AU2003200653B2 - Papilloma virus capsomere vaccine formulations and methods of use - Google Patents

Description

-1-

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant/s: Loyola University of Chicago Actual Inventor/s: Lutz Gissmann and Martin Muller Address for Service: Baldwin Shelston Waters MARGARET STREET SYDNEY NSW 2000 CCN: 3710000352 Invention Title: PAPILLOMA VIRUS CAPSOMERE VACCINE FORMULATIONS AND METHODS OF USE Details of Original Application No. 96846/98 dated 06 Oct 1998 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 27734AUP01 500121114 1.0DC/5844 ks0 -la- C, PAPILLOMA VIRUS CAPSOMERE VACCINE SFORMULATIONS AND METHODS OF USE SFIELD OF THE INVENTION The present application is a divisional application of Australian Application No.

5 96846/98, which is incorporated in its entirety herein by reference. The present ,O 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

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

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 310:880-883 (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 -2human 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 (Arbelt, et al., J. ViroL, 68:4358- 4364 (1994): Auewarakul. et al., Mol. Cell. Biol. 14:8250-8258 (1994); Barbosa. et al.. J. Virol. 65:292-298 (1991); Kaur, et al., J. Gen. Virol.

70:1261-1266 (1989): Schlegel. et al., EMBO 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- 112]. Low risk strains of this type include, for example. HPV 6 and 11.

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, -3- Curr. Top. In Microbiol and Immunol. 186:255-266 (1994): Yindle and Fra.:r, Curr. Top. In Microbiol. an d Immunol. 186;217-253 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, er al., supra: Yindle and Frazer, ei 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 virus late Ll 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 Ll 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.

-4- SUMMARY OF THE INVENTION The present invention provides therapeutic and prophvlaizic 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 Ll and L2 of the papilloma virus. Development of a vaccine of this type faces significant obstacles because papilloma viruses cannot be propagated to adequate liters 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 strdightforward and often results in low protein yield.

Recently. virus-like panicles (VLPs). similar in make up to viral capsid structures. have been described which are fonned in Sf-9 insect cells upon expiession of the viral proteins LI and L2 (or LI on its own) using recombinant vaccinia or baculovirs. Purification of the VLPs can be achieved very simply y means of centrifugation in CsCI or sucrose gradients [Kimbauer. et al.. Proc. Nail. Acad. Sci. (USA), 99:12180-12814 (1992): Kirnbaurer. et al., J. Virol. 67:6929-6936 (1994); Proso, et al., J.

Virol. 6714:1936-1944 (1992): Sasagawa. er al., Virology 2016:126-195 (1995): Volpers, et al.. J. Virol. 69:3258-3264 (1995); Zhou, er al., J.

Gen. Virol. 74:762-769 (1993): Zhou, e al., Virology 185:251-257 (1991)]. WO 93/02184 describes a method in which papilloma virus-like particles (VLPs) are used for diagnostic applications or as a vaccine against infections caused by the papilloma virus. WO 94/00152 describes recombinant production of Ll protein which mimics the conformational neutralizing epitope on human and animal papiiloma 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 virus 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 HPVassociated 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 virus from HPV or BPV is therapeutically effective in the regression. but not in the prevention, of papilloma virus 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 virus capsoineres are provided which comprise either: a first protein that is an intact viral protein expressed as a fusion protein comprised in pan of amino acid residues from a second protein; (ii) a truncated viral protein: (iii) a truncated viral protein expressed as a fusion protein comprised in pan 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 virus (BPV) and human papilloma virus. Preferred -6bovine virus capsomeres comprise protein from bovine papilloma virus type I. Preferred human virus capsomeres comprise proteins from any one of human papilloma virus strains HPV6, HPV11, HPV16, HPV18, HPV33.

and HPV45. The most preferred vaccine formulations comprise capsomeres comprising proteins from HPV16.

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 Ll and L2.

Capsomeres comprised of intact viral protein fusions may be produced using the LI and L2 proteins together or the Ll 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: I. 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 permits formation of capsomeres.

Preferably. addition of the second protein amino acid residues inhibits the ability of the intact viral protein to form virus-like particle structures; 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 virus protein. It also preferred that the second protein be the expression product of papiltonia virus 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 virus strains HVP6, HPV11, HPV18, HPV33, HPV35, or HPV 45. It is most preferred that the second protein be encoded by the HPV16 -7- 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 Ll 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 truncated viral proteins having a deletion of one or more amino acid residues necessary for formation of a virus-like panicle. It is preferred that the amino acid deletion not inhibit formation of capsomeres by the truncated protein, and it is most preferred that the deletion favor capsomere formation. Preferred vaccine formulations of this type include capsomeres comprised of truncated LI with or without L2 viral proteins. Particularly preferred capsomeres are comprised of truncated LI proteins. Truncated proteins contemplated by the invention include those having one or more amino acid residues deleted from the carbox teInninus 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 not from either terminus) of the protein. Preferred capsomere vaccine formulations are comprised of proteins truncated at the carboxy terminus. In formulations including LI protein derived from HPV16, it is preferred that from I 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 -8acid residues be deleted from the LI sequence, corresponding to amino acids 472 to 505 in HPV16 set out in SEQ ID NO: 2, and en..oded by the polynucleotide sequence corresponding to nucleotides 1414 to 1516 in the human HPVI6 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 structures 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 L1 proteins assemble into capsomeres. Capsomeres which result from assembly of truncated 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 fonnulations are provided comprising truncated viral protein expressed as a fusion'protein adjacent amino acid residues from a second protein.

Preferred truncated: viral proteins of the invention are the structural papilloma viral proteins LI and L2. Capsomeres comprised of truncated viral p,'otein fusions niay he produced using LI and L2 protein components together or LI protein alone. Preferred capsomeres are those comprised of LI protein amino acid residues. Truncated viral protein components of the fusion proteins include those having one or more amino acid residues deleted from the carboxy ternninus 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 not from either terminus) of the protein. Preferred capsomere vaccine formulations are comprised of proteins truncated at the carboxy terminus. In those -9formulations including Ll protein derived from HPV16, it is preferred that from I to 34 carboxy terminal amin2 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 L1 sequence.

corresponding to amino acids 472 to 505 in HPVl6 set out in SEQ ID NO: 2. and encoded by the polynucteotide sequence corresponding to nucleotides 1414 to 1516 in the human HPVI6 Ll coding sequence set out in SEQ ID NO: 1. When the vaccine formulation is comprised of capsomeres made up of proteins beanne 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 pennits lonnation of capsomeres. Preferably, addition of the second protein anmino 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 virus protein. It also preferred that the second protein be the expression product of papilloma vinm. early gene. It is most preferred, however, that the second protein be selected from group of early gene products encoding by papilloma vinrs El. E2, E3. E4, E5, E6, and E7 genes. 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 10 a particularly preferred embodiment, LI amino acids comprise approxinma'tely 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 truncated 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 truncated protein subsequently modified to include additional amino acid resides as described above to provide a truncated fusion protein of the invention.

In producing capsoineres. recombinant molecular biology techniques can be utilized to produce DNA encoding either the desired intact protein, the truncated protein. or the truncated fusion protein.

Recoilbinant 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.. MOLECULAR CLONING: A LABORATORY MANL'AL. Cold Spring Harbor Press: Cold Spring Harbor, NY (1989) and Ausebel et al.. leds.). 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 baculoviruses, COS cell specific vectors, vaccinia recombinants, or yeastspecific expression constructs. When recombinant proteins are used to 11 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 imM NaCl. 1 mM CaCI 2 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 mM NaCI. 25 mM Ca+. 10% DMSO (dimethyl sulfoxide). 0.1% Triton-X 100. 10 mM Tris [tris-(hydroxymethyl) aminomethane] acetic acid with a pH value of 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 12 formulation of the invention effective to reduce the level of HPV infection.

The invention also provide methods for prophylactic rcatment 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 tPV 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 virus 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 advaintages when compared to conventional vaccine preparations. As pan 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 virus-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 -13- Smay be buried in the overall structure as a result of internalized positioning within the U VLP itself. Similarly, epitopes of the protein components may be sterically obstructed as a result of capso:nere-to-capsomere contact, and therefore unaccessible for eliciting San 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 n component. This observation is consistent with previous results which indicate that the ND 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 structure, both protein components of the fusion protein substructure 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 virus proteins, expressed as a fusion with LI amino acid sequences.

According to a first aspect, the present invention provides a vaccine formulation comprising a human papilloma virus capsomere, said capsomere comprising a fusion protein comprising a human papilloma virus L1 protein adjacent amino acid residues from a second pro:ein, said L1 protein and said amino acids from said second protein positioned to inhibit virus-like particle formation.

According to a second aspect, the present invention provides a vaccine formulation comprising a human papilloma virus capsomere, said capsomere comprising a truncated human papilloma virus L1 protein having a deletion of one or more amino acid residues necessary for formation of a virus-like particle.

According to a third aspect, the present invention provides a method of treating an individual infected with an HPV virus comprising the step of administering to a patient in need thereof an amount of the vaccine formulation according to the invention effective to reduce the level of HPV infection.

According to a fourth aspect, the present invention provides a method for preventing papilloma virus infection comprising the step of administering to an individual susceptible thereto an amount of the vaccine formulation according to the invention effective to inhibit HPV infection.

According to a fifth aspect, the present invention provides use of a vaccine formulation according to the invention in an amount effective to reduce the level of HPV infection in the manufacture of a medicament for reducing the level of HPV.

13a- SAccording to a sixth aspect, the present invention provides use of a vaccine Sformulation according to the invention in an amount effective to inhibit HPV infection in the manufacture of a medicament for preventing papilloma virus infection.

Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

DETAILED DESCRIPTION OF THE INVENTION The present invention is illustrated by the following examples. Example 1 describes construc:ion of expression vectors to produce fusion, or chimeric, viral proteins. Example 2 relates to generation of recombinant baculoviruses 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.

14- Example 1 Construction of Chimeric L1 Genes DNA encoding the HPV 16 L open reading frame was excised from plasmid 16-114/k-LI/L2-pSynxtVF [Kirbauer et al, J. Virol.

67:6929-6936 (1994)] using BgIl and the resulting fragment subcloned into pUC19 (New England Biolabs. Beverly, MA) previously linearized at the unique BamHI restriction site. Two basic expression constructs were first generated to permit subsequent insertion of DNA to allow fusion protein expression. One construct encoded HPV 16 Lla310 having a nine amino acid deletion: the deleted region was known to show low level homology with all other papilloma virus LI proteins. The second construct, HPV 16 L1 aC. encoded a protein having a 34 amino acid deletion of the carboxy tenninal L residues. Other constructs 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-L1 protein amino acids, aspartate and isoleucine.

A. Generation of an HPV 16 Lla310 expression construct Two primers (SEQ 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 SEQ ID NO: 1. Primers were synthesized to also introduce a unique EcoRV restriction site (underlined in SEQ ID NOs: 5 and 6) at the tennii of the amplification product.

CCCCGATATCGCCTTTAATGTATAAATCGTCTGG

SEQ ID NO:

CCCCGATATCTCAAATTATTITCCTACACCTAGTG

SEQ ID NO: 6 The resulting PCR product was digested with EcoRV to provide complementary ends and the digestion product circularized by ligation.

15 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 a310 was identified as having the appropriate twenty-seven nucleotide deletion and this construct was used to inser DNA fragments encoding other HPV 16 proteins at the EcoRV site as discussed below.

B. Generation of an HPV 16 LI aC expression constructs Two primers (SEQ ID NOs: 7 and 8) were designed complementary to the HPV 16 L1 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.

AAAGATATCTTGTAGTAAAATTTGCGTCCTAAAGGAAAC

SEQ ID NO: 7

AAAGATATCTAATCTACCTCTACAACTGCTAAACGCAAAAAACG

SEQ ID NO: 8 Each primer introduced an EcoRV restriction site at the terminus of the amplification product. In the downstream primer (SEQ ID NO: the EcoRV site was followed by j TAA translational stop codon positioned :.uch that the amplification product. uLpon ligation of the EcoRV ends to circularize, would include deletion of the 34 carboxy terminal LI amino acids. PCR was perfonned 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 a cells. Plasmids from viable clones were analyzed for the presence of an EcoRV site which would linearize the plasmid. One positive 16construct designated pUCHPV16LlaC was identified and used to insert DNA from other HPV In proteins utilizing the EcoRV site.

C. Insertion of DNA fragments into HPV 16 LI A310 and HPV16L1AC DNA fragments of HPV 16 E7 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 L1 a310 and HPV16LIC modified sequence. In the various amplification reactions.

primer E7.1 (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 1with primer E7.3 (SEQ ID NO: 11) 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 paifr E7.5 (SEQ ID NO: 13) and E7.6 fSEQ ID NO: 14) were used to amplify a DNA fragment encoding E7 amino acids 25-75. E7.7 (SEQ ID 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. I SEQ ID NO: 9

AAAAGATATCATGCATGGAGATACACCTACATTGC

Primer E7.2 SEQ ID NO:

TTTTGATATCGGCTCTGTCCGGTTCTGCTTGTCC

Primer 7.3 SEQ ID NO: II TTI'GATATCCTTGCACACAAGG

AATATTGTAATGGGCC

17 Primer E7.4 SEQ ID NO: 12 AAAAGATATCTGGT

CTGAGAACAGATGGGGCAC

Primer E7.5 SEQ ID NO: 13

TTTGATATCGATTATGAGCAATTAAATGACAGCTCAG

Primer E7.6 SEQ ID NO: 14

TITTGATATCGTCTACGTGTGTGCTIGTACGCAC

Primer E7.7 SEQ ID NO:

TTATCGATATCGGTCCAGCTGGACAAGCAGAACCGGAC

Primer E7.3 SEQ ID NO: 16 TTGATATCGATGCCCATTACAATATfGTAACCITITG 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.

TITGATATCGATATGGAATGGCTAAAGACAAGACCAATC

SEQ ID NO: 19 TITTGATATCGTGT7GGATCCCCATTCCCATTG SEQ ID NO: PCR products from each amplification reaction were cleaved with EcoRV and inserted into the EcoRV site of either the HPV 16 Ll

A

3 1 0 and HPVI6LlC sequences previously linearized with the same enzyme. In order to determine the orientation of inserts in piasmids encoding E7-amino acids 25-75 and 50-98 and plasmid including influenza matrix protein, Clal digestion was employed, taking advantage of a 18 restriction site overlapping the newly created EcoRV restriction site (GATATCGAT) and included in the upstream primer. For the thre: expression constructs including the initiating methionine of HPV 16 E7, insert orientation was determined utilizing a Nsil restriction site within the E7 coding region.

Once expression constructs having appropriate inserts were identified, the protein coding region for both LI and inserted amino acids was excised as a unit using restriction enzymes XbaI and Smal and the isolated DNA ligated into plasmid pVLI393 (Invitrogen) to generate recombinant baculoviruses.

D. Elimination of EcoRV Restriction Sites in Expression Constructs The HPV 16 L1 aC 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 constructions was designed in which the artificial EcoRv site was eliminated. The LI sequence for this series of expression constructs was designated HPV 16L1,aC'.

To generate an expression construct containing the HPV 16LlaC* sequence. two PCR reactions were performed to amplify two overlapping fragments from the pUC-HPV16 LaC encoding E7 amino acids 1-50. The resulting DNA fragments overlapped at the position of the LI/E7 boundary but did not contain the two EcoRV restriction sites.

Fragment 1 was generated using primers P1 (SEQ ID NO: 21) and P2 (SEQ ID NO: 22) and fragment 2 using primers P3 (SEQ ID NO: 23) and P4 (SEQ ID NO: 24).

Primer PI SEQ ID NO: 21

GTTATGACATACATACATTCTATG

Primer P2 SEQ ID NO: 22 19

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 primers P1 and P4 only. The resulting amplification product was digested with enzymes EcoNI and HindIII inserted into the HPV I6LIaC expression construct described above following digestion with the same enzymes. The resulting expression construct differed from the original HPV16LIAC construct with DNA encoding LI and E7 amino acids 1-50 by loss of the two internal EcoRV restriction sites. The first EcoRV site was replaced by DNA encoding native LI alanine and glycine amino acids in this position and the second was replaced by a translational stop signal. In addition, the expression construct. designated HPV 16 LIAC* E7 1-52.

contained the first 52 amino acids of HPV 16 E7 as a result of using primer P4 which also encodes E7 amino acids residues histidine at position 51 and tyrosine at position 52. HPV 16 LlaC* E7 1-52 was then used to generate additional HPV 16 LtIC expression constructs further including DNA encoding E7 amino acids 1-55 using primer Pl (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 acidencoding 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:

CATCTGAAGCTTAACAATATTGTAATGGGCTCTGTCCG

Primer P6 SEQ ID NO: 26 CATCTGAAGCTTACTrGCAACAAAAGGTTA-

CAATATTGTAATGGGCTCTGTCCG

Primer P7 SEQ ID NO: 27

CATCTGAAGCTTAAAGCGTAGAGTCACACTTGCAAC-

AAAAGGTTACAATATTGTAATGGGCTCTGTCCG

Similarly. HPV 16 LaC* E7 1-70 was generated using template DNA encoding HPV 16 LIAC* E7 1-66 and the primer pair P1 and P8 (SEQ ID NO: 28).

Primer P8 SEQ ID NO: 28 CATCTGAAGCTTAnGTA CGCACAC .AC- SEQ ID NO: 28 CG AAGCGTAGAGTCACACTTG Followin2 each PCR reaction. the amplification products were digested with EcoNI and HindllI and inserted into IPV16Ll&C previously digested with the same enzymes. Sequences of each constructs were determined using an Applied Biosystems Prism 377 sequencing instrument with fluorescent chain ternninating dideoxynucleotides [Prober et al., Science 238:336-341 (1987)].

Example 2 Generation of Recombinant Baculoviruses Spodoperafrugiperda (Sf9) cells were grown in suspension or monolayer cultures at 27* in TNMFH medium (Sigma) supplemented with fetal calf serum and 2 mM glutamine. For HPV 16 Li-based recombinant baculovirus construction, Sf9 cells were transfected with 10 g of transfer plasmid together with 2 Ag of linearized Baculo-Gold

DNA

-21 (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 baculovirus recombinant at a multiplicity of infection 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 Ll and anti-HPV16 E7 antibodies.

In order to determine which of the chimeric LI protein expression constructs 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 fonnation by electron microscopy. The results are shown in Table I.

The intact HPV LI protein. as well as the expression products HPV 16 LIA310 and HPV 16 L 2C. each were shown to produce capsomeres and virus-like particles in equal proportions. When E7 coding sequences were inserted into the HPV 16 LIA310 vector, only fusion proteins including E7 amino acids I to 50 produced gave rise to detectable capsomere formation.

When E7 encoding DNA was inserted into the HPV 16 LIAC 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 virus-like particle formation was also observed.

The chimeric protein including E7 amino acids 1-60 resulted in nearly equal levels of capsomere and virus-like panicle production.

When E7 sequences were inserted into the HPV 16 LIA*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 causomeres, but equal levels of virus-like particle production werc: observed. While insertion of DNA encoding0 E7 DNA for residues 1-65, 1- 25-75, 40-98, and 1-98 resulted in comparatively lower levels or undetectable levels of capsid. capsomeres were produced in high quantities.

TABLE 1 Capsomeree and Capsid Forming Capacity of Chimeric HPV LI Proteins Li Exprcssrion Capsomere Capsid Yield Yield HVp it Lt Nne +Nonr HPV IbLia3IO N.nc HPV 1b Ll \nc C HPV Ib La310 F- v-Ts HPV' ULIaoIO ii0, I-so HPV 1b LIa310 F-25-7S HPV IOL tJIO r7s5O-Q1S HPV I LiC F2- H1W lP LIaC [7 5O-" HPV 16 L 4 C IF1-no HPV 16 LlaC -DR HPV I6 LaC Iniluenz, HPV 16 LI aC F%-52 .44444. HPV 16 LlI&C E7 1-55+ HPV 1 LIA.C E 7-O HPV 16 Lta'C E7 1-0S HPV IbLlk'C E7 t-70 Example 3 Purification of C:'psomeres Trichopulsia ni (TN) High Five cells were grown to a density of approximately 2 x 10 6 cells/mI in Ex-Cell 405 serum-free medium (JRH Biosciences). Approximately 2 x 108 cells were pelleted by centrifugation at 1000 x g for 15 minutes, resuspended in 20 ml of medium, and infected with recombinant baculoviruses 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 CsCI solution (4.6 g CsCI per 8 ml in extraction buffer). and centrifuged in a Sorval AH629 swinging bucket rotor for 2 hours at 27.000 rpm at The interface region between the CsCI and the sucrose along with the CsCI 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 i Beckman 70 TI rotor. Gradients were fractionated (1 ml per fraction) by puncturing tubes on top and bottom with a 21gauge needle. Fractions were collected from each tube and 2.5 p~ of each fraction were analyzed by a 10% SDS-polyacrylamide gel and Western blotting using an anti-HPV16 LI antibody.

Virus-like particles and capsomeres were separated from the fractions identified above by sedimentation on 10 to 50% sucrose gradients. Peak fractions from CsCI gradients were pooled and dialyzed for 2 hours against 5 mM HEPES (pH Half of the dialysate was used to produce capsomeres by disassembly of intact VLPs overnight by adding EDTA (final concentration 50 mM), EGTA 24 mM), DTT (30 mM). NaCI (100 mM), and Tris/HCI, pH 8.0, (10 mM). As control, NaCI and T:is/HCI 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 ug of HPV chimeric capsomeres mixed 1:1 with complete or incomplete Freund's Adjuvants in a total volume of 100 pl. Six weeks after the third immunization. mice are sacrificed and blood is collected by cardiac puncture.

Example Peptide ELISA to Quantitate Capsomere Formation Microtiter plates (Dynatech) are coated overnight with 50 pl of peptide E701 [Muller er al., 1982] at a concentration of 10 pg/ml in PBS. Wells are blocked for 2 hour at 37"C with 100 pl 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 I of sera diluted 1:5000 in BSA/Tween is added to each well and incubation carried out for 1 hour. Plates are washed again as before and 50 pl of goat-anti-mouse peroxidase conjugate is added at a 1:5000 dilution. After I hour. plates are washed and stained using ABTS substrate (0.2 mg/ml. 2 .2'-Azino-bis(3-ethylbenzhiazoline--sulfonic acid in 0.1 M Na-Acetate-Phosphate buffer (pH 4.2) with 4 .I1 30% H 2 0 2 per 10 ml).

Extinction is measured after 1 hour at 490 nm in a Dynatech automated plate reader.

25 Example 6 Antigen Capture ELISA to Quantitate Capsomere Formation To allow relative quantification of virus-like particles and capsomeres in fractions of CsCI gradients, an antigen capture EUSA was utilized.

Microtiter plates were coated overnight with 50 pl/well of a 1:500 dilution (final concentration of 2 ;g 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 pl of fractions of CsCI 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 pl 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 I hour. Plates were washed again and further incubated with l 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 panicles. Treated particle preparations were assayed in the antigen-capture ELISA and readings compared to untreated controls. For disassembly, 40 pl of VLPs was incubated overnight at 4°C in 500 pl of disruption buffer containing 30 mM DTT. 50 mM EGTA, 60 mM EDTA, 100 mM NaC1, 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 26observations that virus-like particles are capable of ilemagglutinizing 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. HPVI6 LI virus like particles (VLPs) and bovine PVI (BPV) LI VLPs are assayed in parallel with a chimeric capsomere preparation. To establish a positive baseline.

the HPVI6 or BPVI 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 l of the suspension is used per well on a 96-well plate.

Chimeric capsomere aliquots from CsCI gradients are dialyzed for one hour against 10 mM Hepes (pH 7.5) and 100 tl 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 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 HPVI6L AC 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 hu: an VLPs and this differential :eutralization 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.

Claims (17)

1. A vaccine formulation comprising a human papilloma virus capsomere, said capsomere comprising a fusion protein comprising a human papilloma virus LI protein adjacent amino acid residues from a second protein, said LI protein and said amino Cc, acids from said second protein positioned to inhibit virus-like particle formation. In S2. A vaccine formulation comprising a human papilloma virus capsomere, said C capsomere comprising a truncated human papilloma virus L1 protein having a deletion Cc€ Sof one or more am:.no acid residues necessary for formation of a virus-like particle.

3. The vaccine formulation of claim 2 wherein said capsomere comprises a fusion protein comprising a truncated human papilloma virus 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 LI protein is encoded in the genome of a human papilloma virus selected from the group consisting of HPV6, HPV11, HPV16, HPV18, HPV33, HPV35 and The vaccine formulation of claim 4 wherein the papilloma virus is HPV16.

6. The vaccine formulation of any one of claims 2, 3, or 5, wherein carboxy terminal amino acid residues are deleted from the L1 protein.

7. The vaccine formulation of claim 6 wherein 1 to 34 carboxy terminal amino acid residues are deleted from the Ll protein. 41

8. The vaccine formulation of claim 7 wherein 34 ca'oxy terminal amino acid residues are deleted from the L1 prot:in.

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

11. The vaccine formulation of claim 10 wherein the amino acid residues deleted from the LI 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 ftnnutlaion of claim 12 wherein the HPV protein- is an early HPV protein.

14. The vaccine Ik'nntilaiion of claim 12 wherein the early HPV protein is selected from the group consisting of El, E2, E3. E4. E6. and E7. A method of treating an individual infected with an HPV virus comprising the step of administerine to a patient in need thereof an amount of the vaccine fonnulation of claims 1, 2, 3, 5, 7, 8, 11, 13 or 14 effective to reduce the level of HPV infection. IN -42-

16. A method for preventing papilloma virus 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 and 14 effective to inhibit HPV infection.

17. Use of a vaccine formulation of any one of claims 1, 2, 3, 5, 7, 8, 11, 13 or 14 in an amount effective to reduce the level of HPV infection in the manufacture of a medicament for reducing the level of HPV. Cc 18. Use of a vaccine formulation according to claims 1, 2, 3, 5, 7, 8, 11, 13 or 14 in an amount effective to inhibit HPV infection in the manufacture of a medicament for preventing papilloma virus infection.

19. A vaccine formulation according to claim 1 or claim 2, substantially as herein described with refe:rence to any one or more of the examples, but excluding comparative examples. A method of treating an individual infected with an HPV virus according to claim substantially as herein described with reference to any one or more of the examples, but excluding comparative examples.

21. A method for preventing a papilloma virus infection according to claim 16, substantially as herein described with reference to any one or more of the examples, but excluding comparative examples

22. Use of a vaccine formulation according to claim 17 or claim 18, substantially as herein described vith reference to any one or more of the examples, but excluding comparative exanmples. 28 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: (ii) TITLE OF INVENTION: Papilloma Virus Capsomere Vaccine Formulation and Methods of Use (iii) NUMBER OF SEQUENCES: 27 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Marshall, O'Toole, Gerstein, Murray Borun STREET: 233 South Wacker Drive, 6300 Sears Tower CITY: Chicago STATE: Illinois COUNTRY: United States of America ZIP: 60606-6402 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.30 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: CLASSIFICATION: ATTORNEY'/AGENT INFORMATION: NAME: Williams Jr.. Joseph A. REGISTRATION NUMBER: 38,659 REFERENCE/DOCKET NUMBER: 27013/34028 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 312-474-6300 TELEFAX: 312-474-0448 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 151 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: NAME/KEY:: CDS LOCATION: 1..1518 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1- ATG TCT CTT TGG CTG CCT ACT GAG GCC ACT GTC TAC TTG CCT CCT GTC 48 Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 CCA GTA TCT AAG .GTT GTA AGC ACG GAT GAA TAT G-T GCA CGC ACA AAC 56 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr -al Ala Arg Thr Asn -25 29 ATA TAT TAT CAT Ile Tyr Tyr His GOA GG.A ACA Ala Gly Thr AGA OTA OTT GCA Arg Leu Leu Ala GGA CAT COO Gly His Pro TAT rrr Tyr Phe so OCT AOT AAA AAA Pro Ile Lys Lys COT Pro 55 AAO AAT AAC AAA Asn Asn Asn Lys ATA Ile TTA GTrr oCr AAA Leu Val Pro Lys CTA Val T CA GGA TTA CAA Ser Cly Leu Gin TAO Tyr 70 ASC CTA r= AGA Arg Val Phe Arg ATA Ile 75 CAT TI'A COT GAO His Leu Pro Asp 000 Pro AAT AAC rrr CC? Asn Lys Phe Gly Phe 00W GAO ACC TCA Pro Asp Thr 5cr rrr TAT AAT OCA CAT ACA CAG Phe Tyr Asn Pro Asp Thr Gin 90 0CGG TG crr Arg Leu Val TCG Trp 100 GOC TCT GTA GGT Ala Oys Val Cly OTT Val 105 GAG CTA CC? CT Giu Val Cly Arg CTCAG OCA Gly Gin Pro 110, TO CAT GAO Leu Asp Asp TTA OT GTG GCO ATi' ACT GC Leu--G1y Val Gly le 5cr Gly OCT OTA TTA AM' Pro Leu Leu Asn ACA CAA AAT OT ACT OT Thr Ciu Asn Ala Ser Ala 130 TAT Tyr 125 GCA CA AAT GCA Ala Ala Asn Ala GOT Gly 140 CTC CAT AAT AGA Val Asp Asn Arg CAA TOT ATA TOT ATC CAT TAO AAA CAA ACA CAA TO TCT TTA AOT GGT Clu Cys Ile Ser Met Asp Tyr Lys Gin Thr Gin Leu Cys Leu Ile Cly 145 150 155 160 TC AA-A OCA COT Oys Lys Pro Pro ATA Ile 165s CCC GA;A CAC'- TGZU GCC AAA GCA TOO OOA-TGT ACC Gly Ci-u His ?rp CGb Lys Gly Ser Pro Cys Thr 170 175 A.AT CT=? GCA Asn Val Ala ACA OTT AT? Thr Val Ilie 195 AAT OCA GT CAT Asn Pro Gay Asp OOA OCA TTA CAC Pr-o Pro Leu Clu OTA ATA AAO Leu Ile Asn 190 CCT GOT ATh Cly Ala Met OAG CAT CCT CAT Gin Asp Gly Asp ATG Met 200 CG= CAT ACT GC Val Asp Thr Gly Phe GAO TT Asp Phe 210 ACT AC-A TTA OAG Thr Thr.Leu Gin AAC AAA AG? CAA Asn Lys 5cr Clu OCA OTO CAT AT? Pro Leu Asp Ile TCT Oys 225 ACA TOT AT? TGO Thr Ser Ile Oys AAA Lys 230 TAT OOA CAT TAT Ty/r Pro Asp Tyr AT? Ile 235 AAA ATO GTC TO-A Lys Met Val 5cr GAA Giu 240 CZA TAT GCO GAO Pro Tyr Cly Asp AGO Ser 245 TTA T71' TT TAT Leu Phe Phe Tyr CA AGO CAA CAA Arg Arg Clii Gin ATC ri=i Met ?he 255 GTT AGA CAT? Val Ary His GAO CAT TFA Asp Asp Leui 27S TTA Le u 260 =I1~ AAT AGG GOT Phe Asn Arg Ala GCG? Cly 265 CT OTT GOT GAJA Ala Val Gly Clii AAT CTA OCA Aso Val Pro 270 OTA CO ACT Leu Al1a Ser ',AO AT? AAA GGC Tyr Ilie Lys Gly CCC TOT ACT CA Gly Se; Thr Ala TCA AAT Ser Asn 290 TAT 77T CCT ACA CCT Tyr Phe Pro Thr Pro 295 AGT GGT TCT ATC Ser Gly Ser Met Grr Val 300 ACC TCT CAT GCC Thr Ser Asp Ala CAA Gin 305 ATA TTC AAT AAA Ile Phe Asn Lys TAT TGG 7TA CAA Tyr Trp Leu Gin CGA Arg 315 GCA CAG GGC CAC Ala Gin Gly His AAT GGC ATT TGT Asn Gly Ile Cys TGG T=o 325 GGT AAC CA-A CTA Sly Asn Gin Leu fT Phe 330 c=r ACT G=T GTT Val Thr Val Val CAT ACT Asp Thr 335 ACA CCC AGT Thr Arg Ser ACA Thr 340 AAT ATG TCA TTA Asn Met Ser 1eu TGT Cys 345 CCT CCC ATA TCT Ala Ala Ile Ser ACT TCA GAA Thr Ser Glu 350 CAT GGG GAG His Giy Glu ACT ACA TAT A-AA Thr Thr Tyr Lys 355 AAT ACT A-AC Asn Thr Asn rTr Phe 360 AAG GAG TAC CTh Lys Glu Tyr Len CGA Arg 365 CA-A TAT Glu vr 370 CAT TTA CAG 17 Asp Leu Gin Phe TTT CAA CTG TGC Phe Gin Leu Cys AAA Lys 380 ATA ACC TTA ACT Ile Thr Len Thr GCA Ala 385 GAC GTT ATO ACA Asp Val Met Thr TAC Tr 390 ATA C-AT TCT ATG lle His 5cr Met AAT Asn 395 7CC ACT ATT TTG Ser Thr Ile Len 912 960 1008 1056 1104 1152 1200 1242 1296 1344 1392 1440 14 1518 CAC TOG AAT TT Asp Trp Asn Phe GGT Gly 405 CTA C-AA CC.CC-- Leu Gin Pro Pro CCA Pro 410 GGA GGC ACA CTA Cly Cly Thr Len GAA AT Glu Asp 415 ACT TAT AGO Thr Tyr Ary C-CT CCA GCA Pro Pro Ala 435 iT Phe. 420 GTA AC: C7C CAG Val Thr Ser Gin GCA Ala 425 Afi GCT TCT CAA Ile Ala Cys Gin AAA CAT ACA Lys His Thr 430 =17 TGG GA-A Phe Trp, Glu CCT AAA GAA GAT Pro Lys Glu Asp CCC Pro 440 C-T AAA A-A-A TAC Len Lys Lys Tyr ACT Thr 445 OTA Val GGA Gly 465 TTA AAG GA-, A-G Len Lys Glu Lys TCT GCA GAC CTA Ser Ala Asp Len GAT Asp 460 CAG =17 CCT TTA Gin Phe Pro Len CGC AAA 717 2TT Arg Lys Phe Len CTA Len 470 CA- GCA GGA TI-G Gin Ala Gly Len AAG Lys5 475 GCC AAA CCA AAA Ala Lys Pro Lys ACA 7TA GGA AAA Thr Len Gly Lys CGA Arg 485 AAA GCT ACA CCC Lys Ala Thr Pro ACC Thr 490 ACC TCA-TCT ACC Thr Ser Ser Th TCT ACA Ser Tm 495 ACT OCT AA Thr Ala Lys AAA AAA COT AAG Lys Lys Arg Lys CTO TAA Leu C2) INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS- LENGTH; 506 amino acids TYPE: amino acid TOPOLOGY: linear -31 (ii) MOLECULE YPE: protein SEQUENCZE DESCRIPTION: fiQ Met Ser Len Trp Len Pro Ser Glu Ala I Pro Ie Tyr Val Asr. Ary eu Thr Glu 145 Cys Asn Thr Asp Cys 225 Pro Val Asp Val Tyr Phe so Ser Lvs Leu Gly Glu 130 Cys Lys Va1 Val Phe 210 Thr Trr Arg Asp Ser Tyr Pro Gly Phe Val Val 115 Asn Ile Pro Ala lile 195 Thr Ser Gly His Leu 275 Lys His Ile Leu Gly Trp 100 Gly Alla Ser Pro Va1 Gin Thr Ile Asp Leu 260 Tyr val Ala Lys Gin Phe Ala Ile Se Met Ile 165 Asn Asp Leu Cys Ser 245 Phe Ile Val Sly Lys Tvr 70 Pro Cys Ser Al a Asp 150 GSv Pro G I, GIn Lvs 230 Leu Asn Lys Sec Thr Pro 55 Arg Asp Val Glu 135 Gi Asp Ala Phe Arg Gly Thr Ser 40 Asn Val Thr Glv His 120 Ala Lys His Asp Met 200 Asn Pro Phe Ala Ser 280 Asp 25 Arg Asn Phe Ser Va1 105 Pro ~la Gin Trp Cv.s 185 Val Lvs Asp Gly 265 Sly C 1 A A P G L A T G A 54 Le 2! A: ID NO:2: :hr Val ;iu Tyr V 1 eu Leu A Lsn Lys I rg Ile H 75 he Pvr A 90 iu Val G: eu Leu A: an Ala G: 14 hr Sbn Lf 153 ly Lys GI 70 to Pro Le sp Thr C) 2r Glu Va 22 ~r Ile Ly 235 eu Arg Ax 50 la Val GI ar Thr Al yr al la le is sn Ly sn IV L0 YY i '5 -9 1 00 yy aa Len Ala Val Len Len Pro Arg Lys 125 Va1 Cys Sec Glu Phe 205 Pro Met Glu Glu Asn 285 Pro Arg Sly Val Pro Asp Sly 110 Len Asp. Len Pro Len 190 Gly. Len Val Gin Asa 270 Len Pro Thr His Pro Asp Thr Gin Asp Asn Ile Cys 175 Ile Ala Asp Ser Met 255 Val Ala Val Asm Pro Lys Pro Gin Pro Asp Arg Sly 160 Thr Asn Met Ile Glu 240 Phe Pro Ser Sec Asn 290 Tyr Phe Pro Thr Pro Ser Gly Sec Met Val 300 Thr Sec Asp Ala 32 Gin Ile Phe Asn Lys 305 Pro Tyr Trp Leu Gin Asn Thr Thr Glu Ala 385 Asp Thr Pro Val Gly 465 Thr Thr Gly Arg Thr Tvr 370 Asp Trp Pro Asn 450 Arg Leu Ala Ile Ser Tvr 355 Asp Val Asn Arg Al a 435 Leu Lys Gly Lys Cys Thr 340 Lys Leu Met Phe Phe 420 Pro Lys Phe Lys Arg 500 Trp Gly 325 Asn Met Asn Thr Gln Phe Thr Tyr 390 Gly Leu 405 Val Thr Lys Glu Glu Lys Leu Leu 470 Arg Lys 485 Lys Lys Asn Ser Asn Ile 375 Ile Gin Ser Asp Phe 455 Gin Ala Arg Gin Leu Leu Cys 345 Phe Lys 360 Phe Gin His Ser Pro Pro Gin Ala 425 Pro Leu 440 Ser Ala Ala Gly Thr Pro Lys Leu 505 Phe 330 Ala Glu Leu Met Pro 410 Ile Lys Asp Leu Thr 490 Arg 315 Val Ala Tyr Cys Asn 395 -Gly Ala Lys Leu Lys 475 Thr Ala Gin Gly His Asn Thr Val Ile Ser Leu Arg 365 Lys Ile 380 Ser Thr Gly Thr Cys Gin Tyr Thr 445 Asp Gin 460 Ala Lys Ser Ser Val Asp 335 Thr Ser 350 His Gly Thr Leu Ile Leu Leu Glu 415 Lys His 430 Phe Trp Phe Pro Pro Lys Thi Ser 495 320 Thr Glu Glu Thr Glu 400 Asp Thr Glu Leu Phe 480 Thr INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 297 base pairs (8 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..297 (x SEQUENCE DESCRIPTION: SEQ ID NO:3: ATG CAT GGA GAT ACA CCT ACA TTG CAT GAA TAT ATG TTA GAT TTG CAA Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gin 1 5 10 ICA GAG ACA ACT GAT CTC TAC TGT TAT GAG CAA TTA AAT GAC AGC TCA Pro Glu Thr Thr Asp Leu Tyr Cys Tr Glu Gln Leu Asn Asp Se: Ser 25 33 GAG GAG GAG CAT GAA ATA OAT GGT CCA OCT GSA CAA GCA Glu Giu Giu Asp Giu lie Asp Sly Pro Ala Sly Gin Ala 40 CA4A CC GAO. Oiu Pro Asp 1-;4. AGA G00 Arg Ala so CAT TAO AAT A'I7 His Tyr Asn Ile OTA ACC Val Thr

55. 'PIT TOT TGC Phe C3ys Cys AAG Lys TOT LAO TOT7 ACO Cys Asp Ser Thr OTT Leu COG 'TOTOO MCTA Arg Leu Cps Val C.AA G In AGO ACA CAC OTA Ser Thr His Val. ATT COT ACT 'TOG Ile Arg Thr Leu GAO C70 'PTA ATO Asp Leu Leu Met ACA OTA OGA AT Thr Leu Gly Ile 070 Val TOO COO ATO TOT Cys Pro Ile Cys TOT CAG Ser Gin AAA CA. TZJA Lys Pro- INFORMATION FOP. SEQ ID NO:4: Wi SEQUENCE C!AR-AOTERISTIOS: LENGTH: 98 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met I His Gly Asp Pro Thr Leu His Glh Tyr Met Leu Asp 10 Leu Gin is Pro Giu Thr Glu Glu Glu Asp Leu Tyr Cys Glu Gin Leu Asn Asp Ser Ser Asp 0u Ilie Asp Glv 4 0 Pro Ala Gly Gin Ala 0 u Pro Asp Ary Ala s0 His Tyr Asn Ile Thr. Phe Cys Cys Lys Cys Asp Ser Thr Ile Ary Thr Leu Glu Leu Arg Leu Cys Val Ser Thr his Val Asp Leu Leu Met Thr Leu Gly Ilie Val1Cys Pro Ile Cys Ser Gin Lys Pro INFORMATION FOR SEQ ID Wi SEQUENCE r--AOLTEPIJSTICs: CA) LEN. rH-: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TP:DNA (Xi) SEQUENCE DESCRIPTION: SEQ ID 34 CCCCGATATC GCCTTTAATG TATAAATCGT CTGG .34 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE.DESCRIPTION: SEQ ID NO:6: CCCCGATATC TCAAATTATT TTCCTACACC TAGTG 12) INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: AAAGATATCT TGTAGTAAAA ATTTGCGTCC TAAAGGAAAC INFORMATION FOR SEQ ID N3:8: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (1:1 MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AAAGATATCT AATCTACCTC TACAACTGCT AAACGCAAAA AACG 44 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTER:STICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: AAAAGATATC ATGCATGGAG ATACACCTAC ATTGC INFORMATION FOR SEQ TD SEQUENCE CHAPACTERISTICS: LENGTH: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTTTGATATC GGCTCTGTCC GGTTCTGCTT GTCC 34 INFORMATION FOR SEQ ID NO:11: SEQUENCE.CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TTTTGATATC CTTGCAACAA AAGGTTACAA TATTGTAATG GGCC 44 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: AAAAGATATC TGGTTTCTGA GAACAGATGG GGCAC INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 38 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: TTTTGATATC GATTATGAGC AATTAAATGA CAGCTCAG 38 INFORMATION FOP SEQ ID NO:14: SEQUENC' CHARACTERISTICS: LL:;GTH: 35 base pairs TYPE:.nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUEN: D-ESCRIPTION: SEQ ID NO:1: 36 TTTTGATATC GTCTACGTGT GTGCTTTGTA CGCAC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE .DESCRIPTION: SEQ ID TTTATCGATA TCGGTCCAGC TGGACAAGCA GAACCGGAC INFORMATION FOR SEQ ID NO:16: (1i) SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: TTTTGATATC GATGCCCATT ACAATATTGT AACCTTTTG INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 294 base pairs TYPE: nucleic acid STRANDEONESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1..294 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: ATG .AGT CTT CTA ACC GAG GTC GAA ACG CTT ACC AGA Met Ser Leu Leu Thr Glu Val Glu Thr Leu Thr Arg 1 5 10 TGC AAA TGC AGC GAT TCA AGT GAT CCT CTC ATT ATC Cys Lys Cys Ser Asp Ser Ser Asp Pro Leu Ile Ile 25 ATT GGG ATC TTG CAC TTG ATA TTG To-; ATT TTT TAT Ile Gly Ile Leu His Leu 11e Leu Tip Ile Phe Tyr 40 AAA TGC ATT TAT CGT CGC CTT AAA TAC GGT TTG AAA Lys Cys Ile Tyr Arg Arg Leu Lys Tyr Gly Leu Lyl 55 AAC GGA TGG GAG Asn Gly Trp Glu GCA GCG AGT ATC Ala Ala Ser Ile CGT CTT TTC 7TC Arg Leu Phe Phe AGA Arg GGG CCT TCT Gly Pro Ser 37- 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 70 75 CAG AGT GCT GTG GAT GTT GAC GAT GTT CAT TTT GTC AAC ATA GAG CTG 288 Gin Set Ala Val Asp Val Asp Asp Val His Phe Val. Asn Ile Glu Leu 90 GAG TAA 294 Gilu INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids TYPE: amino acid 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 Cys Lys Cys Ser Asp Ser Ser Asp Pro Leu Ile Ile Ala Ala Ser Ile 25 Ile Gly Ile Leu His Leu lie Leu Trp Ile Phe Tyr Arg Leu Phe Phe 40 Lys Cys Ile Tyr Arg Arg Leu Lys T'r Gly Leu Lys Arg Gly Pro Ser 55 Thr Glu Gly Ala Pro Glu Ser Met Arg Glu Glu Tyr Arg Gin Glu Gin 70 75 Gin Ser Ala Val Asp Val Asp Asp Val His Phe Val Asn Ile Glu Leu 90 Glu INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TTTGATATC GATATGGAAT GGCT:AAGAC AAGACCAATC INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single 38 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTTGATATC GTTGTTTGGA TCCCCATTCC CATTG INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GrIATGACAT ACATACATTC TATG 24 INFORMATION FOR SEQ ID NO:22: SEQUENCE CKARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: COATGCATYC CTGCTTGTAG TAAA,-TTT CGTCC INFORMATION FOR SEQ ID NO:23: i) SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CTACAAGCAG GAATGCATGG AGATACACC INFOrMATION FOP, SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: n..cleic acid STRANDE-LtESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CATCTGAAGC TTAGTAATGC GCTCTGTCCG GTTCTG 39 INFORMATION FOR SEQ ID Ci) SEQUENCE CHARACTERISTICS: LENGTH: 3S8 base pairs TYPE: nucleic acid STPJU7DEDNESS1-: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID0 CATCTGAAGC TATC.AATAT TTAGGCTCTGTCCG 38 INFORM4ATION FOR SEQ? D N0:26: Ci) SEQUENCECHRCrTTS LENGTH: '54 base na-rs TYPE: nue:acid CC) S TRPAND EDN4ES S: S -,nzC>It CD) TOPOLOGY,: linear CiMOLECULE TYPE: D)NA Cxi) SEQUENCE DESCRI'PTION: SEQ 1D NO:26: CAT CTGAAGC flACTTOCA A~ CAA;TATTGTA ATGGGCTCTG TCOr 54 INFORM4ATION FOR SEQ- :D NO:27 i)SEQUENCE C r~; LENGTH: e9 mase nair s TYPE: nu:The-z aczo STRANflED0NECC TOPOLOGY: lznea: (ii) MOLECULE TYPE: DNA, (Xi) SEQUENCE DESCRIP-TON: SEQ. :D NO 27l CATCTG.AAGC TTAA-AGCGTA, GACTCACAC7r TGCAACAAAA GG'I-ACAATA TTGTAATGGG CTCTGTCCG 69: INFORMATION FOP SEQI :D N:2" (2SEQUENCE CKARA1CTER:.-ST!1CS: LENGTH: 4' oase pairs TYPE: nudle:: acid STRAJflEDNESS: stnglE- CD) TOPOLOY: li±near (ii) MOLECULE TYPE: DNA7 (\SEQUE: E: DESCRIP.TION: SEQ ID NO::S:- CN'NZ-TGAAGC IT.ATTGTACG CACAACCGAA GCGTAGAGTC ACACTTG 47