WO1999018220A1

WO1999018220A1 - Papilloma virus capsomere vaccine formulations and methods of use

Info

Publication number: WO1999018220A1
Application number: PCT/US1998/020965
Authority: WO
Inventors: Lutz Gissmann; Martin Müller
Original assignee: Loyola University Of Chicago
Priority date: 1997-10-06
Filing date: 1998-10-06
Publication date: 1999-04-15
Also published as: ATE449852T1; EP1021547B1; EP1021547A1; DE69841342D1; PT1021547E; IL135528A0; DE69836753D1; EP1690941A1; PL195332B1; CZ20001244A3; US7371391B2; US20090028894A1; NO328128B1; JP2001519161A; CA2305382A1; US6228368B1; PL339735A1; HUP0004360A3; CZ302351B6; DE69836753T2

Abstract

Vaccine formulations comprising viral capsomeres are disclosed along with methods for their production. Therapeutic and prophylactic methods of use for the vaccine formulations are also disclosed.

Description

PAPILLOMA VIRUS CAPSOMERE VACCINE FORMULATIONS AND METHODS OF USE

FIELD OF THE INVENTION

The present invention relates to vaccine formulations comprising papilloma virus proteins, either as fusion proteins, truncated proteins, or truncated fusion proteins. The invention further embraces methods for producing capsomeres of the formulations, as well as prophylactic and therapeutic methods for their use.

BACKGROUND Infections with certain high-risk strains of genital papilloma viruses in humans (HPV) — for example. HPV 16, 18, or 45 — are believed to be the main risk factor for the formation of malignant tumors of the anogenital tract. Of the possible malignancies, cervical carcinoma is by far the most frequent; according to an estimate by the World Health Organization (WHO), almost 500.000 new cases of the disease occur annually. Because of the frequency with which this pathology occurs, the connection between HPV infection and cervical carcinoma has been extensively examined, leading to numerous generalizations.

For example, precursor lesions of cervical intraepithelial neoplasia (CIN) are known to be caused by papilloma virus infections

[Crum. New Eng. J. Med. 570:880-883 ( 1984)]. DNA from the genomes of certain HPV types, including for example, strains 16, 18, 33, 35, and 45, have been detected in more than 95 % of tumor biopsies from patients with this disorder, as well as in primary cell lines cultured from the tumors. Approximately 50 to 70% of the biopsied CIN tumor cells have been found to include DNA derived only from HPV 16.

The protein products of the HPV 16 and HPV 18 early genes E6 and E7 have been detected in cervical carcinoma cell lines as well as in human keratinocytes transformed in vitro [Wettstein, et al. , in PAPILLOMA VIRUSES AND HUMAN CANCER, Pfister (Ed.),CRC Press: Boca Raton, FL 1990 pp 155-179] and a significant percentage of patients with cervical carcinoma have anti-E6 or anti-E7 antibodies. The E6 and E7 proteins have been shown to participate in induction of cellular DNA synthesis in human cells, transformation of human keratinocytes and other cell types, and tumor formation in transgenic mice [Arbeit, et al. , J. Virol. , (55:4358- 4364 (1994): Auewarakul. et al. , Mol. Cell. Biol. 74:8250-8258 (1994); Barbosa. et al . J. Virol, 65:292-29% (1991); Kaur, et al. , J. Gen. Virol. 70: 1261 -1266 (1989): Schlegel. et al. , EMBO J. . 7:3181-3187 (1988)]. The constitutive expression of the E6/E7 proteins appears to be necessary to maintain the transformed condition of HPV-positive tumors.

Despite the capacity of some HPV strains to induce neoplastic phenotypes in vivo and in vitro, still other HPV types cause benign genital warts such as condylomata acuminata and are only rarely associated with malignant tumors [Ikenberg. In Gross, et al. , (eds.) GENITAL PAPILLOMAVIRUS INFECTIONS. Springer Verlag: Berlin, pp. , 87- 1 12]. Low risk strains of this type include, for example, HPV 6 and 1 1.

Most often, genital papilloma viruses are transmitted between humans during intercourse which in many instances leads to persistent infection in the anogenital mucous membrane. While this observation suggests that either the primary infection induces an inadequate immune response or that the virus has developed the ability to avoid immune surveillance, other observations suggest that the immune system is active during primary manifestation as well as during malignant progression of papilloma virus infections [Altmann et al. in VIRUSES AND CANCER, Minson et al. , (eds.) Cambridge University Press, (1994) pp. 71-80].

For example, the clinical manifestation of primary infection by rabbit and bovine papilloma virus can be prevented by vaccination with wart extracts or viral structural proteins [Altmann, et al. , supra; Campo, Curr. Top. In Microbiol and Immunol. 186:255-266 (1994); Yindle and Frazer, Curr. Top. In Microbiol. and Immunol. 186;2ll-253 (1994)]. Rodents previously vaccinated with vaccinia recombinants encoding HPV 16 early proteins E6 or E7, or with synthetic E6 or E7 peptides, are similarly protected from tumor formation after inoculation of HPV 16 transformed autologous cells [Altman, et al. , supra; Campo, et al. , supra; Yindle and Frazer, et al. supra]. Regression of warts can be induced by the transfer of lymphocytes from regressor animals following infection by animal papilloma viruses. Finally, in immunosuppressed patients, such as, for example, recipients of organ transplants or individuals infected with HIV, the incidence of genital warts. CIN. and anogenital cancer is elevated.

To date, no HPV vaccinations have been described which comprise human papilloma vims late LI protein in the form of capsomeres which are suitable both for prophylactic and therapeutic purposes. Since the LI protein is not present in malignant genital lesions, vaccination with LI protein does not have any therapeutic potential for these patients. Construction of chimeric proteins, comprising amino acid residues from LI protein and, for example E6 or E7 protein, which give rise to chimeric capsomeres, combines prophylactic and therapeutic functions of a vaccine. A method for high level production of chimeric capsomeres would therefore be particularly desirable, in view of the possible advantages offered by such a vaccine for prophylactic and therapeutic intervention. Thus there exists a need in the art to provide vaccine formulations which can prevent or treat HPV infection. Methods to produce vaccine formulations which overcome problems known in the art to be associated with recombinant HPV protein expression and purification would manifestly be useful to treat the population of individuals already infected with HPV as well as useful to immunize the population of individuals susceptible to HPV infection. SUMMARY OF THE INVENTION

The present invention provides therapeutic and prophylactic vaccine formulations comprising chimeric human papilloma capsomeres. The invention also provides therapeutic methods for treating patients infected with an HPV as well as prophylactic methods for preventing HPV infection in a susceptible individual. Methods for production and purification of capsomeres and proteins of the invention are also contemplated.

In one aspect of the invention, prophylactic vaccinations for prevention of HPV infection are considered which incorporate the structural proteins LI and L2 of the papilloma vims. Development of a vaccine of this type faces significant obstacles because papilloma viruses cannot be propagated to adequate titers in cell cultures or other experimental systems to provide the viral proteins in sufficient quantity for economical vaccine production. Moreover, recombinant methodologies to express the proteins are not always straightforward and often results in low protein yield. Recently, vims-like particles (VLPs). similar in make up to viral capsid structures, have been described which are foπned in Sf-9 insect cells upon expression of the viral proteins LI and L2 (or LI on its own) using recombinant vaccinia or baculovinis. Purification of the VLPs can be achieved very simply by means of centrifugation in CsCl or sucrose gradients [Kimbauer. ei al. . Proc Natl. Acad. Sci. (USA), 99: 12180-12814 (1992): Kimbaurer, et al. , J. Virol. 67:6929-6936 (1994); Proso, et al , J. Virol. 6774: 1936-1944 ( 1992): Sasagawa. et al , Virology 2076: 126-195 (1995): Volpers, et al , J. Virol. 69:3258-3264 (1995); Zhou, et al , J. Gen. Virol. 74:762-769 (1993): Zhou, et al , Virology 185:251-251 (1991)]. WO 93/02184 describes a method in which papilloma vims-like particles (VLPs) are used for diagnostic applications or as a vaccine against infections caused by the papilloma vims. WO 94/00152 describes recombinant production of LI protein which mimics the conformational neutralizing epitope on human and animal papilloma virions.

In another aspect of the invention, therapeutic vaccinations are provided to relieve complications of, for example, cervical carcinoma or precursor lesions resulting from papilloma virus infection, and thus represent an alternative to prophylactic intervention. Vaccinations of this type may comprise early papilloma vims proteins, principally E6 or E7. which are expressed in the persistently infected cells. It is assumed that, following administration of a vaccination of this type, cytotoxic T-cells might be activated against persistently infected cells in genital lesions. The target population for therapeutic intervention is patients with HPV- associated pre-malignant or malignant genital lesions. PCT patent application WO 93/20844 discloses that the early protein E7 and antigenic fragments thereof of the papilloma vims from HPV or BPV is therapeutically effective in the regression, but not in the prevention, of papilloma vims tumors in mammals. While early HPV proteins have been produced by recombinant expression in E. coli or suitable eukaryotic cell types, purification of the recombinant proteins has proven difficult due to inherent low solubility and complex purification procedures which generally require a combination of steps, including ion exchange chromatography, gel filtration and affinity chromatography.

According to the present invention, vaccine formulations comprising papilloma vims capsomeres are provided which comprise either: (i) a first protein that is an intact viral protein expressed as a fusion protein comprised in part of amino acid residues from a second protein; (ii) a tmncated viral protein; (iii) a tmncated viral protein expressed as a fusion protein comprised in part of amino acid residues from a second protein, or (iv) some combination of the three types of proteins. According to the invention, vaccine formulations are provided comprising capsomeres of bovine papilloma vims (BPV) and human papilloma vims. Preferred bovine vims capsomeres comprise protein from bovine papilloma vims type I. Preferred human virus capsomeres comprise proteins from any one of human papilloma vims strains HPV6, HPV11 , HPV16, HPV18, HPV33, HPV35, and HPV45. The most preferred vaccine formulations comprise capsomeres comprising proteins from HPV 16.

In one aspect, capsomere vaccine formulations of the invention comprise a first intact viral protein expressed as a fusion protein with additional amino acid residues from a second protein. Preferred intact viral proteins are the structural papilloma viral proteins LI and L2. Capsomeres comprised of intact viral protein fusions may be produced using the LI and L2 proteins together or the LI protein alone. Preferred capsomeres are made up entirely of LI fusion proteins, the amino acid sequence of which is set out in SEQ ID NO: 2 and encoded by the polynucleotide sequence of SEQ ID NO: 1 . Amino acids of the second protein can be derived from numerous sources (including amino acid residues from the first protein) as long as the addition of the second protein amino acid residues to the first protein peπnits formation of capsomeres. Preferably, addition of the second protein amino acid residues inhibits the ability of the intact viral protein to form vims-like particle stmctures; most preferably, the second protein amino acid residues promote capsomere formation. In one embodiment of the invention, the second protein may be any human tumor antigen, viral antigen, or bacterial antigen which is important in stimulating an immune response in neoplastic or infectious disease states. In a preferred embodiment, the second protein is also a papilloma vims protein. It also preferred that the second protein be the expression product of papilloma vims early gene. It is also preferred, however, that the second protein be selected from group of El , E2, E3, E4, E5. E6, and E7 — early gene products encoded in the genome of papilloma vims strains HVP6, HPV1 1 , HPV18, HPV33, HPV35, or HPV 45. It is most preferred that the second protein be encoded by the HPV16 E7 gene, the open reading frame of which is set out in SEQ ID NO: 3. Capsomeres assembled from fusion protein subunits are referred to herein as chimeric capsomeres. In one embodiment, the vaccine formulation of the invention is comprised of chimeric capsomeres wherein LI protein amino acid residues make up approximately 50 to 99 % of the total fusion protein amino acid residues. In another embodiment, LI amino acid residues make up approximately 60 to 90% of the total fusion protein amino acid residues; in a particularly preferred embodiment. LI amino acids comprise approximately 80 % of the fusion protein amino acid residues.

In another aspect of the invention, capsomere vaccine formulations are provided that are comprised of tmncated viral proteins having a deletion of one or more amino acid residues necessary for formation of a vims-like particle. It is preferred that the amino acid deletion not inhibit fomiation of capsomeres by the tmncated protein, and it is most preferred that the deletion favor capsomere formation. Preferred vaccine formulations of this type include capsomeres comprised of tmncated LI with or without L2 viral proteins. Particularly preferred capsomeres are comprised of tmncated LI proteins. Tmncated proteins contemplated by the invention include those having one or more amino acid residues deleted from the carboxy terminus of the protein, or one or more amino acid residues deleted from the amino terminus of the protein, or one or more amino acid residues deleted from an internal region {i.e. , not from either terminus) of the protein. Preferred capsomere vaccine formulations are comprised of proteins tmncated at the carboxy terminus. In formulations including LI protein derived from HPV16, it is preferred that from 1 to 34 carboxy terminal amino acid residues are deleted. Relatively shorter deletions are also contemplated which offer the advantage of minor modification of the antigenic properties of the LI proteins and the capsomeres formed thereof. It is most preferred, however, that 34 amino acid residues be deleted from the LI sequence, corresponding to amino acids 472 to 505 in HPV 16 set out in SEQ ID NO: 2, and encoded by the polynucleotide sequence corresponding to nucleotides 1414 to 1516 in the human HPV16 LI coding sequence set out in SEQ ID NO: 1. When a capsomere vaccine formulation is made up of proteins bearing an internal deletion, it is preferred that the deleted amino acid sequence comprise the nuclear localization region of the protein. In the LI protein of HPV 16. the nuclear localization signal is found from about amino acid residue 499 to about amino acid residue 505. Following expression of LI proteins wherein the NLS has been deleted, assembly of capsomere stmctures occurs in the cytoplasm of the host cell. Consequently, purification of the capsomeres is possible from the cytoplasm instead of from the nucleus where intact LI proteins assemble into capsomeres. Capsomeres which result from assembly of tmncated proteins wherein additional amino acid sequences do not replace the deleted protein sequences are necessarily not chimeric in nature.

In still another aspect of the invention, capsomere vaccine foπnulations are provided comprising tmncated viral protein expressed as a fusion protein adjacent amino acid residues from a second protein. Preferred tmncated viral proteins of the invention are the structural papilloma viral proteins LI and L2. Capsomeres comprised of tmncated viral p otein fusions may be produced using LI and L2 protein components together or LI protein alone. Preferred capsomeres are those comprised of LI protein amino acid residues. Tmncated viral protein components of the fusion proteins include those having one or more amino acid residues deleted from the carboxy terminus of the protein, or one or more amino acid residues deleted from the amino terminus of the protein, or one or more amino acid residues deleted from an internal region (i.e. , not from either terminus) of the protein. Preferred capsomere vaccine formulations are comprised of proteins tmncated at the carboxy terminus. In those formulations including LI protein derived from HPV16, it is preferred that from 1 to 34 carboxy terminal amino acid residues are deleted. Relatively shorter deletions are also contemplated that offer the advantage of minor modification of the antigenic properties of the LI protein component of the fusion protein and the capsomeres formed thereof. It is most preferred, however, that 34 amino acid residues be deleted from the LI sequence, corresponding to amino acids 472 to 505 in HPV16 set out in SEQ ID NO: 2. and encoded by the polynucleotide sequence corresponding to nucleotides 1414 to 1516 in the human HPV 16 LI coding sequence set out in SEQ ID NO: 1. When the vaccine formulation is comprised of capsomeres made up of proteins bearing an internal deletion, it is preferred that the deleted amino acid sequence comprise the nuclear localization region, or sequence, of the protein.

Amino acids of the second protein can be derived from numerous sources as long as the addition of the second protein amino acid residues to the first protein permits formation of capsomeres. Preferably, addition of the second protein amino acid residues promotes or favors capsomere formation. Amino acid residues of the second protein can be derived from numerous sources, including amino acid residues from the first protein. In a preferred embodiment, the second protein is also a papilloma vims protein. It also preferred that the second protein be the expression product of papilloma vims early gene. It is most preferred, however, that the second protein be selected from group of early gene products encoding by papilloma vims El . E2, E3. E4, E5, E6, and E7 genes. In one embodiment, the vaccine foπnulation of the invention is comprised of chimeric capsomeres wherein LI protein amino acid residues make up approximately 50 to 99 % of the total fusion protein amino acid residues. In another embodiment, LI amino acid residues make up approximately 60 to 90% of the total fusion protein amino acid residues; in a particularly preferred embodiment, LI amino acids comprise approximately 80% of the fusion protein amino acid residues.

In a preferred embodiment of the invention, proteins of the vaccine formulations are produced by recombinant methodologies, but in formulations comprising intact viral protein, the proteins may be isolated from natural sources. Intact proteins isolated from natural sources may be modified in vitro to include additional amino acid residues to provide a fusion protein of the invention using covalent modification techniques well known and routinely practiced in the art. Similarly, in formulations comprising tmncated viral proteins, the proteins may be isolated from natural sources as intact proteins and hydrolyzed in vitro using chemical hydrolysis or enzymatic digestion with any of a number of site-specific or general proteases, the tmncated protein subsequently modified to include additional amino acid resides as described above to provide a tmncated fusion protein of the invention.

In producing capsomeres. recombinant molecular biology techniques can be utilized to produce DNA encoding either the desired intact protein, the tmncated protein, or the tmncated fusion protein. Recombinant methodologies required to produce a DNA encoding a desired protein are well known and routinely practiced in the art. Laboratory manuals, for example Sambrook. et al . (eds.), MOLECULAR CLONING: A LABORATORY MANL AL. Cold Spring Harbor Press: Cold Spring Harbor, NY (1989) and Ausebel et al . (eds.). PROTOCOLS IN MOLECULAR BIOLOGY. John Wiley & Sons. Inc. ( 1994-1997), describe in detail techniques necessary to carry out the required DNA manipulations. For large-scale production of chimeric capsomeres, protein expression can be carried out using either viral or eukaryotic vectors. Preferable vectors include any of the well known prokaryotic expression vectors, recombinant baculovimses, COS cell specific vectors, vaccinia recombinants, or yeast- specific expression constmcts. When recombinant proteins are used to provide capsomeres of the invention, the proteins may first be isolated from the host cell of its expression and thereafter incubated under conditions which permit self-assembly to provide capsomeres. Alternatively, the proteins may be expressed under conditions wherein capsomeres are formed in the host cell.

The invention also contemplates processes for producing capsomeres of the vaccine formulations. In one method, LI proteins are expressed from DNA encoding six additional histidines at the carboxy terminus of the LI protein coding sequence. LI proteins expressed with additional histidines (His LI proteins) are most preferably expressed in E. coli and the His LI proteins can be purified using nickel affinity chromatography. His LI proteins in cell lysate are suspended in a denaturation buffer, for example. 6 M guanidine hydrochloride or a buffer of equivalent denaturing capacity, and then subjected to nickel chromatography. Protein eluted from the nickel chromatography step is renatured, for example in 150 inM NaCl. 1 niM CaCl₂, 0.01 % Triton-X 100, 10 mM HEPES (N-2-hydroxyethyl piperazine-N'-2 ethane sulfonic acid), pH 7.4. According to a preferred method of the invention, assembly of capsomeres takes place after dialysis of the purified proteins, preferably after dialysis against 150 M NaCl. 25 mM Ca^{2 +} , 10% DMSO (dimethyl sulfoxide). 0.1 % Triton-X 100. 10 mM Tris [tris-(hydroxymethyl) amino- methane] acetic acid with a pH value of 5.0.

Formation of capsomeres can be monitored by electron microscopy, and, in instances wherein capsomeres are comprised of fusion proteins, the presence of various protein components in the assembled capsomere can be confirmed by Western blot analysis using specific antisera.

According to the present invention, methods are provided for therapeutic treatment of individuals infected with HPV comprising the step of administering to a patient in need thereof an amount of a vaccine formulation of the invention effective to reduce the level of HPV infection. The invention also provide methods for prophylactic treatment of individuals susceptible to HPV infection comprising the step of administering to an individual susceptible to HPV infection an amount of a vaccine formulation of the invention effective to prevent HPV infection. While infected individuals can be easily identified using standard diagnostic techniques, susceptible individuals may be identified, for example, as those engaged in sexual relations with an infected individual. However, due to the high frequency of HPV infection, all sexually active persons are susceptible to papilloma vims infection.

Administration of a vaccine formulation can include one or more additional components such as pharmaceutically acceptable carriers, diluents, adjuvants, and/or buffers. Vaccines may be administered at a single time or at multiple times. Vaccine formulation of the invention may be delivered by various routes including, for example, oral, intravenous, intramuscular, nasal, rectal, transdermal. vaginal, subcutaneous, and intraperitoneal administration.

Vaccine formulations of the invention offer numerous advantages when compared to conventional vaccine preparations. As part of a therapeutic vaccination, capsomeres can promote elimination of persistently infected cells in. for example, patients with CIN or cervical carcinoma. Additionally, therapeutic vaccinations of this type can also serve a prophylactic purpose in protecting patients with CIN lesions from re-infection. As an additional advantage, capsomeres can escape neutralization by pre-existing anticapsid antibodies and thereby posses longer circulating half-life as compared to chimeric vims-like particles.

Vaccine formulations comprising chimeric capsomeres can provide the additional advantage of increased antigenicity of both protein components of the fusion protein from which the capsomere is formed. For example, in a VLP, protein components of the underlying capsomere may be buried in the overall stmcture as a result of internalized positioning within the VLP itself. Similarly, epitopes of the protein components may be sterically obstmcted as a result of capsomere-to-capsomere contact, and therefore unaccessible for eliciting an immune response. Preliminary results using L1/E7 fusion proteins to produce VLPs support this position in that no antibody response was detected against the E7 component. This observation is consistent with previous results which indicate that the carboxy terminal region of LI forms inter-pentameric arm structures that allow assembly of capsomeres into capsids [Garcia, et al, J. Virol. 71: 2988-2995 (1997)]. Presumably in a chimeric capsomere stmcture, both protein components of the fusion protein substmcture are accessible to evoke an immune response. Capsomere vaccines would therefore offer the additional advantage of increased antigenicity against any protein component, including, for example, neutralizing epitopes from other vims proteins, expressed as a fusion with LI amino acid sequences.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is illustrated by the following examples. Example 1 describes constmction of expression vectors to produce fusion, or chimeric. viral proteins. Example 2 relates to generation of recombinant baculovimses for expression of viral proteins. Example 3 addresses purification of capsomeres. Example 4 describes an immunization protocol for production of antisera and monoclonal antibodies. Example 5 provides a peptide ELISA to quantitate capsomere formation. Example 6 describes an antigen capture ELISA to quantitate capsomere formation. Example 7 provides a hemagglutinin assay to assay for the induction of neutralizing antibodies. Example 1 Construction of Chimeric LI Genes

DNA encoding the HPV 16 LI open reading frame was excised from plasmid 16-114/k-Ll/L2-pSynxtVT [Kirnbauer et al., J. Virol. 67:6929-6936 (1994)] using BglE and the resulting fragment subcloned into pUC19 (New England Biolabs, Beverly, MA) previously linearized at the unique BamΗI restriction site. Two basic expression constmcts were first generated to permit subsequent insertion of DNA to allow fusion protein expression. One construct encoded HPV 16 L1Δ310 having a nine amino acid deletion: the deleted region was known to show low level homology with all other papilloma vims LI proteins. The second construct, HPV 16 LI ΔC . encoded a protein having a 34 amino acid deletion of the carboxy teπninal LI residues. Other constmcts include an EcoRV restriction site at the position of the deletion for facilitated insertion of DNA encoding other protein sequences. Addition of the EcoRV site encodes two non-Ll protein amino acids, aspartate and isoleucine.

A. Generation of an HPV 16 L1Δ310 expression construct

Two primers (SΕQ ID NOs: 5 and 6) were designed to amplify the pUC19 vector and the complete HPV 16 LI coding sequence, except nucleotides 916 through 942 in SΕQ ID NO: 1. Primers were synthesized to also introduce a unique EcoRV restriction site (underlined in SΕQ ID NOs: 5 and 6) at the termini of the amplification product.

CCCCGATATCGCCTTTAATGTATAAATCGTCTGG SΕQ ID NO: 5

CCCCGATATCTCAAATTATTTTCCTACACCTAGTG

SΕQ ID NO: 6

The resulting PCR product was digested with EcoRV to provide complementary ends and the digestion product circularized by ligation. Ligated DNA was transformed into E. coli using standard techniques and plasmids from resulting colonies were screened for the presence of an EcoRV restriction site. One clone designated HPV 16 LI Δ310 was identified as having the appropriate twenty-seven nucleotide deletion and this constmct was used to insert DNA fragments encoding other HPV 16 proteins at the EcoRV site as discussed below.

B. Generation of an HPV 16 LI ΔC expression constructs

Two primers (SΕQ ID NOs: 7 and 8) were designed complementary to the HPV 16 LI open reading frame such that the primers abutted each other to permit amplification in reverse directions on the template DNA comprising HPV 16 LI -encoding sequences in pUC19 described above.

AAAGATATCTTGTAGTAAAAATTTGCGTCCTAAAGGAAAC

SΕQ ID NO: 7 AAAGATATCTAATCTACCTCTACAACTGCTAAACGCAAAAAACG

SΕQ ID NO: 8

Each primer introduced an EcoRV restriction site at the terminus of the amplification product. In the downstream primer (SΕQ ID NO: 8), the EcoRV site was followed by a TAA translational stop codon positioned ,uch that the amplification product, upon hgation of the EcoRV ends to circularize, would include deletion of the 34 carboxy terminal LI amino acids. PCR was performed to amplify the partial LI open reading frame and the complete vector. The amplification product was cleaved with EcoRV. circularized with T4 DNA ligase, and transformed into E. coli DH5 ex cells. Plasmids from viable clones were analyzed for the presence of an EcoRV site which would linearize the plasmid. One positive constmct designated PUCHPV16L1ΔC was identified and used to insert DNA from other HPV 16 proteins utilizing the EcoRV site.

C. Insertion of DNA fragments into HPV 16 LI Δ310 and HPV16L1ΔC

DNA fragments of HPV 16 Ε7 encoding amino acids 1-50, 1 -60. 1-98. 25-75. 40-98, 50-98 in SEQ ID NO: 4 were amplified using primers that introduced terminal 5' EcoRV restriction sites in order to facilitate insertion of the fragment into either HPV 16 LI Δ310 and HPV 1 6L1 ΔC modified sequence. In the various amplification reactions, primer Ε7. 1 (SEQ ID NO: 9) was used in combination with primer E7.2 (SEQ ID NO: 10) to generate a DNA fragment encoding E7 amino acids 1- 50: with primer E7.3 (SEQ ID NO: 1 1) generate a DNA fragment encoding E7 amino acids 1 -60: or with primer E7.4 (SEQ ID NO: 12) generate a DNA fragment encoding E7 amino acids 1-98. In other amplification reactions, primer pairs E7.5 (SEQ ID NO: 13) and E7.6 (SEQ ID NO: 14) were used to amplify a DNA fragment encoding E7 amino acids 25-75: E7.7 (SEQ ID NO: 15) and E7.4 (SEQ ID NO: 12) were used to amplify a DNA fragment encoding E7 amino acids 40-98; and E7.8 (SEQ ID NO: 16) and E7.4 (SEQ ID NO: 12) were used to amplify a DNA fragment encoding E7 amino acids 50-98.

Primer E7.1 SEQ ID NO: 9

AAAAGATATCATGCATGGAGATACACCTACATTGC

Primer E7.2 SEQ ID NO: 10

TTTTGATATCGGCTCTGTCCGGTTCTGCTTGTCC

Primer E7.3 SEQ ID NO: 11 TTTTGATATCCTTGCAACAAAAGGTTACAATATTGTAATGGGCC Primer E7.4 SEQ ID NO: 12

AAAAGATATCTGGTTTCTGAGAACAGATGGGGCAC

Primer E7.5 SEQ ID NO: 13

TΠTGATATCGATTATGAGCAATTAAATGACAGCTCAG

Primer E7.6 SEQ ID NO: 14

TTTTGATATCGTCTACGTGTGTGCTTTGTACGCAC

Primer E7.7 SEQ ID NO: 15

TTTATCGATATCGGTCCAGCTGGACAAGCAGAACCGGAC

Primer E7.8 SEQ ID NO: 16

TTTTGATATCGATGCCCATTACAATATTGTAACCTTTTG

Similarly, nucleotides from DNA encoding the influenza matrix protein (SEQ ID NO: 17) was amplified using the primer pair set out in SEQ ID NOs: 19 and 20. Both primers introduced an EcoRV restriction site in the amplification product.

TTTTGATATCGATATGGAATGGCTAAAG AC AAGACCAATC

SΕQ ID NO: 19

TTTTGATATCGTTGTTTGGATCCCCATTCCCATTG

SΕQ ID NO: 20

PCR products from each amplification reaction were cleaved with EcoRV and inserted into the EcoRV site of either the HPV 16 LI Δ310 and HPV16L1 ΔC sequences previously linearized with the same enzyme. In order to determine the orientation of inserts in plasmids encoding Ε7 amino acids 25-75 and 50-98 and plasmid including influenza matrix protein, Clal digestion was employed, taking advantage of a restriction site overlapping the newly created EcoRV restriction site CGATATCGAT) and included in the upstream primer. For the three expression constmcts including the initiating methionine of HPV 16 Ε7, insert orientation was determined utilizing a NsH restriction site within the E7 coding region.

Once expression constmcts having appropriate inserts were identified, the protein coding region for both LI and inserted amino acids was excised as a unit using restriction enzymes Xbal and Smal and the isolated DNA ligated into plasmid pVL1393 (Invitrogen) to generate recombinant baculovimses.

D. Elimination of EcoRV Restriction Sites in Expression Constructs

The HPV 16 LI ΔC sequence includes DNA from the EcoRV site that results in translation of amino acids not normally found in wild-type LI polypeptides. Thus, a series of expression constmctions was designed in which the artificial EcσRv site was eliminated. The LI sequence for this series of expression constmcts was designated HPV

To generate an expression constmct containing the HPV 16L1 C sequence, two PCR reactions were performed to amplify two overlapping fragments from the pUC-HPV16 LI ΔC encoding Ε7 amino acids 1-50. The resulting DNA fragments overlapped at the position of the L1/E7 boundary but did not contain the two EcoRV restriction sites. Fragment 1 was generated using primers PI (SΕQ ID NO: 21) and P2 (SΕQ ID NO: 22) and fragment 2 using primers P3 (SΕQ ID NO: 23) and P4 (SΕQ ID NO: 24).

Primer PI SΕQ ID NO: 21

GTTATGACATACATACATTCTATG

Primer P2 SΕQ ID NO: 22 CCATGCATTCCTGCTTGTAGTAAAAATTTGCGTCC

Primer P3 SEQ ID NO: 23

CTACAAGCAGGAATGCATGGAGATACACC

Primer P4 SEQ ID NO: 24 CATCTGAAGCTTAGTAATGGGCTCTGTCCGGTTCTG

Following the first two amplification reactions, the two purified products were used as templates in another PCR reaction using pπmers PI and P4 only The resulting amplification product was digested with enzymes EcoNI and H dIII inserted into the ΗPV 16L1ΔC expression constmct descπbed above following digestion with the same enzymes. The resulting expression constmct differed from the original ΗPV16L1 ΔC constmct with DNA encoding LI and Ε7 amino acids 1-50 by loss of the two internal EcoRV restriction sites The first EcoRV site was replaced by DNA encoding nativ e LI alanine and glycine amino acids in this position and the second was replaced by a translational stop signal. In addition, the expression constmct. designated ΗPV 16 LI ΔC Ε7 1-52, contained the first 52 amino acids of ΗPV 16 Ε7 as a result of using primer P4 which also encodes E7 amino acids residues histidine at position 51 and tyrosine at position 52 ΗPV 16 LI ΔC* E7 1-52 was then used to generate additional ΗPV 16 LI ΔC expression constmcts further including DNA encoding E7 amino acids 1 55 using pπmer PI (SEQ ID NO: 21) in combination with primer P5 (SEQ ID NO 25), E7 amino acids 1-60 with primer pair PI and P6 (SEQ ID NO' 26), and E7 amino acids 1-65 with primer pair PI and P7 (SEQ ID NO. 27) The additional animo acid- encoding DNA sequences in the amplification products arose from design of the primers to include additional nucleotides for the desired amino acids. Primer P5 SEQ ID NO: 25

CATCTGAAGCTTAACAATATTGTAATGGGCTCTGTCCG

Primer P6 SEQ ID NO: 26

CATCTGAAGCTTACTTGCAACAAAAGGTTA-

CAATATTGTAATGGGCTCTGTCCG

Primer P7 SEQ ID NO: 27

CATCTGAAGCTTAAAGCGTAGAGTCACACTTGCAAC-

AAAAGGTTACAATATTGTAATGGGCTCTGTCCG

Similarly. HPV 16 LIΔC E7 1 -70 was generated using template DNA encoding HPV 16 LIΔC* E7 1 -66 and the primer pair PI and P8 (SEQ ID NO: 28).

Primer P8 SEQ ID NO: 28

CATCTGAAGCTTATTGTACGCACAAC- CGAAGCGTAGAGTCACACTTG

Following each PCR reaction, the amplification products were digested with EcoNI and H dIII and inserted into ΗPV16L1 ΔC previously digested with the same enzymes. Sequences of each constmcts were determined using an Applied Biosystems Prism 377 sequencing instmment with fluorescent chain terminating dideoxynucleotides [Prober et al , Science 2iS:336-341 (1987)].

Example 2 Generation of Recombinant Baculoviruses

Spodoptera frugiperda (Sf9) cells were grown in suspension or monolayer cultures at 27° in TNMFΗ medium (Sigma) supplemented with

10% fetal calf semm and 2 mM glutamine. For ΗPV 16 LI -based recombinant baculovims constmction, Sf9 cells were transfected with 10 μg of transfer plasmid together with 2 μg of linearized Baculo-Gold DNA (PharMingen, San Diego, CA). Recombinant viruses were purified by according to manufacturer's suggested protocol.

To test for expression of HPV 16 LI protein, 10⁵ Sf9 cells were infected with baculovims recombinant at a multiplicity of infection (m.o.i) of 5 to 10. After incubation for three to four days at 28 °C, media was removed and cells were washed with PBS. The cells were lysed in SDS sample buffer and analyzed by SDS-PAGE and Western blotting using anti-HPV16 LI and anti-HPV16 E7 antibodies.

In order to determine which of the chimeric LI protein expression constmcts would preferentially produce capsomeres, extracts from transfected cells were subjected to gradient centrifugation. Fractions obtained from the gradient were analyzed for LI protein content by Western blotting and for VLP fomiation by electron microscopy. The results are shown in Table 1. The intact HPV LI protein, as well as the expression products HPV 16 L1 Δ310 and HPV 16 LI AC. each were shown to produce capsomeres and vims-like particles in equal proportions. When E7 coding sequences were inserted into the HPV 16 L1Δ310 vector, only fusion proteins including E7 amino acids 1 to 50 produced gave rise to detectable capsomere formation.

When E7 encoding DNA was inserted into the HPV 16 LIΔC vector, all fusion proteins were found to produce capsomeres; chimeric proteins including E7 amino acid residues 40-98 produced the highest level of exclusively capsomere structures. Chimeric proteins including E7 amino acids 1 -98 and 25-75 both produced predominantly capsomeres, even thorough vims-like particle formation was also observed. The chimeric protein including E7 amino acids 1-60 resulted in nearly equal levels of capsomere and vims-like particle production.

When E7 sequences were inserted into the HPV 16 L1Δ*C vector, all fusion proteins were shown to produce capsomeres. Insertion of DNA encoding E7 residues 1-52, 1-55, and 1-60 produced the highest level of capsomeres, but equal levels of vims-like particle production were observed. While insertion of DNA encoding E7 DNA for residues 1-65, 1- 70, 25-75, 40-98. and 1-98 resulted in comparatively lower levels or undetectable levels of capsid, capsomeres were produced in high quantities.

TABLE l

Capsomeree and Capsid Forming Capacity of Chimeric HPV LI Proteins

Ll Expression Capsomere Capsid

Construct Insert Yield Yield

HVP 16 Ll one + + + + + + + + + +

HPV 16 L 310 one + + + + +

HPV 16 LIΔC None + + + + + + + +

HP\ 16 LIΔIIO f 1 so + + -

HPV 16 L 310 F" :^■> ^s - -

HPV 16 LIΔC F ι-6θ + + + + + + + + + +

HPV 16 LIΔC I " 40 ')h + + + + -

HPV 16 LIΔC Influenza + + + +

HPV 16 L1Δ*C F^" 1 S2 + + + + + + + + + +

HPV 16 L1Δ*C E7 1-SS + + + + + + + + + +

HPV 16 L1Δ*C E7 1-60 + + + + + + +

Example 3 Purification of Capsomeres

Trichopulsia ni (TN) High Five cells were grown to a density of approximately 2 x 10 cells/ml in Ex-Cell 405 serum-free medium (JRH Biosciences). Approximately 2 x 10 cells were pelleted by centrifugation at 1000 x g for 15 minutes, resuspended in 20 ml of medium, and infected with recombinant baculovimses at m.o.i of 2 to 5 for 1 hour at room temperature. After addition of 200 ml medium, cells were plated and incubated for 3 to 4 days at 27°C. Following incubation, cells were harvested, pelleted, and resuspended in 10 ml of extraction buffer.

The following steps were performed at 4°C. Cells were sonicated for 45 seconds at 60 watts and the resulting cell lysate was centrifuged at 10.000 rpm in a Sorval SS34 rotor. The supernatant was removed and retained while the resulting pellet was resuspended in 6 ml of extraction buffer, sonicated for an additional 3 seconds at 60 watts, and centrifuged again. The two supernatants were combined, layered onto a two-step gradient containing 14 ml of 40% sucrose on top of 8 ml of CsCl solution (4.6 g CsCl per 8 ml in extraction buffer), and centrifuged in a Sorval AH629 swinging bucket rotor for 2 hours at 27,000 rpm at 10°C. The interface region between the CsCl and the sucrose along with the CsCl complete layer were collected into 13.4 ml Quickseal tubes (Beckman) and extraction buffer added to adjust the volume 13.4 ml. Samples were centrifuged overnight at 50.000 rpm at 20°C in a Beckman 70 TI rotor. Gradients were fractionated (1 ml per fraction) by puncturing tubes on top and bottom with a 21 - gauge needle. Fractions were collected from each tube and 2.5 μl of each fraction were analyzed by a 10% SDS-polyacrylamide gel and Western blotting using an anti-HPV16 Ll antibody.

Vims-like particles and capsomeres were separated from the fractions identified above by sedimentation on 10 to 50% sucrose gradients. Peak fractions from CsCl gradients were pooled and dialyzed for 2 hours against 5 mM HEPES (pH 7.5). Half of the dialysate was used to produce capsomeres by disassembly of intact VLPs overnight by adding EDTA (final concentration 50 mM), EGTA (50 mM), DTT (30 mM). NaCl (100 mM), and Tris/HCl, pH 8.0, (10 mM). As control, NaCl and Tris/HCl only were added to the other half.

For analysis of capsomeres produced from disassembled VLPs, EDTA. EGTA, and DTT (final concentration 5 mM each) were added to the sucrose cushions which were centrifuged at 250,000 x g for 2 to 4 hours at 4°C. Fractions were collected by puncturing tubes from the bottom. A 1 : 10 dilution of each fraction was then analyzed by antigen capture ELISA.

Example 4 Immunization Protocol for Production of Polyclonal Antisera and Monoclonal Antibodies

Balb/c mice are immunized subcutaneously three times, every four weeks with approximately 60 μg of HPV chimeric capsomeres mixed 1 : 1 with complete or incomplete Freund's Adjuvants in a total volume of 100 μl. Six weeks after the third immunization, mice are sacrificed and blood is collected by cardiac puncture.

Example 5 Peptide ELISA to Quantitate Capsomere Formation

Microtiter plates (Dynatech) are coated overnight with 50 μl of peptide E701 [Muller et al , 1982] at a concentration of 10 μg/ml in PBS. Wells are blocked for 2 hour at 37°C with 100 μl of buffer containing 5 % BSA and 0.05 % Tween 20 in PBS and washed three times with PBS containing 0.05 % Tween 20. After the third wash. 50 μl of sera diluted 1 :5000 in BSA/Tween 20/PBS is added to each well and incubation carried out for 1 hour. Plates are washed again as before and 50 μl of goat-anti-mouse peroxidase conjugate is added at a 1 :5000 dilution. After 1 hour, plates are washed and stained using ABTS substrate (0.2 mg/ml, 2.2'-Azino-bis(3-ethylbenzhiazoline-0-sulfonic acid in 0.1 M Na- Acetate-Phosphate buffer (pH 4.2) with 4 μl 30% H₂O₂ per 10 ml). Extinction is measured after 1 hour at 490 nm in a Dynatech automated plate reader. Example 6 Antigen Capture ELISA to Quantitate Capsomere Formation

To allow relative quantification of vims-like particles and capsomeres in fractions of CsCl gradients, an antigen capture ELISA was utilized. Microtiter plates were coated overnight with 50 μl/well of a 1:500 dilution (final concentration of 2 μg per ml, in PBS) with a protein A purified mouse monoclonal antibody immunospecific for HPV 16 Ll (antibodies 25/C, MM07 and Ritti 1 were obtained from mice immunized with HPV 16 VLPs). Plates were blocked with 5 % milk/PBS for 1 hour and 50 μl of fractions of CsCl gradients were added for 1 hour at 37°C using a 1 :300 dilution (in 5 % milk/PBS). After three washings with PBS/0.05 % Tween 20. 50 μl of a polyclonal rabbit antiserum (1:3000 dilution in milk PBS), raised against HPV 16 VLPs was added and plates were incubated at 37° for 1 hour. Plates were washed again and further incubated with 50 μl of a goat-anti-rabbit peroxidase conjugate (Sigma) diluted 1 :5000 in PBS containing 5 % milk for 1 hour. After final washing, plates were stained with

ABTS substrate for 30 minutes and extinction measured at 490 nm in a Dynatech automated plate reader. As a negative control, the assay also included wells coated only with PBS.

To test monoclonal antibodies for capsomere specificity, VLPs with EDTA/DTT to disassemble particles. Treated particle preparations were assayed in the antigen-capture ELISA and readings compared to untreated controls. For disassembly, 40 μl of VLPs was incubated overnight at 4°C in 500 μl of dismption buffer containing 30 mM DTT. 50 mM EGTA, 60 mM EDTA, 100 mM NaCl, and 100 mM Tris/HCl. pH 8.0. Aliquots of treated and untreated particles were used in the above capture ELISA in a 1 :20-1 :40 dilution.

Example 7 Hemagglutinin Inhibition Assay

In order to determine the extent to which chimeric capsomere vaccines evoke production of neutralizing antibodies, a hemagglutination inhibition assay is carried out as briefly described below. This assay is based on previous observations that vims-like particles are capable of hemagglutinizing red blood cells.

Mice are immunized with any of a chimeric capsomere vaccine and sera is collected as described above in Example 4. As positive controls, HPV16 Ll vims like particles (VLPs) and bovine PVl (BPV) Ll VLPs are assayed in parallel with a chimeric capsomere preparation. To establish a positive baseline, the HPV 16 or BPV1 VLPs are first incubated with or without sera collected from immunized mice after which red blood cells are added. The extent to which preincubation with mouse cera inhibits red blood cell hemagglutinization is an indication of the neutralizing capacity of the mouse sera. The experiments are then repeated using chimeric capsomeres in order to determine the neutralizing effect of the mouse sera on the vaccine. A brief protocol for the hemagglutination inhibition assay is described below.

One hundred microliters of heparin (1000 usp units/ml) are added to 1 ml fresh mouse blood. Red blood cells are washed three times with PBS followed by centrifugation and resuspension in a volume of 10 ml. Next, erythrocytes are resuspended in 0.5 ml PBS and stored at 4°C for up to three days. For the hemagglutinin assay. 70 μl of the suspension is used per well on a 96-well plate. Chimeric capsomere aliquots from CsCl gradients are dialyzed for one hour against 10 mM Hepes (pH 7.5) and 100 μl of two-fold serial dilutions in PBS are added to mouse erythrocytes in round-bottom 96-well microtiter plates which are further incubated for 3- 16 hours at 4°C. For hemagglutination inhibition, capsomeres are incubated with dilutions of antibodies in PBS for 60 minutes at room temperature and then added to the erythrocytes. The level of erythrocyte hemagglutination, and therefore the presence of neutralizing antibodies, is determined by standard methods.

In preliminary results, mouse sera generated against chimeric capsomeres comprising HPV16L1ΔC protein in association with E7 amino acid residues 1-98 was observed to inhibit hemagglutination by HPV16 VLPs, but not by BPV VLPs. The mouse sera was therefore positive for neutralizing antibodies against the human VLPs and this differential neutralization was most likely the result of antibody specificity for epitopes against which the antibodies were raised. Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the invention.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT:

(ii) TITLE OF INVENTION: Papilloma Virus Capsomere Vaccine Formulatio and Methods of Use

(lii) NUMBER OF SEQUENCES: 27

(IV) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Marshall, O'Toole, Gerstein, Murray & Borun

(B) STREET: 233 South ac er Drive, 6300 Sears Tower

(C) CITY: Chicago

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(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppv disk

(B) COMPUTER: IBM PC compatible

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(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi ) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION

(vm) ATTORNEY/AGENT INFORMATION.

(A) NAME- Williams Ji , Joseph A.

(B) REGISTRATION NUMBER: 38,659

(C) REFERENCE/DOCKET NUMBER: 27013/34028

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 312-474-6300

(B) TELEFAX: 312-474-0448

(2) INFORMATION FOR SEQ ID NO : 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1518 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY: lmeai

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1518

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

ATG TCT CTT TGG CTG CCT AGT GAG GCC ACT GTC TAC TTG CCT CCT GTC 4 Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 15

CCA GTA TCT AAG GTT GTA AGC ACG GAT GAA TAT GTT GCA CGC ACA AAC 9 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn ATA TAT TAT CAT GCA GGA ACA TCC AGA CTA CTT GCA GTT GGA CAT CCC 144 lie Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 40 45

TAT TTT CCT ATT AAA AAA CCT AAC AAT AAC AAA ATA TTA GTT CCT AAA 192 Tyr Phe Pro lie Lys Lys Pro Asn Asn Asn Lys lie Leu Val Pro Lys 50 55 60

GTA TCA GGA TTA CAA TAC AGG GTA TTT AGA ATA CAT TTA CCT GAC CCC 240 Val Ser Gly Leu Gin Tyr Arg Val Phe Arg lie His Leu Pro Asp Pro 65 70 75 80

AAT AAG TTT GGT TTT CCT GAC ACC TCA TTT TAT AAT CCA GAT ACA CAG 288 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gin 85 90 95

CGG CTG GTT TGG GCC TGT GTA GGT GTT GAG GTA GGT CGT GGT CAG CCA 336 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gin Pro 100 105 110

TTA GGT GTG GGC ATT AGT GGC CAT CCT TTA TTA AAT AAA TTG GAT GAC 384 Leu Gly Val Gly lie Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125

ACA GAA AAT GCT AGT GCT TAT GCA GCA AAT GCA GGT GTG GAT AAT AGA 432 Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135 140

GAA TGT ATA TCT ATG GAT TAC AAA CAA ACA CAA TTG TGT TTA ATT GGT 480 Glu Cys lie Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu lie Gly 145 150 155 160

TGC AAA CCA CCT ATA GGG GAA CAC TGG GGC AAA GGA TCC CCA TGT ACC 528 Cys Lys Pro Pro lie Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr 165 170 175

AAT GTT GCA GTA AAT CCA GGT GAT TGT CCA CCA TTA GAG TTA ATA AAC 576 Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu lie Asn 180 185 190

ACA GTT ATT CAG GAT GGT GAT ATG GTT GAT ACT GGC TTT GGT GCT ATG 624 Thr Val lie Gin Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 195 200 205

GAC TTT ACT ACA TTA CAG GCT AAC AAA AGT GAA GTT CCA CTG GAT ATT 672 Asp Phe Thr Thr Leu Gin Ala Asn Lys Ser Glu Val Pro Leu Asp lie 210 215 220

TGT ACA TCT ATT TGC AAA TAT CCA GAT TAT ATT AAA ATG GTG TCA GAA 720 Cys Thr Ser lie Cys Lys Tyr Pro Asp Tyr lie Lys Met Val Ser Glu 225 230 235 240

CCA TAT GGC GAC AGC TTA TTT TTT TAT TTA CGA AGG GAA CAA ATG TTT 76 Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gin Met Phe 245 250 255

GTT AGA CAT TTA TTT AAT AGG GCT GGT GCT GTT GGT GAA AAT GTA CCA 81 Val Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro 260 265 270

GAC GAT TTA TAC ATT AAA GGC TCT GGG TCT ACT GCA AAT TTA GCC AGT 86 Asp Asp Leu Tyr lie Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285 TCA AAT TAT TTT CCT ACA CCT AGT GGT TCT ATG GTT ACC TCT GAT GCC 912 Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300

CAA ATA TTC AAT AAA CCT TAT TGG TTA CAA CGA GCA CAG GGC CAC AAT 960 Gin lie Phe Asn Lys Pro Tyr Trp Leu Gin Arg Ala Gin Gly His Asn 305 310 315 320

AAT GGC ATT TGT TGG GGT AAC CAA CTA TTT GTT ACT GTT GTT GAT ACT 100 Asn Gly lie Cys Trp Gly Asn Gin Leu Phe Val Thr Val Val Asp Thr 325 330 335

ACA CGC AGT ACA AAT ATG TCA TTA TGT GCT GCC ATA TCT ACT TCA GAA 105 Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala lie Ser Thr Ser Glu 340 345 350

ACT ACA TAT AAA AAT ACT AAC TTT AAG GAG TAC CTA CGA CAT GGG GAG 110 Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360 365

GAA TAT GAT TTA CAG TTT ATT TTT CAA CTG TGC AAA ATA ACC TTA ACT 115 Glu Tyr Asp Leu Gin Phe lie Phe Gin Leu Cys Lys lie Thr Leu Thr 370 375 380

GCA GAC GTT ATG ACA TAC ATA CAT TCT ATG AAT TCC ACT ATT TTG GAG 120 Ala Asp Val Met Thr Tyr lie His Ser Met Asn Ser Thr lie Leu Glu 385 390 395 400

GAC TGG AAT TTT GGT CTA CAA CCT CCC CCA GGA GGC ACA CTA GAA GAT 124 Asp Trp Asn Phe Gly Leu Gin Pro Pro Pro Gly Gly Thr Leu Glu Asp 405 410 415

ACT TAT AGG TTT GTA ACC TCC CAG GCA ATT GCT TGT CAA AAA CAT ACA 129 Thr Tyr Arg Phe Val Thr Ser Gin Ala lie Ala Cys Gin Lys His Thr 420 425 430

CCT CCA GCA CCT AAA GAA GAT CCC CTT AAA AAA TAC ACT TTT TGG GAA 134 Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu 435 440 445

GTA AAT TTA AAG GAA AAG TTT TCT GCA GAC CTA GAT CAG TTT CCT TTA 139 Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gin Phe Pro Leu 450 ^' 455 460

GGA CGC AAA TTT TTA CTA CAA GCA GGA TTG AAG GCC AAA CCA AAA TTT 144 Gly Arg Lys Phe Leu Leu Gin Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 475 480

ACA TTA GGA AAA CGA AAA GCT ACA CCC ACC ACC TCA TCT ACC TCT ACA 148 Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495

ACT GCT AAA CGC AAA AAA CGT AAG CTG TAA 151

Thr Ala Lys Arg Lys Lys Arg Lys Leu * 500 505

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 506 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 15

Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 20 25 30 lie Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 35 40 45

Tyr Phe Pro lie Lys Lys Pro Asn Asn Asn Lys lie Leu Val Pro Lys 50 55 60

Val Ser Gly Leu Gin Tyr Arg Val Phe Arg lie His Leu Pro Asp Pro 65 70 75 80

Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gin 85 90 95

Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gin Pro 100 105 110

Leu Gly Val Gly lie Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp 115 120 125

Thr Glu Asn Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg 130 135 140

Glu Cys lie Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu lie Gly 145 150 155 160

Cys Lys Pro Pro lie Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr 165 170 175

Asn Val Ala Val Asn Pro Gly Asp Cys Pro Pro Leu Glu Leu lie Asn 180 ^* 185 190

Thr Val He Gin Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met 195 200 205

Asp Phe Thr Thr Leu Gin Ala Asn Lys Ser Glu Val Pro Leu Asp He 210 215 220

Cys Thr Ser He Cys Lys Tyr Pro Asp Tyr He Lys Met Val Ser Glu 225 230 235 240

Pro Tyr Gly Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gin Met Phe 245 250 255

Val Arg His Leu Phe Asn Arg Ala Gly Ala Val Gly Glu Asn Val Pro 260 265 270

Asp Asp Leu Tyr He Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser 275 280 285

Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala 290 295 300 Gln He Phe Asn Lys Pro Tyr Trp Leu Gin Arg Ala Gin Gly His Asn 305 310 315 320

Asn Gly He Cys Trp Gly Asn Gin Leu Phe Val Thr Val Val Asp Thr 325 330 335

Thr Arg Ser Thr Asn Met Ser Leu Cys Ala Ala He Ser Thr Ser Glu 340 345 350

Thr Thr Tyr Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu 355 360 365

Glu Tyr Asp Leu Gin Phe He Phe Gin Leu Cys Lys He Thr Leu Thr 370 375 380

Ala Asp Val Met Thr Tyr He His Ser Met Asn Ser Thr He Leu Glu 385 390 395 400

Asp Trp Asn Phe Gly Leu Gin Pro Pro Pro Gly Gly Thr Leu Glu Asp 405 410 415

Thr Tyr Arg Phe Val Thr Ser Gin Ala He Ala Cys Gin Lys His Thr 420 425 430

Pro Pro Ala Pro Lys Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu 435 440 445

Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gin Phe Pro Leu 450 455 460

Gly Arg Lys Phe Leu Leu Gin Ala Gly Leu Lys Ala Lys Pro Lys Phe 465 470 ^• 475 480

Thr Leu Gly Lys Arg Lys Ala Thr Pro Thr Thr Ser Ser Thr Ser Thr 485 490 495

Thr Ala Lys Arg Lys Lys Arg Lys Leu * 500 ^{' '} 505

(2) INFORMATION FOR SEQ ID NO : 3 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 297 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..297

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

ATG CAT GGA GAT ACA CCT ACA TTG CAT GAA TAT ATG TTA GAT TTG CAA 4 Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gin 1 5 10 15

CCA GAG ACA ACT GAT CTC TAC TGT TAT GAG CAA TTA AAT GAC AGC TCA 9 Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gin Leu Asn Asp Ser Ser 20 25 30 GAG GAG GAG GAT GAA ATA GAT GGT CCA GCT GGA CAA GCA GAA CCG GAC 144 Glu Glu Glu Asp Glu He Asp Gly Pro Ala Gly Gin Ala Glu Pro Asp 35 40 45

AGA GCC CAT TAC AAT ATT GTA ACC TTT TGT TGC AAG TGT GAC TCT ACG 192 Arg Ala His Tyr Asn He Val Thr Phe Cys Cys Lys Cys Asp Ser Thr 50 55 60

CTT CGG TTG TGC GTA CAA AGC ACA CAC GTA GAC ATT CGT ACT TTG GAA 240 Leu Arg Leu Cys Val Gin Ser Thr His Val Asp He Arg Thr Leu Glu 65 70 75 80

GAC CTG TTA ATG GGC ACA CTA GGA ATT GTG TGC CCC ATC TGT TCT CAG 288 Asp Leu Leu Met Gly Thr Leu Gly He Val Cys Pro He Cys Ser Gin 85 90 95

AAA CCA TAA 297

Lys Pro *

(2) INFORMATION FOR SEQ ID NO 4

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 98 ammo acids

(B) TYPE amino acid (D) TOPOLOGY linear

( ) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO ^• 4

Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gin 1 5 10 15

Pro Glu Thr Thr Asp Leu Tyr Cvs Tyr Glu Gin Leu Asn Asp Ser Ser 20 ^* 25 30

Glu Glu Glu Asp Glu He Asp Gly Pro Ala Gly Gin Ala Glu Pro Asp 35 40 45

Arg Ala His Tyr Asn He Val Thi Phe Cys Cys Lys Cys Asp Ser Thr 50 55 60

Leu Arg Leu Cys Val Gin Ser Thr His Val Asp He Arg Thr Leu Glu 65 70 75 80

Asp Leu Leu Met Gly Thr Leu Gly He Val Cys Pro He Cys Ser Gin 85 90 95

Lys Pro *

(2) INFORMATION FOR SEQ ID NO 5

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 34 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ll) MOLECULE TYPE DNA

(xi ) SEQUENCE DESCRIPTION. SEQ ID NO : 5 CCCCGATATC GCCTTTAATG TATAAATCGT CTGG 34

(2) INFORMATION FOR SEQ ID NO : 6 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 : CCCCGATATC TCAAATTATT TTCCTACACC TAGTG 35

(2) INFORMATION FOR SEQ ID NO : 7 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : : AAAGATATCT TGTAGTAAAA ATTTGCGTCC TAAAGGAAAC 40

(2) INFORMATION FOR SEQ ID NO ^• 8.

(i) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 44 base pairs

(B) TYPE- nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi J SEQUENCE DESCRIPTION SEQ ID NO : 8 : AAAGATATCT AATCTACCTC TACAACTGCT AAACGCAAAA AACG 44

(2) INFORMATION FOR SEQ ID NO : 9

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO : 9 :

AAAAGATATC ATGCATGGAG ATACACCTAC ATTGC 3

(2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: TTTTGATATC GGCTCTGTCC GGTTCTGCTT GTCC 34

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TTTTGATATC CTTGCAACAA AAGGTTACAA TATTGTAATG GGCC 44

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 12: AAAAGATATC TGGTTTCTGA GAACAGATGG GGCAC 35

(2) INFORMATION FOR SEQ ID NO: 13.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: lmeai

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TTTTGATATC GATTATGAGC AATTAAATGA CAGCTCAG 38

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: DNA

(xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TTTTGATATC GTCTACGTGT GTGCTTTGTA CGCAC 35

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TTTATCGATA TCGGTCCAGC TGGACAAGCA GAACCGGAC 39

(2) INFORMATION FOR SEQ ID NO: 16:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 39 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: TTTTGATATC GATGCCCATT ACAATATTGT AACCTTTTG 39

(2) INFORMATION FOR SEQ ID NO: 17: d) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 294 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii ) MOLECULE TYPE: DNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..294

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

ATG AGT CTT CTA ACC GAG GTC GAA ACG CTT ACC AGA AAC GGA TGG GAG 48 Met Ser Leu Leu Thr Glu Val Glu Thr Leu Thr Arg Asn Gly Trp Glu 1 5 10 15

TGC AAA TGC AGC GAT TCA AGT GAT CCT CTC ATT ATC GCA GCG AGT ATC 96 Cys Lys Cys Ser Asp Ser Ser Asp Pro Leu He He Ala Ala Ser He 20 25 30

ATT GGG ATC TTG CAC TTG ATA TTG TGG ATT TTT TAT CGT CTT TTC TTC 144 He Gly He Leu His Leu He Leu Trp He Phe Tyr Arg Leu Phe Phe 35 40 45

AAA TGC ATT TAT CGT CGC CTT AAA TAC GGT TTG AAA AGA GGG CCT TCT 192 Lys Cys He Tyr Arg Arg Leu Lys Tyr Gly Leu Lys Arg Gly Pro Ser 50 55 60 ACG GAA GGA GCG CCT GAG TCT ATG AGG GAA GAA TAT CGG CAG GAA CAG 240 Thr Glu Gly Ala Pro Glu Ser Met Arg Glu Glu Tyr Arg Gin Glu Gin 65 70 75 80

CAG AGT GCT GTG GAT GTT GAC GAT GTT CAT TTT GTC AAC ATA GAG CTG 288 Gin Ser Ala Val Asp Val Asp Asp Val His Phe Val Asn He Glu Leu 85 90 95

GAG TAA 294

Glu *

(2) INFORMATION FOR SEQ ID NO: 18:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 97 ammo acids

(B) TYPE: amino acid (D) TOPOLOGY linear

(ii) MOLECULE TYPE protein

(xi) SEQUENCE DESCRIPTION SEQ ID NO: 18:

Met Ser Leu Leu Thr Glu Val Glu Thr Leu Thr Arg Asn Gly Trp Glu 1 5 10 15

Cys Lys Cys Ser Asp Ser Ser Asp Pro Leu He He Ala Ala Ser He 20 25 30

He Gly He Leu His Leu He Leu Trp He Phe Tyr Arg Leu Phe Phe 35 40 45

Lys Cys He Tyr Arg Arg Leu Lys Tyx Gly Leu Lys Arg Gly Pro Ser 50 55 ^' 60

Thr Glu Gly Ala Pro Glu Ser Met Arg Glu Glu Tyr Arg Gin Glu Gin 65 70 75 80

Gin Ser Ala Val Asp Val Asp Asp Val His Phe Val Asn He Glu Leu 85 ^* 90 95

Glu * (2) INFORMATION FOP SEQ ID NO 19

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 40 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE DNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO: 19: TTTTGATATC GATATGGAAT GGCTAAAGAC AAGACCAATC 4

(2) INFORMATION FOR SEQ ID NO: 20-

(l) SEQUENCE CHARACTERISTICS -

(A) LENGTH 35 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS Single (D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA

(Xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 20: TTTTGATATC GTTGTTTGGA TCCCCATTCC CATTG 35 (2) INFORMATION FOR SEQ ID NO: 21:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: GTTATGACAT ACATACATTC TATG 24 (2) INFORMATION FOR SEQ ID NO: 22:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE. DNA

(xi) SEQUENCE DESCRIPTION SEQ ID NO.22: CCATGCATTC CTGCTTGTAG TAAAAATTTG CGTCC 35 (2) INFORMATION FOR SEQ ID NO 23

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH. 29 case pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(ii) MOLECULE TYPE DNA

(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 23: CTACAAGCAG GAATGCATGG AGATACACC 29 (2) INFORMATION FOR SEQ ID NO: 24:

(l) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24 CATCTGAAGC TTAGTAATGG GCTCTGTCCG GTTCTG 3 (2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: CATCTGAAGC TTATCAATAT TGTAATGGGC TCTGTCCG 38

(2) INFORMATION FOR SEQ ID NO: 26:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26: CATCTGAAGC TTACTTGCAA CAAAAGGTTA CAATATTGTA ATGGGCTCTG TCCG 54

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 69 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: CATCTGAAGC TTAAAGCGTA GAGTCACACT TGCAACAAAA GGTTACAATA TTGTAATGGG 6 CTCTGTCCG 6

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 47 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CATCTGAAGC TTATTGTACG CACAACCGAA GCGTAGAGTC ACACTTG 4

Claims

WHAT IS CLAIMED IS:

1. A vaccine formulation comprising a human papilloma vims capsomere, said capsomere comprising a fusion protein comprising a human papilloma vims Ll protein adjacent amino acid residues from a second protein.

2. A vaccine formulation comprising a human papilloma vims capsomere. said capsomere comprising a tmncated human papilloma vims Ll protein having a deletion of one or more amino acid residues necessary for fomiation of a vims-like particle.

3. The vaccine formulation of claim 2 wherein said capsomere comprises a fusion protein comprising a tmncated human papilloma vims Ll protein adjacent amino acid residues from a second protein.

4. The vaccine formulation of any one of claims 1 ,2, or 3 wherein the Ll protein is encoded in the genome of a human papilloma vims selected from the group consisting of HPV6, HPV11 , HPV16, HPV 18. HPV33. HPV35. and HPV45.

5. The vaccine formulation of claim 4 wherein the papilloma vims is HVP16.

6. The vaccine formulation of any one of claims 2, 3, or 5 wherein carboxy terminal amino acid residues are deleted from the Ll protein.

7. The vaccine formulation of claim 6 wherein 1 to 34 carboxy terminal amino acid residues are deleted from the Ll protein.

8. The vaccine formulation of claim 7 wherein 34 carboxy terminal amino acid residues are deleted from the Ll protein.

9. The vaccine formulation of any one of claims 2, 3, or 5 wherein amino terminal amino acid residues are deleted from the Ll protein.

10. The vaccine formulation of any one of claims 2, 3. or 5 wherein internal amino acid residues are deleted from the Ll protein.

1 1. The vaccine formulation of claim 10 wherein the amino acid residues deleted from the Ll protein comprise a nuclear localization signal.

12. The vaccine formulation of claims 2 or 3 wherein the amino acids residues from the second protein are derived from an HPV protein.

13. The vaccine formulation of claim 12 wherein the HPV protein is an early HPV protein.

14. The vaccine l rmulation of claim 12 wherein the early HPV protein is selected from the group consisting of El , E2, E3, E4, E5. E6. and E7.

15. A method of treating an individual infected with an HPV vims comprising the step of administering to a patient in need thereof an amount of the vaccine fo╧Ç ulation of claims 1 , 2, 3, 5, 7, 8, 1 1 , 13 or 14 effective to reduce the level of HPV infection.

16. A method for preventing papilloma vims infection comprising the step of administering to an individual susceptible thereto an amount of the vaccine formulation of claims 1 , 2, 3, 5, 7, 8, 11 , 13 or 14 effective to inhibit HPV infection.