AU628337B2 - Antibodies to human papillomavirus latent proteins, diagnostic systems and methods - Google Patents

Description

9

Y

~-ilC~II rss~ 1 628337 COMMONWEALTH OF AUSTRALIA PATNTS ACT 1952 COMPLETE SPECIFICATION FOR OFFICE USE Form '#1 snort uitle: Int. Cl: Application Number: Lodged: Complete Specification-Lodged: Accepted: Lapsed: Published: S Priority: S' Related Art: TO BE COMPLETED BY APPLICANT Name of Applicant: Address of Applicant: Actual Inventor: Address for Service: SCRIPPS CLINIC AND RESEARCH

FOUNDATION

10666 North Torrey Pines Road, La Jolla, CALIFORNIA 92037, U.S.A.

JOAKIM DILLNER; RICHARD A. LERNER; RICHARD SMITH and D. ELLIOT PARKS GRIFFITH HACK CO.

71 YORK STREET SYDNEY NSW 2000

AUSTRALIA

0 .D Complete Specification for the invention entitled: "ANTIBODIES TO HUMAN PAPILLOMAVIRUS LATENT PROTEINS, DIAGNOSTIC SYSTEMS AND

METHODS"

SOCCi745the folio g t'mnifis a full description of this invention, including the best method of performing it known to us:- 1204A/bmr 1 -1A- ANTIBODIES TO HUMAN PAPILLOMAVIRUS LATENT PROTEINS, DIAGNOSTIC SYSTEMS AND METHODS Description Cross Reference to Related Application '00 0 a 0 0 o 0 0 0 00 o0 0 0a 00 oo o 0 0 0 o 0 20 O 0 0 This is a continuation-in-part application of copending application Serial Number 194,407, filed May 16, 1988, the disclosures of which are herein incorporated by reference.

Technical Field The present invention relates to antibody and monoclonal antibody compositions containing antibody molecules that immunoreact with latent papillomavirus (PV) proteins. The present invention also relates to methods of preparing these antibody molecules and to methods of detecting these latent PV proteins and latent PV infection.

Background Papillomaviruses induce benign, dysplastic and malignant hyperproliferations of skin or mucosal epithelium. Pfister, Rev. Physiol.

Biochem. Pharmacol., 99:111-181 (1984). According to Nuovo et al., J. Virol, 62:1452-1455 (1988) 51 types (strains) of human papillomavirus (HPV) have been identified.

a ou 0 0 0 e o In humans, different papillomavirus types are known to cause distinct diseases, Pfister, Adv.

Cancer Res., 48:113-147 (1987), Syrjanen, Obstet.

Gynecol. Survey, 39:252-265 (1984). For example, human papillomavirus (HPV) types 1 and 2 cause common warts, and types 6 and li cause condylomas and genital flat warts. In contrast, HPV types 16, 18 and 33 are carried in a majority of cervical cancers and do not cause the usual condyloma but rather persist diffusely in the cervical S-2- I endothelium exhibiting only minimal pathologic changes. It is believed that the HPV types associated with cervical cancer are maintained in a latent state in cervical endothelium tissues for years after initial infection and then progress in jsome cases to cause cervical cancer.

The genome of many of the presently identified HPV types has been cloned and sequenced.

See, for example, Baker, "Sequence Analysis of Papillomavirus Genomes", in The Papovaviridae- Volume 2: The Papillomaviruses, Salzman et al., eds., Plenum Press, New York, pp. 321-386 (1987); and Chow et al., Cancer Cells, 5:55-72 (1987).

o •Historically, the open reading frames o 0 15 (ORFs) of papillomavirus genomes have been 0 0 o0 o0 designated LI and L2 and El to E7, where and 0 denote late and early, respectively. LI, L2 o oo and E4 code for viral capsid proteins and E region 0 00 °o °°ORFs are thought to be associated with functions such as viral replication, transformation and plasmid maintenance. Howley et al., "Molecular Aspects of Papillomavirus-Host Cell Interactions", °o in Viral Etiology of Cervical Cancer, Peto et al., o eds., Banrury Report 21, Cold Spring Harbor S0 0 0 O. 25 Laboratory, pp. 261-272 (1986); and Doorbar et al., EMBO 5:355-362 (1986).

o Presently, there are no papillomavirus- 00 specific antigens that have been unambiguously identified as being either expressed during, or indicative of, latent HPV infection.

This is in contrast to HPV infected tissues where there are actively replicating viruses. In those tissues the presence of some HPV-encoded replication-related antigens viral capsid antigen) has been demonstrated.

Schneider, "Methods of Identification of Human

C.

-3- Papillomaviruses," in Papillomaviruses and Human Disease, Syrjanen et al., eds., Springer-Verlag, pp. 19-39 (1987).

Several studies have reported attempts to identify the protein products of HPV-containing cell lines. Fusion proteins were expressed in Escherichia coli in which various HPV ORF region nucleotide sequences were operatively linked to heterologous genes. The resulting fusion protein product contained a non-HPV amino terminus and part or all of the putative ORF-encoded amino acid residues at the carboxy terminus. The expressed fusion protein was used as an immunogen to raise So" polyclonal antisera, and the sera was then used to a.o 15 detect putative HPV-encoded proteins in vitro in o a .o HPV-containing cell lines.

or instance, Seedorf et al., EMBO J_.,6:139-144 (1987) raised antibodies to a fusion S' protein containing El ORF sequences and detected a 70 kilodalton (kd) protein after in vitro translation of mRNA isolated from HeLa cells containing HPV type 18. Using antisera raised against a fusion protein containing E4 ORF sequences, a 10 kd protein was detected by in vitro translation of mRNA from HPV type 16-containing CaSki cells. Seedorf et al., EMBO 6:139-144 (1987). Similarly, antisera directed against an E6 ORF sequence-derived fusion protein detected an 11 kd protein by in vitro translation of mRNA from HPV type 16-containing CaSki cells. Seedorf et al., EMBO 6:139-144 (1987).

Antisera raised to various fusion proteins that contained E7 ORF sequences have detected several proteins depending on the HPV type studied. In HPV 16 infected cells, a 15 kd protein has been detected using Western immunoblotting and -4radioimmuno-precipitation methodologies using CaSki or SiHa cells as the HPV source. Seedorf et al., EMBO 6:139-144 (1987); and Firzlaff et al., Cancer Cells, 5:105-113 (1987). Smotkin et al., [Proc. Natl. Acad. Sci. USA, 83:4680-4684 (1987)] have described using antibodies raised against an E7 ORF sequence-derived fusion protein to detect a kd protein by immunoprecipitation of HPV type 16-containing CaSki or SiHa cells.

Monoclonal antibodies have been prepared against an E7 ORF-containing fusion protein that detect a 15 kd protein in HPV 16-containing cells by using both Western and immunoprecipitation 4* l j methodologies. Oltersdorf et al., J. Gen. Virol., 15 68:2933-2938 (1987).

Recently, Li et al., Gen Virol., 62:606-609 (1988)] described an antisera raised against an E2 ORF containing fusion protein which Swas used to detect proteins present in primary biopsy tissues known to contain HPV genomic sequences. A 50 kd protein was detected by Western i immunoblotting of lysates from several tissues diagnosed as condylomas and demonstrated by Southern blotting to contain HPV types 6, 11 or 16.

By way of further background, seventeen synthetic polypeptides have been described whose amino acid residue sequences correspond to portions of the HPV type 16 El, E2, E4, E6, or E7 ORFs, or to a portion of the E6 ORF region of HPV type 6.

Schoolnick et al., EPO patent application no.

0257754A2, published March 2, 1988. These polypeptides were used as immunogens to prepare rabbit antisera, and four of the prepared antipeptide antibodies raised against an E6 region of HPV-16 were shown to immunoreact with patient biopsy tissue shown to contain HPV-16 DNA and that i I? Ii o 9 0 O 0 o 000 00 0 0 @0 0a 0 were assessed as having known dysplasias. However, none of the Schoolnik et al. peptide! was demonstrated as having the ability to react as an antigen with antibodies induced as a result of HPV infection.

Brief Summary of the Invention The present invention contemplates a polypeptide represented by a formula selected from the group consisting of:

MADPAGTNGEEGTGC,

HEDEDKENDGDSLPTC,

RPFKSNKSTCC,

CCDWCIAAFGLTPSI,

TYDSEWQRDQFLSQVKIPC,

15 HKSAIVTLTYDSEWQRDQC, and

CINCQKPLCPEEKQRH.

Further contemplated is a polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence having the formula:

-TYDSE-.

Another embodiment contemplates a polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue 25 sequence represented by the formula selected from the group consisting of:

-TGILTVTYHSE-,

-HAIVTVTYDSE-,

-NAIVTLTYSSE-,

NGIVTVTFUTE-, and

-ILTVT-.

Also contemplated is a composition comprising a substantially pure human papillomavirus 54 kd filamentous protein, said protein containing a first epitope having the capacity to immunoreact with anti-polypeptide a ao o 00 0 00 O DO

O

00 4 004 O 0 Clo o OtO 00 00 0

L

I

-6antibodies induced by a polypeptide represented by the formula:

MADPAGTNGEEGTGC;

and containing a second epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polypeptide represented by the formula:

CINCQKPLCPEEKQRH.

Further contemplated is a composition comprising a substantially pure human papillomavirus 48 kd filamentous protein, said protein containing a first epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polypeptide represented by the formula:

MADPAGTNGEEGTGC;

and containing a second epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polypeptide represented by the formula: o 9 9 00 9 9 oc 9 9999 o 9 9 9 0 0 0 0o o 25

CINCQKPLCPEEKQRH.

Another aspect is composition comprising a substantially pure human papillomavirus 112 kd diffuse protein, said protein containing a first epitope having the capacity to immunoreact with anti-peptide antibodies induced by a polypeptide represented by the formula:

HEDEDKENDGDSLPTC;

and containing a second epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polypeptide represented by the formula:

Q

oa** a

HKSAIVTLTYDSEWQRDQC.

A further aspect is a composition comprising a substantially pure human papillomavirus 51 kd nuclear protein, said protein 15 o 0 o 0 020 0 0 0 o 00 0 a0 S 35 0o 0 0 0 00 0 containing an epitope having the capacity to immunoreact with anti-peptide antibodies induced by a polypeptide represented by the formula:

HKSAIVTLTYDSEWQRDQC.

Also contemplated is an anti-polypeptide antibody that immunoreacts with only one of the polypeptides selected from the group consisting of:

MADPAGTNGEEGTGC,

HEDEDKENDGDSLPTC,

RPFKSNKSTCC,

CCDWCIAAFGLTPSI,

TYDSEWQRDQFLSQVKIPC,

HKSAIVTLTYDSEWQRDQC, and

CINCQKPLCPEEKQRH.

Further contemplated is a monoclonal antibody containing antibody molecules that immunoreact with a human papillomavirus latent protein selected from the group consisting of: i) the 112 kd diffuse protein, ii) the 54 kd filamentous protein, iii) the 48 kd filamentous protein, iv) the 51 kd nuclear protein; and v) the 58 kd nuclear protein.

In another aspect the present intention contemplates an antibody containing substantially isolated or substantially pure antibody molecules that immunoreact with a human papillomavirus latent protein selected from the group consisting of: i) the 112 kd diffuse protein, ii) the 54 kd filamentous protein, iii) the 48 kd filamentous protein, iv) the 51 kd nuclear protein; and v) the 58 kd nuclear protein.

Further contemplated are antibody and monoclonal antibody molecules that immunoreact with the polypeptides of the present invention, in i -8addition to compositions containing the contemplated polypeptides or contemplated antibody molecules.

Diagnostic systems, in kit form, containing, is an amount sufficient to perform at least one assay, one or more of the above described polypeptides, protein compositions and antibodies are also contemplated.

Methods for assaying for the presence of papillomavirus infection and type of papillomavirus present using the above described polypeptides, protein compositions and antibodies are further contemplated.

Brief Description of the Drawings Figure 1 shows a schematic representation of the open reading frames (ORFs) deduced from the nucleotide sequence of HPV type 16. Using the numbering system of the HPV type 16 nucleotide C, 4sequence described by Seedorf et al., Virol, O 145-181-185 (1985), which disclosure is hereby incorporated by reference, the ORFs shown in Figure 1 include the nucleotide sequences contained in that disclosure for each ORF as follows: 0 44 ORF Nucleotide Sequence Included E6 65 556 E7 544 855 0 04 Ela 859 1167 Elb 1104 2810 E2 2725 3849 E4 3332 3616 3862 4096 L2 4133 5653 Li 5526 7151 -9- The translational phase of the ORF is indicated by the designation on the left wherein "R1" indicates phase 1, "R2" indicates phase 2 and "R3" indicates phase 3. A scale measured in nucleotide kilobases (kb) is located below the ORFs to indicate their relative positions.

Figure 2 illustrates an immunoblot analysis of human papillomavirus latent proteins present in HPV-containing tissue cultures and biopsy tissue samples. Cell lysates were prepared, electrophoresed in 7.5% polyacrylamide gels and immunoblotted as described in Example 5 using labbit anti-polypeptide 236 antisera.

S o 15 Lanes 1 through 4 show the results S oo obtained using cell lysates prepared from the o t cervical carcinoma cell lines CaSki, HeLa, SiHa and So C-33a, respectively. This antisera immunoreacts A with an 112 kilodalton (kd) protein present in HeLa and SiHa ce-ls, and also immunoreacts nonspecifically with a protein having a molecular weight of about 70,000 present in all cell lysates oO" analyzed (lanes 1-4, 6, Lane 5 contains the S following protein standards electrophoresed as 25 markers having the following molecular weights indicated in kd; lysozyme, 14.4 kd.; trypsin inhibitor, 21.5 kd; carbonic anhydrase, 31 kd; ovalbumin, 42.7 kd; bovine serum albumin, 66.2 kd; o* phosphorylase b, 97.4 kd; beta-galactosidase, 116.25; and myosin, 200 kd. Lanes 6 and 7 show the results obtained using cell lysates prepared from two different condyloma biopsy tissue samples. One condyloma biopsy lysate (lane 6) contains both the 54 kd and 46 kd filamentous proteins, whereas the other condyloma biopsy lysate contains only the 54 kd species.

I i W~™sW^^ o o e a o oo tO r a S0 Q O 00 0 BO oD a Figure 3 illustrates a type-specific immmunoblot analysis of human papillomavirus latent proteins present in HPV-containing cervical carcinoma cells. Cell lysates were prepared, electrophoresed in 7.5% polyacrylamide gels and immunoblotted as described in Example 5 using rabbit anti-polypeptide 236 antisera. In addition to all the non-specific proteins detectable using the polyclonal antisera, the 58 kd and 54 kd filamentous proteins were detected in cell lysates prepared from CaSki cells (lane 2) but not in cell lysates prepared from HeLa cells (lane 1).

Although not shown, the same molecular weight marker proteins as described for Figure 2 were 15 included on the immunoblot, which inclusion provided a means to determi;e the molecular weights of the observed proteins.

Figure 4 illustrates an immunoblot analysis of human papillomavirus latent proteins present in HPV-containing cervical carcinoma cells.

Cell lysates were prepared, electrophoresed in polyacrylamide gels and immunoblotted as described in Example 7 using monoclonal antibody 247:4D11.

Lane 1 contains the same molecular weight marker proteins as described in Figure 2. Lanes 2 through show the results obtained using cell lysates prepared from cervical carcinoma cell lines SiHa, HeLa, CaSki and HT-3, respectively. HPV latent proteins detected in CaSki cells include the 58 kd, 54 kd and 48 kd filamentous proteins (lane 4), whereas only the 54 kd protein was detected in HeLa cells. All the other proteins detected are nonspecific immunoreaction products observed when using monoclonal antibody 247:4D11.

Figure 5 illustrates an immunoblot analysis of n papillomavirus latent proteins -o 0 L'e 0 *040 I i 1 i I S r -11a* a09 o g 00 0 000 BO0 00 0 B 00 00 B B 00D 0a 0 1' 0a 0,0 B prese:'- in HPV-containing cervical Pr=cinoma cells.

Cell lysates were prepared, electropaioresed in 7% polyacrylamide gels and immunoblotted as described in Example 16a using human anti-HPV latent protein antibody molecules affinity isolated on polypeptide 245 (Panel hybridoma 245:11E3 culture supernatant (Panel or rabbit affinity isolated anti-polypeptide 245 antibody molecules (Panel C).

Each cell lysate analyzed is listed at the top of its respective gel lane. Numerals at the right and left margins of the figure denote the molecular weight in kilodaltons (kd) of the major immunoreacting species, 58 kd and 48 kd.

Arrowheads indicate the positions of the marker 15 proteins having a molecular weight of 200, 116, 92, 66, 44 and 31 kd, respectively. The left part of Panel A and the entire Panel B were developed for 12 hours; whereas the right part of Panel A and the entire Panel C were developed for 30 minutes.

Detailed Description of the Invention A. Definitions Amino Acid: All amino acid residues identified herein are in the natural Lconfiguration. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3557-59, (1969), abbreviations for amino acid residues are as shown in the following Table of Correspondence: 0 BQ o~ oa 060 0 O 0 0 L t- -12- TABLE OF CORRESPONDENCE

SYMBOL

1-Letter 3-Letter Y Tyr I G Gly c F Phe L- M Met A Ala S Ser I Ile L Leu T Thr V Val P Pro K Lys H His Q Gin E Glu L-c W Try R Arg D Asp L- N Asn C Cys AMINO ACID L-tyrosine glycine -phenylalanine L-methionine L-alanine L-;-erine L-isoleucine L-leucine L-threonine L-valine L-proline L-lysine L-histidine L-glutamine glutamic acid L-tryptophan L-arginine aspartic acid L-asparagine L-cysteine o0 o 0 0 0 o 0 00 0 0 00 4 6 0000 0 0000 0 00 0 co o 0 0 0 0 0 0 It should be noted that all amino acid residue sequences are represented herein by formulae whose left to right orientation is in the conventional direction of amino-terminus to carboxy-terminus.

Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a bond to a further sequence of one or more amino acid residues up to a total of about fifty residues in the polypeptide chain.

-13- Polypeptide and Peptide: Polypeptide and peptide are terms used interchangeably herein to designate a linear series of no more than about amino acid residues connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

Protein: Protein is a term used herein to designate a linear series of greater than 50 amino acid residues connected one to the other as in a polypeptide.

B. Papillomavirus Latent Proteins Papillomavirus infections can result in the virus being maintained in the infected tissue in a latent state. As it is presently understood for human papilloma viruses (HPVs), viral latency occurs for those HPV types associated with genital papillomavirus infection, particularly those which cause various dysplasias such as cervical cancer.

*Ga Dysplasia-associated HPV types include types 16, 0 0 18, 31 and 33, 35, 52 and the like.

Prior to the making of the present o* o invention, the presence of papillomavirus genome E region ORF-encoded proteins had not been detected in papillomavirus-infected tissues that maintained 4 o 25 the virus in a latent state. It is now demonstrated herein that papillomavirus specific proteins are expressed in infected tissues 0! harboring the virus in a latent, non-replicative e state.

Broadly, therefore, one embodiment of the present invention contemplates a papillomavirus latent protein in substantially pure form. As used herein, the phrases "papillomavirus latent protein," "latent papillomavirus protein" and the like refer to a protein encoded by a HPV E ORF that is expressed in tissue latently infected with HPV.

~~i~Yn~j~r i -14- Tissues latently infected with HPV r-ntain HPV genomic material but do not contain HPV viral capsid antigen at levels detectable by immunologic methods.

1. The Papillomavirus Filamentous Latent Proteins HPV-infected cells that maintain the virus in a latent state are now known to produce a papillomavirus-specific filamentous protein a protein found associated with filamented components of the cell) of about 54 kilodaltons (kd) in molecular weight when measured by polyacrylamide gel electrophoresis in the presence

S

i Q* of sodium dodecyl sulfate (SDS-PAGE) as described in Example 5. For instance, the 54 kd filamentous S protein is detectable in CaSki and SiHa cells as described in Example 5. CaSki and SiHa cells are cervical carcinoma-derived cell lines that contain OS 4 HPV type 16 and are available from the American Type Culture Collection (ATCC; Rockville, MD) as CRL 1550 and HTB 35, respectively.

I 6 The 54 kd filamentous protein is further O 0. characterized as possessing epitopes that are immunologically cross-reactive with the HPV S 25 polypeptides 235 and 247 as shown in Table 1. That is, the 54 kd filamentous protein contains amino acid residue sequences homologous to the sequences 0o V of polypeptides 235 and 247.

Lo Tissue latently infected with HPV also produces a filamentous protein of 48 kd, as determined by SDS-PAGE. The 48 kd filamentous protein is detectable in CaSki cells using the immunoblotting method described in Example The 48 filamentous protein is further characterized as possessing epitopes that are immunologically cross-reactive with polypeptides

II

235, 247 and 245, and thus contains amino acid residue sequences homologous to those polypeptides.

2. The Papillomavirus Latent Nuclear Protein Cells latently infected with HPV expressed a HPV specific protein of about 51 kd as measured by SDS-PAGE, that is detectable in the nucleus of Caski cells. The 51 kd nuclear protein is further characterized as possessing an epitope that is immunologically cross-reactive with polypaptide 245 derived from the amino acid residue sequence of the E2 ORF internal region.

lbo Cells latently invected with HPV express 0 a 15 additional nuclear proteins of 26 kd, 48 kd and 58kd as determined by SDS-PAGE.

So0 The 26 kd, 48 kd and 58 kd proteins are detectable in HPV infected cells using the o* o immunoblotting method described in Example 16a, and are furthe characterized as possessing epitopes that are immunologically cross-reactive with 00o polypeptide 245.

o" 3. The Papillomavirus Latent Diffuse Protein 0 «a 00 0 4 OO 0 0 25 The diffuse protein is a papillomavirus latent protein having an apparent molecular weight of about 112 rd when measured by SDS-PAGE as described in Example 5. The 112 kd diffuse protein is detectable in HeLa and SiHa cells using the immunoblotting method also described in Example HeLa cells are cervical carcinoma tissue culture cells that contain HPV type 18 and are available from the ATCC as CCL2.

The 112 kd diffuse protein is further characterized as possessing epitopes that are immunologically cross-reactive with polypeptides _i l-rrll~--3l~i3L~ -16- 236, 245, 235, 238 and 247 derived from the amino acid residue sequence of the El ORF internal region, the E2 ORF internal region, the Ela ORF amino terminal region, the El ORF internal region and the E6 ORF internal region, respectively.

The various species of latent papillomavirus proteins described hereinabove are useful in substantially pure form as proteinaceous immunogens in an inoculum of the present invention or as antigens in a diagnostic system of the present invention.

Thus, the present invention contemplates each of the above described filamentous, nuclear and diffuse papillomavirus latent proteins in S' 15 substantially pure form. By "substantially pure lt form" is meant that the particular HPV latent I ,C protein is present in a composition that is substantially free of other papillomavirus-related .proteins.

Methods for producing a characterized protein in substantially pure form are well known in the art. Typically, those methods include 4 4 isolating the protein from cells containing the protein using well known biochemical techniques.

For instance, the methods of gel filtration, gel chromatography, ultrafiltration, electrophoresis, ion exchange, affinity chromatography and the like, Ssuch as are known for protein fractionations, can ti be used to isolate the papillomavirus latent proteins found in latently infected HPV containing cultures. Because each of the latent proteins described herein are characterized in part by their immunologic cross-reactivity to defined polypeptides, immunochemical purification methods, such as immunoaffinity, immunoadsorption and the like, are particularly well adapted to producing -r I i: t9 -17the latent proteins in substantially,pure form.

Preferably, the composition is also sdbstantially free of entities such as ionic detergents, e.g., sodium dodecyl sulfate (SDS), polyacrylamide and tissue or cell culture-derived proteins haying an apparent molecular weight of less than about 40 kd as determined by SDS-PAGE.

C. Polypeptides A polypeptide of the present invention contains no more than about 50, more usually fewer than about 35 and preferably fewer than about amino acid residues, and contains at least about residues. In addition, a polypeptide of the present invention is characterized by its amino acid residue sequence and novel functional "j properties.

o Amino acid residues present in a polypeptide of the invention in addition to a sequence specifically enumerated hereinafter up to a total of no more than about 50 amino acid residues can be any residues that do not materially o affect the basic and novel characteristics of a polypeptide as are discussed hereinafter. Such additional residues are usually added to one or o 025 both termini of an enumerated polypeptide and can include repeats and partial repeats of an enumerated polypeptide sequence.

o0 o Broadly, the present invention 0 contemplates a polypeptide that includes an amino o t acid residue sequence capable of producing (inducing) antibody molecules that immunoreact with a papillomavirus latent protein. Preferably a polypeptide of this invention immunoreacts with antibodies induced by a latent papillomavirus infection, anti-latent papillomavirus protein antibodies. Further, the polypeptide contains an 4 -L -I I -qC I- -CL I I i_ 1111 1~~ 3 -18amino acid residue sequence that corresponds to a portion of the amino acid residue sequence deduced from the nucleic acid sequence of those open reading frame (ORF) regions of the papillomavirus genome known to encode latent papillomavirus proteins.

It should be understood that a polypeptide of the present invention need not be identical to the amino acid residue sequence of a latent papillomavirus protein, so long as it is able to produce, upon immunization, an antisera that contains antibody molecules that immunoreact with a latent papillomavirus protein. Preferably, the subject polypeptide is able to immunoreact with 15 antibodies induced by a latent papillomavirus infection. Therefore, a polypeptide of the present invention can be subjected to various changes, such as insertions, deletions and substitutions, either conservative or non-conservative, where such 20 changes provide for certain advantages in their use.

4: 4 44 it 4 4 I c 4 Ir C 44'' 4 Ii IC 4(1 1 t e 25 t nc t( <a 0 4e Conservative substitutions are those where one amino acid residue is replaced by another, biologically similar residue. Examples of conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another such as between arginine and lysine, between glutamic and aspartic acids or between glutamine and asparagine and the like. The term "conservative substitution" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that such a polypeptide also displays the requisite antibody inducing activity.

I'- j -19- When a polypeptide of the present invention has a sequence that is not identical to the sequence of a latent papillomavirus protein because one or more conservative or nonconservative substitutions have been made, usually no more than about 20% and more usually no more than 10% of the amino acid residues are substituted, except where additional residues have been added at either terminus for the purpose of providing a "linker" by which the polypeptides of this invention can be conveniently affixed to a label or solid matrix, or antigenic carrier.

Labels, solid matrices and carriers that can be used with the polypeptides of this invention are S. 15 described hereinafter.

Amino acid residue linkers are usually at least one residue and can be 40 or more residues, f more often 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, t 20 lysine, glutamic and aspartic acid, or the like.

In addition, a polypeptide sequence of this r invention can differ from the natural sequence by the sequence being modified by terminal-NH 2 Sacylation, acetylation, or thioglycolic acid amidation, terminal-carboxlyamidation, e.g., S" ammonia, methylamine, etc.

The peptides of the invention can contain at least one cysteine residue, and in certain S' instances two of such residues. Accordingly, the subject peptides can exist in various oxidative forms. In addition to the monomeric form in which the sulfhydryl group of the cysteine residue(s) is reduced, there can also exist dimeric or polymeric forms in which sulfhydryl groups on two or more peptide molecules become oxidized and form interand intrapeptide disulfide bonis. While subject A further aspect is a composition comprising a substantially pure human papillomavirus 51 kd nuclear protein, said protein r: I:

UI

peptides that possess only one cyste-ne residue can form only linear dimers, those that possess two cysteine residues can form cyclic monomers or linear or cyclic dimers and linear polymers of various lengths. These various oxidative forms are considered part of the subject invention and are included in the terms "polypeptides" and "peptides".

When coupled to a carrier via a linker to form what is known in the art as a carrier-hapten conjugate, a polypeptide of the present invention is capable of inducing antibodies that immunoreact with a latent papillomavirus protein when said protein is present in a sample that contains a 15 latent papillomavirus infection. Representative immunoreactions between a latent papillomavirus protein and antibodies that were induced using polypeptides of the present invention are described in Example a 0 4I 0 #0.

10#4 0 0 S 20 0 0 00 0 q 0 0 A 0 0 4 2 In view of the well established principle of immunologic cross-reactivity, the present invention therefore contemplates antigenically related variants of the polypeptides of the .present invention. An "antigenically related variant" is a polypeptide that includes at least a six amino acid residue sequence portion of a latent papillomavirus protein and which is capable of inducing antibody molecules that immunoreact with a latent papillomavirus protein when said protein is present in a sample that contains a latent papillomavirus infection.

A polypeptide of the present invention can be synthesized by any of the techniques that are known to those skilled in the polypeptide art, including recombinant DNA techniques. Synthetic chemistry techniques, such as a solid-phase

-I

Further contemplated are antibody and monoclonal antibody molecules that immunoreact with the polypeptides of the present invention, in i and the like. An excellent summary of the many techniques available can be found in J.M. Steward ,and J.D. Yound, "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969; M.

-21- Bodanszky, et al., "Peptide Synthesis", John Wiley Sons, Second Edition, 1976 and J. Meienhofer, "Hormonal Proteins and Peptides", Vol. 2, p. 46 W.H. Freeman Co., San Francisco, 1969; Academic Press (New York), 1983 for solid phase peptide synthesis, and E. Schroder and K. Kubke, "The Peptides", Vol. 1, Academic Press (New York), 1965 for classical solution synthesis, each of tt 't 15 which is incorporated herein by reference.

SAppropriate protective groups usable in such synthesis are described in the above texts and in J.F.W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, New York, 1973, which is incorporated herein by reference.

Preferred polypeptides of the present i .invention are deduced from the nucleotide sequence of the El, E2 or E6 ORFs of papillomaviruses, preferably HPVs.

More preferably, polypeptides of the present invention are deduced from the nucleotide Ssequence of specific HPVs known to cause genital papilloma virus infections, including HPV types 6, 11, 16, 18, 33, 35, 52 and the like.

1. HPV Type 16-Related Polypeptides A HPV type 16-related polypeptide of the present invention contains at least 5, preferably at least 12, amino acid residues and includes a sequence that corresponds to portions of the amino acid residue sequence deduced from the El, E2 or E6 ORFs of HPV type 16 as shown in Figure 1.

i IIYI t~ I -i -I -i -Irl -22- *1 SC U U 1.F Ui Preferably, the HPV Type-16 related polypeptides of this invention do not contain the amino acid residue sequence WRQRDQFLSQV.

Preferred polypeptides of the present invention include those whose amino acid residue sequences are shown in Table 1.

Table 1 Human Papillomavirus Polypeptides

POLYPEPTIDE

DESIGNATION ORF 1

SEQUENCE

Ela El El El E2 E2 E6 AMINO ACID RESIDUE 235 236 238 246 237 245 247

MADPAGTNGEEGTGC

HEDEDKENDGDSLPTC

RPFKSNKSTCC

CCDWCIAAFGLTPSI

TYDSEWQRDQFLSQVKIPC

HKSAIVTLTYDSEWQRDQC

CINCQKPLCPEEKQRH

1 The nomenclature for open reading frames (ORFs) corresponds to the ORFs shown in Figure 1 from which the polypeptide amino acid sequence was derived.

A preferred HPV type 16-related polypeptide contains an amino acid residue sequence that corresponds to a portion of the amino acid sequence deduced from the E2 ORF of HPV type 16 and includes an amino acid residue sequence represented by the formula -TYDSE-. Preferably, the included sequence is represented by the formula -LTYDSE-.

More preferably, a HPV type 16-related polypeptide of the present invention is one defined by the amino acid residue sequence represented by -23the formula:

-BYDSBI-;

wherein B is at least one of the following sequence of amino acid residues:

-SAIVTLT,

SAIVTLT,

AIVTLT,

IVTLT,

VTLT,

TLT,

LT, or T; and wherein B' is at least one of the following sequence of amino acid residues: or

E.

*0000 0.00 000 Ot.

W0 0 0cc A more preferred HPV type 16-related polypeptide of the present invention includes an amino acid residue sequence represented by the 20 formula -SAIVTLTDYSE- or -HKSAIVTLTDYSE-.

Still more preferred is a HPV type 16related polypeptide defined by the amino acid residue sequence represented by the formula: -BKSAIVTLTYDSB' wherein B is at least one of the following sequence of amino acid residues:

SSTWHWTGHNVKH,

S TWHWTGHNVKH, 30

TWHWTGHNVKH,

WHWTGHNVKH,

HWTGHNVKH,

WTGH-NVKH,

TGHNVXI{,

GHNVKH,

HNVKH,

0 00 0 C 9 0 0 0 using monoclonal antibody 247:4D11.

Figure 5 illustrates an immunoblot analysis of h. in papillomavirus latent proteins -24-

NVKH,

VKH,

KH, or H; and wherein B' is at least one of the following sequence of amino acid residues:

EWQRDQ,

EWQRD,

EWQR,

EWQ,

EW, and

E.

In a related embodiment, a HPV type 16oo o. related polypeptide contains an amino acid residue 0 15 sequence that corresponds to a portion of the amino acid sequence deduced from the E2 ORF of HPV type 0 04 4 16 and includes at least one of the following amino acid residue sequences o a

-HKSAIV-,

-SAIVTL-, and

-IVTLTD-.

•y Preferred HPV type 16-related 4' polypeptides contain no more than about 30 amino acid residues, have as a part of their amino acid •i 25 residue sequence at least one of the following sequences:

-IVTLTD-,

-SAIVTL-,

-HKSAIV-,

0

-HKSAIVTLTDYSE-,

-SAIVTLTDYSE-,

-LTDYSE-, and -TDYSE-; and are homologous, preferably without insertion or deletion, and more preferably are identical, to a portion of the I-PV type 16 sequence represented by the formula:

-SSTWHWTGHNVKHKSL.IVTLTYDSEWQRDQ.

Preferred specific HPV type 16-related polypeptides include those whose amino acid residue sequences are shown in Table 2.

Table 2

POLYPEPTIDE

DESIGNATION AMINO ACID RESIDUE SEQUENCE 66 SSTWHWTGHNVKHKSAIVTLTYD 71 HKSAIVTLTYDS EWQRDC 72 HKSAIVTITYDSEWQRC 015 73 HKSAIVTLTYDSEWQC Gof.74 HKSAIVTLTYDSEWC

HKSAIVTLTYDSEC

00 0 78 HI(SAIVTLTYC 79 KSAIVTLTYDSEWQRDQC

SAIVTLTYDSEWQRDQC

o a81 AIVTLTYDSEWQRDQC 6*82

IVTILTYDSEWQRDQC

83 VTLTYDSEWQRDQC 84TTDSWR6 84 TLTYDSEWQRDQC Another preferred HPV type 16 related 6030 polypeptide of the present invention is as a polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence represented by the formula: xzxg, wherein Z is an amino acid residue sequence containing at least 5 amino acid residues having a -26sequence corresponding to a portior, pf the sequence represented by the formula:

HKSAIVTLTYDSE,

wherein X is hydrogen or at least one amino acid residue, and wherein X' is hydroxyl or at'least one amino acid residue, said polypeptide being capable of immunoreacting with anti-HPV latent protein antibodies.

Another preferred HPV type 16-related polypeptide is as a polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence having the formula:

XTYDSEX',

wherein X is hydrogen or at least one or more amino acid residue, and wherein X' is hydroxyl or at .least one amino acid residue with the proviso that X' does not include the amino acid residue sequence WQRDQFLSQV. In one embodiment, X' is an amino acid residue sequence represented by a formula selected from the group consisting of:

W,

i

WQ,

opV" WQR, WQRD, and a 25 WQRDQ.

In a preferred embodiment, X' is an amino acid residue sequence represented by a formula selected 0 o from the group consisting of: °OP WQRDQF, and

WQRDQFL.

In another preferred embodiment X' is an amino acid residue sequence represented by a formula selected from the group consisting of: WQRDQFLS, and

WQPDQFLSQ.

-27- Still another way of defining a preferred HPV type 16-related polypeptide is as a polypeptide comprising no more than about 50 amino acid residues and including at least one of the following amino acid residue sequences:

-TDYSE-,

-LTDYSE-,

-SAIVTLTDYSE-,

-HKSAIVTLTDYSE-,

-HKSAIV-,

-SAIVTL-, and -IVTLTD-; and wherein said polypeptide does not contain the amino acid residue sequence WRQRDQFLSQV.

15 2. HPV Type 6-Related Polypeptides A HPV type 6-related polypeptide of the S, present invention contains an amino acid residue sequence that corresponds to a portion of the amino I acid residue sequence deduced from the E2 ORF of j 20 HPV type 6 and includes an amino acid residue sequence represented by the formula -HAIVTVTYDSE-.

^A preferred HPV type 6-related polypeptide has an amino acid residue sequence represented by the formula HKHAIVTVTYDSEEQRQQC.

3. HPV Type 11-Related Polypeptides A HPV type 11-related polypeptide of the present invention contains an amino acid residue Ssequence that corresponds to a portion of the amino acid residue sequence deduced from the E2 ORF of HPV type 11 and includes an amino acid residue sequence represente by the formula -NAIVTLTYSSE-.

A preferred HPV type 11-related polypeptide has a amino acid residue sequence represented by the formula HKNAIVTLTYSSEEQRQQC.

;I 1. i' ii -I i IEP I I~ I_ _II -28- 4. HPV Type 18-Related Polypeptides A HPV type 18-related polypeptide of the present invention contains an amino acid residue sequence that corresponds to a portion of the amino acid residue sequence deduced from the E2 ORF of HPV type 18 and includes an amino acid residue sequence represented by the formula -ILTVT-, and more preferably includes a sequence represented by the formula -TGTLTVTYHSE-.

A preferred HPV type 18-related polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: 4@ 44 I :O 4 I I I 4 I It j (f 44* 4

I

Ii I EKTGILTVTYHSETQRTKC, and

NEKTGILTVTYHSETQRTKC,

5. HPV Type 33-Related Polypeptides A HPV type 33-related polypeptide of the present invention contains an amino acid residue sequence that corresponds to a portion of the amino 20 acid residue sequence deduced from the E2 ORF of HPV type 33 and includes an amino acid residue sequence represented by the formula -NGIVTVTFVTE-.

A preferred HPV type 33-related polypeptide has an amino acid residue sequence 25 represented by the formula SKNGIVTVTFVTEQQQQMC Preferred HPV related polypeptides deduced from the E2 ORF of HPV types 6, 11, 18 and 33 are shown in Table 3.

I I -29- Table 3 Polypeptide HPV Amino Adid Designation Type Residue Sequence 6 HKHAIVTVTYOSEEQRQQC K71 11 HKNAIVTLTYSSEEQRQQC K69 18 EKTGILTVTYHSETQRTRC K68 18 NEKTGILTVTYHSETQRTRC K72 33 SKNGIVTVTFVTEQQQQMC The present invention also contemplates a composition containing a polypeptide of the present invention admixed in a physiologically tolerable diluent. Such compositions typically contain the 15 polypeptide at a concentration in the range of micromolar to molar, preferably millimolar.

In addition, the present invention 9 .4 L contemplates fusion proteins, and composition o thereof, comprising a polypeptide of the present invention operatively linked (fused) to at least one amino acid residue sequence, wherein said sequence is heterologous to a sequence deduced from 44 A a papillomavirus latent protein ORF.

D. Inocula s o 25 In another embodiment, a polypeptide of this inventicn, an antigenically related variant thereof or a substantially pure papillomavirus g latent protein of this invention is used in a .o pharmaceutically acceptable aqueous diluent composition to form an inoculum that, when administered in an effective amount, is capable of inducing antibodies that immunoreact with papillomavirus latent protein.

The word "inoculum" in its various grammatical forms is used herein to describe a composition containing a polypeptide or a .lr 4( 4 .4 4 4 44 44 4

C(

4 1 4 L.4 4 II 4 b 4 substantially pure papillomavirus latent protein of this invention as an active ingredient used for the preparation of antibodies against a papillomavirus latent protein.

When a polypeptide is used to induce antibodies it is to be understood that the polypeptide can be used alone, or linked to a carrier as a conjugate, or as a polypeptide polymer, but for ease of expression the various embodiments of the polypeptides of this invention are collectively referred to herein by the term "polypeptide", and its various grammatical forms.

For a polypeptide that contains fewer than about 35 amino acid residues, it is preferable to use the peptide bound to a carrier for the purpose of inducing the production of antibodies as already noted.

As previously noted, one or more additional amino acid residues can be added to the amino- or carboxy-termini of the polypeptide to assist in binding the polypeptide to a carrier.

Cysteine residues added at the amino- or carboxytermini of the polypeptide have been found to be particularly useful for forming conjugates via 25 disulfide bonds. However, other methods well known in the art for preparing conjugates can also be used. Exemplary additional linking procedures include the use of Michael addition reaction products, di-aldehydes such as glutaraldehyde, Klipstein et al., J. Infect. Dis., 147, 318-326 (1983) and the like, or the use of carbodiimide technology as in the use of a water-soluble carbodiimide to form amide links to the carrier.

For a review of protein conjugation or coupling through activated functional groups, see Aurameas, a a -31et al., Scand. J. Immunol., Vol. 8, Supp-l. 7, 7- 23 (1978).

Useful carriers are well known in the art, and are generally proteins themselves.

Exemplary of such carriers are keyhole limpet hemocyanin (KLH), edestin, thyroglobulin, albumins such as bovine serum albumin (BSA) or human serum albumin (HSA), red blood cells such as sheep erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well as polyamino acids such as poly (D-lysine: D-glutamic acid), and the like.

The choice of carrier is more dependent upon the ultimate use of the inoculum and is based upon criteria not particularly involved in the S' 15 present invention. For example, a carrier that does not generate an untoward reaction in the particular animal to be inoculated should be selected.

1 The present inoculum contains an effective, immunogenic amount of a polypeptide or latent papillomavirus protein of this invention, and for a polypeptide it is typically as a conjugate linked to a carrier. The effective amount of polypeptide or protein per unit dose depends, among other things, on the species of animal inoculated, the body weight of the animal and the chosen inoculation regimen as is well known in the art. Inocula typically contain polypeptide or protein concentrations of about 10 micrograms to about 500 milligrams per inoculation (dose), preferably about 50 micrograms to about milligrams per dose.

The term "unit dose" as it pertains to the inocula of the present invention refers to physically discrete units suitable as unitary dosages for animals, each unit containing a r_ I- -32predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent; carrier, or vehicle. The specifications for the novel unit dose of an inoculum of this invention are dictated by and are directly dependent on the unique characteristics of the active material and the particular immunologic effect to be achieved, and the limitatioi.s inherent in the art of compounding such active material for immunologic use in animals, as disclosed in detail herein, these being features of the present invention.

Inocula are typically prepared from the S'e* 15 dried solid polypeptide-conjugate by dispersing the polypeptide-conjugate in a physiologically 4 tolerable (acceptable) diluent or vehicle such as o O water, saline or phosphate-buffered saline to form a an aqueous composition. Similarly, inocula o oo 0 20 containing latent papillomavirus protein are typically prepared from substantially pure latent papillomavirus protein by dispersion in the same physiologically tolerable diluents. Such diluents S* are well known in the art and are discussed, for S 25 example, in Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing Company, Easton, PA (1980) at pages 1465-1467.

a 00 Inocula can also include an adjuvant as a 0 S. part of the diluent. Adjuvants such as complete S0 30 Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum are materials well known in the art, and are available commercially from several sources.

E. Antibodies and Anti- Polypeptide Antibodies The term "antibody" in its various ici- i- i i l- *n 84 .4 00 o 0 40 0 0 0 4 0 O S 8 a 0 0 0oo 0 0 6 -33grammatical forms is used herein to refer to a composition containing a population of immunoglobulin molecules and/or immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antibody combining site or paratope.

An "antibody combining site" is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that specifically binds (immunoreacts with) antigen.

The phrase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule.

Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contain the paratope, including those portions known in the art as Fab, Fab', F(ab') 2 and F(v).

Fab and F(ab') 2 portions of antibodies are prepared by the proteolytic reaction of papain and 25 pepsin, respectively, on substantially intact antibodies my methods that are well known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous and Dixon. Fab' antibody portions are also well known and are produced from F(ab') 2 portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules are preferred, and are utilized as illustrative herein.

-i I -ii- iii wei -34- The term "immunoreact" in its various forms means binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.

An immunoreaction forms an immunoreaction product that contains an antibody combining site and the bound antigenic determinant. An immunoreaction is substantial if the binding results in the production of an amount of immunoreaction product that is measurable by methods such as ELISA, immunoblotting, immunostaining or the like as described herein.

S "Antigenic determinant" refers to the actual structural portion of the antigen that is Simmunologically bound by an antibody combining site. The terms is also used interchangeably with ,i "epitope".

*Vo° An antibody of the present invention is characterized as containing substantially isolated or substantially pure antibody molecules that so I immunoreact with one of the following 0 a papillomavirus latent proteins: a) the 112 kd diffuse protein; So 25 b) the 54 kd filamentous protein, c) the 48 kd filamentous protein, d) the 51 kd nuclear protein, i e) the 58 kd nuclear protein, f) the 26 kd nuclear protein, or g) the 48 kd nuclear protein.

By "substantially isolated" is meant that at least about 10%, preferably at least about and more preferably at least about 50%, of the antibody molecules present in the antibody are directed against a papillomavirus latent protein or papillomavirus related polypeptide.

WN ,i ~I l sequence that corresponds to portions of the amino acid residue sequence deduced from the El, E2 or E6 ORFs of HPV type 16 as shown in Figure 1.

By "substantially pure" is meant that at least preferably at least 10%, and more preferably at least 50%, of the protein present in the antibody is protein molecules that form antibody combining sites.

In preferred embodiments, a contemplated antibody does not immunoreact with: a) a 70 kd protein present in HeLa cells, b) a 20 kd protein present in CaSki cells, c) a 15 kd protein present in CaSki cells, d) a 11 kd protein present in CaSki cells, or e) a 10 kd protein present in CaSki 0 C cells.

oaC, In another embodiment, the present invention contemplates an anti-polypeptide antibody a 20 containing antibody molecules that immunoreact with a polypeptide, and preferably only one o polypeptide, of the present invention, and at C C least one of the papillomavirus latent proteins selected from the group consisting of: Co 25 a) the 112 kd diffuse protein; b) the 54 kd filamentous protein; c) the 48 kd filamentous protein; So1. d) the 51 kd nuclear protein; and C e) the 58 nuclear protein.

In preferred embodiments, a contemplated anti-polypeptide antibody does not substantially immunoreact With: a) a 70 kd protein present in HeLa cells, b) a 20 kd protein present in CaSki cells, JV a= L JLUJ.LA U XLQUJC More preferably, a HPV type 16-related polypeptide of the present invention is one defined by the amino acid residue sequence represented by L~

F

-36- 00 4 o ao r o o06Q o 09 0 o a o 00 0 00 0 0 0 0 0 o 0o 00 0 o 0 o a c) a 15 kd protein present in CaSki cells, d) a 11 kd protein present in CaSki cells, or e) a 10 kd protein present in CaSki cells; More preferred is a polyclonal antipolypeptide antibody wherein the antibody molecules immunoreact with a polypeptide, preferably only one of the polypeptides, selected from the group consisting of: liADPAGTNGEEGTGC,

HEDEDKENDGDSLPTC,

RPFKSNKSTCC,

CCDWCIAAFGLTPSI,

TYDSEWQRDQFLSQVKIPC,

HKSAIVTLTYDSEWQRDQC, and CIN QKPLCPEEKQRH.

Still further preferred are anti- 20 polypeptide antibodies prepared by immunizing a non-human mammal, such as a goat, horse, rabbit and the like. Exemplary anti-polypeptide antibodies are those prepared in rabbits, designated herein as anti-235, anti-236, anti-237, anti-238, anti-245, 25 anti-246 and anti-247.

An antibody of the present invention is typically produced by immunizing a mammal with an inoculum of the present invention and thereby induce in the mammal antibody molecules having the 30 appropriate polypeptide immunospecificity. The antibody molecules are*then collected from the mammal and isolated or purified to the extent desired by well known techniques such as, for example, by immunoaffinity chromatography. The isolated antibody molecule-containing compositions are then evaluated for their ability to immunoreact

TGHNVKH,

GHNVKH,

HNVKH,

-37according to the described immunospecificity, and those compositions so prepared having the appropriate immunospecificity are retained as antibody compositions of the present invention.

The antibody so produced can be used in, inter alia, the diagnostic methods and systems of the present invention to assay for the presence of latent papillomavirus proteins in a body sample.

The antibodies of this invention induced by a polypeptide of this invention, can be described as being oligoclonal as compared to naturally occurring polyclonal antibodies since they are raised to an immunogen (the relatively small polypeptide) having relatively few epitopes as compared to the epitopes mimicked by an intact viral latency-associated papillomavirus-encoded protein. Consequently, antibody molecules of this invention bind to epitopes of the polypeptide, whereas naturally occurring antibodies raised to Pj 20 whole latent papillomavirus protein molecules bind °°to epitopes throughout those protein molecules and are referred to as being polyclonal.

In another embodiment, an antibody of the present invention is characterized as containing substantially isolated antibody molecules that immunoreact with a latent papillomavirus protein Srelated polypeptide, a polypeptide deduced from a latent protein ORF, preferably El, E2 or E6.

Preferred are substantially isolated antibody '30 molecules that immunoreact with a polypeptide having the formula:

SSTWHWTGHNVKHKSAIVTLTYDSEWQRDC,

EKTGILTVTYHSETQRTRC,

HKHAIVTVTYOSEEQRQQC,

HKNAIVTLTYSSEEQRQQC, or

SKNGIVTVTFVTEQQQQMC.

-TDYSE-; and are homologous, preferably without insertion or deletion, and more preferably are identical, to a -38- These antibodies are typic ,lly produced by immunoaffinity chromatography, using immobilized latent papillomavirus protein-related polypeptides, from anti-latent papillomavirus protein antibodycontaining sera, such as is found in a patlient having a latent papillomavirus infection.

Preferrad antibodies of this embodiment are human antibodies, isolated from the sera of a patients having a latent papillomavirus infection, preferably an infection caused by a type 16, 18, 6, 11 or 33 human papillomavirus, or the like.

Particularly preferred are the human antibodies prepared in Example F. Monoclonal Antibody Compositions A monoclonal antibody contains antibody molecules that immunoreact with papillomavirus latent protein is also contemplated. The phrase "monoclonal antibody" in its various gramiratical forms refers to a population of antibody molecules that contains only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody composition thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, a bispecific monoclonal antibody.

it 44 41 4 4 4 4r6 44 4I 4 4 1 4444 4t I I (C 4b 4 4 (1 4r 4 4 4 A monoclonal antibody composition is typically composed of antibodies produced by clones of a single cell called a hybridoma that secretes (produces) but one kind of antibody molecule. The hybridoma cell is formed by fusing an antibodyproducing cell and a myeloma or other selfperpetuating cell line. Such antibodies were first 1- -39described by Kohler and Milstein, Nature, 256:495- 497 (1975), which description is incorporated by reference.

In one embodiment, monoclonal antibody composition of the present invention is characterized as containing antibody molecules that immunoreact with one of the following papillomavirus latent proteins: a) the 112 kd diffuse protein, b) the 54 kd filamentous protein, c) the 48 kd filamentous protein, d) the 51 kd nuclear protein, e) the 58 nuclear protein, f) the 26 kd nuclear protein, or g) the 48 kd nuclear protein.

Preferably, a monoclonal antibody of this o, invention does not substantially immunoreact with: o a) a 70 kd protein present in HeLa °°cells, 040 20 b) a 20 kd protein present in CaSki 0

P

c) a 15 kd protein present in CaSki cells, d) a 11 kd protein present in CaSki 25 cells, or e) a 10 kd protein present in CaSki cells.

In another embodiment, the present So, invention contemplates an anti-polypeptide o 0 0 o" o 30 monoclonal antibody containing antibody molecules o that immunoreact with a polypeptide of the present invention and a papillomavirus latent protein.

Preferably, an anti-polypeptide monoclonal antibody does not substantially immunoreact with: a) a 70 kd protein present in HeLa cells, i b) a 20 kd protein present in CaSki cells, c) a 15 kd protein present in CaSki Icells, d) a 11 kd protein present in CaSki cells, or e) a 10 kd protein present in CaSki cells.

In preferred embodiments, a monoclonal antibody i~munoreacts with a polypeptide whose amino acid residue sequence corresponds to a polypeptide shown in Table 1, 2 or 3. A particularly preferred monoclonal antibody contains antibody molecules capable of being produced by a hybridoma shown in Table 4.

Table 4 Monoclonal Antibody Producing Hybridomas 20 POLYPEPTIDE 2

HYBRIDOMA

1 ORF' DESIGNATION DESIGNATION El 235 235:B9 1 El 238 238:8E9 SE2 245 245:11E3 25 E6 247 247:4D11 E6 247 247:10F7 E6 247 247:11D11 S1 The nomenclature for open reading frames (ORFs) corresponds to the ORFs shown in Figure 1 from which the polypeptide amino acid sequence was derived.

2 Polypeptides have amino acid sequences as shown in Table 1.

L-ill ii. U -41- Preferred monoclonal antibody producing hybridomas designated 235:B9, 245:113 and 247:4D11 were deposited as hybridoma cultures with the American Type Culture Collection (ATCC), Rockville, MD, on May 12, 1988 and were assigned accession numbers HB 9720, HB 9718 and HB 9719, respectively.

In another embodiment, the present invention contemplates an anti-polypeptide monoclonal antibody containing antibody molecales that immunoreact with a polypeptide having the formula:

SSTWHWTGHNVKHKSAIVTLTYDSEWQRDC,

EKTGILTVTYHSETQRTRC,

HKHAIVTVTYOSEEQRQQC,

HKNAIVTLTYSSEEQRQQC, or i SKNGIVTVTFVTEQQQQMC.

.A monoclonal antibody of the present invention can be produced by initiating a ,o monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma of the present Sinvention that secretes antibody molecules of the appropriate immuno specificity. The culture is S. maintained under conditions and for a time period 0° sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody- S .a containing medium is then collected. The antibody molecules can then be further isolated by well Sknown techniques.

SO" To produce a much greater concentration 0 30 of slightly less pure monoclonal antibody, the S desired hybridoma may Le injected into mice, preferably syngenic or semisyngenic mice. "The hybridoma will cause formation of antibodyproducing tumors after a suitable incubation time, which will result in a high concentration of the desired antibody (about 5-20 mg/ml) in the L- L ir~leaaagsswc'^^-*- i r1 -42bloodstream and peritoneal exudate (ascites) of the host mouse.

Media useful for the preparation of these compositions are both well known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium [DMEM; Dulbecco et al., Virol.

8:396 (1959)] supplemented with 4.5 gm/l glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

The monoclonal antibody compositions produced by the above method can be used, for example, in diagnostic and immunopurification modalities wherein formation of a papillomavirus latent protein-containing immunoreaction product is desired.

G. Hybridomas and Other Monoclonal °Antibody Producing Cells, and o 20 Methods of Preparation Go 00 Hybridomas of the present invention are those which are characterized as having the capacity to produce a monoclonal antibody of the present invention.

°°oo 25 Methods for producing hybridomas producing (secreting) antibody molecules having a °o desired immunospecificity, having the ability to immunoreact with a particular protein, an identifiable epitope on a particular protein 30 and/or a polypeptide, are well known in the art.

9*9°9 Particularly applicable is the hybridoma technology described by Niman et al., Proc. Natl. Acad. Sci.

USA, 80:4949-4953 (1983), and by Galfre et al., Meth. Enzymol., 73:3-46 (1981), which descriptions are incorporated herein by reference.

i Yil c r I~I I~ papillomavirus latent protein.

The word "inoculum" in its various grammatical forms is used herein to describe a composition containing a polypeptide or a -43- Typically, hybridomas of the present invention are produced by using, in the above techniques as an immunogen, a substantially pure latent papillomavirus protein or a pclypeptide of the present invention.

H. Diagnostic Systems A diagnostic system in kit form of the present invention includes, in an amount sufficient for at least one assay, a substantially pure papillomavirus latent protein, polypeptide, antibody, anti-polypeptide antibody, monoclonal antibody or anti-polypeptide monoclonal antibody of the present invention, as a separately packaged reagent. Instructions for use of the packaged reagent are also typically included.

S. As used herein, the term "package" refers Sto a solid matrix or material such as glass, plastic, paper, foil and the like capable of holding within fixed limits a polypeptide, antibody 20 composition or monoclonal antibody composition of the present invention. Thus, for example, a package can be a glass vial used to contain milligram quantities of a contemplated polypeptide Sor it can be a microtiter plate well to which microgram quantities of a contemplated polypeptide have been operatively affixed, linked so as to be capable of being immunologically bound by an antibody.

S, "Instructions for use" typically include a tangible expression describing the reagent concentration or at least one assay method parameter such as the relative amounts of reagent and sample to be admixed, maintenance time periods for reagent/sample admixtures, temperature, buffer conditions and the like.

I i i ;II -44- In one embodiment, a diagnostic system for assaying for the presence of a latent papillomavirus infection in a body sample comprises a package containing an antibody of the present invention that immunoreacts with a latent< papillomavirus protein. Preferably, the antibody is a monoclonal antibody of the present invention.

More preferably, the antibody molecules are those of the antibody produced by a hybridoma of the present invention. Further preferred are kits wherein the antibody molecules are linked to an enzyme label.

Thus, in preferred embodiments, a diagnostic system of the present invention further includes a label or indicating means capable of Asignaling the formation of a complex containing an antibody molecule or polypeptide of the present invention.

The word "complex" as used herein refers .a 20 to the product of a specific binding reaction such as an antibody-antigen reaction. Exemplary complexes are immunoreaction products.

As used herein, the terms "label" and 4 "indicating means" in their various grammatical t 25 forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in a 30 substantially pure latent papillomavirus protein, ,polypeptide, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic c_ II I .iiiliiiii;Li~i-l.- 1-i chemistry and constitute a part of this invention only insofar as they are utilized with otherwise novel protein methods and/or systems.

The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein isothiocyante (FITC), naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB 200 SC) and the like. A description of immunofluorescence analysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis, et ai., eds., John Wiley Sons, Ltd., pp. 189-231 (1982), which is S 20 incorporated herein by reference.

In preferred embodiments, the indicating So croup is an enzyme, such as horseradish peroxidase (HRP), alkaline phosphatase, glucose oxidase, or 2 the like. In such cases where the principal 25 indicating group is an enzyme such as HRP or o° glucose oxidase, additional reagents are required 0 0, to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP include hydrogen o. 30 peroxide and an oxidation dye precursor such as diaminobenzidine or orthophenylenediamine. An *additional reagent useful with glucose oxidase is 2,2'-azino-di-(3-ethyl-benzthiazoline-G-sulfonic acid) (ABTS).

Radioactive elements are also useful labeling agents. An exemplary radiolabeling agent

L':

E. Antibodies and Anti- Polypeptide Antibodies The term "antibody" in its various -46is a radioactive element that produces gamma ray emissions. Elements which themselves emit gamma rays, such as 124I, 1251, 28I, 132 and 51 Cr represent one class of gamma ray emission-producing radioactive element indicating groups.

Particularly preferred is 125I. Another group of useful labeling means are those elements such as 1 C, 18 F, 150 and 3N which themselves emit positrons.

The positrons so emitted produce gamma rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such as "'indium or 3

H.

The linking of labels, labeling of, polypeptides and proteins is well known in the art. For instance, antibody molecules produced by S a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino 4 t So acids provided as a component in the culture oo medium. See, for example, Galfre et al., Meth.

0 20 Enzvmol., 73:3-46 (1981). The techniques of sr o o protein conjugation or coupling through activated functional groups are particularly applicable.

s< See, for example, Aura;aeas, et al., Scand. J.

0 0 0 0o Immunol., Vol. 8 Suppl. 7:7-23 (1978), Rodwell et S" 25 al., Biotech., 3:889-894 (1984), and U.S. Pat. No.

o 4,493,795.

I The diagnostic systems can also include, preferably as a separate package, a specific binding agent. A "specific binding agent" is a S 30 molecular entity capable of selectively binding a o 0 reagent species of the present invention but is not itself a substantially pure protein, polypeptide, or antibody molecule of the present invention.

Exemplary specific binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A and the ii II resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules are preferred, and are utilized as illustrative herein.

-47- 00 0 000000 go o 0 boo b¢ o o 0000 0 0 0 0 0 0o 0 00 00c 0 0 0 00 0o 0 0 like. Preferably, the specific binding agent can bind the antibody molecule or polypitide of this invention when it is present as part of a complex.

In preferred embodiments the specific binding agent is labeled. However, when the diagnostic system includes a specific binding agent that is not labeled, the agent is typically used as an amplifying means or reagent. In these embodiments, the labeled specific binding agent is capable of specifically binding the amplifying means when the anplifying means is bound to a reagent species-containing complex.

The diagnostic kits of the present invontion can be used in an "ELISA" format to detect the presence or quantity of antibody molecule that immunoreact with a latent papillomavirus protein present in a body fluid sample such as serum, plasma or urine. "ELISA" refers to an enzyme-linked immunosorbent assay that employs an antibody or antigen bound to a solid phase and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antigen or antibody present in a sample. A description of the ELISA technique is found in 25 Chapter 22 of the 4th Edition of Basic and Clinical Immunology by D.P. Sites et al., published by Lange Medical Publications of Los Altos, CA in 1982 and in U.S. Patents No. 3,654,090; No. 3,850,752; and No. 4,016,043, which are all incorporated herein by reference.

Thus, in preferred embodiments, the substantially pure protein, polypeptide, or antibody molecule of the present invention can be affixled to a solid matrix to form a solid support that is separately packaged in the subject diagnostic systems.

p..

and more preferably at least about 50%, of the antibody molecules present in the antibody are directed against a papillomavirus latent protein or papillomavirus related polypeptide.

-48- The reagent is typically affixed to the solid matrix by adsorption from an aqueous medium although other modes of affixation, well known to those skilled in the art can be used.

Useful solid matrices are well known in the art. Such materials include the cross-linked dextran available under the trademark SEPHADEX from Pharmacia Fine Chemicals (Piscataway, NJ); agarose; latex; beads of polystyrene beads about 1 micron to about 5 millimeters in diameter available from Abbott Laboratories of North Chicago, IL; polyvinyl chloride, polystyrene, cross-linked polyacrylamide, nitrocellulose- or nylon-based webs such as sheets, strips or paddles; or tubes, plates or the wells of a microtiter plate such as those made from polystyrone or polyvinylchloride.

S, The reagent species, labeled specific binding agent or amplifying reagent of any diagnostic system described herein can be provided 20 in solution, as a liquid dispersion or as a 4a 4o substantially dry power, in lyophilized form.

S° Where the indicating means is an enzyme, the enzyme's substrate can also be provided in a separate package of a system. A solid support such o, 25 as the before-described microtiter plate and one or S" more buffers can also be o0 included as separately packaged elements in this diagnostic assay system.

The packaging materials discussed herein in relation to diagnostic systems are those 0customarily utilized in diagnostic systems. Such materials include glass and plastic polyethylene,' polypropylene and polycarbonate bottles, vials, plastic and plastic-foil laminated envelopes and the like.

L, b- I -49- In another embodiment a diagnostic system of the present invention is useful for assaying for the presence of antibodies induced by a latent papillomavirus infection, anti-latent papillomavirus protein antibodies. Such a system comprises, in kit form, a package containing a latent papillomavirus protein or a polypeptide of this invention. Preferably the included polypeptide contains a sequence homologous to a portion of a deduced amino acid residue sequence derived from the E region open reading frames (ORFs) of a sequenced papillomavirus genome. More preferably, the included polypeptide contains a sequence deduced from the El, E2 or E6 ORF of a

HPV.

e, In one embodiment, it is preferred to include in a contemplated diagnostic system a polypeptide that is related to a particular HPV type, such as is disclosed herein for types 6, 11, I 20 16, 18, 33, and 35. It is particularly preferred to include a HPV type 6, 11, 16, 18, or 13 related polypeptide of the present invention, preferably one of those whose sequence is shown before in I t Tables 1, 2 or 3.

25 In view of the results discussed in the Examples, it is clear that a significant antigenic determinant of the human papillomavirus which reacts with HPV type 16 latent protein-induced .antibodies is defined by (contained within) the 30 five amino acid residue sequence -TYDSE- described before. Moreover, even though each of the HPV type 16 related polypeptides of the present invention reacts with most anti-HPV type 16 latent protein antibody containing sera, individual patient sera have been observed to react specifically with one of the HPV type 16 related polypeptides but not

L~

desired by well known techniques such as, for example, by immunoaffinity chromatography. The isolated antibody molecule-containing compositions are then evaluated for their ability to immunoreact another. This observation indicates that additional antigenic determinants exist in other peptides containing a sequence that includes the formula -LTYDSE-, -SAIVTLTDYSE-, -HKSAIVTLTDYSE-, HKSAIV-, -SAIVTL-, or -IVTLTD-, as described before.

Therefore, the present invention further contemplates the discovery that recognition of antibodies to HPV type 16 latent proteins in immunological assays is significantly enhanced if the above described HPV type 16-related polypeptides are used in combination with a different species of HPV type 16-related polypeptide. An exemplary and preferred embodiment includes in combination polypeptides 66 and 245, or 78 and 85, or 66 and 78 and 85, and the like combinations.

t t I fC C; C t I

CC

Thus in one embodiment a diagnostic system contains more than one species of HPV type 16 related polypeptide of the present invention.

Preferably the polypeptide species are present in the system as an admixture, although individual species may be present in separate packages or segregated into separate locations in the system.

This combination format provides the ability to detect in a single kit, or preferably on a single solid support if admixed, anti-HPV latent protein antibodies having different immunospecificities, thereby improving the screening capabilities of such a system.

Ci I When it ip desired to provide a diagnostic system capable of being used to detect and distinguish between exposure to different papillomavirus types, the kit contains more than one polypeptide wherein the additional polypeptides are selected on the basis of their ability to 1.-4 L~ I I r ±TULTVTYHSETQRTRC,

HKHAIVTVTYOSEEQRQQC,

HKNAIVTLTYSSEEQRQQC, or

SKNGIVTVTFVTEQQQQMC.

-51produce antibody molecules, upon immunization, that immunoreact with the latent papillomavirus proteins from a second virus type that is different from the virus type from which the first polypeptide was derived. Furthermore, the additional polypeptides induce antibody molecules that do not immunoreact with the latent papillomavirus proteins expressed by the first virus type. Thus, as used herein, "different" means that there is a substantial 1 10 measurable difference in the ability of the two polypeptide-induced antibody molecule compositions to immunoreact with latent papillomavirus proteins produced in a latent infection by a single papillomavirus type. Thus, it is said that the polypeptides are type-specific in their ability to induce antibody compositions that do not both Simmunoreact with the latent papillomavirus proteins of a single virus type.

Stated alternatively, polypeptides are 0« 20 "different" and therefore type-specific where there is a substantial measurable difference in their ability to immunoreact with antibodies induced by a papillomavirus latent protein of one type when I compared to their ability to immunoreact with the protein of another type. A difference in immunoreaction when measured by ELISA, as in Example 14, is substantial if there is more than a 0.05, more preferably a 0.1 and still more S. preferably a 0.4 difference in optical density.

Preferred type specific polypeptides for inclusion in this embodiment of the present diagnostic system include the HPV type 16 related, type 18 related, type 6 related, type II related, and type 33 related polypeptides described before.

Exemplary uses of type specific polypeptide of the i 'Ilr e I IC~ ~III~

I

-52present invention in a diagnostic system are shown in Example 14.

In this embodiment of a diagnostic kit utilizing type specific polypeptides it is contemplated that the polypeptides may be provided physically separated within the kit thereby allowing for distinguishing between the presence of antibodies that immunoreaL- with one or the other of the included polypeptides. An exemplary kit of this type includes a first solid support having operatively affixed thereto a first polypeptide and a second solid support having operatively affixed thereto a secon' polypeptide, in which the two separated polypeptides are type-specific for 15 different papillomavirus types. Of course, the two 00 an 0 o" solid supports can be on the same or different bulk medium, as in the case where the solid supports are 0 microtiter wells, and the wells are on the same or 0 0 different microtiter plates. In addition, this S 20 embodiment can include a third solid support having 0 0 affixed thereto a third type-specific polypeptide whose specificity is different from both the first 0"0, and second, and so on.

.0 0 o 2 In another embodiment, different typespecific polypeptides may be included in a diagnostic kit as an admixture of all the peptides desired to be included, thereby creating the ability to screen for the presence of antibodies induced by latent papillomavirus infections caused o 30 by more than one papillomavirus type using one solid support. A kit of this type typically comprises a solid support such as a microtiter plate having operatively affixed thereto in an individual well an admixture of the polypeptides being type-specific for more than one papillomavirus type.

invention and a papillomavirus latent protein.

Preferably, an anti-polypeptide monoclonal antibody does not substantially immunoreact with: a) a 70 kd protein present in HeLa cells, -53- I. Assay Methods The present invention contemplates any method that results in detecting latent papillomavirus proteins, particularly those proteins as are found in a tissue sample such as a biopsy, urethral smear or pap smear, by producing an immunciomplex containing a substantially pure latent papillomavirus protein, a polypeptide or antibody molecule contained in an antibody or monoclonal antibody composition of the present invention.

In addition, the present invention contemplates any method that results in detecting antibody molecules that immunoreact with latent 15 papillomavirus proteins or polypeptides deduced Sfrom the nucleotide sequence of a papillomavirus 009000 wo genome, particularly those antibody molecules in a o vascular body fluid such as are found in serum or oo vaginal secretions from a patient or other animal :o,0O, 20 species carrying a latent papillomavirus infection, O by producing an immunocomplex containing a substantially pure latent papillomavirus protein, a 0 00 polypeptide or antibody molecule contained in an antibody or monoclonal antibody composition of the 0 0 0 present invention.

;o Those skilled in the art will understand that there are numerous well known clinical diagnostic chemistry procedures that can be 0 Oa0 utilized to form these complexes. Thus, while 00ooo 30 exemplary assay methods are described herein, the invention is not so limited.

1. Immunohistochemical Labeling of Tissue Samples A method for detecting the presence of a latent papillomavirus infection in tissue samples is contemplated. In this embodiment, I -54antibody molecules of the present invention are used to detect latent papillomavirus infection by means of their ability to immunoreact with the latent papillomavirus proteins present in papillomavirus infected tissue samples such as cervical epithelial biopsy, condyloma biopsy, urethral smears and pap smears. In preferred embodiments, the antibody molecules are present as a monoclonal antibody composition and more preferably are produced by a hybridoma listed in Table 4.

For example, a biopsy sample is obtained and prepared by fixation for immunohistochemical analysis by well known techniques. See, for example, Tubbs, Atlas of S' Immunohistology, American Society of Clinical *3a Pathology Press, Chicago. The prepared biopsy r sample is admixed with an antibody moleculet" containing composition of the present invention to form an immunoreaction admixture. The admixture thus formed is maintained under biological assay conditions for a time period sufficient for any latent papillomavirus proteins present in the Ir 2, sample to immunoreact with the added antibody molecules to form an immunoreaction product. The 4 0presence of an immunoreaction product is then assayed.

In this embodiment, antibody molecules o utilized for detecting a latent papillomavirus o 30 infection in tissue samples can include substantially isolated antibody molecules that immunoreact with a latent papillomavirus protein related polypeptide, preferably a polypeptide deduced from the E2 ORF. Exemplary antibody molecules of this class are those isolated by affinity chromatography, using immobilized i c; eo 4 oo 9 r a i A 0 polypeptides having papillomavirus related sequences, and isolated from anti-latent papillomavirus protein antibody containing sera, such as is found in a patient having a latent papillomavirus infection. Exemplary are the detection methods described in Example 16 using antibody molecules affinity isolated from a HPV infected patient antisera.

2. Detection of Antibodies to Latent Papillomavirus Infection Various heterogeneous and homogeneous assay protocols can be employed, either competitive or non-competitive, for detecting the presence and preferably amount of antibodies that immunoreact with latent papillomavirus proteins in a vascular body fluid, anti-latent papillomavirus protein antibodies.

In particular this invention contemplates an "ELISA" format as discussed herein to detect the 20 presence and quantity of antibody molecules in a body fluid sample such as serum, plasma, vaginal secretions or urine, said antibody molecules being those that immunoreact with a latent papillomavirvs protein. In this format, the method employs an antigen or antibody bound to a solid phase (solid support) and an enzyme-antigen or enzyme-antibody conjugate to detect and quantify the amount of an antibody present in the sample. In preferred embodiments the antigen bound to the solid phase is 30 a polypeptide of the present invention.

For example, a human blood sample and a solid support containing a polypeptide of the present invention attached thereto are admixed.

The admixture thus formed is maintained under biological assay conditions for a time period sufficient for any antibodies to immunoreact with 00*0 0*r 00 0 0 *0 0 4, D 0 *0 0I 0 100 0 0 00 O a 0*0 r_ -56- K the solid phase polypeptide and for" an immunoreacticn product. A second labeled antibody, K such as horseradish peroxidase labeled anti-human IgA antibodies, is then admixed with the first immunoreaction product containing-solid sipport and maintained under biological assay conditions for a time period sufficient for any first immunoreaction product to immunoreact with the labeled antibodies and form a labeled second immunoreaction product.

The labeled second immunoreaction products are th'n separated from the non-reacted labeled-antibodies, typically by washing the solid support sufficient to remove the unbound label. The amount of labeled immunoreaction product is then assayed.

15 Biological assay conditions are those that maintain the biological activity of the 8 88 antibody molecules and polypeptide molecules of "0 this invention and the antibody molecules sought to ^be assayed. Those conditions include a temperature o° 0 20 range of about 4 degrees C to about 45 degrees C, preferably about 37 degrees C, a pH value range of about 5 to about 9, preferably about 7 and an ionic 0 8 ostrength varying from that of distilled water to that of about one molar sodium chloride, preferably about that of physiological saline. Methods for 8 08 o optimizing such conditions are well known in the art.

In preferred embodiments, the present 0 04

S

0 o oELISA formatted assay method utilizes polypeptides 0o44 30 deduced from the El, E2 or E6 ORFs of HPV type 16, 0 8 particularly the polypeptides whose amino acid residue sequences are those listed in Table 1.

Still more preferred are the HPV type 16 polypeptides 237, 245 and 246. Additional HPV type 16-related polypeptides preferred for use in detecting anti-latent papillomavirus proteins -57antibodies are those listed in Table 2.

In another embodiment, the present assay contemplates the detection of anti-HPV latent protein antibodies of human papillomaviruses of types other than type 16 by the use of HPV-related polypeptides deduced from the E2 ORF region of other genital papillomaviruses. Preferred are the HPV-related polypeptides deduced from type 6, 11, 18 and 33, particularly those shown in Table 3.

Exemplary ELISA methods utilizing various polypeptides are shown in the Examples.

3. Detection of Antibodies to Latent Papillomavirus Protein by Competition ELISA The present invention contemplates a competition assay method for detecting the presence and preferably amount of anti-latent papillomavirus Sprotein antibodies that uses a basic FLISA in two different competition formats.

S 20 In one format, a method is contemplated S°for assaying a body fluid sample for the presence of anti-latent papillomavirus protein antibodies

C

0 comprising the steps of: S«0 substantially simultaneously admixing a body fluid sample with a polypeptide °s0 of the present invention that is affixed to a solid support and a predetermied amount of a liquidphase labeled anti-polypeptide antibody of the S" present invention that immunoreactes with the 30 affixed polypeptide to form a competition immunoreaction admixture having a solid and a liquid phase. Preferably the body fluid sample is a known amount of blood, serum, plasma, urine, saliva, semen or vaginal secretion.

Maintaining the admixture under biological assay conditions for a predetermined I- an antibody or monoclona) antibody composition of the present invention, or used separately, and those atoms or molecules can be used alone or in conjunction with additional reagents. Such labels are themselves well-known in clinical diagnostic -58time period such as about 10 minutes to about 16 to hours at a temperature of about 4 degrees C to about 45 degrees C that is sufficient for any antilatent papillomavirus protein antibodies present in the sample to immunoreact with the polypeptide, and also sufficient for the labeled anti-polypeptide antibody to compete for immunoreaction with the same polypeptides to form solid phase labeled antipolypeptide-containing immunoreaction product.

Assaying for the presence of any labeled anti-polypeptide-containing immunoreaction product in the solid phase and thereby the presence of any anti-latent papillomavirus protein antibodies in the 15 immunoraction admixture is also determined.

Preferably, the amount of any labeled anti- :2 polypeptide-containing immunoreaction product formed is determined, and thereby the amount of So: anti-latent papillomavirus protein antibodies 20 present in the sample.

In another format, a competitive ELISA of this invention comprises the steps of: substantially simultaneously 8 o 2 admixing a body fluid sample, as in the previous format, with a polypeptide of the present o invention that is affixed to a solid support, and a predetermined amount of the same, or an imxmunologically cross-reactive polypeptide in *o liquid phase to form a competition immunoreaction 30 admixture having a solid and a liquid phase; maintianing the admixture under biological assay conditions for a time period sufficient for any anti-latent papillomavirus protein antibodies present in the sample to immunoreact with either the solid phase or liquid phase polypeptide to form both a solid phase and a -59liquid phase polypeptide-contaiing. imunoreaction product; and assaying for the presence of any solid-phase polypeptide-containing immunoreaction product that formed, and t1ereby the presence of any anti-latent papillomavirus protein antibodies in the immunoreaction admixture is also determined.

In a different embodiment of the second format, the polypeptide in the liquid phase is not the same nor substantially immunologically cross reacting with the polypeptides in the solid phase.

Thus, the two polypeptide included in the assay are different papillomavirus type-specific polypeptides .o 15 in so far as "different" has been defined herein.

4 For example, a HPV type 16-related polypeptide is present in the solid phase and a HPV 6-related polypeptide is present in the li_ ,id phase, such as .o polypeptides 245 and K-70 respectively. Such a V.6.o 20 competition assay format provides for a method to distinguish the HPV type present that has induced the detected anti-HPV latent protein antibodies.

4O44 O° Examples The following examples are intended to 25 illustrate, but not limit, the present invention.

o a S1 i. Polypeptide Synthesis Polypeptides corresponding in amino acid residue sequence to portions of latent proteins boo encoded by various HPV type 16 E ORFs were 30 chemically synthesized according to the solid-phase methods disclosed in U.S. Patent No. 4,631,211, which disclosure is hereby incorporated by reference.

The amino acid residue sequence of the polypeptides synthesized and their location within the deduced amino acid sequence of HPV E region i I L tscmyIbI J tifcJii zw vTne present. invention Du- is nuitself a substantially pure protein, polypeptide, or antibody molecule of the present invention.

Exemplary specifi binding agents are second antibody molecules, complement proteins or fragments thereof, S. aureus protein A and the ORFs are listed in Table 1.

Additional HPV-related polypeptides were chemically synthesized by above solid-phase methods, each polypeptide having as an amino acid residue sequence one of the sequences listed in Tables 2 and 3.

2. Preparation of Polyclonal Anti-Polypeptide Antiserum a. Preparation of Reduced Polypeptide Polypeptides prepared as described in Example 1 were analyzed to determine their cysteine content. A one milliliter (ml) volume solution of PE buffer [0.1 M sodium phosphate 4' 15 buffer, pH 7.2, 5 mM ethylenediaminetetraacetic acid (EDTA)] containing 125 micrograms tug) of polypeptide was admixed with 100 microliters (ul) of DTNB solution (1 mM dithio nitrobenzoic acid in methanol) and maintained at room temperature for 1 20 minutes. The optical density of the resulting maintained admixture was measured at 412 ir nanometers (nm) against a control solution of PE %i buffer alone. By comparison to a standard curve Susing glutathione, in which a 28 ug/ml solution in PE buffer exhibits about 1.14 O.D. units at 412 nm, the amount of free cysteine was determined for each polypeptide measured. ?olypeptides having less Sthan 75 percent of the available, cysteine residues I oxidized were considered reduced polypeptides.

S 30 Those polypeptides having more than 75 percent of the available cysteine residues oxidized were subjected to reduction as described below.

Polypeptides were reduced by admixing 10 milligrams (mg) of polypeptide with mg dithiothreitol (DTT; Sigma Chemical Co., St.

Louis, MO) in 1 ml of 50 mM phosphate buffer (pH ii aL.JLaz.y pure protein, polypeptide, or antibody molecule of the present invention can be affi>:pd to a solid matrix to form a solid support that is separately packaged in the subject diagnostic systems.

-61and maintaining the admixture at room temperature with continuous stirring agitation.

After 60 minutes of agitation, 50 ul concentrated acetic acid was further admixed and the resulting admixture was then applied to a 15 ml bed volume Sephadex G-10 (Pharmacia, Piscataway, NJ) column prewashed with 5 percent acetic acid in water.

The column was rinsed with 5% acetic acid and the resulting column eluant was collected in fractions, The optical density at 206 nm was determined for each fraction, the O.D. 206-defined fractions of the first protein peak were pooled and the pooled fractions were lyophilized to yield dried reduced polypeptide. The dried polypeptide was 15 dissolved in acetate buffer (pH 4.0) at 5 mg/ml to t yield reduced polypeptide.

b. Preparation of Polypeptide-KLH Coniugated Immunogen Reduced polypeptide, prepared in 20 Example 1, was conjugated to keyhole limphet hemocyanin protein (KLH; Pacific Bio-Marine Laboratories, Venice, CA) using the coupling 1 t C reagent m-maleimidobenzoyl N-hydroxysuccinimide ester (MBS; Sigma). Two hundred ul of KLH solution, dialyzed against PB (10 mM phosphate buffer, pH 7.2) at 20 mg KLH/ml PB, was admixed /1 |with 55 ul PB and then 85 ul of MBS solution (6 mg/ml in dimethyl formamide) was admixed slowly by S! dropwise addition while stirring the admixture at 30 room temperature. The resulting admixture was maintained for 30 minutes with stirring at room temperature and was then applied to a Sephadex column (Pharmacia) with a 15 ml bed volume that was previously rinsed with PB50 (50 mM phosphate buffer, pH The column was rinsed with and the resulting column eluant was collected -62dropwise in fractions (35 drops per-, raction). The O.D. 260 was determined for each fraction and the peak fractions were pooled. The pool was admixed with 1 ml of reduced polypeptide [5 mg/ml in acetate buffer (pH the resulting acmixture was monitored for pH and adjusted as needed with sodium hydroxide or hydrochloric acid to maintain the admixture between pH 7.0 and 7.5, while stirring at room temperature for 3 hours. The maintained and stirred admixture was then combined with sufficient PBS (phosphate buffered saline) to make a 2.0 ml final volume to produce KLHconjugated polypeptide solution.

c. Immunization of Rabbits to 15 Produce Polyclonal Anti- Polypeptide Antisera New Zealand white rabbits obtained from the SCRF Vivarium (Research Institute of ,o0: Scripps Clinic, La Jolla, CA) were immunized using KLH-conjugated polypeptide solutions prepared in Example 2b by the following schedule of inoculations. The first inoculation comprised four subcutaneous injections administered at locations o along the back, each injection having approximately 375 ul of a solution prepared by admixing 3 mg S0 mycobacterium (DIFCO Laboratories, Detroit, MI), ml incomplete Freund's adjuvant (IFA; Sigma) and 1.5 ml PBS containing 250 ul KLH-conjugated o polypeptide solution, said admixture having been S"'oo 30 emulsified for five minutes prior to inoculation.

The second inoculation was similarly administered 14 days after the first inoculation, using the same admixture but omitting the mycobacterium. The third inoculation was administered 21 days after the first inoculation, comprising 1 ml of a well shaken admixture of 250 ul KLH-conjugated I 16 related polypeptides of the present invention reacts with most anti-HPV type 16 latent protein antibody containing sera, individual patient sera have been observed to react specifically with one of the HPV type 16 related polypeptides but not -63polypeptide solution, 950 ul PBS and 800 ul aluminum hydroxide solution (10 mg/ml sterile water), injected intraperitoneally.

Antisera was obtained from the above-immunized rabbits by bleeding the ear veins 28 and 35 days after the first injection to yield rabbit anti-polypeptide antisera.

Rabbit anti-polypeptide antisera raised to the polypeptides whose amino acid residue sequences are shown in Table 1 were then screened by the ELISA assay described in Example 3. All of the rabbit antisera so prepared and screened by the ELISA assay immunoreacted with the immunizing polypeptide at titers in excess of 1:2560.

15 3. ELISA Assays Antibody molecules contained in antibody compositions and monoclonal antibody compositions were examined for their ability to immunoreact with S, various polypeptides using an enzyme-linked oo 20 immunoassay (ELISA) procedure.

One hundred ul of coating solution (0.1 M sodium carbonate buffer, pH 9.2) containing .It o polypeptide prepared as described in Example la at a concentration of 10 ug per ml coating solution was added to each well of a flat-bottom 96-well Somicrotiter plate, and the plate was maintained overnight at room temperature to permit the polypeptide to adsorb onto the walls of the wells.

SoThereafter the coating solution was removed by o:o 30 inversion and shaking, the wells were rinsed twice with distilled water and 150 ul of blocking solution [3%'bovine serum albumin (BSA; w/v) in PBS] were admixed into each well (solid support) to block excess protein sites.

The wells were maintained for 20 minutes at room temperature and then the blocking solution -64was removed by shaking. Into each well was admixed 100 ul of a solution containing rabbit anti-peptide antisera, prepared as described in Example 2c and serially diluted in blocking buffer. The resulting solid/liquid phase immunoreaction admixtures were maintained at room temperature for 60 minutes to permit formation of a first solid phase-bound immunoreaction product between the solid phasebound polypeptide and admixed antibodies. The solid and liquid phases were then separated, the wells were rinsed 5 times with distilled water and excess iquid was removed by shaking.

One hundred ul of a solution containing glucose oxidase labelea goat anti-rabbit IgG 15 (Cooper Biomedical, Malvern, PA), diluted 1:1000 in blocking solution, was admixed into each well to form a second solid/liquid phase immunoreaction admixture (labeling immunoreaction admixture). The II wells were maintained for one hour at 37 degrees C to permit formation of a second immunoreaction product between the labeled antibody and any solid phase-bound antibody of the first immunoreaction product and then rinsed 5 times with distilled S. water to isolate the solid phase-bound label 25 containing immunoreaction products. Excess liquid was then removed from the wells.

A chromogenic substrate solution was freshly prepared before use by admixing 28 mls of a prepared glucose solution containing 2.1% glucose in PB [0.1 M phosphate buffer (pH said glucose solution having been maintained overnight to allow the glucose to mutarotate, (2) 200 ul of a solution containing 0.1% (w/v) horseradish peroxidase in PB, and 200 ul of an ABTS solution containing freshly prepared ABTS dye (2,2'-azino-di[3-ethylbenzthiazolinesulfonate(6)] iL diammonium salt; Boehringer-Mannheim, Indianapolis, IN) at a concentration of 45 mg per ml PB. 100 ul of the chromogenic substrate solution were then admixed into each well to form a color developingreaction admixture. After maintaining the developing-reaction admixture in the dark for 1 hour at room temperature, the O.D. of the solution admixture was measured directly in the well using a multiskan microtiter plate reader (Bio-Tek Instr., Winooski, VT) with a 415 nm filter.

The results of the above ELISA procedure were expressed as a dilution of the antibody composition that provided approximately 50% of the maximum O.D. that the chromogenic substrate II 15 solution produced when using undiluted, positively reacting antigen. Antibody molecules contained in those compositions were considered as having the capability to immunoreact with a solid phase sa polypeptide if the dilution to achieve 50% maximal 20 O.D. was greater than 1:4.

,0 4. Polypeptide-Ligand Affinity Isolation of Rabbit Polyclonal Antisera f 0 ,To increase the specificity of rabbit anti-polypeptide antisera, these antisera were affinity isolated using solid phase polypeptide ligands as described herein.

Five milligrams of polypeptide, prepared St as described in Example 1, was dissolved in water c04«« 30 and subsequently coupled to AH-Sepharose 4B (Pharmacia) according to the manufacturer's instructions to form a polypeptide-agarose solid support. A column having a 3 ml bed volume was prepared using polypeptide-agarose solid support and equilibrated by rinsing with NET buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH About -66- I mis of a rabbit anti-polypeptide antiserum, prepared as described in Example 2c, was applied to ;the equilibrated column and the column was then washed with 30 column volumes of NET buffer.

Thereafter 100 mM citrate buffer (pH 3.0) was applied to the column and the eluate was collected in fractions. The O.D. of the fractions was measured at 280 nm, the peak-containing fractions were determined and pooled to yield an antibody containing pool. The pH of the pool was measured and adjusted to 7.0 using Tris-base and was then dialyzed against PBS to yield a solution containing affinity purified rabbit antibody molecules.

The resulting affinity purified rabbit S* 15 antibody molecule containing solution represents a substantially isolated antibody because greater L than 50% of the antibody molecules contained in the solution have the capacity to immunoreact with an It HPV 'latent protein.

20 Each rabbit anti-polypeptide antiserum affinity-isolated by the above procedure was isolated using the same polypeptide as was used in S the preparation of the immunogen that raised that t particular antiserum. The rabbit anti-polypeptide antisera that was affinity-isolated (AI) by this V method includes rabbit anti-polypeptide 235 (rabbit anti-235), hereinafter referred to as rabbit AI S anti-235, rabbit AI anti-236, rabbit AI anti-245 l and rabbit AI anti-247.

S 30 5. Western Immunoblot Detection of Latent Papillomavirus Proteins Using Polyclonal Anti-Polypeptide Antibodies Using Western immunoblot assays, HPVcontaining tissue culture cell lysates and various biopsy samples were prepared and examined for the -67-

I.,

I

4.

ii 14 44 *r I Ir eoe .4 4 tI 41 II I 9a o te 49( 0 presence of latent human papillomavirus proteins.

The human cervical carcinoma cell lines, C-33A (HTB-31), HT-3 (HTB-32), Hela (CCL2), CaSki (CRL 1550), MS 751 (HTB 34) and SiHa (HTB 35) were 5 obtained from the American Type Culture Collection (ATCC; Rockville, MD), have the ATCC designations indicated in parenthesis after their names, and were cultured using ATCC recommended media and methods.

Cells grown in monolayer culture were harvested and washed twice in PBS-EDTA (PBS containing 0.02% EDTA), pelleted to recover the washed cells and the PBS-EDTA was removed to yield a packed cell pellet. The packed cell pellet was 15 weighted, resuspended by vortexing in PBS at 4 0 C at a concentration of 0.1 gms packed cell pellet per mis PBS, and lysed by the addition of 0.5 mls of 2XSB a buffer prepared having twice the concentration of sample buffer) to yield cell 20 lysates. Sample buffer (SB) contains 2% SDS, 50 mM dithiolthreitol, 10% glycerol, 125 mM Tris-HCl pH 6.8 and 1 mM phenyl methyl sulfonyl flouride

(PMSF).

Tissue biopsies of condyloma, obtained from Dr. Z. Bekassy (Department of Gynecology, Lund University Hospital, Lund, Sweden), were weighed, minced and suspended in SB at a concentration of 0.1 mg per ml. The minced suspension was disrupted by three strokes in a loose fitting pestle of a 30 dounce homogenizer, and then sonicated for hours in a waterbath sonicator. The sonicated suspension was frozen to -70 degrees C and thawed through 4 cycles of freeze-thaw, and the resulting suspension was centrifuged at abut 12,000 xg in a microcentrifuge to remove tissue debris. The 'hl 19"' 9*s 4 94

I

191 49*

I

-68resulting supernatant was retained to yield a condyloma tissue lysate.

Cell lysates were subjected to SDSpolyacrylamide gel electrophoresis (SDS-PAGE) on a 7.5% slab gel using the discontinuous buffer system described by Laemmli, Nature, 226:680-685 (1970) as modified by Blake et al., Infect. Immun., 33:212- 272 (1981), which method is hereby incorporated by reference, using 100 microliter (ul) of cell lysate per gel lane, flanked on either side by a lane containing prestained molecular weight marker proteins (Bio-Rad Laboratories, Richmond, CA). The proteins present in the marker preparation include: lysozyme, 14.4 kd; trypsin inhibitor, 21.5 kd; Sc. 15 carbonic anhydrase, 31 kd; ovalbumin, 42.7 kd; bovine serum albumin, 66.2 kd; phosphorylase b, S 97.4 kd; beta-galactosidase, 116.25; and myosin, 200 kd, all in units of one thousand daltons.

After electrophoresis and electroblotting 20 using a Bio-Rad transfer unit (BioRad, Richmond, CA) onto nitrocellulose, as described by Towbin et I al., Proc. Natl. Acad. Sci. USA, 76:4350-4354 t (1979) which method is hereby incorporated by reference, the blot was blocked with a solution of BLOTTO powdered nonfat milk in PBS containing 0.025% antifoam A (sigma Chemical Corp. St. Louis, MO)] by immersion of the blot into BLOTTO for 1 hour with agitation. Blocked blots were then maintained in BLOTTO containing rabbit antiti 30 polypeptide antisera or AI rabbit anti-polypeptide antisera as indicated at a dilution of 1:100 in BLOTTO for 2 hours at room temperature to allow an immunoreaction product to form between the admixed antibody compositions and the latent papillomavirus protein present as solid phase on the blots.

Thereafter the blots were washed in BLOTTO three I- 1 -69times for about 1 minute, 20 minutes and minutes, respectively, to remove unbound antipeptide antisera. The washed blots were then maintained for 30 minutes in BLOTTO containing alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma) diluted to 1:1000 to allow a second immunoreaction product to form between the second admixed antibody and the first formed immunoreaction product present on the solid phase of the blot. The blot was then washed in PBS-T (PBS containing 0.5% Tween 20) once for 5 minutes and 4 times for 30 minutes each to remove the unbound second admixed antibody. The washed blot was then maintained in a solution of chromogenic d 15 substrate containing developer for about 4 hours at S* room temperature to visualize the immunoreaction products present on the blot.

Developer solution was prepared by Sadmixing 5 ml 1.5 M Tris (pH 45 ml water, S o 20 mg nitro blue tetrazolium, 0.2 ml 1 M MgCl 2 and mg 5-bromo-4-chloroindoxylphosphate.

Results using the Western immunoblot f"t assay to detect latent papillomavirus protein are shown in Figures 2 and 3.

For instance lanes 1-4 of Figure 2 f .illustrated that the diffuse protein having a molecular weight of about 112 kd was detected in SiHa and HeLa cell lysates, but not in CaSki or CrIA ft 33A cell lysates, using polyclonal rabbit anti-236 tiI 30 antisera. The same 112 kd diffuse protein was also detected by the formation of an immunoreaction product on immunoblots using rabbit anti-245 antisera.

Lanes 6 and 7 of Figure 2 show that the HPV 54 kd filamentous protein was detected by immunoblotting cell lysates of condyloma biopsy -8.

-83- 3 moc h+-inpa fron natients havina tissues with rabbit anti-236 antise a. The HPV 48 kd filamentous protein was also detected in one of the condyloma biopsy cell lysates using rabbit anti-236 antisera (Fig. 2, lanes 6) but not in the other condyloma lysate (lane The 48 kd filamentous protein has also been detected by immunoreaction on blots using rabbit anti-235 rabbit anti-245 and rabbit anti-247.

Figure 3 demonstrates that a polyclonal antisera raised against polypeptide 236 can be used as a type-specific reagent for distinguishing between HPV type 16 and HPV type 18 infections. As shown in Figure 3, the 54 kd and 58 kd filamentous proteins were both detected by immunoblotting CaSki 15 cell lysates (lane 2) but were not detected tin HeLa cell lysates (lane 1) using rabbit anti-236 antisera.

4 if The 51 kd nuclear protein was detected by immunoblotting CaSki cell lysates with affinity 20 isolated rabbit anti-245.

4 0 The above results demonstrate that antipeptide antisera raised to polypeptides deduced from E region ORFs of papillomaviruses have the capacity to immunoreact with papillomavirus latent proteins. In some cases an anti-polypeptide S* antisera has the capacity to immunoreact with a latent protein produced by one but not another HPV type, a type-specific antisera.

0 6. Preparation of Hybridomas and Anti- Polypeptide Monoclonal Antibodies a. Mouse Immunizations All hybridomas were produced using spleen cells from immunized 129 Gx mice obtained from the SCRF Vivarium (La Jolla, CA) having an age of about 3 weeks at the beginning of immunization.

I

-71- Each of the polypeptides listed in Table 1 was KLH-conjugated and used as an immunogen to produce the hybridomas described herein.

Each mouse to be immunized with a particular polypeptide was first injected intraperitoneally (IP) with a suspension that contained an emulsified admixture of about 62.5 ul of a KLH-conjugated polypeptide solution, prepared as in Example 2b, 0.43 ml PBS and 0.5 ml complete Freund's adjuvant (CFA). About two weeks later each mcuse received an injection IP of a suspension containing about 32 ul of the same KLH-conjugated polypeptide solution as previously received, 0.47 ml PBS and 0.5 ml alum suspension (aluminum 15 hydroxide at 10 mg/ml in PBS). About 7 to 10 days aftsr the second injection, mouse antisera was collected by eyebleed and the titre of the antisera was determined using the ELISA procedure described 1 in Example 3 except using the modifications 20 described hereinbelow.

After the microtiter wells were coated with the same polypeptide as used in the mouse immunization, the wells were blocked as described and then serial dilutions of mouse eyebleed antisera diluted in blocking buffer were Sadmixed and the admixture was maintained as described. Where glucose oxidase conjugated antibody is required in the ELISA method, goat C C anti-mouse IgG conjugate was used in place of anti- 30 rabbit IgG (Cooper Biomedical). If the eyebleed titer was determined to be less than 1:3200 to achieve 50% maximal OD at 415 nm, then an additional injection was administered to the mouse 2 weeks after the second injection containing the same inoculum as for the second.

-72- About one month after the titer of the eyebleed reaches greater than 1:3200, the mouse was given a final injection of a suspension containing about 32 ul of the same KLH-conjugated polypeptide solution as previously received and 0.47 ml PBS, administered intravenously (IV) into a tail vein.

b.Hybridoma Fusion Mouse spleenocytes were harvested from the mouse immunized in Example 6a about 3 days after the final injection and fusion with myeloma cells was conducted as described herein. About 1X10 8 spleen cells (ATCC CRF 1581) from each mouse were admixed with about 2X10 7 SP2/0-Ag 14 myeloma 0* 15 cells in a fusion medium comprising 40% PEG 0 (Boehringer-Mannheim, Indianapolis, IN). After "cell fusion, the resulting hybridoma cells were seeded into 96 well microtiter plates, cultured in HAT medium (hypoxanthine, aminopterin and thymidine) as is well known, and the surviving hybridoma cultures resulting therefrom were screened for the ability to produce antibody .4 4 1 molecules that immunoreact with the immunizing polypeptide using the modified ELISA procedure described in Example 6a except that hybridoma culture supernatants were used in place of the mouse eyeblead antisera.

Hybridoma culture supernatants <t screened for the presence of anti-polypeptide antibody molecules in the ELISA assay were considered positive if the optical density at 414 nm of 1:2 diluted culture supernatant was greater than four times the O.D. obtained for control culture medium. A typical fusion was plated onto thirty 96-well microtiter plates and yielded from 10 to 60 hybridoma cultures per fusion -73that inmmunoreacted with the immunizing polypeptide in the ELISA assay.

Antibody molecules produced by a particular hybridoma selected by the foregoing screening methods are referred herein by characters that indicate 1) the polypeptide used to immunize the mouse that donated spleen cells to a particular fusic-, and 2) the 96 well culture plate, row and well number from which the particular HAP medium resistant hybridoma cell was isolated. The specific referring character is listed herein as one word, where the number preceding the colon is the polypeptide designation and the symbols following designate the microtiter well 247:11D12).

S, Isolated hybridomas are shown in 'Table 5 below.

I

4 4 t t «a S 4 6 a t t t t -74- Table Hybridomas Total Immunizing Hybridomas Polvpeptide Hybridomas Produced 235 1G5,2A5,2C9,2F9,2G7 31 3C5,3E9,3Fl2,3G5,4F5, 4F7,4G12,5A6,5D3,5D7, 5G11,6Dll,6G9,6H4,7BS,7E9, 7Fl2,7Gl,8G4,8Gl0,8Hl0, 9C7,9F5,9G10,l1El,B9 31 236 1C4,1Cl2,lF6,2All,2D5, 2Fl1,3G4,4Dl,6H5,8C11, 8BG6,9Cl1,9H5, 10C3,11C4, *&el lG2,llG6,11H8,12D5 19 238 2D4,4D4,5A10,8E9,8Gl0, 9420 1OC10,10C11,1OElO 8 245 2Al,2Fl1,4E3,4F7,8D4, if*-11E3 3D11, 14C6, 15F8, 15F10, 17C6 11 246 lD1Q,2H4,3F2,4GH,7D8,9E1 6 247 lB8,1C2,lFll,2C9,2Gl2, it 4D11,5F7,5F8,7G7,9E3, Ei 30 9G2,9G6,9G9,l0F7,11D11, 12BI1, 12C10, 13E11,M, 17D10

II

4~ @1 4 I I 14*1* I (I I I

LI''

4101 1* II I I I 1 4 1141 C I I 41 4 I II t I I 4 41 C. FPLC Purification of Monoclonal Antibodies A monoclonal antibody composition was prepared by harvesting the ascites fluid of a mouse that had been injected IP with a hybridoma culture and maintained by methods well known. Two ml of the resulting ascites fluid was centrifuged for 15 min at 12,000 xg, the supernatant was collected and filtered through a 0.2 u Acrodisc filter (Gelman) to yield filtered Ascites fluid.

A Superose 6 and a Superose 12 column (Pharmacia) were connected in series on an FPLC apparatus and equilibrated with PBS for 60 min at a flow rate of 0.5 ml/min. Five hundred ul of the 15 filtered ascites fluid were applied to the equilibrated columns and chrortatographed at a flow rate of 0.4 ml per min using PBS. The resulting eluant was monitored for optical density at 280 nm and 1 ml fractions of the eluant were 20 collected. Fractions were assayed by the ELISA procedure for the presence of anti-polypeptide antibody molecules using the immunizing polypeptide as described in Example 6b. Typically two fractions were determined to contain the majority 25 of the anti-polypeptide antibodies and the two ml fractions were then pooled to yield an FPLC purified monoclonal antibody solution having a anti-polypeptide titer greater than 1:2560 as determined by ELISA described in Example 6b.

30 By this procedure monoclonal antibodies 247:4D11, 235:B9 and 245:11E3 were FPLC purified, having a known protein concentration based on the O.D. 280 measurements taken.

Each of the resulting FPLC purified monoclonal antibody molecule containing solutions represent a substantially pure antibody because I '.f

U

''4

''II

-*-LUUPUn~lC4L~: -76- Ir I 4 41 0 00 0 4O 4 greater than 50% of the protein cont 'Ined in the solution is composed of protein molecules that form antibody combining sites.

7. Western Immunoblot Detection of Papillomavirus Latent Proteins Using Anti-Polypeptide Monoclonal Antibodies Monoclonal antibodies prepared as described in Example 6 were used to detect human papillomavirus '.atent proteins present in HPVcontaining tissues by the same Western immunoblot procedures described in Example 5 with the exceptions noted herein.

An electroblotted and blocked 15 nitrocellulose blot prepared as in Example 5 was admixed with a solution containing FPLC purified antibody solution, prepared as described in Example 6c, diluted to a concentration of 32 ug per ml of BLOTTO for 247:4D11 or to a concentration of 25 ug 20 per ml of diluted BLOTTO (to 2% milk) for 235:B9, and maintained in that solution as previously described to allow an immunoreaction product to form. As before the blot was washed, and then was maintained for 45 minutes in a solution of BLOTTO 25 containing rabbit anti-mouse antibodies (Cooper Biomedical) diluted to a concentration of about 1:500 to allow the formation of an immunoreaction product. Thereafter the blot was washed in BLOTTO 3 times for 1, 20 and 20 minutes and then maintained in a BLOTTO solution containing alkaline phosphatase conjugated goat anti-rabbit IgG as previously described. Subsequent washes and development were also as before.

The resulting immunoblot, shown in Figure 4, demonstrates the detection of the 54 kd and 48 kd filamentous proteins, in CaSki cell lysates rc 4e 4 0% 4 0\ 0 00 4 4 4~ *0 -77- (Lane 4) when using the monoclonal antibody 247:4D11. Also observed in CaSki cell lysates is an additional HPV-specific protein having a molecular weight of about 58 kd. The 58 kd protein 'A believed to be a product of cellular processing (glycosylation) of the 54 kd filamentous protein.

Monoclonal antibody 247:4D11 also immunoreacts with the 54 kd filamentous protein present in HeLa cell lysates, but does not immunoreact significantly with the shown preparation of SiHa or HT-3 cell lysates. (lanes 2 and 5, respectively) By the above immunoblotting methods monoclonal antibodies were demonstrated to immunoreact with all the papillomavirus latent proteins. For example, the 54 kd filamentous protein was detected by immunoblotting HeLa, CaSki and SiHa cell lysates using mionoclonal antibodies 235:B9 or 247:4D11. The 48 kd filamentous protein was similarly detected in CaSki cell lysates using the.e same monoclonal antibodies. The nuclear protein was detected by immunoblotting CaSki cell lysates usingmonoclonal antibody 245:I1E3. The diffuse protein was detected by immunoblotting HeLa and SiHa cell lysates using monoclonal antibodies 25 235:B9, 238:8E9, 247:10F7 and 247:11D11.

8. Immunohistochemical Detection of 0o 0 0o 4 0 *0 0 0000 O f 0 0 4 0 00 0 0 a0 644 t Latent Papillomavirus Proteins a. Detection in HPV-Containing i Tissue Culture Cells S 30 Cervical carcinoma cell lines HT-3, MS751, C-33A, SiHa, HeLa, and CaSki were cultured as described in Example 5. Semi-confluent monolayer cultures were selected, rinsed with PBS to remove excess culture media and air dried for minutes. Air dried cultures were then fixed by flooding the culture with cold (-20 degrees C) 6 C SJU U C U JLAL.CL. L.CL kz)~ 200 ul of a solution containing 0.1% (w/v) horseradish peroxidase in PB, and 200 ul of an ABTS solution containing freshly prepared ABTS dye (2,2'-azino-di[3-ethylbenzthiazolinesulfonate(6)] -78- 00 44 0 9 4 4 4 0 acetone for 5 min. The fixed cultures were then maintained in 3% hydrogen peroxide for 15 minutes followed by fifty rapid immersions into and out dips) of PBS at room temperature. The cultures were then maintained first in PBS for 2 minutes and then maintained in a continuously rocking PBS solution containing 8% normal horse serum and 0.01% thimerosal for 1 hr at room temperature to form non-specific protein binding site-blocked samples.

The blocked samples were maintained at room temperature for 60 minutes with a solution containing an anti-peptide antibody composition to form an immunoreaction product containing the admixed antibody and the blocked sample. As described below, several different antibodies were used in these assays at different dilutions.

Thereafter the immunoreacted sample was dipped times in PBS, maintained for 2 minutes in PBS, 20 dips plus 2 min) and this was repeated twice 3 times, 20 dips each plus 2 min).

The sample was then maintained in a solution of 0.5% BLOTTO non-fat powdered milk, 0.01% thimerosal, 0.025% antifoam A in PBS) containing 25 biotinylated horse anti-mouse IgG (Vector Labs, Burlingame, CA) at a concentration of 7 ug per ml for 45 minutes at room temperature to allow the formation of a second immunoreaction product between the admixed biotinylated IgG and the bound mouse antibodies present on the sample. Thereafter the sample was rinsed in PBS three times, 20 dips each plus 2 minutes, and then maintained at room temperature in NHT buffer (0.3 M NaCl, 20 mM Hepes, pH 6.5, 0.01% thimerosal) containing avidin Dperoxida'-e (Vector labs) at 12 ug per ml for minutes .o allow the avidin reagent to complex with

I

9 4' 44 4 4 40 O 4 0 O 04 0.0 4 0 I 010 9 40 004 0

LL_

support. A column having a 3 ml bea volume was prepared using polypeptide-agarose solid support and equilibrated by rinsing with NET buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl, pH About -79the biotin present in the second immunoreaction product. Thereafter the sample was rinsed in PBS three times, 20 dips each plus 2 minutes, and then maintained in AEC buffer, prepared by admixing (1) 4 mls dimethyformamide containing 50 mg of aminoethyl carbazole (Sigma), 80 ul hydrogen peroxide, and 200 ml 100 mM sodium acetate buffer pH 5.5 for 10 minutes at room temperature to allow the color-developing reaction to occur on the a sample. After development, the samples were shaken to remove excess liquid, rinsed in water and then maintained in Mayer's hematoxylin stain (Sigma) for three minutes followed by a water rinse. The stained sample was then mounted in 50% glycerol in water and viewed by light microscopy.

o° I° Results of immunohistochemical staining I (immunostaining) of HPV-containing cell lines is shown in Table 6.

t to 20 Table 6 0 Immunostaining of HPV-Containing Tissue Culture Cell Lines' 25 Cell Line Antibody Composition 2 4. 235:B9 245:11E3 247:4D11 S HeLa CaSki SI SiHa 30 MS751 i HT-3 C33-A 1 A significant and positive immunoreaction was determined by the presence of the characteristic rust colored staining pattern Antibodies Using Western immunoblot assays, HPVcontaining tissue culture cell lysates and various biopsy samples were prepared and examined for the as compared to the blue-grey color of the hematoxylin counterstain that predominates in the absence of antibody dependent peroxidase staining In i some cases, a weak immunoreaction was observed 2 Monoclonal antibody 235:B9 was immunoreacted with the blocked sample using a solution containing FPLC purified antibody, prepared as described in Example 6c, at a concentration of ug per ml of 0.2% BLOTTO non-fat powdered milk, 0.01% thimerosal, 0.025% antifoam A, in PBS). Monoclonal antibody 245:11E3 was immunoreacted with the blocked sample using a solution of ascites fluid that o 6 was prepared as described in Example 6c and diluted 1:40 in 10% normal horse serum v/v in PBS). Monoclonal antibody 247:4D11 was immunoreacted with the blocked sample using a 20 solution containing FPLC purified antibody at o a concentration of 100 ug per ml of 10% normal horse serum.

o6 4 goo The results in Table 6 demonstrate the ability of a monoclonal antibody of this invention to immunoreact with latent HPV proteins in an immunostaining format using HPV infected cell lines. The specificity for papillomavirus latent proteins is demonstrated by the ability of these antibodies to immunoreact with cells known to contain HPVs (HeLa, CaSki, SiHa and MS751) but not with cells that do not contain HPV (HT-3 and C33-

A).

LLLLUULJ 'k L;.yL;.Lb UL X.CUUZU-_IIay UAA'A suspension was centrifuged at abut 12,000 xg in a microcentrifuge to remove tissue debris. The 6-81b. Detection of Papillomavirus Latent Protein in Tissue Samples Formalin-fixed, paraffin-embedded tissue biopsies of human cervical carcinoma and human condyloma were obtained from Dr. Carpenter (UCSD Medical Center, San Diego, CA), Dr. J. Robb (Dept. of Pathology, Green Hospital, La Jolla, CA) and Dr. W. Lancaster (Georgetown University, Washington, DC). Papanicolaou smears were obtained from Dr. J. Willems (OB/GYN, Scripps Clinic, La Jolla, CA). These tissue samples were subjected to the same immunohistochemical staining procedure described in Example 8a with exceptions as hereby noted.

Formalin-fixed, embedded tissues *a were first deparaffinized in xylene by 50 dips S. followed by a 2 minutes soak in xylene. Thereafter the fixed tissue was dipped 50 times into 20 ethanol followed by a 2 minute soak, then dipped o* times into 80% ethanol followed by a 2 minute soak, then dipped into 50% ethanol 50 times followed by a 2 minute soak, and finally dipped 50 times in PBS o* followed by a 2 minute soak to form a rehydrated 25 sample. Thereafter the rehydrated sample was processed as described in Example 8a beginning with the step of maintaining the sample in 3% hydrogen peroxide solution.

Pap smear-containing slides were air 30 dried for 10 minutes, and then fixed by maintenance t 0! in 67% acetone/33% methanol for 10 minutes at degrees C. The fixed pap smears were then processed as described in Example 8a beginning with the step of maintaining the sample in the 3% peroxide solution.

I

1 -82- Results of immunostai, Lng various tissue biopsy samples using monoclonal antibodies of the present invention are shown in Table 7.

Table 7 Immunostaining of HPV-Containing Tissue Biopsies' Antibody Composition 2 No.

Tissue Samples 235:B9 245:11E3 247:4D11 Pap Smear 3 Condylomatous 3 3/3 N.T. N.T.

Not infected 1 0/1 N.T. N.T.

44 4 S' Biopsy t Cervical Dysplasia HPV Type 16 14 14/14 14/14 11/11 HPV Type 31 3 3/3 2/2 2/2 o4 HPV Type 11 2 2/2 0/2 0/2 Untyped 2 2/2 N.T. 1/1 Cervical Cancer 8 5/8 5/8 2/2 0. "Normal" epithelium 2 1/2 0/1 0/2 Uninfected epithelium 1 0/1 0/1 0/1 1 Results of immunostaining are reported as the number of tissues that tested positive over o the total number of samples tested. A 30 positive reaction was scored if the staining was significant as described in note 1 of Table 6.

2 Monoclonal antibodies 235:B9, 245:11E3 and 247:4D11 were immunoreacted using the conditions described in note 2 of Table 6.

-83- S Pap smears obtained from patients having colposcopically verified cervical condylomas were designated condylomatous. The pap smear designated "not infected" was derived from a verified virgin, believed to have had no risk of exposure to genital HPV infection.

4 Biopsy samples included biopsies of cervical dysplasia that were screened for the presence of a particular HPV type by nucleic acid hybridization [DeVilliers et al., Lancet, ii:703 (1987)] where indicated. Cervical cancer biopsies were screened having unknown HPV status. Two seemingly normal epithelium biopsies from surgery were screened, having unknown HPV status. A control uninfected epithelium biopsy was obtained from a six-year old female believed to have no risk of exposure to genital HPV infection.

o The results in Table 7 demonstrate the ability of monoclonal antibody molecules to immunoreact with HPV latent proteins present in 8, biopsy tissue samples. These samples showed 3.00% o. positive reactivity with no false negatives when using 235:B9 on samples known by independent means to contain latent HPV infection.

The results in Table 7 also demonstrate that certain monoclonal antibodies have .a the ability to immunoreact with a latent protein S30 produced by one but not another HPV type, a 4 4 type specific antibody molecule. For example, whereas monoclonal antibody 235:B9 immunoreacted by immunostaining with the HPV-typed dysplasia tissues having an 11, 16 or 31 type designation, the monoclonal antibodies 245:11E7 and 247:4D11 immunoreacted with HPV type 15 and type 31 L' __i

I

-84containing dysplasias but did not immunoreact with type 11 containing dysplasias.

In addition, detection of HPV latent proteins by immunostaining indicates the cellular localization of the latent proteins, thus providing additional characterization of these proteins. For example, the monoclonal antibody 247:4D11 produced an imunostaining pattern on CaSki cells or on various HPV-containing biopsy tissues that was distributed over cytoplasmic filament-associated components of the cell. Therefore, the 46 kd, 54 kd and 58 kd proteins, detected by immunoblotting with monoclonal antibody 247:4D11 as shown in Example 7, have been further characterized as being "filamentous' type latent proteins based on their distribution in immunostained cells. A similar filamentous staining pattern was observed using monoclonal antibody 235:B9 to immunostain HPVcontaining pap smears, biopsy samples and tissue 048 20 culture cells.

88 a Monoclonal antibody 245:11E3 produced a characteristic immunostaining pattern in CaSki cells that was localized to the nuclei.

4 a 8 Thus, the 51 kd protein, detected by immunoblotting 25 with this monoclonal antibody as described in Example 7, has been further characterized as being Hit 1 a "nuclear" type latent protein.

Monoclonal antibodies 238:8E9, 1 247:10F7 and 247:11Dll each produced a 30 characteristic immunostaining pattern in HeLa cells and SiHa cells that was localized in a diffuse manner throughout both the nucleus and the cytoplasm of the stained cells. Because these monoclonal antibodies detected the 112 kd protein in the immunoblotting assay described in Example 7.

The 112 kd protein has been further characterized aaaltional injection was administered to the mouse 2 weeks after the second injection containing the same inoculum as for the second.

0 0 00 0 o 9 a o f 0 t e oo

*C

S0 0 6 a a o o 0000 o o o O Q 00 0 00 0 b 0 0 00 0 0 a 0 0 0 0 0 as being a "diffuse" type latent protein.

9. Detection of Anti-HPV Latent Protein Antibody Molecules in Human Blood Antisera from patients diagnosed as having latent HPV infection, in the form of histologically confirmed condyloma lesions, were obtained from Drs. R. Robb and J. Willems (Scripps Clinic, La Jolla, CA).

Following an ELISA procedure similar to that described in Example 3, these antisera were evaluated for the ability to bind to polypeptides 237, 245 and 246, with the following exceptions as noted.

Fifty ul of coating solution containing 1 ug of polypeptide 237, 245 or 246 were added to the wells of a microtiter plate, and maintained at 4 degrees C overnight to allow the polypeptide to adsorb to the walls of the wells. Thereafter the wells were rinsed as before and blocked by adding 20 and maintaining NGS Buffer (10% normal goat serum in PBS) for 90 minutes at 37 degrees C. The wells were then emptied by inversion and shaking to remove the excess liquid, and were dried by maintaining the wells at 37 degrees C for 1 hour to 25 form dried plates.

One hundred ul of solution containing a patient antisera diluted in NGS buffer was admixed to each to form an immunoreaction admixture, and the admixture was maintained at 37 degrees C for 1 30 hour to allow the antibodies in the antisera to immunoreact with the polypeptides adsorbed onto the well walls and form a polypeptide-containing immunoreaction product. The wells were then washed times with PBS-T Tween 20 in PBS) to remove the unbound antisera, and the excess liquid was removed by shaking.

-86- 00 00 o a 0 0 ft o 4 e 004404 04 0 4 4 a €o a a *0 4 oo 0 a o .a 1 •O o 1f 040 010 0 o a44a o 4 One hundred ul of a solution containing horseradish peroxidase labeled monoc.onal antihuman immunoglobulin IgA conjugate (Janssen, Piscataway, NJ) diluted 1:5000 in NGS buffer was admixed into each well and maintained for 1 hr at 37 degrees C to permit formation of a second immunoreaction product between the bound human antibodies and the added labeled conjugate. The added solution was then removed, the wells ril.sed as before and excess liquid was removed by shaking.

A peroxidase substrate solution was freshly prepared by admixing 50 ml developing buffer [0.12 M Citric Acid, 0.26 M dibasic sodium phosphate (pH 1 ml OPD (1 mg 15 orthophenylenediamine per ml water), and 25 ul 30% hydrogen peroxide. One hundred ul of the peroxidase substrate solution were then admixed into each well to form a color developing-reaction admixture. After maintaining the developing- 20 reaction admixture in the dark for 20 minutes at room temperature, the O.D. of the solution admixture was measured using a multiskan plate reader equipped with a 492 nm filter.

Antisera from 6 different condylomatous patients when tested in the above ELISA procedure, demonstrated elevated levels of immunoglobulin IgA antibody molecules that immunoreacted with both polypeptides 237, 245 and 246. In contrast antisera from three healthy control patients failed to immunoreact with polypeptides 237, 245 or 246 Further, antisera from a patient with HPV type 11 condyloma failed to immunoreact with either polypeptide.

Antisera from a HPV-carrying patient was also show to immunoreact with HPV latent proteins using an immunoblotting format.

-87- Antisera that immunoreacted in the above ELISA format using polypeptide 245 was affinity isolated as described in Example 4. Affinity isolated anti-polypeptide 245 antisera (AI anti- 245) was then used in the immunoblot assay described in Example 5 on blots that contained cell lysates prepared from CaSki, SiHa, HeLa, C-33A and HT-3 cells when this immunoblot assay was conductad using alkaline phosphatase labeled monoclonal antihuman immunoglobulin IgA antibodies in place of the goat anti-rabbit IgG antibodies as described for immunoblotting in Example 5, a 58 kd HPV latent protein was detected in the CaSki cell lysates only. These results indicate that the presence of human anti-HPV latent protein antibodies can be o demonstrated using the immunoblotting format.

a 10. Isolation of a Papillomavirus Latent Protein One hundred mg of each of Macrosphere 20 Amino 300 AO beads (Alltech Associates, Deerfield, IL) were dispersed in 15 mls of 50 mM phosphate ibuffer (pH and the dispersion was degassed.

I, The beads were recovered from the degassed S.dispersion by centrifugation and the resulting pellet was recovered and resuspended in 13.5 ml of mM phosphate buffer (pH 7.5) to form a degassed bead suspension. While stirring the degassed bead suspension, 1.5 ml of 25% aqueous glutaraldehyde (FM Science, Cherry Hill, NJ) was admixed and the 30 stirring continued for 3 minutes to form glutaraldehyde coupled beads. Water was then added to the coupled beads while stirring to bring the final volume up to 5C ml, the beads were collected by centrifugation and washed in 50 ml water three times to form activated beads.

-88- An amount of rabbit anti-R-'ypeptide 236 antisera, prepared as described in Example 2c and containing about 500 mg of total protein, was diluted with an equal volume of PBS and centrifuged at 12,000 rpm at 4 degrees C for 15 min. in. a JA- 200 rotor (Beckman). The resulting supernatant was extracted with an equal volume of chloroform, and the aqueous phase was dialyzed overnight against buffer A (100 mM Tris-HCl, pH 8.0) to form dialyzed rabbit antisera.

About 9 mis of dialyzed rabbit antisera was applied to a PD-10 column (Pharmacia Fine Chemicals, Piscataway, pre-equilibrated with mis of buffer 7A. Of the eluant that exited the column, the first 2.5 mis were discarded and the remaining eluant was retained. Thereafter four mis o0. of buffer A were then added to the column and the a r o t resulting eluant was again retained and admixed with the previously retained eluant. The admixture 20 was passed through a 0.2u nitrocellulose acrodisc 9 filter (Gelman Sciences, Ann Arbor, MI) to form a filtered rabbit antisera.

9o The filtered rabbit antirera was applied to a Mono Q anion exchanger column equipped on an 25 automated FPLC apparatus (Pharmacia) using buffer A as the equilibrating buffer. The column was then washed in buffer A and then an elution gradient was applied comprising a 0-30% gradient of buffer D 9. (0.5 M NaCl in buffer The resulting gradientof 30 eluted fractions were monitored for antipolypeptide antibody immunoreactivity as measured in the ELXISA and the assay described in Example 3antibody-containing fractions were pooled to form an FPLC purified anti-polypeptide 236 antibody solution (FPLC anti-236).

Each of the resulting FPLC purified monoclonal antibody molecule containing solutions represent a substantially pure antibody because -89- One ml of FPLC anti-236 determined by O.D. 280 to have a concentration of 6 mg/ml, was admixed with 10 mg activated beads and the admixture was maintained at 4 degrees C for minutes in continuous agitation. Thereafter the agitated beads were isolated away from the unbound antibody molecules by centrifugation, resuspended in 1 ml of 1M glycine buffer (pH 7.0) and maintained for 30 minutes at room temperature to block the excess activated sites present on the beads. The blocked beads were then isolated away from the glycine buffer by centrifugation to form anti-236 conjugated beads.

The anti-236 conjugated beads were washed first in 1 ml of 5 mM Citrate buffer, pH 3.0, to 0 9elute excess glycine and were then washed twice in 1 ml PBS followed by 2 washes in RIPA buffer i. (prepared by admixing 2 ml nonidet P-40 (NP40), 2 gm sodium deoxycholate, 0.2 rm SDS, 2 ml 0.5M EDTA 0 and sufficient PBS to make the final volums 200 ml) to form equilibrated anti-236 conjugated beads.

A 0.12 gm packed cell pellet of HeLa cells was prepared as described in Example frozen to -70 degrees C, thawed and then suspended in 1 ml of RIPA buffer The HeLa cell suspension 'was then agitated by three strokes with a loosefitting pestle in a dounce homogenizer, and the agitated suspension was centrifuged at 12,000 x g for 15 minutes in a microcentrifuge. The resulting supernatant was collected and admixed with equilibrated anti-236 conjugated beads, and the admixture was maintained for 2 hr at 4 degrees C in continuous agitation to allow formation of an immunoreaction product between the conjugated antibodies and the latent papillomavirus diffuse protein. The admixture was then placed in a column

IN

and the beads contained in the admixture were washed by rinsing the column with 1 ml of RIPA buffer, 1 ml of LB buffer NP 40, 20 mM Tris HCL, pH 7.5, 150 mM PTaCl), 1 ml of 1 M KC1 and 1 ml of 2.5 mM CaCl2 in 5% PBS. To the rinsed column was then added 200 ul of 50 mM sodium citrate, pH and the eluant collecced. Sufficient 1 M sodium phosphate buffer (pH 7.5) was added to the eluant to neutralize the citrate buffer to about pH and the neutralized eluant was then admixed with sufficient 100% trichloroacetic acid to achieve TCA in order to precipitate the proteins present in the eluant. The protein precipitate was collected and washed with acetone, and the acetone washed protein was then resuspended in SB and analyzed by SDS-PAGE as described in Example SDS-PAGE analysis of the acetone washed protein isolated from HeLa cells demonstrated a protein having an apparent molecular weight of about 112 kd, that immunoreacted in the immunoblot 0 assay with rabbit anti-245 antisera prepared in Example 2c.

11. Detection of Anti-HPV Latent Protein ,j Antibody Molecules Using HPV 16- 25 Related Nested Polypeptides The polypeptides shown in Table 2, and polypeptides 245 and a control polypeptide 65 were used in an ELISA assay similar to that described in Example 9 to detect anti-HPV latent protein antibody molecules with the following exceptions as noted.

Fifty ul of coating solution containing polypeptide was added to the wells of a 96 wellflat bottom microtiter plate (Immunolon II, Dynatech, Chantilly, VA), so that each well contains 1 ug of only one of the polypeptides shown a, c to remove excess culture media and air dried for minutes. Air dried cultures were then fixed by flooding the culture with cold (-20 degrees C) i

I

-91in Table 8. The wells were maintained, rinsed and blocked as before. The plates were then dried as before and thereafter 100 ul of patient sera diluted 1/10 in NGS buffer was added to each well to form an immunoreaction admixture, that was maintained and then washed as before.

Imunoreaction product was detected using a horseradish peroxidase labeled monoclonal antihuman Ig-A conjugte (Janssen) diluted 1:3000 in NGS as before. A second set of wells were similarly prepared except that the labeled conjugate was anti-IgG (Ortho Diagnostics, Ontario, Canada) rather than IgA. The resulting colored reaction admixture was measured for optical density at about 490 nm (OD 90 o) and the measurements are shown in o Table 8.

0 0 0 0900 0 0 0 0 0 60 0 0 0 4 0 C 0 L I -i I remperature in NHT buffer (0.3 M NaCi, 20 MM Hepes, pH 6.5, 0.01% thimerosal) containing avidin Dperoxidaqe (Vector labs) at 12 ug per ml for minutes .o allow the avidin reagent to complex with -92- Table 8 Immunoreaction of H-PV-Infected Patient Antisera With HPV Type 16 Polypept Oes Hll-1 -Polypeptide Sequence Patient No.

653 66 245 71 72 73 74 75 79 80 go, 81 82 83 84 Blan]

SSEWQRDQFLSQV

SSTWHWTGHNVKHKSAIVTLTYD

HKSAIVTLTYDSEWQRDQC

HKSAIVTLTYDSEWQRDC

HKSN~IVTLTYDSEWQRC

HKSAIVTLTYDSEWQC

HKSAIVTLTYDSEWC

HKSAIVTLTYDSEC

KSAIVTLTYDSEWQRDQC

SAIVTLTYDSEWQRDQC

AIVTLTYDSEWQRDQC

IVTLTYDSEWQRDQC

VTLTYDSEWQRDQC

TLTYDSEWQRDQC

LTYDSEWQRDQC

1 I CA Jg .077 .028 .129 .049 .343 04'F .307 ND 2 .126 ND .421 .040 ND .082 ND .032 .201 .021 .360 .020 .303 .031 .252 .045 .123 .041 .090 .03-, 2 ZqgA I A .081 .038 .149 .060 .308 .911 .280 ND .120 ND .329 1.012 .436 .863 .396 1.361 .178 .386 .365 .796 .283 .287 .251 .066 .189 .065 .092 .049 .083 .056 .010 .028 .123 014 .034

ND

4 44 4 4- 4 444 4 404444 1 "Blank" indicates that NGS buffer was added in place of a polypeptide.

2 "IND" indicates not determined.

3 Polypeptide 65 is a control polypeptide whose sequence was described by Schoolnick et al., in published Application No. EPO 0257754A2.

S

-93- The results in Table 8 show that all the polypeptides that included either the sequence -LTYDSF-, or in the case of polypeptide 66, the sequence -HKSAIVTLTYD-, immunoreacted more strongly with IgG or IgA present in the patient's antisera than did control peptide 65. Patient 1 was diagnosed as having a type 18 latent HPV infection, and patient 2 was histologically confirmed to have squamous cell carcinoma of the cervix.

These results show that antisera from a HPV-infected patient immunoreacts with HPV type 16related polypeptides. The results demonstrate that the immunoreaction is dependent on the presence of either -LTYDSE- or in the case of polypeptide 66 a less well characterized epitope.

12. Determination of the Binding Epitope for An Anti-HPV Polypeptide Monoclonal Antibody S' Monoclonal antibody 245:11E3 was 20 evaluated for its ability to immunoreact with HPV H t 4 type 16-related polypeptides following a solidphase ELISA procedure similar to that described in Example 3, with the following exceptions as noted.

The polypeptides shown in Table 9 were 44, 25 adsorbed onto the walls of each well of a 96-well 4 microtiter plate (one polypeptide species per l C well), and thereafter the wells were blocked with NHS buffer (10% normal horse serum in PBS) rather than with blocking solution. Thereafter, 50 ul of monoclonal antibody 245:11E3 diluted serially in two-fold dilutions using NHS buffer was admixed to each blocked well. The admixture was maintained as before and an immunoreaction product was formed.

Solid phase bound immunoreaction product was detected using the goat anti-mouse IgG conjugate as described in Example 6a, and the results shown in -94- Table 9 are expressed as a titer nee -id to obtain maximal OD at 415 nm.

I

Table 9 Epitope Mapping Of Mab 245:11E3

NO.

235 237 245 74 Pentide Seguence M4AD PAGTN GEE GT GC TYDSEWQRDQFLSQVKI PC

HKSAIVTLTYDSEWQRDQC

HKSAIVTLTYDSEWC

HKSAIVTLTYDSEC

HKSAIVTLTY.DSC

HKSAIVTLTYDC

HKSAIVTLTYC

Titre 8 1:512 1:512 1:512 1:512 1: 256 1:128 8 'r 4 C 4 ri 4 4 4 44 4.

ft t Ct 4 4 C C 4 C C 4.4 Ct C C 4 Ctc C L:t 444 4 ~44 (4 -96- The results in Table 9 indicate that the epitope for HPV type 16-related polypeptide binding to monoclonal antibody 245:11E3 includes the amino acid residue sequence -TYDSE-.

13. Detection of Anti-HPV Latent Protein Antibody Molecules in Human Blood Using a Combination of HPV Type 16- Related Polypeptides HPV type 16-related polypeptides 237, 245 and 246 were included in an ELISA assay similar to that described in Example 9 to detect anti-HPV latent protein antibody molecules in patient's blood, with the following exceptions as noted.

Antisera was obtained from 46 patients 15 diagnosed as having latent HPV infections, in the 0 form of various histologically confirmed condyloma lesions or grades of cervical dysplasia as indicated in Table 10. These antisera were each admixed in individual wells having polypeptide 237, 245 or 246 adsorbed therein and were assayed as described in Example 9 for the presence of anti-HPV latent protein antibody molecules (immunoglobulin I IgA) capable of immunoreaction with the adsorbed HPV type 16-related polypeptides. The results are shown in Table -97- Table Detection of Patient IA That Immunoreacts With HPV Type 16 Polypeptides 1 4a a 4 0 t 9 4 a «c 4 a« 64 o a~ *0 0 4 a 0 o Co 94 8 0 640 04« 0 Patient Number 1 2 3 4 6 7 8 9 11 12 13 14 16 17 18 19 20 21 22 23 24 26 27 28 29 31 32 33 34 36 37 38 39 41 42 43 44 46 00 00 00 237 .441 .273 .148 .091 .155 .281 .150 .157 .279 .271 .196 .332 .528 .352 .320 .947 .159 .136 .181 .515 .232 .105 .517 .101 .515 .349 .102 .035 .080 .101 .052 .005 .026 .000 .020 .174 .017 177 .216 .186 .046 .000 .073 .074 .007 .000 .012 .000 .006 245 .550 .413 .243 .211 .333 .349 .198 .227 .615 .290 .228 .271 .608 .431 .318 1.052 .588 .477 .390 .639 .281 .271 .539 .128 .654 .525 .207 .067 .078 .142 .007 .072 .050 .027 .598 .243 .066 .361 .437 .211 .141 .042 .211 .114 .059 .057 .000 .000 .010 246 .273 .144 .095 .066 .099 .307 .111 077 .278 .152 .083 .057 .311 .253 .224 .393 .064 .078 .198 .299 .138 .105 .134 .037 .285 .254 .068 .026 .018 107 .024 .107 .000 .000 023 .022 .000 .136 .108 .145 .024 .000 .064 .083 .040 .000 .000 .012 .019 Blank .159 .066 .050 .026 .061 .070 .133 .031 .119 .073 .011 .058 .041 .068 .048 .048 .008 .026 .034 .096 .023 .022 .120 .065 .187 .146 .054 .026 .027 .029 .011 .000 .000 .012 .023 .048 .027 .098 .037 .046 .037 .019 .054 .023 .044 .034 .000 .003 .019 Histology 2 SC CANCER GRADE 2/3 CIN2 CIN3 UTERUS BIOPSY NEGATIVE CIN2 CIN3 CIN2 CIN1 CIN1 CIN1 CIN3 CIN2 CIN1 CIN2 CIN2 CIN3 CIN3 CIN2 CIN3+AM CIN2 CIN3 CIN3 CIN1 CIN1 CIN2 CIN2 SC CANCER, MICROINVASIVE MALE LAB WORKER MALE LAB WORKER CIN1 OB/GYN NO VISIBLE LESION OB/GYN NO VISIBLE LESION OB/GYN VISIBLE WART CIN1 OB/GYN NO VISIBLE LESION CIN2 COM1 CIN2 CIN2 CIN2 OB/GYN VISIBLE WART OB/GYN VISIBLE WART OB/GYN NO VISIBLE LESION OB/GYN VISIBLE WART OB/GYN VISIBLE WART OB/GYN VISIBLE WART NO SERUM ADDED NO SERUM ADDED NO SERUM ADDED 0 00 0 480 4 4 444444 4 4 cytoplasm of the stained cells. Because these monoclonal antibodies detected the 112 kd protein in the immunoblotting assay described in Example 7.

The 112 kd protein has been further characterized -98- SThe results are expressed as 0D 492 for wells having polypeptide 237, 245 or 246 adsorbed per well, and also for control wells having no polyepptide adsorbed (Blank).

2 Histology is reported for each antisera donor.

CIN indicates cervical intraepithelial neoplasia.

Borderline CIN CIN) is usually carrying HPV of some type, CIN1 is mild dysplasia, CIN2 is moderate dysplasia, and CIN3 is severe dysplasia or carcinoma in situ prior to basal cell layer penetration. Other histological characterizations are also noted.

o t ts i i' o t t o i 4 i t 4 4 t l t t I I t44 t tI The results in Table 10 indicate that patients harboring latent HPV infections and exhibiting different stages of cervical dysplasia or condyloma contain in their blood IgA antibody molecules that immunoreact with not only one but several different species of HPV type 16-related 20 polypeptide.

Thus the present invention contemplates the use of different species of polypeptide whose sequences are all deduced from one HPV type, in combinations with one another. These different 25 polypeptides can be included in separate wells of the practiced ELISA assay or diagnostic kit, as above, or can be combined together and adscrbed onto a single solid support, such as in a single well. A preferred combination includes 30 polypeptides 237, 245 and 246 in separate wells of a single microtiter plate.

14. Detection of Anti-HPV Latent Protein Antibody Molecules in Human Blood Using a Combination of HPV Type Specific Polypeptides Panels of antisera obtained from patients 4' I 4 4 1 4 c ht -99having latent HPV infections diagnosed histologically at various grades of dysplasia were analysed in an ELISA assay similar to that described in Example 9 with the following exceptions as noted.

Fifty ul of coating solution containing 1 ug of either polypeptide K69, K70, K72 or 245, or containing 1 ug of PV (purified papillomavirus viri-ns isolated by standard virological procedures from moose warts containing conserved virion papillomavirus proteins) as a control, were added to the wells of a 96 well, half area, flat bottom microtiter plate (Costar, Cambridge, MA), and maintained as before to adsorb the added material 15 to the walls of the wells. The wells were blocked *it Susing NHS buffer rather than NGS buffer.

Thereafter 50 ul of patient sera diluted 1:20 in NHS buffer was admixed to each well to form an ximmunoreaction admixture, and the admixture was 20 maintained at 37 degrees for 2 hours to alliw 4 formation of a polypeptide-containing immunoreaction product.

Fifty ul of a solution containing alkaline phosphatase-labeled polyclonal affinity 1 25 purified anti-human immunoglobulin IgA conjugate (Dakopatts, Copenhagen, Denmark) diluted 1:800 in NHS buffer was admixed into each well and B maintained for 2 hrs at 37 degrees to permit U formation of a second immunoreaction product between the bound human antibodies and the added labeled conjugate. The added solution was then removed, the wells rinsed as before using NHS buffer and excess liquid was removed by shaking.

Fifty ul of a PNPP substrate solution [pnitrophenyl phosphate; SIGMA Chemical Corp. St.

Louis, MO; at a concentrationof 1 mg per ml of 'f u~l-W~ hlSCIIIILP*NI

C

-100diethanolamine buffer, 9.8% p containing 0.01% MgCl] were then admixed into each well tc form a color developing-reaction solution.

After maintaining the admixture for 45 minutes at room temperature, the O.D. of the solution was measured using a multiskan plate reader equipped with a 405 nm filter.

The results measuring immunoglobulin IgA antibody molecules is shown in Table 11.

t t r t c

S(

I vo±ume up to 5c ml, the beads were collected by centrifugation and washed in 50 ml water three times to form activated beads.

-101- Table 11 Type-Specific Detection Of IA In HPV-Infected Patient Antisera Patient Number 1 2 3 4 6 7 8 11 12 13 14 17 18 21 22 24 26 27 28 So30 4 o 31 32 33 4 34 36 44 37 38 44 39 42 43 4 44 45 47 54 61 62 63 64 66 67 68 69 HPV Tvne Specific Polvoeptide Histology' SC CANCER CIN2 CIN3

NORMAL

CIN3 CIN2

CIN

CIN

CIN3 CIN2

CINI

CIN2 CIN2 CIN3 CIN2 CIN2 CIN3 CIN3

CIN

CIN2 SC CANCER

N.D.

CINI

NORMAL

NORMAL

GENITAL WART

CINI

CIN2

CIN

CIN2 CIN2 CIN2 GENITAL WART

NORMAL

GENITAL WART GENITAL WART

MONONUCLEOSIS

SC CANCER

UVI

GLAUCOMA

N.D.

CONVULS IONS

N.D.

CINi

CINI

N.D.

N.D.

CIN2 Pv 2 0 0 .128 .048 .071 0 .045 .057 0 0 0 .023 0 .012 0 .020 .036 .008 029 .030 .082 .142 .035 .0031 .055 .028 .010 .151 .088 .040 .006 .032 .030 .011 .011 .018 .004 .064 .037 0 .052 0 .003 .009 .059 .011 0 .021 16' .614 .277 .622 .154 .638 .018 .273 .696 .086 .524 .156 .462 .358 .088 .362 .342 .143 .046 .277 .368 .439 .071 N. D.

.007 .089 .071 .022 .041 .104 .495 .304 .301 .108 .026 .064 .040 .071 .384 .569 .084 .198 .189 .050 .026 .267 .060 .030 .030 6 0 N. D.

.134 .052 .182 0 .016 .099 0 0 .014 .115 .10 .050 .026 .079 .017 .035 0 .220 0 .037 .080 .041 .077 0 .083 .077 .073 .010 0 017 .043 .003 .036 .030 .043 0.

.297 .041 .035 0 .031 .046 .001 .039 .050 .043 185 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .052 0 0 0 0 0 0 0 .010 .038 0 0 .415 0 0 0 0 0 0 0 .007 0 0 0 0 0 0 0 0 0 0 0 0 3" 36 .025 0.005 .057 037 .153 .005 .049 .046 0 .027 .039 .072 .070 .023 .003 .064 .022 .026 0 .153 .034 .008 .049 .024 .020 .028 .060 .064 .058 .218 .047 .006 .017 0 .023 .021 .037 .036 .168 0 .038 0 .068 .028 .018 .029 .056 .043 3antibody-containing fractions were pooled to form an FPLC purified anti-polypeptide 236 antibody solution (FPLC anti-236).

-102- VAGINAL WARTS .016 CIN .086 N.D. .038 NORMAL .002 METAPLASIA .042 CIN 2-3 .020 CIN1 0 CIN2 .004 AUTOIMMUNE DISEASE .013 WRIST FIX 0 N.D. .022 JUVENILE DIABETES 0.009 MIGRAINE 0 SINUITIS .027 N.D. 0 N.D. .640 N.D. .014 N.D. .019 N.D. .028 N.D. .007 N.D. .002 N.D. .067 N.D. .045 N.D. .031 .029 .066 .059 .017 .096 .070 .022 .040 .454 .238 .219 .346 .131 .315 .035 .364 .069 .067 .056 .086 .100 .106 .052 .082 .052 .076 .045 .066 .265 .058 0 .035 0 0 .026 0 0 0 .012 0 .040 0.028 .029 .035 .039 .720 .132 .076 0 0 0 0 0001 0 0 0 0 0 .004 .007 .026 0 0 0 0 0 0 0 0 0 0 0 .029 .025 .037 .042 .115 .030 .046 .080 0 .017 .058 0 0 0 .040 0 .061 .038 .022 .057 .023 .032 .054 .054 003 00 9 o .o I t t t I 1 I It 119 It 1 «I ujIIn..IL4UUu agayli-Lon To a±Low zormar-Lon or an immunoreaction product between the conjugated antibodies and the latent papillomavirus diffuse protein. The admixture was then placed in a column -103- 1 Histology is reported for each antisera donor as in note 2 to Table 2 PV is control papilloma virions isolated from moose warts.

3 "16' indicates polypeptide 245 was included in the well having a sequence deduced from HPV type 16.

4 indicates polypeptide K70 was included in the well and having a sequence deduced fromHPV type 6.

5 "18" indicates polypeptide K69 was included in the well and having a sequence deduced from HPV type 18.

6 "33" indicates polypeptide K72 was included in the well having a sequence deduced from HPV type 33.

fI The results in Table 11 show that patients harboring various latent HPV infections 'c contain in their blood immunoglobulin IgA antibody 20 molecules that im-unoreact preferentially with one o over another HPV type-related polypeptide. For example, patient 1 is a confirmed HPV type 16 infected individual, as determined using ViraType DNA Typing Kit (Molecular Diagnostics, 25 Gaithersburg, MD), and his blood contained IgA antibody molecules that immunoreact 'substantially with HPV type 16-related polypeptide 245, and immunoreacted only to a small degree with type 33- S. related polypeptide K72. Patient 36 contained in 30 his blood IgA antibody molecules that preferentially immunoreact with HPV type 18-related polypeptide K69. Patients 75 and 92 contained in their blood IgA antibody molecules preferentially immunoreacting with HPV type 6-related polypeptide polypeptide was added to the wells of a 96 wellflat bottom microtiter plate (Immunolon II, Dynatech, Chantilly, VA), so that each well contains 1 ug of only one of the polypeptides shown -104- The results in Table 11 demonstrate one embodiment of the present invention h which HPV related-polypeptides, whose amino acid residue sequences are deduced from different HPV types, can be used to detect and distinguish, in a type specific manner, antibody molecules that are induced by one HPV type but not another. HPVrelated type specific polypeptides can be included in seperate wells of the practiced ELISA assay or diagnostic kit, as above, or can be combined together and adsorbed onto a single solid support, such as in a single well.

A similar assay was conducted using labeled antibody conjugates to detect both IgA and IgG immunoglobulins in a panel of donor antisera.

,In that assay, the results were first obtained as above for antisera containing immunoglubulin IgA antibf ly molecules that immunoreacted with the HPV type specific polypeptides shown in Table 12.

t i 20 Thereafter a similar ELISA assay was run on the same panel of antisera except using 50 ul of a solution containing horseradish peroxidase labeled polyclonal anti-human immunoglobulin IgG conjugate (Dakopatts) diluted 1:800 in NHS buffer in place of the IgA conjugate. After formation of a second immunoreaction product, and then rinsing as before, 50 ul of an ABTS substrate solution (ABTS at a concentration of 0.2 mg per ml of 0.002 SM Citrate, pH 5.0, 0.009% hydrogen peroxide) was admixed into each well to form a color developingreaction admixture. After maintaining the developing-reaction admixture in the dark for minutes at room temperature, the OD. of the solution admixture was measured using a multiskan plate reader equipped with a 415 nm filter.

-105- The results of detecting both IgA and IgG antibody molecules in human donor blood that immunoreact with HPV-related polypeptides is shown in Table 12, with the same headings as for Table 11.

9 0 209 9AS": t r

C)

a)

O

OR

W-)

9) .n 0,-I r_) ra( 'a 0 a)

H

0 E) M r- -iio 4r r r. _1LO o 0 C)000 c04 a -,4 r4 >1 0n 0 a) 'do-I 0 V. 0 4 Sa)0 n 4

I

C_1 M 10 to P4 o Table 12 Type-Specific Detection Of I A and I G In HPV-Infected Patient Antisera Patient Number Pv I A I -g 16 -]g

A

19

G

6 6 G 18 .034 .035 .027 .000 .067 .014 .152 .054 0 .024 .045 .031 .032 0 .019 .070 .040 .065 0 0 .044 .032 .023 .041 .031 S150 .380 0 0 .034 .032 .192 0 .407 .275 0 0 0 0 0 .080 .056 0 0 .069 .029 N. D 0 .060 ,189 .851 0 .442 .378 .012 .053 .048 .294 122 .066 .253 .619 .048 .034 .123 .082 .181 .370 018 .103 .020 0 .162 .055 .052 0 .056 .046 .032 .177 .284 0 .046 .239 .156 .789 .374 .654 .367 .117 .064 .064 .196 .119 .274 .520 .220 0

N.D.

.093 .5 78 0 .051 .311 1.056 .007 .568 .886 .106 .082 .063 .304 .112 .086 .093 .112 .024 .054 .033 .089 .152 0 .029 .122 0 .092 .607 .104 .073 .052 .016 .012 .123 .297 0 .345 .202 .247 .286 .360 .626 1.009 1.101 1.494 .192 .442 .376 .215 .019 .520 .048 .074 .117 .201 .818 0 .549 .555 0 .094 1.900 0 _IgA .013 .001 .004 0 .001 0 0 .368 0 0 0 0 0 0 .006 0 0 0 0 0 0 .020 0 0 .012 .161 0 33 I G I IG .007 .038 .117 .032 .160 0 0 .210 .326 0 .114 .192 0 0 .107 0 0 0 0 0 0 .036 1.456 0 0 .766 0 .062 .038 .025 .085 .084 .095 .042 .075 .086 .054 .058 .061 .098 .043 .014 .106 0 .048 .105 .020 .071 .038 .029 .046 .035 .265 0 .192 .247 .384 .248 .586 .389 .527 .556 .394 .233 .374 .748 .207 .438 .520 .106 0 .051 .209 0.009 0 .286 .209 0 .138 .982 0 O~e a a before and an immunoreaction product was formed.

Solid phase bound immunoreaction product was detected using the goat anti-mouse IgG conjugate as described in Example 6a, and the results shown in -107- Polypeptide Ligand Affinity Isolation of Anti-"'" Latent Protein Antibody Molecules From Human Blood Ten mg of polypeptide 245, prepared as described in Example 1, were dissolved in water and subsequently coupled to 4 ml of packed CH-Sepharose beads (Pharmacia) according to the manufacturer's instructions to form a polypeptide 245-agarose solid support. The prepared support was first washed with 10 mis of 4 M KSCN, then washed with 400 mls of PBS to form an equilibrated 245-support.

A second support was similarly prepared using a control polypeptide that had no sequence homology to HPV E region ORFs to form an equilibrated control support.

Antisera from a CIN patient having antibody molecules immunoreactive with polypeptide 245, as determined using the procedure described in Example 9, were collected. Two mls of the collected antisera was applied at a flow rate of Sml per hour to the control support, and the eluant off of the support was collected. Thereafter the Sa collected eluant was similarly applied to the Sequilibrated 245-support and the support was then 25 washed with about 80 mis of PBS containing 0.5 M NaCl to rinse off any material that was not 44 t specifically immunoreacted with the polypeptide contained on the 245-support.

Immunoreacted antibody molecules were a< 30 then eluted off of the 245-support by adding 4 M KSCN at a flow rate of 5 ml per hour to the support, and collecting the eluant in fractions.

The O.D. of the fractions was measured at 280 nm, the peak-containing fractions were determined and pooled to yield an antibody-containing pool. The pool was then dialysed against PBS to yield a -I a I r d i -108solution containing polypeptide 245-isolated purified human anti-HPV latent protein antibody molecules. The antibody molecules contained in the solution so prepared are referred to as affinity purified or affinity isolated human anti-HPV latent protein antibody molecules.

The resulting affinity isolated antibody molecules represents a substantially isolated antibody because greater than 50% of the antibody molecules contained in the solution have the capacity to immunoreact with a HPV type 16-related polypeptide. As demonstrated herein, these antibody molecules also have the capacity to immunoreact with a HPV latent protein.

16. Detection of Nuclear HPV Latent a 9 ~Proteins Using Human Anti-HPV #.fo Latent Protein Antibody Molecules a. Western Immunoblotting f, The cell line NIH3T3/HPV16 is a 20 mouse fibroblast NIH3T3 cell line stably transected with HPV type 16 (Yasumoto it al., J. Virol., 57:572-577, 1986), and was obtained from Dr. J.

DiPaolo. C4II is a HPV type 18-carrying cervical carcinoma cell line (Yee et al., Am. J. Pathol., 25 119:361-366,1985), and was obtained from the ATCC and cultured according to ATCC specifications.

The cell lines HT-3, CaSki and SiHa, described in Example 5, NIH3T3/HPV16 and C4II, and <the normal cell line NIH3T3 (ATCC) were subjected 30 to Western immunoblot assay as described in Example with the exceptions as noted.

Cell lysates were subjected to SDS-PAGE as before, but using 7% polyacrylamide gels and the molecular weight marker proteins indicated in the legend to Figure 5. After transfer of the electrophoresed cell lysates to nitrocellulose and I 1 -109blocking, the blocked blots were maintained for 12 hours .n a solution of polypeptide 245-affinity isolated human anti-HPV latent protein antibody molecules, prepared in Example 15 and diluted 1:10 in BLOTTO, undiluted culture supernatant from hybridoma 245:11E3 prepared as in Example 6b, or a solution containing rabbit affinity isolated anti-polypctide 245 antibody molecules prepared in Example 4 and diluted 1:32 in BLOTTO to allow an immunoreaction product to form between the admixed.

antibody compositions and the latent papillomavirus proteins prestnt as a solid phase on the blots.

The washed blots were then maintained in BLOTTO containing alkaline phosphatase conjugated to either &nti-human IgA, anti-mouse IgG, or anti-rabbit IgG, respectively, each diluted 1:1000, to allow a second immunoreaction product to I form between the second admixed antibody and the first formed immunoreaction product present on the t 20 solid phase of thti blot. The blots were then washed and the solid phase immunoreaction products were visualized usi.ng the chromogenic substrate developer solution for the developing times indicated in the le:end to Figure 25 Results using the Western immunoblot assay to detect latei.t papillomavirus proteins with l i t human antibody molecu..es are shown in Figure For instance, the human antibody molecules immunoreacte' with a 48 kd protein in 30 C4II cells, with a 48 Yi and 26 kd protein in NIH3T3/HPV16 calls, and with a 58 kd protein in CaSki cells on long exp~cnre (left portion of Figure 5A), but no immunreaction was seen with control cells HT-3 or NIHlT3.

As a further chaacterization, monoclonal anitbody 245:11E3 immunore cted with the 58 kd i -110protein in CaSki cells but not in HT-3 of SiHa cells (Figure 5b). The affinity isolated rabbit antibody immunoreacted predominantly with the 48 kd protein, and minimally with the 51 kd and 58 kd proteins in CaSki cells (Figure b. Immunohistochemical Detection CaSki, C-33A, HT-3, SiHa and Hela Tissue culture cells were prepared for immunochistochemical detection as described in Example 8a except that the fixed cells were blocked in 8% NHS for 30 minutes and were immunoreacted for 90 minutes using a solution containing either polypeptide 245-affinity isolated human anti-HPV latent protein antibody molecules, prepared in Example 15 and diluted in BLOTTO, or antibody molecules present in a supernatant from hybridoma 245:11AE3 culture,prepared in Example 6b and diluted 1:18 in 20 BLOTTO. The results showed that both antibody molecules immunoreacted with HPV type 16-infected CaSki cell but did not immunoreact with the other i cell lines tested. Visualized immunoreaction product in CaSki cells showed strong staining in the cell nucleus. The nuclear localization was confirmed by subcellular fractionation of CaSki cells to isolate the nuclei, and subsequent analysis of the isolated nuclei to identify a 58 kd protein by Western immunoblotting according to Example 16a.

These analyses indicate that both the human and the mouse polypeptide 245-affinity isolated antibody molecules immunoreacted with a nuclear HPV latent 58 kd protein and also with a 26 kd and 48 kd HPV latent protein.

_f .000 .073 .074 .007 .000 .012 .000 .006 .042 .211 .114 .059 .057 .000 .000 .010 .000 .064 .083 .040 .000 .000 .012 .019 .019 .054 .023 .044 .034 .000 .003 .019 OB/GYN VISIBLE WART OB/GYN NO VISIBLE LESION OB/GYN VISIBLE WART OB/GYN VISIBLE WART OB/GYN VISIBLE WART NO SERUM ADDED NO SERUM ADDED NO SERUM ADDED i h -111- 17. Correlation of Dysplasia Severity With IgA Anti-HPV Latent Protein Antibody Molecules in Human BloodyStatistical analysis was performed on ELISA immunoreaction results obtained by the ELISA assay in Example 9, using the antisera samples obtained from patients having dysplasias hystologically confirmed as CIN1, CIN2 or CIN3 as defined in note 2 to Table Table 13 shows the results of the statistical analysis.

*4 41 0* 4 4 F

CIN

Severity CIN1 CIN2 CIN3 Sample Size 32 20 14 Table 13 Mean OD 492 0.137 0.162 0.320 Standard Deviation 0.198 0.126 0.174 20 a fi 0 4.04 4 4,4 25 i 4 44 (444 4n 4 1 OD at 492nm was measured by immunoreaction of patient IgA antibody molecules with polypeptide 245 in ELISA assay.

The results show a correlation between dysplasia severity and anti-HPV latent protein antibody immunoreactivity with polypeptide 245.

Therefore, the present methods and diagnostic systems for detecting anti-papillomavirus latent protein antibody molecules can be utilized to correlate patient IgA immunoreactivity and titres with the severity of papillomavirus induced genital lesions and dysplasia.

The foregoing specification, including the specific embodiments and examples, is intended to be illustrative of the present invention and is i I- I L-w;Launor Anti-HPV Latent protei~n Antibody Molecules in Human Blood Using a Combination of HPV Type Specific oyetie Panels of antisera obtained from patients -112not to be taken as limiting. Numero s other variations and modifications can be effected without departing from the true spirit and scope of the present invention.

4 0

Claims (52)

1. A polypeptide represented by a formula selected from the group consisting of: MADPAGTNGEEGTGC, HEDEDKENDGDSLPTC, RPFKSNKSTCC, CCDWCIAAFGLTPSI, TYDSEWQRDQFLSQVKIPC, HKSAIVTLTYDSEWQRDQC, and CINCQKPLCPEEKQRH.

2. A polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence having the formula: -TYDSE-.

3. The polypeptide of claim 2 wherein ,the included amino acid residue sequence has a Kttt formula selected from the group consisting of: -LTYDSE-, -SAIVTLTYDSE-, and t 20 -HKSAIVTLTYDSE-. t t 4 1 4. A polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence having a formula selected from the group consisting of: 25 -HKSAIV- -SAIVTL- and -IVTLTD-. A polypeptide comprising no more S i than about 50 amino acid residues and including an amino acid residue sequence represented by a formula selected from the group consisting of: -TGILTVTYHSE-, -HAIVTVTYDSE-, -NAIVTLTYSSE-, -NGIVTVTFVTE-, and -ILTVT-. i; VJL UV.L Dov .304 1Iw- 62 GLAUCOMA 0 .084 .041 0 0 63 N.D. .052 .198 .035 0 .038 64 CONVULSIONS 0 .189 0 0 0 N.D. .003 .050 .031 0 .068 66 CIN1 .009 .026 .046 0 .028 67 CIN1 .059 .267 .001 0 .018 68 N.D. .011 .060 .039 0 .029 69 N.D. 0 .030 .050 0 .056 CIN2 .021 .030 .043 0 .043 a s a t t a f i *0^ -114-

6. A polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence represented by the formula: XZX', wherein Z is an amino acid residue sequence containing at least 5 amino acid residues having a sequence corresponding to a portion of the sequence represented by the formula: HKSAIVTLTYDSE, wherein X is hydrogen or at least one amino acid residue, and wherein X' is hydroxyl or at least one amino acid residue, said polypeptide being capable of immunoreacting with anti-human papilloma virus latent protein antibodies.

7. The polypeptide of claim 6 wherein Z is an amino acid residue sequence that includes an amino acid residue sequence represented by a formula selected from the group consisting of: oo 20 -TYDSE-, S°-LTYDSE-, -SAIVTLTYDSE-, and -HKSAIVTLTYDSE-. o, 8. The polypeptide of claim 6 wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: SSTWHWTGHNVKHKSAIVTLTYD, ,o HKSAIVTLTYDSEWQRDC, HKSAIVTLTYDSEWQRC, SHKSAIVTLTYDSEWQC, HKSAIVTLTYDSEWC, HKSAIVTLTYDSEC, HKSAIVTLTYDSC, HKSAIVTLTYDC, HKSAIVTLTYC, i -115- KSAIVTLTYDSEWQRDQC, SAIVTLTYDSEWQRDQC, AIVTLTYDSEWQRDQC, IVTLTYDSEWQRDQC, VTLTYDSEWQRDQC, TLTYDSEWQRDQC, and LTYDSEWQRDQC.

9. A polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence having the formula: XTYDSEX', wherein X is hydrogen or at least one amino acid residue, and wherein X' is hydroxyl or at least one amino acid residue with the proviso that X' does not include the amino acid residue sequence WQIRDQFLSQV. I *1 10. The polypeptide of claim 9 wherein X' is an amino acid residue sequence represented by 1 a formula selected from the group consisting of: W, WQ, WQR, t WQRD, and WQRDQ.

11. The polypeptide of claim 9 wherein X' is an amino acid residue sequence represented by a formula selected from the group consisting of: WQRDQF, and WQRDQFL.

12. The polypeptide of claim 9 wherein X' is an amino acid residue sequence represented by a formula selected from the group consisting of: WQRDQFLS, and WQRDQFLSQ.

13. A polypeptide comprising no more than about 50 amino acid residue and including an j !i| their blood IgA antibody molecules preferentially immunoreacting with HPV type 6-related polypeptide -116- amino acid residue sequence represented by a formula selected from the group consisting of: -TDYSE-, -LTDYSE-, -SAl VTLTDYSE-, -HRSAIVTLTDYSE-, -HKSAIV-I -SAl VTL- ,and -IVTLTD- ;and wherein said polypeptide does not contain the amino acid residue sequence -WRQRDQFLSQV.

14. A polypeptide having an amino acid residue sequence represented by the formula selected from the group consisting of: SSTWHWTGHNVKHKSAIVTLTYD, HKAITLYSEcRC HKSAI'vTLTYDSEWQRC, HI(SAIVTLTYDSEWQC, HKSAIVTLTYDSEWC, HKSAIVTLTYDSEWC, HKSAIVTLTYDSC, HKAITTYC HKSAIVTLTYDC, HKSAIVTLTYDCQRC HSAIVTLTYCWQDC SAIVTLTYDSEWQRDC, S~lVTLTYDSEWQRDC, AVTLTYDSEWQRDC, ITLTYDSEWQRDC,an LTYDSEWQRDC. A Composition comprising a substantially pure human papillomavirus 54 kd filamentous protein, said protein containing a first epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polype; -ide represented by the formula: %U A I I 6-1A= JG11 L J. 6% minutes at room temperature, the O.D. of the solution admixture was measured using a multiskan plate reader equipped with a 415 nm filter. -117- MADPAGTNGEEGTGC; and containing a second epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polypeptide represented by the formula: CINCQKPLCPEEKQRH.

16. A composition comprising a substantially pure human papillomavirus 48 kd filamentous protein, said protein containing a first epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polypeptide represented by the formula: MADPAGTNGEEGTGC; and containing a second epitope having the capacity to immunoreact with anti-polypeptide antibodies I "t induced by a polypeptide represented by the formula: CINCQKPLCPEEKQRH.

17. A composition comprising a substantially pure human papillomavirus 112 kd diffuse protein, said protein containing a first epitope having the capacity to immunoreact with anti-peptide antibodies induced by a polypeptide represented by the formula: HEDEDKENDGDSLPTC; and containing a second epitope having the capacity to immunoreact with anti-polypeptide antibodies induced by a polypeptide represented by the formula: HKSAIVTLTYDSEWQRDQC.

18. A composition comprising a substantially pure human papillomavirus 51 kd nuclear protein, said protein containing an epitope having the capacity to immunoreact with anti- peptide antibodies induced by a polypeptide represented by the formula: c HKSAIVTLTYDSEWQRDQC.

19. An antibody containing substantially isolated antibody molecules that innunoreact with a polypeptide having the formula; SSTWHWTGHNVXHKSAIVTLTYDSEWRDC, EKTGILTVTYHSETQRTRC, HKHAIVTVTYQSEEQRQQC, HKNAIVTLTYSSEEQRQQC, or SKNGIVTVTFVTEQQQQMC.

20. The antibody of claim 19 wherein said antibody molecule is a human antibody moleule.21. An anti-polypeptide antibody that immiunoreacts with only one of the polypeptides selected from the group consisting of: I4ADPAGTNGEEGTGC, HEDEDKENDGDSLPTC, 20 RPFXSNI<STCC, TYDSEWQRDQFLSQVKIPC, HKSAIVTLTYDSEWQRDQC, and CINCQKPLCPEEKQRH. 44t~t22. A monoclonal antibody containing a as. antibody molecules that iminunoreact with a polypeptide having the formula: 4 g MADPAGTNGEEGTGC, HEDEDKENDGDSLPTC, RPFKSNKSTCC, CCDWCIAAFGLTPSI, TYDSEWQRDQFLSQVKIPC, HKSAIVTLTYDSEWQRDQC, or CINCQKPLCPEEKQRH.

23. A monoclonal antibody containing antibody molecules that immunoreact with a polypeptide having the formula: SSTWHWTGHNVXHKSAIVTLTYDSEWQRDC, i I (D CO W 00 0 0 U 0 j 0 tb. W WWW wwL oco ooui 0t~W0 ~W~L71Mc4P.O3 1 119 EKTGILTVTYHSETQRTRC, HKHAIVTVTYOSEEQRQQC, HKNAIVTLTYSSEEQRQQC, or SKNGIVTVTFVTEQQQQMC

24. A monoclonal antibody containing antibody molecules that immunoreact with one of the following human papillomavirus latent proteins: i) the 112kd diffuse protein, ii) the 54 kd filamentous protein, iii) the 48 kd filamentous protein, iv) the 51 kd nuclear protein, or v) the 58 kd nuclear protein wherein said human papillomavirus latent proteins are as hereinbefore defined.

25. A monoclonal antibody comprising from the group of hybridomas consisting of 235:39, 245:11E3 and 247:4D11, wherein said hybridomas are as hereinbefore defined, said antibody molecules being capable of immunoreacting with the following latent human papillomavirus proteins: i) the 112kd diffuse protein ii) the 54kd filamentous protein iii) the 48kd filamentous protein iv) the 51 kd and 58kd nuclear protein 25 the 58 kd nuclear protein wherein said human papillomavirus latent proteins are as Shereinbefore defined.

26. A hybridoma that produces antibody molecules that immunoreact with a latent human papillomavirus protein, said hybridoma selected from the group of hybridomas I consisting of 235:B9, which reacts with the 112 kd S. ,diffuse protein, the 54kd filamentous protein and the 48kd filamentous protein; 245:11E3 which reacts with the 51 kd and 58 kd nuclear protein; and 247:4D11 which reacts with the 54kd and 48 kd filamentous protein.

27. A polyclonal antibody containing substantially isolated antibody molecules that immunoreact with a human papillomavirus latent protein selected from. the group consisting of: O r- -119A- i) the 112 kd diffuse protein, ii) the 54 kd filamentous protein, iii) the 48 kd filamentous protein iv) the 51 kd nuclear protein; and v) the 58 kd nuclear protein wherein said human papillomavirus latent proteins are as hereinbefore defined.

28. A polyclonal antibody containing substantially pure antibody molecules that irmunoreact with a human papillomavirus latent protein selected from the group consisting of: i) the 112 kd diffuse protein ii) th 54 kd filamentous protein, iii) the 48 kd filamentous potein iv) the 51 kd nuclear protein; and v) the 58 kd nuclear protein wherein said human papillomavirus latent proteins are as hereinbefore defined. 00 o a 00 e t a 00 o t 1 41 -A D, witn tne exceptions as noted. Cell lysates were subjected to SDS-PAGE as before, but using 7% polyacrylamide gels and the molecular weight marker proteins indicated in the legend to Figure 5. After transfer of the electrophoresed cell lysates to nitrocellulose and

29. A method of assaying fbr the presence of a papillomavirus infection in a human subject. comprising the steps of: a) forming an immunoreaction admixture by admixing a body fluid sample of said subject with a polypeptide represented by a formula selected from the group consisting of: HKSAIVTLTYDSEWQRDQC, and CCDWCIAAFGLTPSI; b) maintaining said. immunoreaction admixture under biological assay conditions for a time period sufficient for any anti-papillomavirus latent protein antibodies present in the sample to immunoreact with said polypeptide to form a polypeptide-containing immunoreaction product; and c) assaying for the presence of any polypeptide-containing immunoreaction product that f: formed, and thereby the presence of any papillomavirus infection in said subject.

30. The method according to claim 29 o wherein said polypeptide-containing immunoreaction Sproduct is labeled prior to assaying according to step c) by: i) forming a labeling reaction S 25 admixture by admixing with said polypeptide- S: containing immunoreaction product a labeled specific binding agent cdaable of binding to any S'human immunoglobulin product, and ii) maintaining said labeling reaction admixture so formed under' biological assay conditions for a time period sufficient for said L I i i i 3 -T1 4labeled binding agent to immunoreact with any anti- papillomavirus latent protein antibodies present as polypeptide-containing immun-C,-eaction product to form a labeled complex.

31. The method according to claim 29 wherein prior to said admixtu-,- of step a) said polypeptide is affixed to a saAid matrix.

32. A method of aselying for the presence cki a latent human pap.M.lomavirus infection in a tissue sample comprising: a) forming an immu-r -,.eaction adimixture by admixing the tissue sample vi~th an anti- polypeptide antibody containin; antibody molecules that immunoreact with a polypeptide selected from g 15 the group consisting of: It MADPAGTNGEEI, rMc, 0 HEDEDI(ENDGD4I'i.TPTC, RPFKSNKSTCC, I CCDWCIAAFGL'I:)?SI, 120 TYDSEWQRDQF:E[,,,3QVKIPC, HRSAIVTLTYDE::EWQRDQC, and ~1 CINCQKPLCPELKQRHi; b) maintaining the dmixture for a time period sufficient for said ant,," ody molecules to innunoreact with any papilloma'rvirus latent protein present in the sample and for,,, an immunoreaction pro~duct; and c) detecting the~ presence of any imaunoreaction product form~lu in step b) and thereby the presence of a lav1~ent human papillomavirus infection in m~aid sample.

33. The method ac(,.,-rding to claim 32 wherein said anti-polypeptide antibody is a monoclonal antibody.

34. The method according to claim 33 wherein said monoclonal antibody is produced by a -122- hybridoma selected from the group consisting of 235:B9, 245:11E3 and 247:4D11. A method of assaying for the presence of anti-human papillomavirus latent protein antibodies in a human subject comprising the steps of: a) forming an immunoreaction admixture by admixing a body fluid sample of said subject with a polypeptide comprising no iio!e than about 50 amino acid residues and including an amino acid residue sequence represented by the formula: XZX', wherein Z is an amino acid residue sequence containing at least 5 amino acid residues having a sequence corresponding to a portion of the sequence represented by the formula: .V HKSAIVTLTYDSE, t wherein X is hydrogen or at least one amino acid residue, and wherein X' is hydroxyl or at least one amino acid residue, said polypeptide being capable of immunoreacting with anti-human papilloma virus latent protein antibodies; o O b) maintaining said immunoreaction admixture under biological assay conditions for a time period sufficient for any anti-papillomavirus latent protein antibodies present in the sample to immunoreact with said polypeptide to form a Io polypeptide-containing immunoreaction product; and 0oD 0 .o c) assaying for the presence of o 0 any polypeptide-containing immunoreaction product that formed, and thereby the presence of any anti- human papillomavirus latent protein in said subject.

36. The method according to claim wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the -123- group consisting of: SSTWHWTGHNVKHKSAIVTLTYD, HKSAIVTLTYDSEWQRDC, HKSAIVTLTYDSEWQRC, HKSAIVTLTYDSEWQC, HKSAIVTLTYDSEWC, HKSAIVTLTYDSEC, HKSAIVTLTYDSC, HKSAIVTLTYDC, HKSAIVTLTYC, KSAIVTLTYDSEWQRDC, SAIVTLTYDSEWQRDC, AIVTLTYDSEWQRDC, o IVTLTYDSEWQRDC, S' 15 VTLTYDSEWQRDC, TLTYDSEWQRDC, and LTYDSEWQRDC.

37. The method according to claim wherein said polypeptide-containing immunoreaction product is labeled prior to assaying according to step c) by: forming a labeling reaction admixture by admixing with said polypeptide- containing immunoreaction product a labeled specific binding agent capable of binding to any human immunoglobulin product, and ii) maintaining said labeling reaction admixture so formed under biological assay tt: conditions for a time period sufficient for said labeled binding agent to immunoreact with any anti- papillomavirus latent protein antibodies present as polypeptide-containing immunoreaction product to form a labeled complex.

38. The method according to claim wherein prior to said admixture of step a) said polypeptide is affixed to a solid matrix. -I- JU 4 O 0 4rro ill -124-

39. A method fo assaying .or the presence of a papillomavirus infection anti-human papillomavirus latent protein antibodies in a human subject comprising the steps of: a) forming an immunoreaction admixture by admixing a body fluid sample of said subject with a polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence having a formula selected from the group consisting of: -TGILTVTYHSE-, -HAIVTVTYDSE-, -NAIVTLTYSSE-, -NGIVTVTFVTE-, and -ILTVT-; S..t b) maintaining said immunoreaction admixture under biological assay conditions for a time period sufficient for any anti-papillomavirus latent protein antibodies present in the sample to 20 immunoreact with said polypeptide to form a polypeptide-containing immunoreaction product; and c) assaying for the presence of any polypeptide-containing immunoreaction product "that formed, and thereby the presence of any anti- human papillomavirus latert protein in said subject. The method according to claim 39 S wherein said, polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: EKTGILTVTYHSETQRTKL, HKHAITVVTYDIEEQRQQC, HKNAIVTLTYSSEEQRQQC, and SKNGIVTVTFVTEQQQMC.

41. The method according to claim 39 wherein said polypeptide-containing immunoreaction L iIC I~ I -125- product iE labeled prior to assaying according to step c) by: i) forming a labeling reaction admixture by admixing with said polypeptide- containing immunoreaction product a labeled specific binding agent capabe of binding to any human immunoglobulin product, and ii) maintaining said labeling reaction admixture so formed under biological assay conditions for a time period sufficient for said labeled binding agent to immunoreact with any anti- papillomavirus latent protein antibodies present'as polypeptide-containing immunoreaction product to form a labeled complex.

42. The method according to claim 39 wherein prior to said admixture of step a) said polypeptide is affixed to a solid matrix.

43. A diagnostic s' 'tem, in kit form, comprising a package containing, in an amount sufficient to perform at least one assay, a substantially pure human papillomavirus latent protein selected from the group consisting of i) the 112 kd diffuse protein, ii) the 54 kd filamentous protein, iii) the 48 kd filamentous protein, iv) the 51 kd nuclear protein; and v) the 58 kd nuclear protein wherein said .human papillomavirus latent proteins are as hereinbefore defined.

44. A diagnostic system in kit form for C30 assaying for the presence of a latent human papillomavirus infection in a tissue sample, which system comprises a package containing, in an amount sufficient to perform at least one assay, an anti- polypeptide antibody that immunoreacts with only one of the polypeptides selected from the group consisting of: A 2 MADPAGTNGEEGTGC, u ,uecruuea trom the group consisting of: -TGILTVTYHSE-, -HAIVTVTYDSE-, -NAIVTLTYSSE-, -NGIVTVTFVTE-, and -ILTVT-. -126- HEDEDKENDGDSLPTC, RPFKSNKSTCC, CCDWCIAAFGLTPSI, TYDSEWQRDQFLSQVKI3PC, HKSAIVTLTYDSEWQRDQC, and CINCQKPLCPEEKQRH. The diagnostic system according to claim 44 wherein said anti--polypeptide antibody is a monoclonal antibody.

46. A diagnostic system, in kit form for assaying for'the presence of a latent papillomavirus infection in a human subject, which system comprises a package containing, in an amount sufficient to perform at least one assay, an antibody containing substantially pure or substantially isolated antibody molecules that immunoreact with a human papillomavirus latent protein selected from the group consisting of: a) the 112 kd diffuse protein, 0 b) the .54 kd filamentous protein, c) the 48 kd filamentous protein, d) the 51 kd nuclear protein, and e) the 58 kd nuclear protein wherein said human papillomavirus latent proteins are as hereinbefore defined.

47. The diagnostic system according to claim 46 wherein said antibody is a monoclonal antibody.

48. A diagnostic system in kit form for assaying for the presence of anti-papillomavirus 30 latent protein antibodies in a body fluid sample comprising a package containing, in an amount sufficient to perform at least one assay, a polypeptide represented by a formula selected from the group consisting of: HKSAIVTLTYDSEWQRDQC, and CCDWCIAAFGLTPSI, m 6 0 HKSAIVTLTYDSEWQC, HKSAIVTLTYDSEWC, HKSAIVTLTYDSEC, HKSAIVTLTYDSC, HKSAIVTLTYDC, HKSAIVTLTYC, r- c -127- said polypeptide being capable of immunoreacting with antibodies induced by a latent papillomavirus infection.

49. The diagnostic system according to claim 48 wherein said polypeptide is affixed to a solid matrix. A diagnostic system in kit form for assaying for the presence of anti-papillomavirus latent protein antibodies in a body fluid sample comprising a package containing, in an amount sufficient to perform at least one assay, at least one species of polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence represented by the formula: 6o 4 P 4 4 4 4 4. 4 4r 4, 4~ 4 4 (-4 4 4 15 XZX', wherein Z is an amino acid residue sequence containing at least 5 amino acid residues having a sequence corresponding to: HKSAIVTLTYDSE, wherein X is hydrogen or at least one amino acid residue, wherein X' is hydroxyl or at least one amino acid residue, said polypeptide being capable of immunoreacting with anti-human papilloma virus latent protein antibodies.

51. The diagnostic system according to claim 50 wherein said polypeptide is affixed to a solid matrix.

52. The diagnostic system according to claim 50 wherein said polypeptide comprises more than one species of polypeptide and wherein said species are present as an admixture.

53. The diagnostic system according to claim 50 further including, in a separate package, a labeled specific binding agent for signaling the presence of a polypeptide-containing immunoreaction product. v wnerein X' is an amino acid residue sequence represented by a formula selected from the group consisting of: WQRDQFLs, and WQRDQFLSQ. 13. A polypeptide comprising no more than about 50 amino acid residue and including an C, k -128-

54. The diagnostic system .according to claim 50 wherein said polypeptide includes an amino acid residue sequence represented by a formula selected from the group consisting of: -TYDSE-, -LTYDSE-, -SAl VTLTYDSE-, and -I-KSAIVTLTYDSE-. The diagnostic system according'to claim 50 wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: SSTWHWTGHNVKHKSAIVTLTYD, HKSAIVTLTYDSEWQRDC, 00 1 40AVLYDEQC 215 HKSAIVTLTYDSEWQRC, 000110HKSAIVTLTYDSEWQC, HKSAIVTLTYDSEWC, to S4HKSAIVTLTYDSC, HKSAIVTLTYDC, HKSAIVTLTYC, 0SAVTLYDEWIsC O KSAIVTLTYDSEWQRDC, P 0 aSITTYSWRC 00 AIVTLTYDSEWQRDC, .06.2 IVTLTYDSEWQRDC, VTLTYDSEWQRDC, TLTYDSEWQRDC, C LTYDSEWQRDC,

56. The dAganostic system according to 0430 claim 50 further including at least one species of polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence represented by a formula selected from the group consisting of: -TGILTVTYHSE-, -HAIVTVTYDSE-, -129- -NAIVTLTYSSE-, -HGIVTVTFVTE-, and -ILTVT-.

57. The diagnostic system according to claim 56 wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: EKTGILTVTYHSETQRTKL, HKHAIVTVTYDSEEQRQQC, HKNAIVTLTYSSEEQRQQC, and SKNGIVTVTFVTEQQQQMC.

58. The diagnostic system of claim 56 wherein said recited polypeptides are present as an admixture. 15 59. A diagnostic system in kit form for assaying for the presence of anti-papillomavirus latent protein antibodies in a body fluid sample t comprising a package containing, in an amount ~sufficient to perform at least one assay, at least 0 20 one species of polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence represented by a formula 0 «o selected from the group consisting of: -TGILTVTYHSE-, o 25 -HAIVTVTYDSE-, -NAIVTLTYSSE-, -HGIVTVTFVTE-, and So0 -ILTVT-; o said polypeptide being capable of immunoreacting o 30 with antibodies induced by a latent papillomavirus infection. The diagnostic system according to claim 59 wherein said polypeptide is affixed to a solid matrix.

61. The diagnostic system according to claim 59 wherein said polypeptide comprises more 18. A composition comprising a substantially pure human papillomavirus 51 kd nuclear protein, said protein containing an epitope having the capacity to immunoreact with anti- peptide antibodies induced by a polypeptide represented by the formula: -130- than one species of polypeptide and jerein said species are present as an admixture.

62. The diagnostic system according to claim 59 further including, in a separate package, a labeled specific binding agent for signaling the presence of a polypeptide-containing immunoreaction product.

63. The diagnostic system accordingto claim 59 wherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: EKTGILTVTYHSETQRTKL, HKHAIVTVTYDSEEQRQQC, HKNAIVTLTYSSEEQRQQC, and S 15 SKNGIVTVTFVTEQQQMC. oo

64. A diagnostic system in kit form 4 It p comprising: a) a first solid support comprised of a i solid matrix having operatively affixed thereto, a polypeptide comprising no more than about 50 amino acid residues and including an amino acid residue sequence represented by the formula: SXZX', wherein Z is an amino acid residue sequence containing at least 5 amino acid residues having a sequence corresponding to: HKSAIVTLTYDSE, r 3 wherein X is hydrogen or at least one amino acid residue, and wherein X' is hydroxyl or at least one amino acid residue, said polypeptide being capable of immunoreacting with anti-human papilloma virus latent protein antibodies; and b) a second solid support comprised of a solid matrix having operatively affixed thereto, a polypeptide comprising no more than about amino acid residues and including an amino acid -131- residue sequence represented by a formula selected from the group consisting of: -TGILTVTZHSE-, -HAIVTVTYDSE-, -NAIVTLTYSSE-, -HGIVTVTFVTE-, and -ILTVT-; said polypeptide being capable of immunoreacting with antibodies induced by a latent papillomavirus infection. The diagnostic system according to claim 64 wherein said first solid support affixed- polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: '0 4 SSTWHWTGHNVKHKSAIVTLTYD, HKAIT YSEQRC 8 HKSAIVTLTYDSEWQRC, 2. HKSAIVTLTYDSEWQRC, HKSAIVTLTYDSEWC, o *VTTDSC HKSAIVTLTYDS~C, HKSAIVTLTYDSc, HKSAIVTLTYDC, HKSAIVTLTYDCQRC HSAIVTLTYCWQDC ~*25 KAIVTLTYDSEWQRDC, SIVTLTYDSEWQRDC, AVTLTYDSEWQRDC, LTYDSEWQRDC,

66. The diagnostic system according to claim 64 wherein said solid support af fixed- polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: -132- EKTGILTVTYSETQRTKI, HIHArTVTYDSEEQRQQC, HKNA=VLTYS SEEQRQQC, and SKNGIVTVTFVTEQQQQ4C.

67. A polypeptide substantially as disclosed herein in conjunction with any one of the examples. DATED this 10th day ofE June 1992 SCRIPPS CLINIC AND RESEARCH FOUNDATION By their Patent Attorneys GRIFFITH HACK CO.