AU714533B2

AU714533B2 – DNA encoding human papilloma virus type 18
– Google Patents

AU714533B2 – DNA encoding human papilloma virus type 18
– Google Patents
DNA encoding human papilloma virus type 18

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Publication number
AU714533B2

AU714533B2
AU53141/96A
AU5314196A
AU714533B2
AU 714533 B2
AU714533 B2
AU 714533B2
AU 53141/96 A
AU53141/96 A
AU 53141/96A
AU 5314196 A
AU5314196 A
AU 5314196A
AU 714533 B2
AU714533 B2
AU 714533B2
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Australia
Prior art keywords
protein
dna molecule
recombinant
virus
papillomavirus
Prior art date
1995-03-22
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Expired, expires 2021-03-18

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AU53141/96A
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AU5314196A
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Inventor
Hugh A George
Kathryn J Hofmann
Kathrin U Jansen
Joseph G Joyce
Michael P. Neeper
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Merck Sharp and Dohme LLC

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Merck and Co Inc
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1995-03-22
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1996-03-18
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2000-01-06
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1995-03-22
Priority claimed from US08/408,669
external-priority
patent/US5840306A/en

1995-03-22
Priority claimed from US08/409,122
external-priority
patent/US5820870A/en

1996-03-18
Application filed by Merck and Co Inc
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Merck and Co Inc

1996-10-08
Publication of AU5314196A
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patent/AU5314196A/en

2000-01-06
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2000-01-06
Publication of AU714533B2
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2010-01-28
Assigned to MERCK SHARP & DOHME CORP.
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MERCK SHARP & DOHME CORP.
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Assignors: MERCK & CO., INC.

2012-11-01
Assigned to MERCK SHARP & DOHME CORP.
reassignment
MERCK SHARP & DOHME CORP.
Request to Amend Deed and Register
Assignors: MERCK SHARP & DOHME CORP.

2021-03-18
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C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof

C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

A61P31/12—Antivirals

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

A61P31/12—Antivirals

A61P31/20—Antivirals for DNA viruses

C—CHEMISTRY; METALLURGY

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C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor

C12N15/09—Recombinant DNA-technology

C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

C12N15/90—Stable introduction of foreign DNA into chromosome

C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast

C—CHEMISTRY; METALLURGY

C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof

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C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING

C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

C12N9/10—Transferases (2.)

C12N9/1048—Glycosyltransferases (2.4)

C12N9/1051—Hexosyltransferases (2.4.1)

C—CHEMISTRY; METALLURGY

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C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes

C12N9/14—Hydrolases (3)

C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)

C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)

C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi

C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES

A61K39/00—Medicinal preparations containing antigens or antibodies

C—CHEMISTRY; METALLURGY

C07—ORGANIC CHEMISTRY

C07K—PEPTIDES

C07K2319/00—Fusion polypeptide

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C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA

C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses

C12N2710/00011—Details

C12N2710/20011—Papillomaviridae

C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

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C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses

C12N2710/00011—Details

C12N2710/20011—Papillomaviridae

C12N2710/20023—Virus like particles [VLP]

Abstract

The present invention relates to expression vectors for HPV18 L1 proteins.

Description

DNA Encoding Human Papilloma Virus Type 18 Field of the Invention The present invention is directed to DNA molecules encoding purified human papillomavirus type 18 and derivatives thereof.
Brief Description of the Drawings Figure 1 shows the HPV 18 L1 nucleotide and deduced amino acid sequences.
Figure 2 is a list of amino acid variations within the L1 protein of HPV 18: Figure 3 shows the HPV 18 L2 nucleotide and deduced amino acid sequences.
Figure 4 shows an immunoblot of HPV 18 L1 protein expressed in yeast.
Figure 5 shows an immunoblot of HPV 18 L2 protein expressed in yeast.
Figure 6 is an electron micrograph of virus-like particles formed by HPV 18 L1 protein expressed in yeast.
Background of the Invention Papillomavirus (PV) infections occur in a variety of animals, including humans, sheep, dogs, cats, rabbits, monkeys, snakes and cows. Papillomaviruses infect epithelial cells, generally inducing benign epithelial or fibroepithelial tumors at the site of infection.
PV are species specific invective agents; a human papillomavirus does not infect a nonhuman animal.
S” o **o o* [N:\LIBAA0120:TAB WO 96/29413 PCT/US96/03649 -2- Papillomaviruses may be classified into distinct groups based on the host that they infect. Human papillomaviruses (HPV) are further classified into more than 70 types based on DNA sequence homology. PV types appear to be type-specific immunogens in that a neutralizing immunity to infection by one type of papillomavirus does not confer immunity against another type of papillomavirus.
In humans, different HPV types cause distinct diseases.
HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts in both normal and immunocompromised individuals. HPV types 5, 8, 9, 12, 14, 15, 17, 19-25, 36 and 46-50 cause flat lesions in immunocompromised individuals. HPV types 6, 11, 34, 39, 41-44 and 51-55 cause benign condylomata of the genital or respiratory mucosa. HPV types 16 and 18 cause epithelial dysplasia of the genital mucosa and are associated with the majority of in situ and invasive carcinomas of the cervix, vagina, vulva and anal canal.
Papillomaviruses are small (50-60 nm), nonenveloped, icosahedral DNA viruses that encode for up to eight early and two late genes. The open reading frames (ORFs) of the virus genomes are designated El to E7 and LI and L2, where denotes early and “L” denotes late. LI and L2 code for virus capsid proteins. The early (E) genes are associated with functions such as viral replication and cellular transformation.
The LI protein is the major capsid protein and has a molecular weight of 55-60 kDa. The L2 protein is a minor capsid protein which has a predicted molecular weight of 55-60 kDa and an apparent molecular weight of 75-100 kDa as determined by polyacrylamide gel electrophoresis. Immunological data suggest that most of the L2 protein is internal to the LI protein within the viral capsomere. The LI ORF is highly conserved among different papillomaviruses. The L2 proteins are less conserved among different papillomaviruses.
The LI and L2 genes have been identified as good targets for immunoprophylaxis. Studies in the cottontail rabbit papillomavirus (CRPV) and bovine papillomavirus (BPV) systems have shown that immunizations with the LI and L2 proteins expressed in bacteria or by WO 96/29413 PCT/US96/03649 -3using vaccinia vectors protected animals from viral infection. Expression of papillomavirus LI genes in baculovirus expression systems or using vaccinia vectors resulted in the assembly of virus-like particles (VLP) which have been used to induce high-titered virus-neutralizing antibody responses that correlate with protection from viral challenge.
Following HPV type 16, HPVI8 is the second most prevalent HPV type found in cervical carcinomas. HPVI8 was detected in 5-20% of cervical cancer biopsies collected from various parts of the world (Ikenberg, H. 1990. Human papillomavirus DNA in invasive genital carcinomas. In Genital Papillomavirus Infections, G. Gross et al., eds. p. 85-112). There appears to be a geographic dependence of infection with HPV 18 since tumor biopsies from African and South American women harbor HPV 18 more frequently than similar biopsies from European and North American women. The underlying reasons for these geographic differences are not known. The development of a vaccine against HPV 18 infection becomes extremely relevant since HPV 8I is also associated with more aggressively growing cancers and is rarely found in the milder precursor lesions, CIN I-II.
SUMMARY OF THE INVENTION The present invention is directed to DNA molecules encoding purified human papillomavirus type 18 (HPV type 18 HPV 18) and uses of the DNA molecules.
DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to DNA molecules encoding purified human papillomavirus type 18 (HPV type 18: HPV 18) and derivatives thereof. Such derivatives include but are not limited to peptides and proteins encoded by the DNA, antibodies to the DNA or antibodies to the proteins encoded by the DNA. vaccines comprising the DNA or vaccines comprising proteins encoded by the DNA.
immunological compositions comprising the DNA or the proteins encoded by the DNA, kits containing the DNA or RNA derived from the DNA or proteins encoded by the DNA.
WO 96/29413 PCT/US96/03649 -4- Pharmaceutically useful compositions comprising the DNA or proteins encoded by the DNA may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington’s Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the DNA or protein or VLP. Such compositions may contain DNA or proteins or VLP derived from more than one type of HPV.
Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose PV infections. The effective amount may vary according to a variety of factors such as the individual’s condition, weight, sex and age. Other factors include the mode of administration. Generally, the compositions will be administered in dosages ranging from about I tg to about 1 mg.
The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral, mucosal, intravenous and intramuscular.
The vaccines of the invention comprise DNA, RNA or proteins encoded by the DNA that contain the antigenic determinants necessary to induce the formation of neutralizing antibodies in the host.
Such vaccines are also safe enough to be administered without danger of clinical infection; do not have toxic side effects; can be administered by an effective route; are stable; and are compatible with vaccine carriers.
The vaccines may be administered by a variety of routes, such as orally, parenterally, subcutaneously, mucosally intravenously or intramuscularly. The dosage administered may vary with the condition.
sex, weight, and age of the individual: the route of administration: and the type PV of the vaccine. The vaccine may be used in dosage forms such as capsules, suspensions. elixirs, or liquid solutions. The vaccine may he formulated with an immunologically acceptable carrier.
The vaccines are administered in therapeutically effective amounts, that is, in amounts sufficient to generate a immunologicaly protective response. The therapeutically effective amount may van WO 96/29413 PCTIUS96/03649 according to the type of PV. The vaccine may be administered in single or multiple doses.
The DNA and DNA derivatives of the present invention may be used in the formulation of immunogenic compositions. Such compositions, when introduced into a suitable host, are capable of inducing an immune response in the host.
The DNA or its derivatives may be used to generate antibodies. The term “antibody” as used herein includes both polyclonal and monoclonal antibodies, as well as fragments thereof, such as, Fv, Fab and F(ab)2 fragments that are capable of binding antigen or hapten.
The DNA and DNA derivatives of the present invention may be used to serotype HPV infection and HPV screening. The DNA, recombinant proteins, VLP and antibodies lend themselves to the formulation of kits suitable for the detection and serotyping of HPV.
Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as HPV18 DNA, recombinant HPV protein or VLP or anti-HPV antibodies suitable for detecting a variety of HPV types. The carrier may also contain means for detection such as labeled antigen or enzyme substrates or the like.
The DNA and derived proteins therefrom are also useful as molecular weight and molecular size markers.
Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and therefore, the amino acid sequence can be encoded by any of a set of similar DNA oligonucleotides. Only one member of the set will be identical to the HPV 18 sequence but will be capable of hybridizing to HPV 18 DNA even in the presence of DNA oligonucleotides with mismatches under appropriate conditions. Under alternate conditions, the mismatched DNA oligonucleotides may still hybridize to the HPVI8 DNA to permit identification and isolation of HPV 18 encoding DNA.
The purified HPV 18 DNA of the invention or fragments thereof may be used to isolate and purify homologues and fragments of HPV 18 from other sources. To accomplish this, the first HPV 18 WO 96/29413 PCT/US96/03649 -6- DNA may be mixed with a sample containing DNA encoding homologues of HPV 18 under appropriate hybridization conditions.
The hybridized DNA complex may be isolated and the DNA encoding the homologous DNA may be purified therefrom.
It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids.
Therefore, this invention is also directed to those DNA sequences which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not substantially alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for lysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the DNA sequences include, but are not limited to site-directed mutagenesis.
As used herein, a “functional derivative” of HPV18 is a compound that possesses a biological activity (either functional or structural) that is substantially similar to the biological activity of HPVI8. The term “functional derivatives” is intended to include the “fragments,” “variants,” “degenerate variants,” “analogs” and “homologues” or to “chemical derivatives” of HPV 18. The term “fragment” is meant to refer to any polypeptide subset of HPV 18. The term “variant” is meant to refer to a molecule substantially similar in structure and function to either the entire HPV 18 molecule or to a fragment thereof. A molecule is “substantially similar” to HPV 18 if both molecules have substantially similar structures or if both molecules possess similar biological activity. Therefore, if the two molecules possess substantially similar activity, they are considered to be variants __III_ l111~-~LL~.ILI~ i_ I WO 96/29413 PCTIUS96/03649 -7even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
The term “analog” refers to a molecule substantially similar in function to either the entire HPV 18 molecule or to a fragment thereof.
A variety of procedures may be used to molecularly clone HPV18 DNA. These methods include, but are not limited to, direct functional expression of the HPV18 genes following the construction of a HPV 18-containing cDNA or genomic DNA library in an appropriate expression vector system. Another method is to screen HPV 18-containing cDNA or genomic DNA library constructed in a bacteriophage or plasmid shuttle vector with a labeled oligonucleotide probe designed from the amino acid sequence of the HPV 18. An additional method consists of screening a HPV 18-containing cDNA or genomic DNA library constructed in a bacteriophage or plasmid shuttle vector with a partial DNA encoding the HPV 18. This partial DNA is obtained by the specific polymerase chain reaction (PCR) amplification of HPV 18 DNA fragments through the design of degenerate oligonucleotide primers from the amino acid sequence of purified HPV 18. Another method is to isolate RNA from HPV 18producing cells and translate the RNA into protein via an in vitro or an in vivo translation system. The translation of the RNA into a peptide or a protein will result in the production of at least a portion of HPV 18 protein which can be identified by, for example, the activity of HPV 18 protein or by immunological reactivity with an anti-HPV 18 antibody.
In this method, pools of RNA isolated from HPV 18-producing cells can be analyzed for the presence of an RNA which encodes at least a portion of the HPV18. Further fractionation of the RNA pool can be done to purify the HPV 18 RNA from non-HPV18 RNA. The peptide or protein produced by this method may be analyzed to provide amino acid sequences which in turn are used to provide primers for production of HPV 18 cDNA. or the RNA used for translation can be analyzed to provide nucleotide sequences encoding HPV 18 and produce probes for the screening of a HPV 18 cDNA library. These WO 96/29413 PCT/US96/03649 -8methods are known in the art and can be found in, for example, Sambrook, Fritsch, Maniatis, T. in Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 1989.
It is apparent that other types of libraries, as well as libraries constructed from other cells or cell types, may be useful for isolating HPV 18-encoding DNA. Other types of libraries include, but are not limited to, cDNA libraries derived from other cells or cell lines containing HPV type 18 and genomic DNA libraries.
Preparation of cDNA libraries can be performed by a variety of techniques. cDNA library construction techniques can be found in Sambrook, et al., supra. It is apparent that DNA encoding HPV 18 may also be isolated from a suitable genomic DNA library.
Construction of genomic DNA libraries can be performed by a variety of techniques. Genomic DNA library construction techniques can be found in Sambrook, et al. supra.
The cloned HPV 18 DNA or fragments thereof obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant HPV18. Techniques for such manipulations are fully described in Sambrook, et al., supra, and are known in the art.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria-yeast or bacteria-animal cells or bacteria-fungal cells or bacteria-invertebrate cells. An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy WO 96/29413 PCT/US96/03649 -9number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
A variety of mammalian expression vectors may be used to express HPV18 DNA or fragments thereof in mammalian cells.
Commercially available mammalian expression vectors which may be suitable for recombinant HPV 18 expression, include but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTI (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12)
(ATCC
37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2dhfr (ATCC 37146), pUCTag (ATCC 37460), and XZD35 (ATCC 37565).
A variety of bacterial expression vectors may be used to express HPV 18 DNA or fragments thereof in bacterial cells.
Commercially available bacterial expression vectors which may be suitable include, but are not limited to pETI la (Novagen), lambda gtl 1 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to express HPV18 or fragments thereof in fungal cells. Commercially available fungal cell expression vectors which may be suitable include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen), and Hansenula expression (Rhein Biotech, Dusseldorf, Germany).
A variety of insect cell expression vectors may be used to express HPV 18 DNA or fragments thereof in insect cells.
Commercially available insect cell expression vectors which may be suitable include but are not limited to pBlue Bac III (Invitrogen) and pAcUW51 (PharMingen, Inc.).
An expression vector containing DNA encoding HPV 18 or fragments thereof may be used for expression of HPV 18 proteins or WO 96/29413 PCT/US96/03649 fragments of HPV 18 proteins in a cell, tissues, organs, or animals (including humans). Host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC CCL L cells L-M (ATCC CCL 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-I (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce HPV18 protein. Identification of HPV18 expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-HPV18 antibodies, and the presence of host cell-associated HPV18 activity, such as HPV 18specific ligand binding or signal transduction defined as a response mediated by the interaction of HPVI8-specific ligands with the expressed HPV18 proteins.
Expression of HPV DNA fragments may also be performed using in vitro produced synthetic mRNA or native mRNA.
Synthetic mRNA or mRNA isolated from HPV18 producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocvtes being preferred.
I
WO 96/29413 PCT/US96/03649 11 Following expression of HPV 18 protein(s) in a host cell, HPV 18 protein may be recovered to provide HPV 18 in purified form.
Several HPV18 purification procedures are available and suitable for use. As described herein, recombinant HPV18 protein may be purified from cell lysates and extracts by various combinations of, or individual application of salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography and hydrophobic interaction chromatography.
In addition, recombinant HPV 18 may be separated from other cellular proteins by use of an immunoaffinity column made with monoclonal or polyclonal antibodies specific for full length nascent HPVI 8, or polypeptide fragments of HPVI 8. Monoclonal and polyclonal antibodies may be prepared according to a variety of methods known in the art. Monoclonal or monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogenous binding characteristics for HPVI 8.
Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope.
It is apparent that the methods for producing monospecific antibodies may be utilized to produce antibodies specific for HPV18 polypeptide fragments, or full-length nascent HPV 18 polypeptide. Specifically, it is apparent that monospecific antibodies may be generated which are specific for the fully functional HPV I8 or fragments thereof.
The present invention is also directed toward methods for screening for compounds which modulate the expression of DNA or RNA encoding HPVI8 as well as the function(s) of HPVI8 protein(s) in vivo. Compounds which modulate these activities may be DNA.
RNA, peptides, proteins, or non-proteinaceous organic molecules.
Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding HPV 18. or the function of HPV 18 protein.
Compounds that modulate the expression of DNA or RNA encoding HPV 18 or the function of HPV 18 protein may be detected by a variety
I
WO 96/29413 PCT/US96/03649 -12of assays. The assay may be a simple “yes/no” assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample.
Kits containing HPV 18 DNA, fragments of HPV 18 DNA, antibodies to HPV 18 DNA or HPV 18 protein, HPV 18 RNA or HPV 18 protein may be prepared. Such kits are used to detect DNA which hybridizes to HPV18 DNA or to detect the presence of HPV 18 protein(s) or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic analyses and epidemiological studies.
Nucleotide sequences that are complementary to the HPV 18 encoding DNA sequence may be synthesized for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates,
RNA,
stable derivatives of RNA such as 2′-O-alkylRNA, or other HPV 18 antisense oligonucleotide mimetics. HPVI 8 antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. HPV18 antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce HPV 18 activity.
The term “chemical derivative” describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, halflife, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington’s Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal inhibition of the HPVI8 or its activity while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.
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WO 96/29413 PCT/US96/03649 13- Advantageously, compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in several divided doses. Furthermore, compounds for the present invention may be administered via a variety of routes including but not limited to intranasally, orally, transdermally or by suppository.
For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, the active agents can be administered concurrently, or they each can be administered at separately staggered times.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug’s availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
In the methods of the present invention, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers (collectively referred to herein as “carrier” materials) suitably selected with respect to the intended form of administration, that is. oral tablets, capsules. elixirs syrup, suppositories, gels and the like, and consistent with conventional pharmaceutical practices.
For instance, for oral administration in the form of a tablet or capsule. the active drug component can be combined with an oral. non-toxic pharmaceutically acceptable inert carrier such as WO 96/29413 PCT/US96/03649 -14ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum and the like.
For liquid forms the active drug component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methyl-cellulose and the like. Other dispersing agents which may be employed include glycerin and the like. For parenteral administration, sterile suspensions and solutions are desired. Isotonic preparations which generally contain suitable preservatives are employed when intravenous administration is desired.
Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form. e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
The compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present invention may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the WO 96/29413 PCT/US96/03649 present invention may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidephenol, polyhydroxy-ethylaspartamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
The following examples illustrate the present invention without, however, limiting the same thereto.
EXAMPLE 1 Cloning of HPV 18 genome Total genomic DNA was prepared from the human cervical carcinoma-derived cell line, SW756 (Freedman, R. et al., 1982, In Vitro, Vol 18, pages 719-726) by standard techniques. The DNA was digested with EcoR and electrophoresed through a 0.8% low-melting temperature, agarose preparative gel. A gel slice was excised corresponding to DNA fragments approximately 12 kbp in length. The agarose was digested using AgaraseTM enzyme (Boehringer Mannheim, Inc.) and the size-fractionated DNA was precipitated, dephosphorylated and ligated with EcoR digested lambda EMBL4 arms (Stratagene, Inc.).
The lambda library was packaged using Gigapack II Gold packaging extract (Stratagene, Inc.). HPV1 8 -positive clones were identified using a 700 bp, HPV18 LI DNA probe that was generated by polymerase chain reaction (PCR) using SW756 DNA as template and oligonucleotide primers that were designed based on the published HPVI8 LI DNA sequence (Cole and Danos. 1987, J. Mol. Biol., Vol. 193:599-608: Genbank Accession #X05015). A HPV18-positive. lambda clone was isolated that contained a 12 kbp EcoR I fragment insert and was designated as #187-1.
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WO 96/29413 PCT/US96/03649 -16- EXAMPLE 2 Construction of Yeast Expression Vectors The HPV18 LI open reading frame (ORF) was amplified by PCR using clone #187-1 as template, Vent polymeraseTM (New England Biolabs, Inc.), 10 cycles of amplification (94’C, 1 min; 50 0 C, 1 min; 72 0 C, 2 min) and the following oligonucleotide primers which contain flanking BglII sites (underlined): sense primer, CGGCCTAGTG-3′, antisense primer, ACGCACACGC-3′.
The sense primer introduces a yeast non-translated leader sequence (Kniskem, et al., 1986, Gene, Vol. 46:135-141) immediately upstream to the HPV18 LI initiating methionine codon (highlighted in bold print).
The 1.5 kbp LI PCR product was digested with BglII and gel purified.
The pGALl-10 yeast expression vector was constructed by isolating a 1.4 kbp Sphl fragment from a pUCI 8/bidirectional
GAL
promoter plasmid which contains the Saccharomyces cerevisiae divergent GAL1-GAL1 promoters from the plasmid pBM272 (provided by Mark Johnston, Washington University, St. Louis, MO). The divergent promoters are flanked on each side by a copy of the yeast ADHI transcriptional terminator, a BamHI cloning site located between the GAL] promoter and the first copy of the ADH1 transcriptional terminator and a Smal cloning site located between the GAL10 promoter and the second copy of the ADHI transcriptional terminator. A yeast shuttle vector consisting of pBR322, the yeast LEU2d gene, and the yeast 2u plasmid (gift of Benjamin Hall, University of Washington, Seattle, WA) was digested with SphI and ligated with the 1.4 kbp SphI divergent GAL promoter fragment resulting in pGAL 1 -10. pGAL 1-10 was linearized with BamHI which cuts between the GAL] promoter and the ADHI transcription terminator. The BamHI digested vector and the BglII digested HPVI8 LI PCR fragment were ligated and used to transfom WO 96/29413 PCT/US96/03649 -17- E. coli DH5 cells (Gibco BRL, Inc.). A pGALI-10 plasmid was isolated which contains the HPV18 LI gene and was designated p191-6.
A yeast expression vector that co-expresses both the HPV 18 L1 and L2 genes was constructed. Plasmid p191-6 (pGALI-10 HPV18 L1) was digested with Smal which cuts between the GALIO promoter and the ADH1 transcription terminator. The 1.4 kbp HPV18 L2 gene was amplified by PCR as described above using the following oligonucleotide primers which contain flanking Smal sites (underlined): sense primer, GTATCCCACCGTGCCGCACGAC-3′, antisense primer, 5′-TCCCCCGGGCTAGGCCGCCACAAAGCCATCTGC-3′.
The sense primer introduces a yeast non-translated leader sequence (Kniskem et al., 1986, supra) immediately upstream to the HPV 18 L2 initiating methionine codon (highlighted in bold print). The PCR fragment was digested with Smal, gel purified and ligated with the Smal digested p191-6 plasmid. A pGAL1-10 plasmid containing both the HPV18 LI and L2 genes was isolated and designated, p195-11.
EXAMPLE 3 Typing of Clinical Samples Cervical biopsy samples were collected at the Veterans Administration Medical Center in Indianapolis, IN (courtesy of Dr.
Darron Brown) and at the Albert Einstein Medical Center in Philadelphia.
PA (courtesy of Dr. Joan Adler) and were frozen at 20 0 C. DNA was isolated as described by Brown et al., 1993 (Brown, D. et al., 1993, J.
Clin. Microhiol., Vol. 31:2667-2673). Briefly. clinical specimens were processed with a Braun mikro-dismembrator II Braun Instruments.
Melsungen, Germany) and solubilized in buffer containing 10 mM EDTA and 0.6% sodium dodecyl sulfate (SDS). Samples were adjusted to mM Tris pH 7.4 and protein was digested with 50 mcg/mL Proteinase K in the presence of 0.1 mcg/mL RNase A followed by extraction with WO 96/29413 PCT/US96/03649 18- -lgphenol/chloroform/isoamyl alcohol. DNA was ethanol precipitated and quantified by spectrophotometry.
The DNA samples were screened for the presence of HPV 18 by PCR and Southern blot analyses. A 256 bp segment of the HPV 18 L1 ORF was amplified by PCR using the following oligonucleotide primers: sense primer, 5′-CAATCCTTATATATTAAAGGCACAGGTATG-3′, antisense primer, 5′-CATCATATTGCCCAGGTACAGGAGACTGTG-3′.
The PCR conditions were according to the manufacturer’s recommendations for the AmpliTaqTM DNA Polymerase/GeneAmpTM kit,(Perkin Elmer Corp.) except that 0.5 til of clinical sample DNA was used as template and 10 pmoles of each primer, 2 mM dNTPs and mM MgC12 were in the final reaction mixture. A 2 min, 94°C denaturation step was followed by 40 cycles of amplification (94’C, 1 min; 45°C, 1 min; 72’C, 1 min).
The PCR products were electrophoresed through a agarose gel, blotted onto nylon membranes and hybridized with a 32p_ labeled HPVI8 Ll-specific oligonucleotide probe.
EXAMPLE 4 DNA Sequencing of LI and L2 genes The HPV18 Ll and L2 genes in clones #187-1, p191-6 and p195-11 were sequenced using the PRIZM Sequencing kit and the automated DNA ABI Sequencer #373A (Applied Biosystems). To obtain a consensus HPV18 sequence, portions of the LI gene DNA were amplified by PCR from human clinical isolates, sequenced and compared to the claimed and published sequences. A 256 bp fragment (nucleotides 817-1072) was amplified from each clinical DNA isolate for this purpose using the oligonucleotides and heating cycles described in Example 3.
The following primers, 3′ and CCTAACGTCCTCAGAAACATTAGAC-3′ were used to amplify an amino-terminal 432 bp portion of LI DNA (nucleotides 1- WO 96/29413 PCT/US96/03649 -19- 431) using the heating cycles described in Example 3. Both PCR products were ligated separately with plasmid pCRII (Invitrogen Corp.) using the reagents and procedures recommended by the manufacturer.
Plasmid DNA was isolated from the transformants and those containing EcoRI inserts were sequenced.
EXAMPLE Analysis of DNA and Deduced Amino Acid Sequences The nucleotide and deduced amino acid (aa) sequences of the claimed HPV18 L1 are shown in Figure 1. The DNA sequence was derived from a consensus of clones #187-1, p191-6 and p 19 5 1 1. A comparison of the claimed HPV 18 LI nucleotide sequence with the published HPV18 LI sequence (Genbank Accession #X05015) identified 20 bp changes out of 1524 bps. Five of the nucleotide changes (C to G at position 89, C to A at 263, C to G at 848, G to A at 967 and C to G at 1013) result in amino acid substitutions. The five residue differences from published are P to R at aa positions 30, 283 and 338, T to N at aa 88 and V to I at aa 323 (Figure Positions 88 and 323 represent conservative changes while the three P to R changes may substantially alter the physical properties of the expressed LI protein.
A comparison of the amino acid sequences derived from clinical isolates (numbers 354, 556, 755, 697, 795 and 23) with the claimed sequence and the published sequence is shown in Figure 2.
There are four locations where the clinical isolates and the claimed sequence differ from the published sequence. Positions 30. 283 and 338 encode arginine in all the isolates found to date, including the claimed sequence. This is in sharp contrast to the published sequence which has prolines at each of these locations. Furthermore, position 88 is an asparagine in the isolates and the claimed sequence but is a threonine in the published sequence. The last difference, position 323, was found to be a valine in many of the clinical isolates and the published strain versus an isoleucine in the claimed sequence and one of the isolates The conclusion is that the claimed sequence reflects the predominant viral sequences that are associated with clinical infections I WO 96/29413 PCTfUS96/03649 and the absence of isolates containing any of the position 30, 283 or 338 prolines of the published sequence suggests that the published clone is either an artefact or an inconsequential subtype.
The nucleotide and deduced aa sequences of HPV 18 L2 were derived from a consensus sequence of clones #187-1 and p 1 95-11 and are shown in Figure 3. A comparison of the L2 nucleotide sequence with the published HPV18 sequence (Genbank Accession #X05015) identified 40 bp changes out of 1389 bps. The bp differences result in 14 changes at the aa level: P to S at aa 29, P to N at aa 33, A to S at aa 177, D to E at aa 266, D to N at aa 270, D to G at aa 346, M to I at 355, V to M at aa 359, S to P at aa 365, F to S at aa 369, F to V at aa 371, F to S at aa 372, K to T at aa 373 and S to P at aa 409.
EXAMPLE 6 Generation of HPV 18 L2 Antiserum HPV18 L2 specific antibodies were prepared in goats using a trpE-HPV18 L2 fusion protein expressed in E. coli. The full-length L2 ORF was amplified by PCR using oligonucleotide primers providing HindIll and BamHI sites flanking the and ends, respectively. The L2 fragment was inserted into the HindIIl-BamHI digested, pATH23 expression plasmid (Koemer et al., 1991, Meth. Enzymol. Vol. 194:477- 490). The fusion protein was expressed in E. coli RRI cells (Gibco BRL, Inc.) after induction with 3-b-indoleacrylic acid. The insoluble fraction was analyzed by SDS-PAGE followed by staining with Coomassie Blue.
The trpE-L2 fusion protein accounted for the major portion of the E. coli insoluble fraction. Goats were immunized with the trpE-L2 fusion protein according to the standard protocol of Pocono Rabbit Farm and Laboratory, Inc. for fusion protein antigens (Protein Rabbit Farm.
Canadensis.
PA).
WO 96/29413 PCT/US96/03649 -21 EXAMPLE 7 Preparation of Yeast Strain U9 Saccharomyces cerevisiae strain 2150-2-3 (MATalpha, leu2- 04, add, cir°) was obtained from Dr. Leland Hartwell (University of Washington, Seattle, WA). Cells of strain 2150-2-3 were propagated overnight at 30C in 5 mL of YEHD medium (Carty et al., J. Ind Micro 2 (1987) 117-121). The cells were washed 3 times in sterile, distilled water, resuspended in 2 mL of sterile distilled water, and 0.1 mL of cell suspension was plated onto each of six 5-fluoro-orotic acid (FOA) plates in order to select for ura3 mutants (Cold Spring Harbor Laboratory Manual for Yeast Genetics). The plates were incubated at 30 0 C. The medium contained per 250 mL distilled water: 3.5 g, Difco Yeast Nitrogen Base without amino acids and ammonium sulfate: 0.5 g Fluoro-orotic acid; 25 mg Uracil; and 10.0 g Dextrose.
The medium was sterilized by filtration through 0.2 .tm membranes and then mixed with 250 mL of 4% Bacto-Agar (Difco) maintained at 50 0 C, 10 mL of a 1.2 mg/mL solution of adenine, and 5 mL of L-leucine solution (180 mg/ 50 mL). The resulting medium was dispensed at 20 mL per petri dish.
After 5 days of incubation, numerous colonies had appeared.
Single colonies were isolated by restreaking colonies from the initial FOA plates onto fresh FOA plates which were then incubated at 30 0 C. A number of colonies from the second set of FOA plates were tested for the presence of the ura3 mutation by replica-plating onto both YEHD plates and uracil-minus plates. The desired result was good growth on YEHD and no growth on uracil-minus medium. One isolate (U9) was obtained which showed these properties. It was stored as a frozen glycerol stock (strain #325) at -70°C for later use.
EXAMPLE 8 Preparation of a Vector for disruption of the Yeast MNN9 gene In order to prepare a vector for disruption of the AMNN9 gene.
it was necessary to first clone the MNN9 gene from S. cercvi.iac genomic WO 96/29413 PCT/US96/03649 -22- DNA. This was accomplished by standard Polymerase Chain Reaction (PCR) technology. A 5′ sense primer and 3′ antisense primer for PCR of the full-length MNN9 coding sequence were designed based on the published sequence for the yeast MNN9 gene (Zymogenetics: EPO Patent Application No. 88117834.7, Publication No. 0-314-096-A2). The following oligodeoxynucleotide primers containing flanking HindIII sites (underlined) were used: sense primer: 5′-CTT AAA GCT TAT GTC ACT TTC TCT TGT ATC G-3′ antisense primer: 5′-TGA TAA GCT TGC TCA ATG GTT CTC TTC CTC-3′.
The initiating methionine codon for the MNN9 gene is highlighted in bold print. The PCR was conducted using genomic DNA from S. cerevisiae strain JRY188 as template, Taq DNA polymerase (Perkin Elmer) and 25 cycles of amplification (94 0 C 1 min., 37°C 2 min., 72 0 C 3 min.). The resulting 1.2 kbp PCR fragment was digested with HindIII, gel-purified, and ligated with HindIII-digested, alkalinephosphatase treated pUC13 (Pharmacia). The resulting plasmid was designated pl 183.
In order to disrupt the MNN9 gene with the yeast URA3 gene, the plasmid pBR322-URA3 (which contains the 1.1 Kbp HindIII fragment encoding the S. cerevisiae URA3 gene subcloned into the HindIIl site of pBR322) was digested with HindIII and the 1.1 kbp DNA fragment bearing the functional URA3 gene was gel-purified, made bluntended with T4 DNA polymerase, and then ligated with Pmll-digested plasmid p1183 (PmlI cuts within the MNN9 coding sequence).
The resulting plasmid p 1199 contains a disruption of the MNN9 gene b\ the functional URA3 gene.
WO 96/29413 PCT/US96/03649 23 EXAMPLE 9 Construction of U9-derivative strain 1372 containing disruption of MNN9 gene For disruption of the MNN9 gene in strain U9 30 tg of plasmid pi 199 were digested with HindIl to create a linear mnn9::URA3 disruption cassette. Cells of strain 325 were transformed with the HindfII-digested p 1 1 99 DNA by the spheroplast method (Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75:1929-1933) and transformants were selected on a synthetic agar medium lacking uracil and containing M sorbitol. The synthetic medium contained, per liter of distilled water: Agar, 20 g; Yeast nitrogen base w/o amino acids, 6.7 g; Adenine, 0.04 g; L-tyrosine, 0.05 g; Sorbitol, 182 g; Glucose, 20 g; and Leucine Minus Solution 10 ml. Leucine Minus Solution #2 contains per liter of distilled water: L-arginine, 2 g; L-histidine, 1 g; L-Leucine, 6 g; L- Isoleucine, 6 g; L-lysine, 4 g; L-methionine, 1 g; L-phenylalanine, 6 g; Lthreonine, 6 g; L-tryptophan, 4 g.
The plates were incubated at 30’C for five days at which time numerous colonies had appeared. Chromosomal DNA preparations were made from 10 colonies and then digested with EcoRI plus HindIII.
The DNA digests were then evaluated by Southern blots Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989) using the 1.2 kbp HindIII fragment bearing the MNN9 gene (isolated from plasmid pi 199) as a probe. An isolate was identified (strain #1372) which showed the expected DNA band shifts on the Southern blot as well as the extreme clumpiness typically shown by mnn9 mutants.
EXAMPLE Construction of a Vector for Disruption of Yeast HIS3 Gene In order to construct a disruption cassette in which the S.
ccrevisiae HIS3 gene is disrupted by the URA3 gene. the plasmid YEp6 Struhl et al.. 1979. Proc. Natl. Acad. Sci., USA 76:1035) was digested with BamHI and the 1.7 kbp BamHI fragment bearing the HIS3 gene was WO 96/29413 PCT/US96/03649 -24gel-purified, made blunt-ended with T4 DNA polymerase, and ligated with pUCI 8 which had been previously digested with BamHI and treated with T4 DNA polymerase. The resulting plasmid (designated p1501 or pUC 8-HIS3) was digested with Nhel (which cuts in the HIS3 coding sequence), and the vector fragment was gel-purified, made blunt-ended with T4 DNA polymerase, and then treated with calf intestine alkaline phosphatase. The URA3 gene was isolated from the plasmid pBR322- URA3 by digestion with HindIII and the 1.1 kbp fragment bearing the URA3 gene was gel-purified, made blunt-ended with T4 DNA polymerase, and ligated with the above pUCI8-HIS3 Nhel fragment. The resulting plasmid (designated pUCI 8-his3::URA3 or p 1 505) contains a disruption cassette in which the yeast HIS3 gene is disrupted by the functional URA3 gene.
EXAMPLE 11 Construction of Vector for Disruption of Yeast PRBI Gene by the HIS3 Gene Plasmid FP8AH bearing the S. cerevisiae PRBI gene was provided by Dr. E. Jones of Carnegie-Mellon Univ. M. Moehle et al..
1987, Genetics 115:255-263). It was digested with HindIII plus Xhol and the 3.2 kbp DNA fragment bearing the PRBI gene was gel-purified and made blunt-ended by treatment with T4 DNA polymerase. The plasmid pUC18 was digested with BamHI, gel-purified and made blunt-ended by treatment with T4 DNA polymerase. The resulting vector fragment was ligated with the above PRBI gene fragment to yield the plasmid pUCI 8- PRB 1. Plasmid YEp6, which contains the HIS3 gene, was digested with BamHI. The resulting 1.7 kbp BamHI fragment bearing the functional HIS3 gene was gel-purified and then made blunt-ended by treatment with T4 DNA polymerase. Plasmid pUC18-PRBI was digested with EcoRV plus Ncol which cut within the PRBI coding sequence and removes the protease B active site and flanking sequence. The 5.7 kbp EcoRV-Ncol fragment bearing the residual 5′ and 3′-portions of the PRBI coding sequence in pUCX8 was gel-purified. made blunt-ended by treatment with T4 DNA polymerase. dephosphorylated with calf intestine alkaline WO 96/29413 PCTIUS96/03649 phosphatase, and ligated with the blunt-ended HIS3 fragment described above. The resulting plasmid (designated pUC8-prbl ::HIS3, stock #1245) contains the functional HIS3 gene in place of the portion of the PRB1 gene which had been deleted above.
EXAMPLE 12 Construction of a U9-related Yeast Strain containing disruptions of both the MNN9 and PRB1 Genes The U9-related strain 1372 which contains a MNN9 gene disruption was described in Example 9. Clonal isolates of strain 1372 were passaged on FOA plates (as described in Example 7) to select ura3 mutants. A number of ura3 isolates of strain 1372 were obtained and one particular isolate (strain 12930-190-S 1-1) was selected for subsequent disruption of the HIS3 gene. The pUCI8-his3::URA3 gene disruption vector (p1505) was digested with XbaI plus EcoRI to generate a linear his3::URA3 disruption cassette and used for transformation of strain 12930-190-S 1-1 by the lithium acetate method [Methods in Enzvmology, 194:290 (1991)]. Ura+ transformants were selected on synthetic agar medium lacking uracil, restreaked for clonal isolates on the same medium, and then replica-plated onto medium lacking either uracil or histidine to screen for those isolates that were both Ura+ and His-. One isolate (strain 12930-230-1) was selected for subsequent disruption of the PRBI gene. The PRB1 gene disruption vector (pUC 8-prbl ::HIS3, stock #1245) was digested with Sad plus Xbal to generate a linear prl ::.HIS3 disruption cassette and used for transformation of strain 12930-230-1 by the lithium acetate method. His+ transformants were selected on agar medium lacking histidine and restreaked on the same medium for clonal isolates. Genomic DNA was prepared from a number of the resulting His+ isolates, digested with EcoRI, and then electrophoresed on 0.8agarose gels. Southern blot analyses were then performed using a radiolabeled 617 bp probe for the PRB1 gene which had been prepared by PCR using the following oligodeoxynucleotide primers: TGG TCA TCC CAA ATC TTG AAA 3′ 5′ CAC CGT AGT GTT TGG AAG CGA 3′ WO 96/29413 PCT/US96/03649 -26- Eleven isolates were obtained which showed the expected hybridization of the probe with a 2.44 kbp prbhl::HIS3 DNA fragment.
This was in contrast to hybridization of the probe with the 1.59 kbp fragment for the wild-type PRBI gene. One of these isolates containing the desired prbl ::HIS3 disruption was selected for further use and was designated strain #1558.
EXAMPLE 13 Expression of HPV 18 LI and L2 in Yeast Plasmids p191-6 (pGAL -10 HPV18 LI) and p195-11 HPV18 LI L2) were used to transform S. cerevisiae strain #1558 (MATa, leu2-04, prbl::HIS3, mnn9::URA3, adel, cir-).
Clonal isolates were grown at 30 0 C in YEHD medium containing 2% galactose for 88 hours. After harvesting the cells, the cell pellets were broken with glass beads and cell lysates analyzed for the expression of HPV18 LI and/or HPV18 L2 protein by immunoblot analysis. Samples containing 25 ug of total cellular protein were electrophoresed through Tris-Glycine gels (Novex, Inc.) under denaturing conditions and electroblotted onto nitrocellulose filters. LI protein was immunodetected using rabbit antiserum raised against a trpE-HPV 1 LI fusion protein as primary antibody (Brown et al., 1994, Virology 201:46-54) and horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG (Amersham, Inc.) as secondary antibody. The filters were processed using the chemiluminescent ECLTM Detection Kit (Amersham, Inc.). A 50-55 KDa LI protein band was detected in both the LI and LI L2 coexpressor yeast clones (strains 1725 and 1727, respectively) and not in the negative control (pGAL -10 without LI or L2 genes) (Figure 4).
The HPV 18 L2 protein was detected by Western analysis using goat polyclonal antiserum raised against a trpE-HPV 18 L2 fusion protein as primary antibody followed by HRP-conjugated, rabbit antigoat IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The filters were processed as described above. The L2 protein was detected as a 75 kDa protein band in the LI L2 coexpressor yeast clone (strain illl_~_~ WO 96/29413 PCTIS96/03649 -27- 1727) but not in either the negative control or the LI expressor clone (Figure EXAMPLE 14 Fermentation of HPV 18 L1 (strain 1725) and 18 L1+AL2 (strain 1727) Surface growth of a plate culture of strains 1725 and 1727 was aseptically transferred to a leucine-free liquid medium containing (per 8.5 g Difco yeast nitrogen base without amino acids and ammonium sulfate; 0.2 g adenine; 0.2 g uracil; 10 g succinic acid; 5 g ammonium sulfate; 40 g glucose; 0.25 g L-tyrosine; 0.1 g L-arginine; 0.3 g L-isoleucine; 0.05 g L-methionine; 0.2 g L-tryptophan; 0.05 g Lhistidine; 0.2 g L-lysine; 0.3 g L-phenylalanine; this medium was adjusted to pH 5.0-5.3 with NaOH prior to sterilization. After growth at 28°C, 250 rpm on a rotary shaker, frozen culture vials were prepared by adding sterile glycerol to a final concentration of 17% prior to storage at 70 0 C (1 mL per cryovial). Inocula were developed in the same medium (500 mL per 2-L flask) and were started by transferring the thawed contents of a frozen culture vial and incubating at 28’C. 250 rpm on a rotary shaker for 29 hr. Fermentations of each strain used a New Brunswick SF-116 fermentor with a working volume of 10 L after inoculation. The production medium contained (per 20 g Difco yeast extract; 10 g Sheffield HySoy peptone; 20 g glucose; 20 g galactose; 0.3 mL Union Carbide UCON LB-625 antifoam; the medium was adjusted to pH 5.3 prior to sterilization. The entire contents (500 mL) of the 2-L inoculum flask was transferred to the fermentor which was incubated at 28 0 C, 5 L air per min, 400 rpm, 3.5 psi pressure. Agitation was increased as needed to maintain dissolved oxygen levels of greater than 40% of saturation. Progress of the fermentation was monitored by off-line glucose measurements (Beckman Glucose 2 Analyzer) and on-line mass spectrometry (Perkin-Elmer 1200). After incubation for 66 hr. cell densities of 9.5 to 9.7 g dry cell weight per L were reached. The cultures were concentrated by hollow fiber filtration (Amicon H5MP01-43 cartridge in an Amicon DC-10 filtration system) to ca. 2 L. diafiltered with 2 L phosphate-buffered saline, and concentrated further (to ca. 1 L) WO 96/29413 PCT/US96/03649 28 before dispensing into 500-mL centrifuge bottles. Cell pellets were collected by centrifugation at 8,000 rpm (Sorval GS-3 rotor) for 20 min at 4°C. After decanting the supernatant, the pellets (total 191 to 208 g wet cells) were stored at 70 0 C until use.
EXAMPLE Purification of Recombinant HPV Type 18 LI Capsid Proteins All steps performed at 4 0 C unless noted.
Cells were stored frozen at -70 0 C. Frozen cells (wet weight 126 g) were thawed at 20-23 0 C and resuspended in 70 mL “Breaking Buffer” (20 mM sodium phosphate, pH 7.2, 100 mM NaCI). The protease inhibitors PMSF and pepstatin A were added to final concentrations of 2 mM and 1.7 jiM, respectively. The cell slurry was broken at a pressure of approximately 16,000 psi by 4 passes in a Ml Y Microfluidizer (Microfluidics Corp., Newton, MA). The broken cell slurry was centrifuged at 12,000 x g for 40 min to remove cellular debris.
The supernatant liquid containing L antigen was recovered.
The supernatant liquid was diluted 1:5 by addition of Buffer A (20 mM MOPS, pH 7.0) and applied to an anion exchange capture column (9.0 cm ID x 3.9 cm) of Fractogel EMD TMAE-650 resin (EM Separations, Gibbstown, NJ) equilibrated in Buffer A. Following a wash with Buffer A, the antigen was eluted with a gradient of 0 1.0 M NaCI in Buffer A. Column fractions were assayed for total protein by the Bradford method. Fractions were then analyzed at equal total protein loadings by Western blotting and SDS-PAGE with silver stain detection.
TMAE fractions showing comparable purity and enrichment of LI protein were pooled. The antigen was concentrated by ammonium sulfate fractionation. The solution was adjusted to 35% saturated ammonium sulfate by adding solid reagent while gently stirring over min. The sample was placed on ice and precipitation allowed to proceed for 4 hours. The sample was centrifuged at 16,000 x g for 45 min. The pellet was resuspended in 20.0 mL PBS (6.25 mM Na phosphate. pH 7.2.
150 mM NaCI).
WO 96/29413 PCT/US96/03649 -29- The resuspended pellet was chromatographed on a size exclusion column (2.6 cm ID x 89 cm) of Sephacryl 500 HR resin (Pharmacia, Piscataway, NJ). Running buffer was PBS. Fractions were analyzed by western blotting and SDS-PAGE with silver stain detection.
The purest fractions were pooled. The resulting pool was concentrated in a 50 mL stirred cell using 43 mm YM-100 flat-sheet membranes (Amicon, Beverly, MA) at a N2 pressure of 4-6 psi.
Final product was analyzed by western blotting and SDS- PAGE with colloidal Coomassie detection. The Ll protein was estimated to be 50-60% homogeneous. The identity of LI protein was confirmed by western blotting. The final product was aliquoted and stored at -70 0
C.
This process resulted in a total of 12.5 mg protein.
Bradford Assay for Total Protein Total protein was assayed using a commercially available Coomassie Plus kit (Pierce, Rockford, IL). Samples were diluted to appropriate levels in Milli-Q-H20. Volumes required were 0.1 mL and mL for the standard and microassay protocols, respectively. For both protocols, BSA (Pierce, Rockford, IL) was used to generate the standard curve. Assay was performed according to manufacturer’s recommendations. Standard curves were plotted using CricketGraph software on a Macintosh IIci computer.
SDS-PAGE and Western Blot Assays All gels, buffers, and electrophoretic apparatus were obtained from Novex (San Diego, CA) and were run according to manufacturer’s recommendations. Briefly, samples were diluted to equal protein concentrations in Milli-Q-H20 and mixed 1:1 with sample incubation buffer containing 200 mM DTT. Samples were incubated min at 100 0 C and loaded onto pre-cast 12% Tris-glycine gels. The samples were electrophoresed at 125V for 1 hr 45 min. Gels were developed using either silver staining by a variation of the method of Heukeshoven and Demick IElectrophoresis., (1985) 103-112\ or WO 96/29413 PCT/US96/03649 colloidal Coomassie staining using a commercially obtained kit (Integrated Separation Systems, Natick, MA).
For western blots, proteins were transferred to PVDF membranes at’25V for 40 min. Membranes were washed with Milli-Q- H20 and air-dried. Primary antibody was polyclonal rabbit antiserum raised against a TrpE-HPV I1Ll fusion protein (gift of Dr. D. Brown).
Previous experiments had shown this antiserum to cross react with HPV type 18 LI on western blots. The antibody solution was prepared by dilution of antiserum in blotting buffer non-fat milk in 6.25 mM Na phosphate, pH 7.2, 150 mM NaCI, 0.02% NaN3). Incubation was for at least 1 hour at 20-23°C. The blot was washed for 1 min each in three changes of PBS (6.25 mM Na phosphate, pH 7.2, 150 mM NaCI).
Secondary antibody solution was prepared by diluting goat anti-rabbit IgG alkaline phosphatase-linked conjugate antiserum (Pierce, Rockford, IL) in blotting buffer. Incubation proceeded under the same conditions for at least 1 hour. Blots were washed as before and detected using a 1 step NBT/BCIP substrate (Pierce, Rockford, IL).
EXAMPLE 16 Electron Microscopic Studies For EM analysis (Structure Probe, West Chester, PA), an aliquot of each sample was placed on 200-mesh carbon-coated copper grids. A drop of 2% phosphotungstic acid (PTA), pH 7.0 was placed on the grid for 20 seconds. The grids were allowed to air dry prior to transmission EM examination. All microscopy was done using a JEOL 100CX transmission electron microscope (JEOL USA, Inc.) at an accelerating voltage of 100 kV. The micrographs generated have a final magnification of 100,000x. Virus-like particles were observed in the 55 nm diameter size range in the yeast sample harboring the HPV I8 LI expression plasmid (Figure No VLPs were observed in the yeast control samples.
WO 96/29413 PCT/US96/03649 -31 EXAMPLE 17 Sub-cloning of the HPV 18 cDNA into expression vectors The cDNA encoding HPV18 is sub-cloned into several vectors for expression of the HPV 18 protein in transfected host cells and for in vitro transcription/translation. These vectors include pBluescript I1 SK+ (where expression is driven by T7 or T3 promoters) pcDNA I/Amp (where expression is driven by the cytomegalovirus (CMV) promoter), pSZ9016-1 (where expression is driven by the HIV long terminal repeat (LTR) promoter) and the baculovirus transfer vector pAcUW51 (PharMingen, Inc.) (where expression is driven by the polyhedrin (PH) promoter) for producing recombinant baculovirus containing the HPV 18 encoding DNA sequence.
a) pBluescript II SK+:HPV18. The full length HPVI8 cDNA clone is retrieved from lambda bacteriophage by limited Eco RI digestion and ligated into Eco RI-cut, CIP-treated pBluescript 11 SK+.
Separate subclones are recovered in which the sense orientation of HPV 18 followed either the T7 or T3 promoters.
b) pcDNA I/Amp:HPV18. To facilitate directional cloning, HPV18 is excised from a purified plasmid preparation of pBluescript II SK+:HPVI8 in which the HPV 1 DNA sequence is downstream of the T7 promoter using Eco RV and Xba I. The resulting Eco RV, Xbu I HPV 18 fragment is purified and ligated into Eco RV-cut, Xba I-cut, CIP-treated pcDNA I/Amp such that the HPV 18 encoding DNA is downstream of the CMV promoter.
c) pSZ9016-l:HPV18. HPV 18 is excised from pBluescript II SK+:HPV18 by limited Eco RI digestion and subsequent purification of the 1.3 Kb fragment from agarose gels. The resulting Eco RI HPV18 fragment is ligated into Eco RI-cut. CIP-treated pSZ9016-1.
Subclones are selected in which the sense orientation of HPV 18 is downstream of the HIV LTR promoter.
d) pAcUW5I:HPVI8 L1. The full-length HPV 1 LI ORF was amplified by PCR from clone #187-1 using oligonucleotide primers providing flanking BglII sites. The LI gene was inserted into the BamHI site of the baculovirus transfer vector. pAcUW51 (PharMingen.
WO 96/29413 PCT/US96/03649 -32- Inc.), under control of the polyhedrin promoter. Recombinant baculoviruses were generated containing the HPV18 LI expression cassette according to the procedures described in the Pharmingen Manual.
Recombinant clones were purified by limiting dilution and dot blot hybridization.
EXAMPLE 18 Expression Of The HPV 18 Polypeptide By In Vitro Transcription/ Translation And By Transfection Into Host Cells Vectors containing HPV DNA sequences are used to drive the translation of the HPV18 polypeptide in rabbit reticulocyte lysates, mammalian host cells, and in baculovirus infected insect cells. The experimental procedures are essentially those outlined in the manufacturers’ instructions.
a) In vitro Transcription/Translation. pBluescript III SK+:HPV18 plasmid DNA (with HPV18 in the T7 orientation) is linearized by Bam HI digestion downstream of the HPV 18 insert. The linearized plasmid is purified and used as a template for run-off transcription using T7 RNA polymerase in the presence of m7G(5′)ppp(5′)G. The resulting capped HPVI 8 transcripts are purified by LiCI precipitation and used to drive the translation of HPV 18 in nuclease-pretreated rabbit reticulocyte lysate in the presence of L- 3 5 S]methionine.
b) Expression in Mammalian Cells. The HPV18 protein is expressed in mammalian host cells following transfection with either pcDNA I/Amp:HPV18 (under control of the CMV promoter) or pSZ9016-1 :HPV 18 (under control of the HIV LTR promoter). In the latter case (pSZ9016-l:HPVI8). cells are co-transfected with the TAT expressing plasmid pSZ90161:TAT. For both HPV 18 expression plasmids. COS-7 cells are transfected using either DEAE-dextran or lipofection with Lipofectamine (BRL).
c) Expression in Insect Cells. The HPVI8 LIcontaining baculovirus transfer vector pAcUW51 :HPV 18 LI is used to produce recombinant baculovirus (Auto,,rapha californica) by in viv WO 96/29413 PCT/US96/03649 -33homologous recombination. Epitope tagged HPV18 LI is then expressed in Sf9 (Spodopterafrugiperda) insect cells grown in suspension culture following infection with the HPV 18 containing recombinant baculovirus.
EXAMPLE 19 Compounds that affect HPV18 activity may be detected by a variety of methods. A method of identifying compounds that affect HPV 18 comprises: mixing a test compound with a solution containing HPV 18 to form a mixture; measuring HPV18 activity in the mixture; and comparing the HPV 18 in the mixture to a standard.
Compounds that affect HPV 18 activity may be formulated into pharmaceutical compositions. Such pharmaceutical compositions may be useful for treating diseases or conditions that are characterized by HPV 18 infection.
EXAMPLE DNA which is structurally related to DNA encoding HPVI8 is detected with a probe. A suitable probe may be derived from DNA having all or a portion of the nucleotide sequence of Figure 1 or Figure 3, RNA encoded by DNA having all or a portion of the nucleotide sequence of Figure 1 or Figure 3 or degenerate oligonucleotides derived from a portion of the sequence of Figure 1 or Figure 3.

Claims (24)

1. The isolated and purified DNA molecule which encodes human papilloma virus type 18, wherein the DNA is selected from the nucleotide sequence shown in Figure 1, the nucleotide sequence shown in Figure 3, a functional derivative of the nucleotide s sequence shown in Figure 1, and a functional derivative of the nucleotide sequence shown in Figure 3.

2. An expression vector for expression of the DNA molecule of Claim 1 in a host.

3. An essentially purified protein encoded by the DNA molecule of Claim 1.

4. An antibody immunologically reactive with the a compound selected from the DNA molecule of Claim 1, RNA complementary to the DNA molecule of Claim 1 or a protein encoded by the DNA molecule of Claim 1. A process for expression of a human papillomavirus type 18 protein in a host, comprising: transferring the expression vector of Claim 2 into a suitable host; and culturing the host of step under conditions which allow expression of the human papillomavirus type 18 protein from the expression vector.

6. The process of Claim 5 wherein the protein is selected from HPV 18 L1i protein, HPV 18 L2 protein and combinations thereof.

7. A composition capable of inducing an immune response in a subject treated with the composition, the composition containing a compound selected from the DNA molecule of Claim 1, at least one peptide encoded by the DNA molecule of Claim 1, 9* ~RNA complementary to the DNA molecule of Claim 1, or combinations thereof together with a pharmaceutically acceptable diluent of carrier. 25 8. A vaccine for the prevention or treatment of human papillomavirus infection, the vaccine containing a compound selected from the group consisting of the DNA molecule of Claim 1, at least one peptide encoded by the DNA molecule of Claim 1, RNA complementary to the DNA molecule of Claim 1, or combinations thereof. 1.9 A method for inducing an immune response against infection or disease caused 30 by human papillomavirus which method comprises introducing into an animal the DNA molecule of Claim 1, RNA complementary to the DNA molecule of Claim 1, protein encoded by the DNA molecule of Claim 1, or combinations thereof. The DNA molecule of Claim 1, RNA complementary to the DNA molecule of Claim 1, protein encoded by the DNA molecule of Claim 1, or combinations thereof 35 when used for inducing an immune response against infection or disease caused by human papillomavirus.

11. Use of a the DNA molecule of Claim 1, RNA complementary to the DNA molecule of Claim 1, protein encoded by the DNA molecule of Claim 1, or combinations thereof in the manufacture of a medicament for inducing an immune response against “4fection or disease caused by human papillomavirus. [N:\LIBAA]0120:TAB

12. Virus-like particles comprised of recombinant LI protein or recombinant LI L2 proteins of human papillomavirus 18, the virus-like particles being at least 60% pure, wherein the L1 protein has an amino acid sequence shown in Figure 1 and the L2 protein has an amino acid sequence shown in Figure 3.

13. The virus-like particles of Claim 12 wherein the recombinant L1 protein or the recombinant LI L2 proteins is produced in yeast.

14. A method of producing the virus-like particles of Claim 12 or Claim 13, the method comprising: transforming yeast with a recombinant DNA molecule encoding papillomavirus LI protein or papillomavirus L2 protein or papillomavirus L1 L2 proteins; cultivating the transformed yeast under conditions that permit expression of the recombinant DNA molecule to produce the recombinant papillomavirus protein; and isolating the recombinant papillomavirus protein to produce to virus-like particles of Claim 1.

15. Recombinant papillomavirus protein produced by the method of Claim 14.

16. A vaccine comprising the virus-like particles of Claim 12 or Claim 13.

17. Pharmaceutical compositions comprising the virus-like particles of Claim 12 or Claim 13.

18. A method for inducing an immune response in an animal comprising administering the virus-like particles of Claim 12 or Claim 13 to an animal.

19. The virus-like particles of Claim 12 or Claim 13 when used for inducing an immune response in an animal.

20. Use of the virus-like particles of Claim 12 or Claim 13 in the manufacture of a i. medicament for inducing an immune response in an animal.

21. A method of preventing papillomavirus infection the comprising administering the vaccine of Claim 16 to a host.

22. A method for producing a yeast-derived recombinant papillomovirus capsid protein assembled into a virus-like particle, comprising: a) cloning a papillomavirus gene that codes for at least one papillomavirus 30 capsid protein into a vector; b) transferring the vector into a host cell to produce a recombinant host cell; c) cultivating the recombinant host cell under conditions that permit production of papillomavirus capsid protein; and d) purifying the papillomavirus capsid protein under conditions that permit formation of a virus-like particle; wherein the papillomavirus capsid protein has an amino acid sequence selected from the sequence shown in Figure 1 and the sequence shown in Figure 3.

23. Virus-like particles produced by the method of Claim 22. [N:\LIBAA]0120:MCC 36

24. An isolated and purified DNA molecule which encodes human papillomavirus type 18 or a functional derivative thereof, substantially as herein before described with reference to any one of the Examples. A process for expression of a human papillomavirus type 18 protein in a host, substantially as hereinbefore described with reference to any one of the Examples.

26. Virus-like particles comprised of recombinant L1 protein or recombinant L1 L2 proteins of human papillomavirus 18, substantially as hereinbefore described with reference to any one of the Examples.

27. A process for producing virus-like particles comprised of recombinant L1 to protein or recombinant L1 L2 proteins of human papillomavirus 18, the virus-like particles being at least 60% pure substantially as hereinbefore described with reference to any one of the Examples

28. Recombinant papillomavirus protein substantially as hereinbefore described with reference to any one of the Examples.

29. A method for producing a yeast-derived recombinant papillomovirus capsid protein assembled into a virus-like particle substantially as hereinbefore described with reference to any one of the Examples. .Dated 20 October, 1999 20 Merck Co, Inc. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON O 9** 9 g** “2222 o* *o o oo o** e* [N:\LIBAA]0120:MCC

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2000-05-04
FGA
Letters patent sealed or granted (standard patent)

2021-04-08
MK14
Patent ceased section 143(a) (annual fees not paid) or expired

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