be designed on the basis of the syndecan nucleotide and syndecan amino acid sequences disclosed here and above. Such fusion proteins include but are not limited to IgFc fusions which stabilize the syndecan protein or peptide and prolong half-life in vivo; or fusions to any amino acid sequence that allows the fusion protein to be anchored to the cell membrane, allowing the ΕCD to be exhibited on the cell surface; or fusions to an enzyme, fluorescent protein, or luminescent protein which provide a marker function.

The polypeptides of the present invention include a polypeptide comprising a polypeptide having the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), and syndecan-4 (accession number #CAA16520); as well as polypeptides which are at least 70% identical, and more preferably at least 80%, 90%, 95% or 99% identical to those described above, and also include domains of such polypeptides (ΕCD) and domains which are at least 70% identical, and more preferably at least 80%, 90%, 95% or 99% identical to those domains described above By a polypeptide having an amino acid sequence at least, for example, 95%

"identical" to a reference amino acid sequence of a syndecan polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the syndecan polypeptide. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 70%, 80%, 90%, 95% or 99% identical to, for instance, the amino acid sequence described above can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

While the syndecan polypeptides and peptides can be chemically synthesized (e.g., see Creighton, 1983 Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y.), large polypeptides derived from the syndecan and the full length syndecan itself may advantageously be produced by recombinant DNA technology using techniques well known in the art for expressing nucleic acid containing syndecan gene sequences and/or coding sequences. Such methods can be used to construct expression vectors containing the syndecan nucleotide sequences described above and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. See, for example, the techniques described in Sambrook et al. 1989 Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.; and Ausubel et al. 1989 Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. Alternatively, RNA capable of encoding syndecan nucleotide sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in Oligonucleotide Synthesis 1984 Gait, M. J. ed. IRL Press, Oxford.

A variety of host-expression vector systems may be utilized to express the syndecan nucleotide sequences of the invention. Where the syndecan peptide or polypeptide is a soluble derivative (e.g., syndecan peptides corresponding ECD; truncated or deleted syndecan in which the TM and/or CD are deleted) the peptide or polypeptide can be recovered from the culture, i.e., from the host cell in cases where the syndecan peptide or polypeptide is not secreted, and from the culture media in cases where the syndecan peptide or polypeptide is secreted by the cells. However, the expression systems also encompass engineered host cells that express the syndecan or functional equivalents in situ, i.e., anchored in the cell membrane. Purification or enrichment of the syndecan from such expression systems can be accomplished using appropriate detergents and lipid micelles and methods well known to those skilled in the art. However, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of the syndecan, but to assess biological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing syndecan nucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the syndecan nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the syndecan sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing syndecan nucleotide sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the syndecan gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of syndecan protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al. 1983 EMBOJ 2:1791), in which the syndecan coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye 1985 Nucleic Acids Res 13:3101-3109; Van Heeke & Schuster 1989 J Biol Chem 264:5503-5509); and the like. pGΕX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S- transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The pGΕX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety. In an insect system, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The syndecan gene coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). Successful insertion of syndecan gene coding sequence will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus, i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene. These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed (e.g., see Smith et al. 1983 J Virol 46:584; Smith, U.S. Pat. No. 4,215,051). In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the syndecan nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region Εl or Ε3) will result in a recombinant virus that is viable and capable of expressing the syndecan gene product in infected hosts (e.g., see Logan & Shenk 1984 PNAS USA 81:3655-3659). Specific initiation signals may also be required for efficient translation of inserted syndecan nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire syndecan gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the syndecan coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc (see Bittner et al. 1987 Methods in Enzymol 153:516-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, epithelial cell lines. For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the syndecan sequences described above may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the syndecan gene product. Such engineered cell lines may be particularly useful in screening and evaluation of mutant syndecans with altered binding affinity for papillomaviruses.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al. 1977 Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski 1962 PNAS USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al. 1980 Cell 22:817) genes can be employed in tk^", hgprt^" or aprt^" cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al. 1980 PNAS USA 77:3567; O'Hare et al. 1981 PNAS USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg 1981 PNAS USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al. 1981 J Mol Biol 150:1); and hygro, which confers resistance to hygromycin (Santerre et al. 1984 Gene 30:147).

Alternatively, any fusion protein may be readily purified by utilizing an antibody specific for the fusion protein being expressed. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al. 1991 PNAS USA 88:8972-8976). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. 5. Screening Assays for Compounds that Modulate Syndecan Expression or Activity

The following assays are designed to identify compounds that interact with (e.g., bind to) syndecan (including, but not limited to the extracellular domain (ECD), or cytoplasmic domain (CD), or transmembrane domain (TM), of syndecan, compounds that interact with (e.g., bind to) intracellular proteins that interact with syndecan (including, but not limited to, TM and CD of syndecan), compounds that interfere with the interaction of syndecan with papillomavirus, as well as with transmembrane or intracellular proteins involved in syndecan-mediated signal transduction, and to compounds which modulate the activity of syndecan gene (i.e., modulate the level of syndecan gene expression) or modulate the level of syndecan. Assays may additionally be utilized which identify compounds which bind to syndecan gene regulatory sequences (e.g., promoter sequences) and which may modulate syndecan gene expression. See e.g., Platt, K. A., 1994, J. Biol. Chem. 269: 28558-28562.

The compounds which may be screened in accordance with the invention include, but are not limited to peptides, antibodies and fragments thereof, and other organic compounds (e.g., peptidomimetics) that bind to the ECD or TM of syndecan and inhibit the activity triggered by papillomavirus (i.e., antagonists); as well as peptides, antibodies or fragments thereof, and other organic compounds that mimic the ECD of the syndecan (or a portion thereof) and bind to and "neutralize" papillomavirus.

Such compounds may include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries; (see, e.g., Lam, K.S. et al. 1991 Nature 354:82-84; Houghten, R. et al. 1991 Nαtwre 354:84-86), and combinatorial chemistry-derived molecular library made of D- and/or L- configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang, Z. et al. 1993 Cell 72: 767-778, antibodies (including, but not limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab')₂ and FAb expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.

Other compounds which can be screened in accordance with the invention include but are not limited to small organic molecules that are able to gain entry into an appropriate cell and affect the expression of the syndecan gene or some other gene involved in the syndecan signal transduction pathway (e.g., by interacting with the regulatory region or transcription factors involved in gene expression); or such compounds that affect the activity of the syndecan (e.g., by inhibiting or enhancing the enzymatic activity of the CD) or the activity of some other intracellular factor involved in the syndecan signal transduction pathway. Computer modeling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate syndecan activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand-binding sites, such as the interaction domains of papillomavirus with syndecan itself. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X- ray crystallographic methods can be used to find the active site by finding where on the syndecan polypeptide the complexed ligand is found. Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intra-molecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures may be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, the methods of computer based numerical modeling can be used to complete the structure or improve its accuracy. Any recognized modeling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.

Finally, having determined the structure of the active site, either experimentally, by modeling, or by a combination, candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. These compounds found from this search are potential syndecan modulating compounds.

Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modeling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.

Further experimental and computer modeling methods useful to identify modulating compounds based upon identification of the active sites of papillomavirus, syndecan, and related transduction and transcription factors will be apparent to those of skill in the art. Examples of molecular modeling systems are the CHARMM and QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMM performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive with specific- proteins, such as Rotivinen, et al. 1988 Ada Pharmaceutical Fennica 97:159-166; Ripka 1988 New Scientist 54-57; McKinaly and Rossmann 1989 Annu Rev Pharmacol Toxicol 29: 111-122; Perry and Davies 1989 OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 Alan R. Liss, Inc.; Lewis and Dean 1989 Proc R Soc Lond 236: 125-140 and 141-162; and, with respect to a model receptor for nucleic acid components, Askew et al. 1989 J Am Chem Soc 111:1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, Calif), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA as well, once that region is identified. Although described above with reference to design and generation of compounds which could alter binding of papillomavirus to syndecan, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which are inhibitors or activators, preferably inhibitors.

Compounds identified via assays such as those described herein may be useful in preventing papillomavirus infection. 5.1. In Vitro Cell-Free Screening Assays for Compounds that Bind to Syndecan

In vitro systems may be designed to identify compounds capable of interacting with (e.g., binding to) syndecan (including, but not limited to, the ECD or CD or TM of syndecan). Compounds identified may be useful, for example, in modulating the activity of wild type and/or mutant syndecan gene products; may be useful in elaborating the biological function of the syndecan; may be utilized in screens for identifying compounds that disrupt syndecan-papillomavirus interactions; or may in themselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to the syndecan involves preparing a reaction mixture of the syndecan and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected in the reaction mixture. The syndecan species used can vary depending upon the goal of the screening assay. For example, where antagonists of the natural ligand are sought, the full length syndecan, or a soluble truncated syndecan, e.g., in which the TM and/or CD is deleted from the molecule, a peptide corresponding to the ECD or a fusion protein containing the syndecan ECD fused to a protein or polypeptide that affords advantages in the assay system (e.g., labeling, isolation of the resulting complex, etc.) can be utilized. Where compounds that interact with the cytoplasmic domain are sought to be identified, peptides corresponding to the syndecan CD and fusion proteins containing the syndecan CD can be used.

The screening assays can be conducted in a variety of ways. For example, one method to conduct such an assay would involve anchoring the syndecan protein, polypeptide, peptide or fusion protein or the test substance onto a solid phase and detecting syndecan/test compound complexes anchored on the solid phase at the end of the reaction.

In one embodiment of such a method, the syndecan reactant may be anchored onto a solid surface, and the test compound, which is not anchored, may be labeled, either directly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solid phase. The anchored component may be immobilized by non-covalent or covalent attachments. Non- covalent attachment may be accomplished by simply coating the solid surface with a solution of the protein and drying. Alternatively, an immobilized antibody, preferably a monoclonal antibody, specific for the protein to be immobilized may be used to anchor the protein to the solid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the nonimmobilized component is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously nonimmobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the previously nonimmobilized component is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the previously nonimmobilized component (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Alternatively, a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected, e.g., using an immobilized antibody specific for syndecan protein, polypeptide, peptide or fusion protein or the test compound to anchor any complexes formed in solution, and a labeled antibody specific for the other component of the possible complex to detect anchored complexes. Alternatively, cell-based assays, membrane vesicle-based assays and membrane fraction-based assays can be used to identify compounds that interact with syndecan. To this end, cell lines that express syndecan, or cell lines (e.g., COS cells, CHO cells, fibroblasts, etc.) that have been genetically engineered to express syndecan (e.g., by transfection or transduction of syndecan DNA) can be used. Interaction of the test compound with, for example, the ECD of syndecan expressed by the host cell can be determined by comparison or competition with papillomavirus. 5.2. Assays for Intracellular Proteins that Interact with the Syndecan

Any method suitable for detecting protein-protein interactions may be employed for identifying transmembrane proteins or intracellular proteins that interact with syndecan. Among the traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns of cell lysates or proteins obtained from cell lysates and the syndecan to identify proteins in the lysate that interact with the syndecan. For these assays, the syndecan component used can be a full-length syndecan, a soluble derivative lacking the membrane-anchoring region (e.g., a truncated syndecan in which the TM is deleted resulting in a truncated molecule containing the ECD fused to the CD), a peptide corresponding to the CD or a fusion protein containing the CD of syndecan. Once isolated, such an intracellular protein can be identified and can, in turn, be used, in conjunction with standard techniques, to identify proteins with which it interacts. For example, at least a portion of the amino acid sequence of an intracellular protein which interacts with the syndecan can be ascertained using techniques well known to those of skill in the art, such as via the Edman degradation technique (see, e.g., Creighton 1983 Proteins: Structures and Molecular Principles W.H. Freeman & Co. N.Y. pp. 34-49). The amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known (see, e.g., Ausubel et al. 1989 Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y.; and PCR Protocols: A Guide to Methods and Applications, 1990, Innis, M. et al. eds. Academic Press, Inc., New York). Additionally, methods may be employed which result in the simultaneous identification of genes which encode the transmembrane or intracellular proteins interacting with syndecan. These methods include, for example, probing expression libraries, in a manner similar to the well known technique of antibody probing of λgtl l libraries, using labeled syndecan protein, or a syndecan polypeptide, peptide or fusion protein, e.g., a syndecan polypeptide or syndecan domain fused to a marker (e.g., an enzyme, fluor, luminescent protein, or dye), or an Ig-Fc domain. One method which detects protein interactions in vivo, the two-hybrid system, is described in detail for illustration only and not by way of limitation. One version of this system has been described (Chien et al. 1991 PNAS USA 88:9578-9582) and is commercially available from Clontech (Palo Alto, Calif). Briefly, utilizing such a system, plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA-binding domain of a transcription activator protein fused to a syndecan nucleotide sequence encoding syndecan, a syndecan polypeptide, peptide or fusion protein, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein which has been recombined into this plasmid as part of a cDNA library. The DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HBS or lacZ) whose regulatory region contains the transcription activator's binding site. Either hybrid protein alone cannot activate transcription of the reporter gene: the DNA-binding domain hybrid cannot becaμse it does not provide activation function and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression of the reporter gene, which is detected by an assay for the reporter gene product. The two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product. By way of example, and not by way of limitation, syndecan may be used as the bait gene product. Total genomic or cDNA sequences are fused to the DNA encoding an activation domain. This library and a plasmid encoding a hybrid of a bait syndecan gene product fused to the DNA- binding domain are cotransformed into a yeast reporter strain, and the resulting transformants are screened for those that express the reporter gene. For example, and not by way of limitation, a bait syndecan gene sequence, such as the open reading frame of syndecan (or a domain of syndecan), can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein. These colonies are purified and the library plasmids responsible for reporter gene expression are isolated. DNA sequencing is then used to identify the proteins encoded by the library plasmids. A cDNA library of the cell line from which proteins that interact with bait syndecan gene product are to be detected can be made using methods routinely practiced in the art. According to the particular system described herein, for example, the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4. This library can be co-transformed along with the bait syndecan gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence. A cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait syndecan gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene. Colonies which express HIS3 can be detected by their growth on Petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait syndecan gene-interacting protein using techniques routinely practiced in the art. 5.3. Assays for Compounds that Interfere with Svndecan/Intracellular or Syndecan/Transmembrane Macromolecule Interaction

The macromolecules that interact with the syndecan are referred to, for purposes of this discussion, as "binding partners". These binding partners are likely to be involved in the syndecan signal transduction pathway, and therefore, in the role of syndecan in papillomavirus infection. Therefore, it is desirable to identify compounds that interfere with or disrupt the interaction of such binding partners with syndecan which may be useful in inhibiting or preventing papillomavirus infection.

The basic principle of the assay systems used to identify compounds that interfere with the interaction between the syndecan and its binding partner or partners involves preparing a reaction mixture containing syndecan protein, polypeptide, peptide or fusion protein as described in Sections above, and the binding partner under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex. In order to test a compound for inhibitory activity, the reaction mixture is prepared in the presence and absence of the test compound. The test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the syndecan moiety and its binding partner. Control reaction mixtures are incubated without the test compound or with a placebo. The formation of any complexes between the syndecan moiety and the binding partner is then detected. The formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the syndecan and the interactive binding partner. Additionally, complex formation within reaction mixtures containing the test compound and normal syndecan protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant syndecan. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal syndecans.

The assay for compounds that interfere with the interaction of the syndecan and binding partners can be conducted in a heterogeneous or homogeneous format. Heterogeneous assays involve anchoring either the syndecan moiety product or the binding partner onto a solid phase and detecting complexes anchored on the solid phase at the end of the reaction. In homogeneous assays, the entire reaction is carried out in a liquid phase. In either approach, the order of addition of reactants can be varied to obtain different information about the compounds being tested. For example, test compounds that interfere with the interaction by competition can be identified by conducting the reaction in the presence of the test substance, i.e., by adding the test substance to the reaction mixture prior to or simultaneously with the syndecan moiety and interactive binding partner. Alternatively, test compounds that disrupt preformed complexes, e.g., compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. The various formats are described briefly below.

In a heterogeneous assay system, either the syndecan moiety or the interactive binding partner, is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly. In practice, microtiter plates are conveniently utilized. The anchored species may be immobilized by non-covalent or covalent attachments. Non- covalent attachment may be accomplished simply by coating the solid surface with a solution of the syndecan gene product or binding partner and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored. In order to conduct the assay, the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g., by washing) and any complexes formed will remain immobilized on the solid surface. The detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed. Where the non-immobilized species is not pre-labeled, an indirect label can be used to detect complexes anchored on the surface, e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody). Depending upon the order of addition of reaction components, test compounds which inhibit complex formation or which disrupt preformed complexes can be detected. Alternatively, the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected, e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes. Again, depending upon the order of addition of reactants to the liquid phase, test compounds which inhibit complex or which disrupt preformed complexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can be used. In this approach, a preformed complex of the syndecan moiety and the interactive binding partner is prepared in which either the syndecan or its binding partners is labeled, but the signal generated by the label is quenched due to formation of the complex (see, e.g., U.S. Pat. No. 4,109,496 by Rubenstein which utilizes this approach for immunoassays). The addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt syndecan/intracellular binding partner interaction can be identified.

In a particular embodiment, a syndecan fusion protein can be prepared for immobilization. For example, the syndecan or a peptide fragment, e.g., corresponding to the CD, can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein. The interactive binding partner can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art. This antibody can be labeled with the radioactive isotope ¹²⁵I, for example, by methods routinely practiced in the art. In a heterogeneous assay, e.g., the GST-syndecan fusion protein can be anchored to glutathione-agarose beads. The interactive binding partner can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur. At the end of the reaction period, unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components. The interaction between the syndecan gene product and the interactive binding partner can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity. Alternatively, the GST-syndecan fusion protein and the interactive binding partner can be mixed together in liquid in the absence of the solid glutathione-agarose beads. The test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione-agarose beads and unbound material is washed away. Again the extent of inhibition of the syndecan/binding partner interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can be employed using peptide fragments that correspond to the binding domains of the syndecan and/or the interactive or binding partner (in cases where the binding partner is a protein), in place of one or both of the full length proteins. Any number of methods routinely practiced in the art can be used to identify and isolate the binding sites. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding. Alternatively, one protein can be anchored to a solid surface using methods described above, and allowed to interact with and bind to its labeled binding partner, which has been treated with a proteolytic enzyme, such as trypsin. After washing, a short, labeled peptide comprising the binding domain may remain associated with the solid material, which can be isolated and identified by amino acid sequencing. Also, once the gene coding for the intracellular binding partner is obtained, short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.

For example, and not by way of limitation, a syndecan gene product can be anchored to a solid material as described, above, by making a GST-syndecan fusion protein and allowing it to bind to glutathione agarose beads. The interactive binding partner can be labeled with a radioactive isotope, such as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin. Cleavage products can then be added to the anchored GST-syndecan fusion protein and allowed to bind. After washing away unbound peptides, labeled bound material, representing the intracellular binding partner binding domain, can be eluted, purified, and analyzed for amino acid sequence by well-known methods. Peptides so identified can be produced synthetically or fused to appropriate facilitative proteins using recombinant DNA technology. 5.4. Cell- and Membrane-Based Screening Assays for Syndecan Inhibitors

Compounds, including but not limited to binding compounds identified via assay techniques such as those described in the preceding sections above can be tested for the ability to treat or prevent papillomavirus infection. The assays described above can identify compounds which affect syndecan activity, e.g., compounds that bind to syndecan, inhibit binding of the natural ligand, and either activate signal transduction (agonists) or block activation (antagonists), and compounds that bind to a natural ligand of syndecan and neutralize ligand activity; or compounds that affect syndecan gene activity (by affecting syndecan gene expression, including molecules, e.g., proteins or small organic molecules, that affect or interfere with splicing events so that expression of the full length syndecan can be modulated). However, it should be noted that the assays described can also identify compounds that modulate syndecan signal transduction (e.g., compounds which affect upstream or downstream signaling events). The identification and use of such compounds which affect another step in the syndecan signal transduction pathway in which the syndecan gene product is involved and, by affecting this same pathway may modulate the effect of syndecan on papillomavirus infection are within the scope of the invention. Such compounds can be used as part of a therapeutic method for the prevention or treatment of papillomavirus infection.

Cell-based systems, membrane vesicle-based systems, and membrane fraction- based systems can be used to identify compounds which may act to treat or prevent papillomavirus infection. Such systems can include, for example, recombinant or non- recombinant cells, such as cell lines, which express the syndecan gene. In addition, expression host cells (e.g., COS cells, CHO cells, fibroblasts) genetically engineered to express a functional syndecan and to respond to activation by papillomavirus, e.g., as measured by a chemical or phenotypic change, induction of another host cell gene, change in ion flux, phosphorylation of host cell proteins, etc., can be used as an end point in the assay.

In utilizing such cell-based systems, cells may be exposed to a compound suspected of exhibiting an ability to treat or prevent papillomavirus infection, at a sufficient concentration and for a time sufficient to elicit chemical or phenotypic change, induction of another host cell gene, change in ion flux, phosphorylation of host cell proteins, etc. in the exposed cells. After exposure, the cells can be assayed to measure alterations in the expression of the syndecan gene, e.g., by assaying cell lysates for syndecan mRNA transcripts (e.g., by Northern analysis) or for syndecan protein expressed in the cell; compounds which regulate or modulate expression of the syndecan gene are good candidates as therapeutics. Still further, the expression and/or activity of components of the signal transduction pathway of which syndecan is a part, or the activity of the syndecan signal transduction pathway itself can be assayed.

For example, after exposure, the cell lysates can be assayed for the presence of phosphorylation of host cell proteins, as compared to lysates derived from unexposed control cells. The ability of a test compound to inhibit phosphorylation of host cell proteins in these assay systems indicates that the test compound inhibits signal transduction initiated by syndecan activation. The cell lysates can be readily assayed using a Western blot format, i.e., the host cell proteins are resolved by gel electrophoresis, transferred and probed using an antibody against a phosphorylated peptide residue (e.g., an antibody labeled with a signal generating compound, such as radiolabel, fluor, enzyme, etc.) (see, e.g., Glenney et al. 1988 J Immunol Methods 109:277-285; Frackelton et al. 1983 Mol Cell Biol 3:1343-1352). Alternatively, an ELISA format could be used in which a particular host cell protein involved in the syndecan signal transduction pathway is immobilized using an anchoring antibody specific for the target host cell protein, and the presence or absence of a phosphorylated peptide residue on the immobilized host cell protein is detected using a labeled antibody (see, King et al. 1993 Life Sciences 53:1465-1472). In yet another approach, ion flux, such as calcium, potassium, sodium, bicarbonate, chloride ion flux, can be measured as an end point for syndecan stimulated signal transduction.

In general, other cell-based screening procedures of the invention involve providing appropriate cells which express a syndecan polypeptide on the surface thereof. Such cells include cells from mammals, yeast, Drosophila or E. coli. In particular, a polynucleotide encoding syndecan is employed to transfect cells to thereby express a syndecan. The expressed syndecan is then contacted with a test compound to observe binding, stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores which are transfected to express a syndecan polypeptide. Such a screening technique is described in PCT WO 92/01810, published Feb. 6, 1992. Such an assay may be employed to screen for a compound which inhibits activation of syndecan by contacting the melanophore cells which encode syndecan with both a ligand, such as a papillomavirus or VLP, and a compound to be screened. Inhibition of the signal generated by the binding of the ligand to syndecan indicates that a compound is a potential antagonist for the syndecan, i.e., inhibits activation of the syndecan.

The technique may also be employed for screening of compounds which activate syndecan by contacting such cells with compounds to be screened and determining whether such compound generates a signal, i.e., activates the syndecan. Other screening techniques include the use of cells which express a syndecan (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation. In this technique, compounds may be contacted with cells expressing a receptor polypeptide of the present invention. A second messenger response, e.g., signal transduction or pH changes, is then measured to determine whether the potential compound activates or inhibits the receptor.

Another screening technique involves expressing a syndecan polypeptide in which the receptor is linked to phospholipase C or D. Representative examples of such cells include, but are not limited to, endothelial cells, smooth muscle cells, and embryonic kidney cells. The screening may be accomplished as hereinabove described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase second signal. Another method involves screening for compounds which are antagonists, and thus inhibit activation of a syndecan receptor polypeptide of the present invention by determining inhibition of binding of labeled ligand, such as a papillomavirus or VLP, to cells which have the receptor on the surface thereof, or cell membranes containing the receptor. Such a method involves transfecting a eukaryotic cell with a DNA encoding a syndecan polypeptide such that the cell expresses the receptor on its surface (or using a eukaryotic cell that expresses the syndecan on its surface). The cell is then contacted with a potential antagonist in the presence of a labeled form of a ligand, such as a papillomvirus or VLP. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity associated with transfected cells or membrane from these cells. If the compound binds to the receptor, the binding of labeled ligand to the receptor is inhibited as determined by a reduction of labeled ligand which binds to the receptors. This method is called a binding assay.

Another such screening procedure involves the use of eukaryotic cells which are transfected to express the syndecan receptor polypeptide of the present invention (or use of eukaryotic cells that express the syndecan on their surface). The cells are loaded with an indicator dye that produces a fluorescent signal when bound to calcium, and the cells are contacted with a test substance and a receptor agonist, such as a papillomvirus or VLP. Any change in fluorescent signal is measured over a defined period of time using, for example, a fluorescence spectrophotometer or a fluorescence imaging plate reader. A change in the fluorescence signal pattern generated by the ligand indicates that a compound is a potential antagonist (or agonist) for the receptor.

Another such screening procedure involves use of eukaryotic cells which are transfected to express the syndecan receptor of the present invention (or use of eukaryotic cells that express the syndecan on their surface), and which are also transfected with a reporter gene construct that is coupled to activation of the receptor (for example, luciferase or beta-galactosidase behind an appropriate promoter). The cells are contacted with a test substance and a receptor agonist, such as a papillomavirus or VLP, and the signal produced by the reporter gene is measured after a defined period of time. The signal can be measured using a luminometer, spectrophotometer, fiuorimeter, or other such instrument appropriate for the specific reporter construct used. Inhibition of the signal generated by the ligand indicates that a compound is a potential antagonist for the receptor. Another such screening technique for antagonists or agonists involves introducing RNA encoding a syndecan receptor polypeptide into Xenopus oocytes to transiently or stably express the receptor. The oocytes are then contacted with a receptor ligand, such as a papillomavirus or VLP, and a compound to be screened. Inhibition or activation of the receptor is then determined by detection of a signal, such as, cAMP, calcium, proton, or other ions.

Another method involves screening for syndecan polypeptide inhibitors by determining inhibition or stimulation of syndecan receptor polypeptide-mediated cAMP and/or adenylate cyclase accumulation or diminution. Such a method involves transiently or stably transfecting a eukaryotic cell with a syndecan receptor polypeptide to express the receptor on the cell surface (or using a eukaryotic cell that expresses the syndecan on its surface). The cell is then exposed to potential antagonists in the presence of a receptor ligand, such as a papillomavirus or VLP. The amount of cAMP accumulation is then measured, for example, by radio-immuno or protein binding assays (for example using Flashplates or a scintillation proximity assay). Changes in cAMP levels can also be determined by directly measuring the activity of the enzyme, adenylyl cyclase, in broken cell preparations. If the potential antagonist binds the receptor, and thus inhibits syndecan receptor polypeptide binding, the levels of syndecan receptor polypeptide-mediated cAMP, or adenylate cyclase activity, will be reduced or increased. 5.5. Potential Syndecan Antagonists

Examples of potential syndecan receptor polypeptide antagonists include antibodies or, in some cases, oligonucleotides, which bind to the receptor but do not elicit a second messenger response such that the activity of the receptor is prevented.

Potential antagonists also include proteins which are closely related to a ligand of the syndecan receptor polypeptide, i.e. a fragment of the ligand, which has lost biological function and when binding to the syndecan receptor polypeptide, elicits no response.

A potential antagonist also includes an antisense construct prepared through the use of antisense technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both methods of which are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the syndecan receptor polypeptide of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix— see Lee, et al., 1979, Nucl. Acids Res., 6: 3073; Cooney, et al, 1988, Science, 241: 456; and Dervan, et al., 1991, Science, 251: 1360), thereby preventing transcription and production of a syndecan receptor polypeptide. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule to a syndecan receptor polypeptide (antisense— Okano, J., 1991, Neurochem., 56: 560; Oligonucleotides as antisense inhibitors of gene expression, 1988, CRC Press, Boca Raton, Fla.). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of a syndecan receptor polypeptide.

Another potential antagonist is a small molecule which binds to a syndecan receptor polypeptide, making it inaccessible to ligands such that normal biological activity is prevented. Examples of small molecules include, but are not limited to, small peptides or peptide-like molecules. Potential antagonists also include soluble forms of a solube receptor polypeptide, e.g., fragments of the polypeptide, which bind to the ligand and prevent the ligand from interacting with membrane bound syndecan receptor polypeptides. 5.6. Assays for in vivo identification of compounds that block binding

Animal-based systems may be used to identify compounds capable of treating or preventing papillomavirus infection. Such animal models may be used as test substrates for the identification of pharmaceuticals, therapies and interventions which may be effective in treating or preventing papillomavirus infection. For example, animal models may be exposed to a compound, suspected of exhibiting an ability to treat or prevent papillomavirus infection, at a sufficient concentration and for a time sufficient to elicit such a treatment or prevention of papillomavirus infection in the exposed animals. The response of the animals to the exposure may be monitored by assessing the treatment or prevention of papillomavirus infection . With regard to intervention, any treatments which reverse any aspect of papillomavirus infection should be considered as candidates for therapeutic or preventive intervention. 6. The Prevention of Papillomavirus Infection

The invention encompasses methods and preparation of compositions for preventing papillomavirus infection. To this end, the administration of soluble peptides, proteins, or fusion proteins that bind to and "neutralize" papillomavirus particles, a ligand for syndecans, can be used to effectuate the prevention of papillomavirus infection. For example, peptides corresponding to the ECD of syndecan, soluble deletion mutants of syndecan (e.g., TM and/or CD mutants), or either of these syndecan domains or mutants fused to another polypeptide (e.g., an IgFc polypeptide) are envisioned. Polypeptides or peptides corresponding to the extracellular domain (ECD) of the syndecans or portions of these domains, advantageously the evolutionarily conserved domains, conveniently the GAG attachment sites and the protease cleavage sites can be used. Truncated syndecans in which one or two of the domains is deleted, e.g., a soluble syndecan lacking the transmembrane domain (TM) or both the TM and cytoplasmic domain (CD) can also be used. Fusion of the syndecan, the syndecan ECD or the truncated syndecan to an IgFc polypeptide should not only increase the stability of the preparation, but will increase the half- life and activity of the syndecan-Ig fusion protein in vivo. Small molecules that inhibit or prevent binding of HPV to syndecans can also be used for preventing of papillomavirus infection. Such syndecan peptides, proteins, fusion proteins, or small molecules are administered to a subject in amounts sufficient to neutralize papillomavirus particles and/or to effectuate the prevention of papillomavirus infection.

7. Pharmaceutical Preparations and Methods of Administration

The compounds that are determined to prevent papillomavirus infection can be administered to a patient at therapeutically effective doses to prevent papillomavirus infection. A therapeutically effective dose refers to the amount of the compound sufficient to result in prevention of papillomavirus infection.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices are preferred.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of concentrations that include the ED₅₀ with little or no toxicity.

The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.

The pharmacologically active compounds of this invention can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans. The compounds of this invention can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application, which do not deleteriously react with the active compounds. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., vitamins.

For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. Ampoules are convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules. A syrup, elixir, or the like can be used wherein a sweetened vehicle is employed.

Sustained or directed release compositions can be formulated, e.g., by inclusion in Hposomes or incorporation into an epidermal patch with a suitable carrier, for example DMSO. It is also possible to freeze-dry these compounds and use the lyophilizates obtained, for example, for the preparation of products for injection. For topical application, there are employed as non-sprayable forms, viscous to semi- solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a freon.

Topical administration is preferred.

It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the compositions formulated, the mode of application, and the particular situs and organism being treated. Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol.

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All patents, patent applications and publications referred to above are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for preventing papillomavirus infection in a mammal comprising, in an amount sufficient to prevent papillomavirus infection, a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, in admixture with conventional excipients for topical or other application.

2. A pharmaceutical composition for preventing papillomavirus infection in a mammal comprising, in an amount sufficient to prevent papillomavirus infection, a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, , in admixture with conventional excipients for topical or other application.

3. A pharmaceutical composition for preventing papillomavirus infection in a mammal comprising, in an amount sufficient to prevent papillomavirus infection, a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, in admixture with conventional excipients for topical or other application.

4. A pharmaceutical composition for preventing papillomavirus infection in a mammal comprising, in an amount sufficient to prevent papillomavirus infection, a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, in admixture with conventional excipients for topical or other application.

5. A method for preventing papillomavirus infection in a mammal comprising administering to the mammal in an amount sufficient to prevent papillomavirus infection a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles.

6. A method for preventing papillomavirus infection in a mammal comprising administering to the mammal in an amount sufficient to prevent papillomavirus infection a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles.

7. A method for preventing papillomavirus infection in a mammal comprising administering to the mammal in an amount sufficient to prevent papillomavirus infection a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles.

8. A method for preventing papillomavirus infection in a mammal comprising administering to the mammal in an amount sufficient to prevent papillomavirus infection a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles.

9. A kit for preventing papillomavirus infection in a mammal comprising a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA 16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, together with instructions for administration to effectuate the prevention of papillomavirus infection.

10. A kit for preventing papillomavirus infection in a mammal comprising a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, together with instructions for administration to effectuate the prevention of papillomavirus infection

11. A kit for preventing papillomavirus infection in a mammal comprising a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%), 90%, 95%o, or 99% identical to the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA 16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, together with instructions for administration to effectuate the prevention of papillomavirus infection.

12. A kit for preventing papillomavirus infection in a mammal comprising a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%), 80%), 90%, 95%, or 99% identical to the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, together with instructions for administration to effectuate the prevention of papillomavirus infection.

13. A method for making a molecular decoy for preventing papillomavirus infection in a mammal comprising recombinantly expressing a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles; and combining it in admixture with conventional excipients for topical or other application.

14. A method for making a molecular decoy for preventing papillomavirus infection in a mammal comprising recombinantly expressing a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles; and combining it in admixture with conventional excipients for topical or other application.

15. A method for making a molecular decoy for preventing papillomavirus infection in a mammal comprising recombinantly expressing a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA 16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles; and combining it in admixture with conventional excipients for topical or other application.

16. A method for making a molecular decoy for preventing papillomavirus infection in a mammal comprising recombinantly expressing a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan- 2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles; and combining it in admixture with conventional excipients for topical or other application.

17. Use of a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, for the preparation of a medicament for preventing papillomavirus infection in a mammal.

18. Use of a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the amino acid sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the amino sequence of syndecan-1 (accession number #A41776), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the amino acid sequence of syndecan-1 (accession number #A41776), under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, , in admixture with conventional excipients for topical or other application, for the preparation of a medicament for preventing papillomavirus infection in a mammal. .

19. Use of a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the extracellular domain of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, in admixture with conventional excipients for topical or other application, for the preparation of a medicament for preventing papillomavirus infection in a mammal.

20. Use of a soluble peptide, protein, or fusion protein that binds to papillomavirus particles as a ligand for syndecans having heparan sulfate glycosaminoglycan chains attached, wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan-2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is at least 70%, 80%, 90%, 95%, or 99% identical to the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan- 2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520), or wherein the peptide, protein, or fusion protein that binds to papillomavirus particles is or comprises a sequence that is encoded by a nucleotide sequence that hybridizes to the complement of the cDNA sequence that encodes the glycosaminoglycan attachment site of syndecan-1 (accession number #A41776), syndecan- 2 (accession number #P34741), syndecan-3 (accession number #075056), or syndecan-4 (accession number #CAA16520) under highly stringent conditions or moderately stringent conditions and encodes a functionally equivalent syndecan gene product as judged by ability to bind papillomavirus particles, in admixture with conventional excipients for topical or other application, for the preparation of a medicament for preventing papillomavirus infection in a mammal.

21. The composition, method, kit, or use of any of the preceding claims wherein the affinity of binding is 1-5 nM.

22. The composition, method, kit, or use of any of the preceding claims wherein the papillomavirus is a member selected from the group consisting of HPV-1, HPV-6, HPV-11, HPV-16, and HPV-18.

23. The composition, method, kit, or use of any of the preceding claims wherein the prevention is of genital warts and the papillomavirus is HPV- 16.

24. The composition, method, kit, or use of any of the preceding claims wherein the prevention is of skin warts and the papillomavirus is HPV-1.

25. A method of identifying a compound that can be used to prevent papillomavirus infection comprising steps of: (a) incubating a cell that expresses a syndecan receptor, and said receptor being associated with a second component that provides a detectable signal in response to binding of a compound to said receptor, in a presence of a ligand which is a papillomavirus or virus-like particle, and additionally in a presence and absence of a test compound;

(b) determining whether said test compound binds to said sydecan receptor by measuring a level of a signal generated from interaction of the test compound with said receptor;

(c) selecting said test compound that binds to said syndecan receptor; and

(d) identifying said selected compound as being a candidate compound useful for the prevention of papillomavirus infection.

26. A method of identifying a compound that can be used to prevent papillomavirus infection comprising steps of:

(a) incubating a cell that expresses a syndecan receptor, or membrane vesicle or membrane fraction thereof, in a presence of a ligand which is a papillomavirus or virus-like particle, and additionally in a presence and absence of a test compound;

(b) determining whether said test compound inhibits binding of said ligand to said syndecan receptor by measuring an amount of said ligand bound to said receptor;

(c) selecting said test compound that causes reduction of binding of said ligand; and

27. The method of Claim 25 or 26 in which the ligand is labeled.

28. A method of making a pharmaceutical composition comprising a step of combining the compound identified by any of Claims 25-27 with a pharmaceutically acceptable carrier.

29. A method of preventing papillomavirus infection comprising a step of administering the pharmaceutical composition of Claim 28 to a patient in need thereof.

30. Use of the pharmaceutical composition of Claim 28 for the preparation of a medicament for preventing papillomavirus infection in a mammal.