AU2007201791A1 - Papilloma virus capsomere vaccine formulations and methods of use - Google Patents

Description

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AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name of Applicant/s: Actual Inventor/s: Loyola University of Chicago Lutz Gissmann and Martin Muller Address for Service is: SHELSTON IP Margaret Street SYDNEY NSW 2000 CCN: 3710000352 Attorney Code: SW Telephone No: Facsimile No.

(02) 97771111 (02) 9241 4666 Invention Title: PAPILLOMA VIRUS CAPSOMERE VACCINE FORMULATIONS AND METHODS OF USE Details of Original Application No. 2003200653 dated 25 Feb 2003 The following statement is a full description of this invention, including the best method of performing it known to me/us:- File: 27734AUP02 5011569961.000C5844 la- PAPILLOMA VIRUS CAPSOMERE VACCINE FORMULATIONS AND METHODS OF USE FIELD OF THE INVENTION The present application is a divisional application of Australian Application No.

2003200653, which is incorporated in its entirety herein by reference. 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 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

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-2human keratinocytes transformed in virro [Wettstein, er 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, er al., J. Virol., 68:4358- 4364 (1994): Auewarakul. et al., Mol. Cell. Biol. 14:8250-8258 (1994); Barbosa. eta!.. 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 er 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.-er, Curr. Top. In Microbiol. a,!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, er al., supra; Campo, er al., supra, Yindle and Frazer, er 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 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 desirahle. 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 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 Sinfection in a susceptible individual. Methods for production and purification of capsomeres and proteins of the invention are also Scontemplated.

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 virus. 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. virus-like panicles (VLPs). similar in make up to viral capsid structures. have been described which are formed in Sf-9 insect cells upon expiession of the viral proteins LI and L2 (or LI on its own) using 20 recombinant vaccinia or baculovinis. Purification of the VLPs can be achieved very simply by means of centrifugation in CsCI or sucrose gradients [Kimbauer. et al.. Proc. Nall. 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. et al., Virology 2016:126-195 (1995): Volpers, et al.. J. Virol. 69:3258-3264 (1995); Zhou, et al., J.

Gen. Virol. 74:762-769 (1993): Zhou, er al., Virology 185:251-257 (1991)]. WO 93/02184 describes a method in which papilloma virus-like panicles (VLPs) are used for diagnostic applications or as a vaccine against infections caused by the papilloma virus. WO 94/00152 describes recombinant production of L1 protein which mimics the conformational neutralizing epitope on human and animal papliloma virions.

In another aspect of the invention, therapeutic vaccinations are provided to relieve complications of, for example, cervical carcinoma 5 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.

Swhich 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 20 require a combination of steps. including ion exchange chromatography. gel filtration and affinity chromatography.

According to the present invention, vaccine formulations comprising papilloma virus capsomeres 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 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 pennrmits 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 2( 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 papilloma 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 L1 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 trnmcated LI proteins. Truncated proteins contemplated by the invention include those having one or more amino acid residues deleted from the carhoxy 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 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 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 -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. 3ded 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 Ll 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 fusionprotein 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 may 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 tenninus 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 L1 protein derived from HPVI6, it is preferred that from I to 34 carboxy terminal amin: acid residues are deleted. Reiitivelv shorter deletions are also contemplated that offer the advantage of minor modification of the antigenic properties of the L1 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 HPV16 Ll 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 pennits lonnation 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 virus protein. It also preferred that the second protein be the expression product of papilloma vinis early gene. It is most preferred, however, that the second protein he 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 alother 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 approxin-ately 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 virro 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 capsomeres. recombinant molecular biology techniques can be utilized to produce DNA encoding either the desired intact protein, the truncated protein, or the truncated 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.. MOLECULAR CLONING: A LABORATORY MANUAL. Cold Spring Harbor Press: Cold Spring Harbor, NY (1989) and Ausebel et al.. 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, Ll 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 mM NaCI. I mM CaCI-,. 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 2 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 confinred 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 -satment 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 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 advaritages 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

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13 may be buried in the overall structure as a result of internalized positioning within the VLP itself. Sinriarly, epitopes of the protein components may be sterically obstructed as a result of capsomere-to-capsomere contact, and therefore unaccessible for eliciting an immune response. Preliminary results using Ll/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. er 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.

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

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

67:6929-6936 (1994)] using Bgll 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 L1a310 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 LI AC. encoded a protein having a 34 amino acid deletion of the carboxy terminal LI 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. aspanate 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 termini of the amplification product.

CCCCGATATCGCCTTAATGTATAAATCGTCTGG

SEQ ID NO:

CCCCGATATCTCAAATTATTTCCTACACCTAGTG

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 a 3 10 was identified as having the appropriate twenty-seven nucleotide deletion and this construct was used to insert DNA fragments encoding other HPV 16 proteins at the EcoRV site as discussed below.

B. Generation of an HPV 16 L1 AC expression constructs Two primers (SEQ 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.

AAAGATATCTTGTAGTAAAAATTGCGTCCTAAAGGAAAC

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 a TAA translational stop codon positioned .uch that the amplification product. upon 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 cr cells. Plasmids from viable clones were analyzed for the presence of an EcoRV site which would linearize the plasmid. One positive 16 construct designated pUCHPV16LlAC was identified and used to insert DNA from other HPV 1. proteins utilizing the EcoRV site.

C. Insertion of DNA fragments into HPV 16 L1 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 LI a310 and HPVI6L1aC 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 pairs E75 (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. (SEQ ID NO: 16) and E7j4 (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:

TITTGATATCGGCTCTGTCCGGTTCTGCTTGTCC

Primer E7.3 SEQ ID NO: 11

"ITTGATATCCTTGCAACAAAAGGTACAATATTGTAATGGGCC

17- Primer E7.4 SEQ ID NO: 12

AAAAGATATCTGGTITCTGAGAACAGATGGGGCAC

Primer E7.5 SEQ ID NO: 13

TTTGATATCGATTATGAGCAATAAATGACAGCTCAG

Primer E7.6 SEQ ID NO: 14

TIGATATCGTCTACGTGTGTGCTITGTACGCAC

Primer E7.7 SEQ ID NO:

TITATCGATATCGGTCCAGCTGGACAAGCAGAACCGGAC

Primer E7.8 SEQ ID NO: 16 TITGATATCGATGCCCATTACAATATIrGTAACCTIITG 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 sire in the amplification product.

TTTGATATCGATATGGAATGGCTAAAGACAAGACCAATC

SEQ ID NO: 19

TTTGATATCGTTGTTTGGATCCCCATTCCCATG

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

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3 10 and HPVI6LlaC sequences previously linearized with the same enzyme. In order to determine the orientation of inserts in plasmids encoding E7 amino acids 25-75 and 50-98 and plasmid including influenza matrix protein, ClaI digestion was employed, taking advantage of a 18restriction site overlapping the newly created EcoRV restriction site (GATATCGAT) and included in the ,tpstream primer. For the thre: expression constructs including the initiating methionine of HPV 16 E7, insert orientation was determined utilizing a NslI 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 Snma and the isolated DNA ligated into plasmid pVL1393 (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 16LIaC*.

To generate an expression construct containing the HPV 16LIaC* sequence, two PCR reactions were performed to amplify two overlapping fragments from the pUC-HPVI6 LIaC encoding E7 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 (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

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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 PI and P4 only. The resulting amplification product was digested with enzymes EcoNI and HindIII inserted into the HPV 16LlaC expression construct described above following digestion with the same enzymes. The resulting expression construct differed from the original HPV16LIC construct with DNA encoding Ll 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 constirct. designated HPV 16 LlaC* 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 LIAC* E7 1-52 was then used to generate additional HPV 16 L1,C expression constructs further including DNA encoding E7 amino acids 1-55 using primer P1 (SEQ ID NO: 21) in combination with primer P5 (SEQ ID NO: 25), E7 amino acids 1-60 with primer pair P1 and P6 (SEQ ID NO: 26). and E7 amino acids 1-65 with primer pair P1 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: CATCTGAAGCTTAACAATAITrGTAATGGGCTCTGTCCG Prmer P6 SEQ ID NO: 26 CATCTGAAGCTrACTrGCAACAAAAJGG'rA-

CAATATTGTAATGGGCTCTGTCCG

Primer P7 SEQ ID NO: 27 CATCTGAAGC1rAAGCGTAGAGTCACACTTJGCACc-i AAAAGGTTrACAATATITGTAATGGGCTCTGTCCG Similarly. HPV 16 L1AC E7 1-70 was generated using template DNA encoding HPV 16 LleAC* E7 1-66 and the primer pair P1 and P8 (SEQ ID NO: 2 8).

Primer P8 SEQ ID NO: 28 CATCTGAAGCT17ATrGTACGCACAAC-

CGAAGCGTAGAGTCACACTTG

Followvine each PCR reaction, the amplification products were digested with EcoNI and Hindlfland inserted into HPV16LIt&C previously digested with the same enzymes. Sequences of each constructs were determined using an Applied Biosystems Prism 377 sequencing instrument with chain terminatins! d ideox vnuc leot ides [Prober er al., Science 238:336-341 (1987)].

Example 2 Generation of Recombinant Baculoviruses Spodopterafruigiperda (Sf9) cells were grown in suspension or monolayer cultures at 270 in TNMFH medium (Sigma) supplemented with fetal calf serum and 2 mM glutamine. For HPV 16 LI- based recombinant baculovirus construction, Sf cells were transfected with 10 Atg of transfer plasmid together with 2 gtg of linearized Baculo-Gold DNA C -21 (PharMingen, San Diego, CA). Recombinant viruses were purified by O according to manufacturer's suggested protocol.

To test for expression of HPV 16 L1 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 0was removed and cells were washed with PBS. The cells were lysed in SDS sample buffer and analyzed by SDS-PAGE and Western blotting using Santi-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 fornation by electron microscopy. The results are shown in Table 1.

The intact HPV LI protein, as well as the expression products HPV 16 L12310 and HPV 16 LIlC. 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 L1AC 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 vinis-like panicle formation was also observed.

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

When E7 sequences were inserted into the HPV 16 L1A*C vector, all fusion proteins were shown to produce capsomeres. Insertion of 22 DNA encoding E7 residues 1-52, 1-55, and 1-60 produced the highest level of caosoreres, but equal levels of virus-like paricle production wer: observed. While insertion of DNA encoding 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 Exressictn Capsomere Capsid Con-nj..: hI'.ert Yield Yield HVP ib LI HP' IbALI 310 Nonc HPVI LUC HPV ib LIA110 F- I-'1S HP' L LI0IO F 50 HP'' lb LIa100 F HP\' 6b LI 3 IO O.,S HP'. IoLlaC E' HP' Io LI&C L 2S77S HP' Io L&C E7 50-49 HPV 16 Ll&C I -CIO HPV 16 aC HPV 1b LIaC Influvnl HPV 1b Lla*C -52 HPV 16 LlsC E7 1-55 HPV It)LlaC E' 1-00 HPV 16 Ll&C E- I-5 HPV I6LI&*C E7 1-70 Example 3 Purification of Capsomeres Trichopulsia ni (TN) High Five cells were grown to a density of approximately 2 x 106 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 0 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 a 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 .4l of each fraction were analyzed by a 10% SDS-polyacrylamide gel and Western blotting using an anti-HPV16 Ll 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/HCl, pH 8.0, (10 mM). As control, NaCI and 7: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 4l. 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 /l of peptide E701 [Muller et al., 1982] at a concentration of 10 tpg/ml in PBS. Wells are blocked for 2 hour at 37 0 C with 100 1l 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 /Al 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 1 hour. plates are washed and stained using ABTS substrate (0.2 mg/ml. 2 2 '-Azino-bis(3-ethylbenzhiazoline-f-sulfonic acid in 0.1 M Na-Acetate-Phosphate buffer (pH 4.2) with 4 pl 30% H202 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 ELISA was utilized.

Microtiter plates were coated overnight with 50 pl/well of a 1:500 dilution (final concentration of 2 ug per ml, in PBS) with a protein A purified mouse monoclonal antibody immunospecific for HPV 16 L1 (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 ul of fractions of CsCI gradients were added for 1 hour at 37 0 C using a 1:300 dilution (in 5% milk/PBS). After three washings with PBS/0.05% Tween 20. 50 /l 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 I 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 #l of VLPs was incubated overnight at 4°C in 500 Al of disruption buffer containing 30 mM DTT. 50 mM EGTA, 60 mM EDTA, 100 mM NaCI, and 100 mM Tris!HCI. 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 detenmine 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 CI -26observations that virus-like particles are capable of hemagglutinizing red blood cells.

Mice are immunized with any of a chimeric capsomere vaccine and Ssera is collected as described above in Example 4. As positive controls. HPV16 LI virus like panicles (VLPs) and bovine PV1 (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 O 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 ml PBS and stored at 4 0 C for up to three days. For the hemagglutinin assay. 70 /l of the suspension is used per well on a 96-well plate.

:20 Chimeric capsomere aliquots from CsCI gradients are dialyzed for one hour against 10 mM Hepes (pH 7.5) and 100 ~l of two-fold serial dilutions in PBS are added to mouse erythrocyvts in round-bottom 96-well microtiter plates which are further incubated for 3-16 hours at 4 0 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 HPV16LIAC 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;:nan VLPs and this differential ,utralization 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 (14)

1. 01. A vac ine formulation comprising a Iuman papilloma virus capsomere. said capsomere comprising a fusion protein comprising a human papilloma virus LI protein adjacent amino acid residues from a 5 second protein.
2. 2. A vaccine formulation comprising a human papilloma Svirus capsomere. said capsomere comprising a truncated human papilloma Svirus Ll protein having a deletion of one or more amino acid residues necessary for formation of a virus-like panicle.
3. 3. The vaccine fonnulation of claim 2 wherein said capsomere comprises a fusion protein comprising a truncated human papilloma virus LI protein adjacent amino acid residues from a second protein.
4. 4. The vaccine fonnulation of any one of claims 1,2. or 3 wherein the LI protein is encoded in the genome of a human papilloma vins selected from the group consisting of HPV6. HPV11. HPV16, HPV 8. HPV33. HPV35. and The vaccine fonlulation of claim 4 wherein the papilloma virus is HVP16.
5. 6. The vaccine lonnulation of any one of claims 2, 3, or wherein carboxy tenninal amino acid residues are deleted from the LI protein.
6. 7. The vaccine formulation of claim 6 wherein 1 to 34 carboxy terminal amino acid residues are deleted from the L1 protein. -41
7. 8. The vaccine formulation of claim 7 wherein 34 ca<.oxy terminal amino acid residues are deleted from the LI pro>:;in.
8. 9. The vaccine formulation of any one of claims 2. 3. or wherein amino terminal amino acid residues are deleted from the LI protein. The vaccine formulation of any one of claims 2, 3. or wherein internal amino acid residues are deleted from the Ll protein.
9. 11. The vaccine formulation of claim 10 wherein the amino acid residues deleted from the LI protein comprise a nuclear localization signal.
10. 12. The vaccine fonnulation of claims 2 or 3 wherein the amino acids residues from the second protein are derived from an HPV protein.
11. 13. The vaccine fonulation of claim 12 wherein the HPV protein, is an early HPV protein.
12. 14. The vaccine irnmilation 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 administering 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. S- 42-
13. 16. A method for preventing papilloma virus infection comprising the step of administering to an .ndividual susceptible thereto a; amount of the vaccine formulation of claims 1, 2. 3. 5, 7, 8, 11, 13 or 14 Seffective to inhibit HPV infection.
14. 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: (virl; ATTORNEY.AGENT INFOPMATION: 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: 1512 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 AGT 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 96 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr val Ala Arg Thr Asn 25 29 ATA TAT TAT CAT GCA GGA AC-A TCC AGA CTA CTT Ile Tyr Tyr His A-1a Gly Thr Ser Arg Leu Leu TAT Tyr GTA Val AAT Asn CG A-rg TTA Leu ACA Thr GAA Giu 145 TGC Cys AAT Asri ACA Thr GAC Asp TOT Cys 225 C CA Prco GTT Val. Phe TCA Se r AAG Lys CTG Leu GGT 0 1 ly GAA Glu 130 TGT Cys AAA Lys OTT Val1 OTT Val TTT Phe 210 ACA Thr TAT Tyr AGA Arg AAA Lys CAA Gin Phe GCC Ala ATT Ile AGT Ser ATG Met .ATA Ile 165 AAT Asn GAT Asp TTA Leu TGC Cys AGC Ser 245 TTT Phe AAA Lys TAC Tyr 70 CCr Pro TOT Cys AGT Ser OCT Ala OAT Asp 150 GGG G ly CCA Pro GGT Gly CAG Gin AAA Ly s 230 TTA Le u AAT As n CCT Pro 55 AGG Ar g GAC Asp) GTA Val1 GGC Gly TAT Tyr 135 TACZ Tyr G"A G I GAT G C Ala D TAT Ty r TTT~ Phe AGG Arg 40 AAC AAT AAC Asn Asn Asn GTA TTT AGA Val Phe Arg ACC TCA TTT- Thr Ser Phe 90 GGT GTT GAG Gly Val Glu 105 CAT CCT TrA His Pro Leu 12C GCA GCA AAT Ala Ala Asn AAA CAA ACA Lys Gin Thr C,;AC TG3 GGC- His Tr-p Gly 1-70 GAT TGT CCA Asp Cys Pro 185 ATG GTT GAT Met Val Asp 200 7-'C AAA; AGT Asn Lys Ser CCA GAT TAT Pro Asp Tyr TTT TAT TTA Phe Tyr L.eu 250 GCT GuOT OCT Ala Gly Ala 265 AAA Lys ATA Ile 75 TAT Tyr GTA Val TTA Leu GOCA Ala CAA Gin 155 AAA Lys CCA Pro ACT Thr GAA Giu ATT Ile 235 CGA Arg G71' Val1 GCA Ala AT). Ile 60 CAT His AAT Asn GGT Oly AAT Asn GT Gly 140 Le u GGA Oly TTA Le u GGC Gly GTT Val1 220 AAA Lys AGO Arg GT Gly TTA Leu TTA Leu CCA Pro COT Arg AAA Lys 125 GTG Val TGT Cys TCC Ser GAG Giu Phe 205 CCA Pro ATG Met GAA Giu GA 01 u- OTT OGA CAT CCC Val Oly His Pro OTT Val CCT Pro OAT Asp GGT Gly 110 TTG Leu GAT Asp TTA Leu CCA. Pro TTA Leu 190 GGT Gly CTO Leu OTG Val CAA Gin P.AT Asn 270 CCT Pro GAC Asp ACA Thr CAG Gin OAT Asp AAT Asn ATT Ile TOT Cys 175 ATA Ile OCT Ala OAT Asp TCA Ser ATO Met 255 GTA AAA) Lys CCC Pro s0 CAG Gin Pro GAC Asp AGA Arg GT Gly 160 ACC Th r AAC Asn ATO Met Ile GAA Olu 240 Ph e CC). Pro GAC OAT 7TA ATT AAA GOC TCT GOG TC'. ACT OCA AAT TTA 0CC AOT Asp Asp Leu 275 Tyr Ile Lys Oly Ser Oly Ser Thr Ala Asn Leu Ala Ser 280 285 30 'rCA AAT TAT TTr CCT ACA COT AGT GGT TCT ATG OTT ACO- TCT OAT GCC Ser Asn Tyr Phe Pro Thr Pro Ser Gly Ser CAA Gin 305 AAT Asn ACA Thr ACT Thr GIA Giu- GCA Al a 385 GAC Asp ACT Thr CT Pro GTA Val GGA Gly 465 ACA Thr ACT Thr 290 ATA Ile GC Gly CGC Ar g ACA Thr TPAT Th'r 370 GAC Asp TGG TrD TAT Tv r CCA Pro AAT Asn 450 CGC Arg Le u OCT Al a TT-C Phe ATT Ile AGT Ser TAT Tryr 355 GAT Asp GTT Val AAT Asn AGG Arg GCA Ala 435 TTA Leu AAA Lys GGA Gly AAA L-ys AAT Asn TGT Cys ACA Thr 340 AA Lys TTA Le u ATG Met Phe Phe 420 CT Pro AAG Lys Phe AAA Lys CGC Arg 500 AAA Lys TG Trp 325 AAT Asn AAT Asn CAG Gin ACA Th r GGT Gly 405 GTA Val1 AAA Lvs GAA Glu TTA Le u CGA Arg 485 AAA Lys CCT Pro 310 GOT Giy ATG Met ACT Thr Phe TAO 390 OTA Le u AC C- Tb r GAA Gi u AAG Ly s CTA Leu 470 AAA Ly s AAA Lys TOO C.AA TTA Le u Phe 360 Phe CAT Pis COT1 Pro CAG Gin CCC Pro 440 TCT Ser- OCA Ala ACA Tb r AAG Lys TTA Leu CTA Leu TOGT Cys 345 AAG Ly s C-A Gin TCT Ser C CC Pro GCA Ala 425 CTT Le u GCA Ala GA G ly CCC Pro CTG Leu SOS CAA Gin Phe 330 GOT Al a GAG Glu Leu ATG Metr CA Pro 410 ATT lie A.AA Lys GAC Asp T-1G Le u ACC Thr 490 TAA Met Val 300 CGA GC:A Arg Ala 315 OTT ACT Val Thr GCC ATA Ala Ile TAC CTA TPyr Leu TOC AAA Cys Lys 380 AAT TCC Asn Ser 395 GOA GGC Oly Oly OCT TOT Ala Cys AAA TAC Lys Tyr CTA GAT Leu Asp 460 AAG 0CC Lys Ala 475 ACC T CA Thr Ser Thr CAG Gin OTT Val TCT Ser CGA Arg 365 ATA Ile ACT Thr ACA Thr CAA Gin ACT Thr 445 CAG Gin AAA Lys TOT Ser Ser GOC Gly OTT Val ACT Thr 350 CAT His ACC* Thr ATT Ile CTA Leu AAA Lys 430 Phe Phe CCA Pro ACC Thr Ala ART Asn 320 ACT Thr GAA Giu GAG 01u ACT Thr GAG Olu 400 GAT Asp AC:A Thr GAA Glu TTA Leu Phe 480 ACA Thr 912 960 1008 1056 1104 1152 1200 1248 1296 1344 1392 1440 14 1518 INFORMATION FOR SEQ ID NO:2: SEQUENCL CHARACTERISTICS: LEN-.1GTH: 506 amino acids TYPE: arnino acid TOPOLOGY: linear -31 (ii) MOLECULE TYPE: protein SEQUENCE DESCRIPTION: S£Q ID NO:2: Met Ser Leu Trp Leu Pro Ser Glu Ala Thr Val Tyr Leu Pro Pro Val 1 5 10 Pro Val Ser Lys Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn 25 Ile Tyr Tyr His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro 40 Tyr Phe Pro Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys 55 Val Ser Gly Leu Gin Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro 70 75 Asn Lys Phe Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gin 90 Arg Leu Val Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gin Pro 100 105 110 Leu Gly Val Gly Ile 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 130 125 140 Glu Cys Ile Ser Met Asp Tvr Lys Gin Thr Gin Leu Cys Leu Ile Gly 145 150 155 160 Cys Lys Pro Pro Ile Gly Giu 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 Ile Asn 180 185 190 Thr Val Ile 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 Ile 210 215 220 Cys Thr Ser Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu 225 230 235 240 Pro Tyr Gly Asp Ser Leu Phe Phe Tvr 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 Ile 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 32 Gin Ile Phe Asn Lys Pro Tyr Trp Leu Gin 305 310 Gly Arg Thr Tyr 370 Asp Trm Pro Asn 450 Arg Le u Aila Ile Ser Trvr 355 Asp Val1 Asn Arg Aila 435 Leu Lys Gly Ly s Cys Thr 340 Lys Leu Met Phe Phe 420 Pro Lys Phe Lys Arg 500 Trp, 325 Asn Asn Gin Thr Gly 405 Vai Lys Giu Le u Arg 485 Lvs Asn Se r Asn Ile 375 Ile G In Se r Asp Phe 455 Gin Al a Arg Gin Leu Phe 360 Phe His Pro Gin Pro 440 Ser Ala Th r Ly s Leu cysr 345 Lys Gin Se r Pro Al a 425 Leu Al a Gly Pro Le u 505 Phe 330 Ala Giu Leu Met Pro 410 Ile Lys Asp Leu Thr 490 Arg Ala 315 Vai Thr Ala Ile Tyr Leu Cys Lys 380 Asn Ser 395 Gly Gly Aia Cys Lys Tyr Leu Asp 460 Lys Ala 475 Thr Ser Gin Val Se r Axrg 365 Ile Thr Thr Gin Thr 445 Gin Lys Ser Gly Vai Thr 350 His Thr Ile Leu Lys 430 Phe Phe Pro Th~k Hi_4s Asp 335 Ser Gly Leu Leu Giu 415 His Trp Pro Lys Se r 495 Asn 320 Thr Giu Giu Thr Glu 400 Asp Thr Giu Leu Phe 480 Thr INFORMATION FOR SEQ ID NO:3: i)SEQUENCE CHARACTER.ISTICS: LENGTH: 297 base pairs 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: CAT GSA SAT ACA CCT ACA T1G CAT SAA TAT ATG ITA GAT TrG CAA His Sly Asp Thr Pro Thr Leu His Slu Tyr Met Leu Asp Leu Gin 10 GAG A\CA ACT GAT CTC TAG TGT TAT GAG CAA TrA AAT GAC AGC TCA Slu Thr Thr Asp Leu Tyr Cys TrGlu Gin Leu Asn Asp Ser Ser 25 33 GAG GAG GAG Giu Giu Giu GAT GAA ATA GAT Asp Giu lie Asp GGT Giy 40 CCA GCT GGA CAA GCA GAA CCG GAC Pro Ala Gly Gin Ala Giu Pro Asp AGA GCC Arg Ala CAT TAC AAT ATI' His Tyr Asn Ile GTA Val ACC =r TGT TGC Thr Phe Cys Cys TGT GAC TCT ACG Cys Asp Ser Thr CTr CGG TTG TGC GTA Leu Arg Leu Cys Val C.AA Gin AGC ACA CAC GTA Ser Thr His Val. GAC Asp) 75 ATT CGT ACT1 TTG Ile Arg Thr Leu GAA Giu GAC CTG ITA ATG Asp Leu Leu Met G Giy ACA CTA GGA ATT Thr Leu Gly Ile TGC CCC ATC TGT Cys Pro Ile Cys TCT CAG Ser Gin AACCA TAA 297 Lys Pro" INFORMATION FOP. SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 98 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: prote in (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met His Giy Asp Thr 1 5 Pro Thr Leu His Tyvr Met Leu Asp Leu Gin Pro Giu Thr Thr Asp Leu Tyr Cx's Tv,.r 25 Giu Giu Giu Asp Glu Ile Asp Gly Pro 3S C Giu Gin Leu Asn Asp Ser Ser Giu Pro Asp Ala Giy Gin Arg Ala H-is Tyr Asn Ile Val Thr Phe Cys Cys Cys Asp Ser Thr Leu Arg Leu Cys Val Ser Thr His Val Asp 75 Ile Arg Thr Leu Giu Asp Leu Leu Met Gly Thr Leu Gly Ile Val1 90 Cys Pro Ile Cys Ser Gin 9S Lys Pro INFORMATION FOP SEQ ID SEQUENCE CHARACTERISTICS: LEN(.:rHi: 34 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE-:: 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 MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CCCCGATATC TCAAATrATr 7-rCCTACACC TAGTG IN4FORMATION FOR SEQ ID NO: 7: SEQUENCE CHARACTERISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: A;.AGATATCT TGTAGTA;AAA AT7TG-CGTCC ?rAA;:*GGAAAC INFORMATION FOR SEQ 1D N3:8: SEQUENCE CHARACTEISTICS: LENGTH: 44 base pairs TYPE: nucleiz acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA, (xi) SEQUENCE DESCRIP7ION: SEQ ID NO:8: AAAGATATCT AATCTACCTrC TACAACTGC-T AAACGCAAAA. AACG 44 INFORMATION FOR SEQ 1D NO:9: SEQUENCE CHARACTEP.:STICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESE: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA, (xi) SEQUENCE DEScRIPTION: SEQ ID NO:9: AAAAGATATC ATGCATGGAG ATACACCTAC ATTGC INFORM4ATION FOR SEQ ID NO:l0: SEQUENCE CHARPACTERISTICS: LENGTH: 34 base pairs 35 TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: TTTTGATATC GGCTCTGTCC GGTTCTGCTT GTCC 34 INFORMATION FOR SEQ 1D NO:1l: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TTTrGATATC CTTGC.AACAA 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: DN.; (xi) SEQUENCE DESCRIPTION: SEQ WD NO: AAAAGATATC TGGTYTCTGA GAACAGATGG GGCAC INFORMATION FOR SEQ ID NO:l3: Ai) SEQUENCE CHARACTER.ISTICS: 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 FOR SEQ ID NO:14: SEQUEN-- CHARACTERISTICS: LL:GTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE 7YPE: DNA (xi SEQUENCY DESCRIPTION: SEQ ID 36 ~T1TGATATC GTCTACGTGT GTGC'TrGTA 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 TCGGTCC.AGC TGGACAAGCA GAACCGGAC INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:16: T'ITGATATC GATGCCC.ArI' ACAATATTGTL AACCITG INFORM4ATION FOR SEQ ID NO: 17: SEQUENCE CHARACTERISTICS: LENGTH: 294 base pairs TYPE: nucleic acid STR-ANDEDNESS: single TOPOLOGY: linear (1i) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CIDS 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 Met Ser Leu Leu Thr Glu Val Glu Thr Leu Thr Arg Asn Gly Trp Glu 1 5 10 TGC AAA TGC AGC GAT TCA ACT GAT CCT CTC A~r ATC GCA GCG AGT ATC Cys Lys Cys Ser Asp Ser Ser Asp Pro Leu Ile Ile Ala Ala Ser Ile 25 ATT GGG ATC TTG CAC TTG ATA TTG TC -,ATT =r TAT CGT CTT TIC TTC Ile Gly Ile Leu His Leu lie Leu T,j Ile Phe Tyr Ary Leu Phe Phe 40 AAA TGC ATT TAT CGT CGC CIT AAA TAC GG TTG AAA~ AGA GGG CCT TCT Lys Cys Ile Tyr Arg 1-Arg Leu Lys Tyr C, y Leu Arg Gly Pro Ser 55 37 ACG Thr CAG Gin GAG Giu (2) GAA GGA GCG CCT GAG TCT ATG AGG GAA GAA TAT CGG CAG GAA CAG Giu Gly Ala Pro Giu Ser Met Arg Giu Glu Tyr Arg Gin Glu Gin 70 75 AGT GCT GTG GAT GTT GAC GAT G71' CAT TTr GTC AAC ATA GAG CTC. Ser Ala Val Asp Val Asp Asp Val His Phe Val Asn Ile Glu Leu 90 TAA INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 97 amiuno acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein Met Cvs (xi) SEQUENCE Ser Leu Leu Thr Lys Cys Ser Asp Ile Gly Ile Leu His Lys Cys Ile Tyr Ary Thr Glu Gly Ala Pro DESCRIPTION: SEQ ID Giu Val Giu Thr Leu 10 Ser Ser Asp Pro Leu 25 Leu lie Leu Trp Ile 40 Arg Leu Lys Ty;r Gly Glu Ser Met Ara Giu '70 Val Aso Asp Val His NO: 18: Thr A-rg Asn Ile Ile Ala Gly Trp Glu Ala Ser Ile Leu Phe Phe Phe Tyr Leu Lys Glu Tyr Arg Arg Gly Pro Ser Arg Gin Glu Gin Giu (2) 75 Phe Gin Leu Ser Ala Val Asp Val Asn Ile INFORMATION FOPR SEQ ID NO:19: SEQUENCE CHARACTER.ISTICS: LENGTH: 40 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:i9: ZT=TGATATC GATATGGAAT GGCT. -AAGAC AAGACCAATC INFORMATION FOR SEQ ID N0:20: SEQUETNCE CHARACTERISTICS: LENGTH: 35 base pairs TYPE: nucleic acid STRANDEDNESS: single 38 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID TTGATATC GTTGTTTGG.A TCCCCAITCC CATTG INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DMA (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:21: G7ITATGACAT ACATACATC TATG *24 INFORMATION FOR SEQ ID NO:22: C)SEQUENCE CHARACTERISTICS: LENGTH: 3S base pairs TYPE: nuclei-c acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ 1D NO:22: CCATGCXITC CTGCTTGTAC TAAAAATTTG CGTCC INFORMATION FOR SEQ ID NO:23: ()SEQUENCE CHAACTER.ISTICS: LENGTH: 219 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (11) MOLECULE TYPE: DNA;- (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CTACAAGCAG GATGCATGG AGATACACC INFOIYMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 36 base pairs TYPE: nrcleic acid ()STRANDEL'NESS: single TOPOLOGY: linear (ii) MO0LECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CATCTGAAGC TTAGTAATGC GCTCTGTCCG GTTCTG 3 39 INFORMATION FOP. SEQ ID NO:2S: SEQUENCE CHARACERIST-ICS: LENGTH: base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ -D CATCTGAAGC TTATCAATAT TGTAAT,-GGGC TCTGTCCG 38 INFORM4ATION FOR SEQ N0:26: SEQUENCE CHARACTERISTICS: LENGTH: 54 oase p~airs TYPE: nuce:7acid STRANDEDN-ESS: TOPOLOGY: ieai- MOLECULE TYPE: DNA,; (xi) SEQUENCE DESCRIPTION: SEQ) 1D NO:26: CATCTGAAGC TTACTTGCAACAAGA CAAITA-.rGTA ATGGGCTCTG TCCG 54 INFORM4ATION FOP. SE-, :D N~ SEQUENCE CFA7:!'S T ~Sj LENGTP: 69 oase oaiis TYPE: nuclez- az:-,c STRANDMEDNESS re TOPOLOGY: linea: (ii) MOLECULE TYPE:DN (xi) SEQUENCE DESCRIPTION: SEQ-e :D NO: '7: CATCTGA6AGC TTAAAGCGTA GAZTTACACT TGC;AACAAAA GGTTACAATA TT'GTAATGGG CTCTGTCCG 69: INFORMATION FOP SEQ DN.b Ci) SEQUENCE CHARC--:TCS LENGTH: 4" oase pairxs TYPE: nuclei acid STPANDEDNESS- single TOPOLOGY: linear MOLECULE TYPE: ONA7 SEQUEN-.E DESCRIPTION: SEQ ID NO:.;8: CATCTGAAGC TTATTGTACG CAC;AACCGAA GCGTAGAGTC ACACTTG 47