EP0972047A2

EP0972047A2 - Papilloma virus main capsid protein and the use thereof in diagnosis, therapy and vaccination

Info

Publication number: EP0972047A2
Application number: EP98928070A
Authority: EP
Inventors: Ethel-Michele De Villiers-Zur Hausen; Harald Zur Hausen; Donna Lavergne; Claire Benton
Original assignee: Deutsches Krebsforschungszentrum DKFZ
Current assignee: Deutsches Krebsforschungszentrum DKFZ
Priority date: 1997-03-25
Filing date: 1998-03-24
Publication date: 2000-01-19
Also published as: WO1998042847A3; JP2001520519A; DE19712541C1; US6610303B1; WO1998042847A2

Abstract

The invention relates to a DNA that codes the peptide of a papilloma virus main capsid protein or a papilloma virus genome. The invention further relates to the proteins coded by the papilloma virus genome and the antibodies directed against said proteins as well as to their use in diagnosis, therapy and vaccination.

Description

PAPILLOMVIRUS MAIN CAPSID PROTEINS AND THEIR USE IN DIAGNOSIS, THERAPY AND VACCINATION

The invention relates to a DNA which codes for a peptide of a papillomavirus main capsid protein or a papillomavirus genome. Furthermore, the invention relates to proteins encoded by the papilloma virus genome and antibodies directed against them, and to their use in diagnosis, therapy and vaccination.

Papillomaviruses are known to infect human and animal epithelial tissue. Human papilloma viruses (hereinafter referred to as HP viruses) are found in benign, e.g. Warts, genital condylomas, and malignancies, e.g. Carcinomas of the skin and uterus, epithelial neoplasms (see Zur Hausen, H., Biochimica et Biophysica Acta (BBA) 1 288, (1 996), pages 55-78). HP viruses are also being considered for the development of malignant tumors in the oropharyngeal area (cf. Zur Hausen, H., Curr. Top. Microbiol. Immunol. 78, (1 977), pages 1-30).

Papilloma viruses have an icosahedral capsid without a shell, in which a circular, double-stranded DNA molecule of approximately 7900 bp is present. The capsid comprises a major capsid protein (L1) and a minor capsid protein (L2). Both proteins, coexpressed or L1 expressed alone, lead to the formation of virus-like particles in vitro (cf. Kirnbauer, R. et al., Journal of Virology, (1 993), pages 6929-6936).

Papilloma viruses cannot be propagated in monolayer cell culture. Their characterization is therefore extremely difficult, and the detection of papilloma viruses already creates considerable problems. This is particularly true for papilloma viruses in skin carcinomas. The object of the present invention is therefore to provide an agent with which papilloma viruses, in particular in carcinomas of the skin, can be detected. A means should also be provided to treat these papillomaviruses therapeutically.

According to the invention, this is achieved by the provision of the subject matter in the claims.

The invention thus relates to a DNA coding for a peptide of a papillomavirus main capsid protein (L1), the peptide representing the amino acid sequence of FIGS. 1, 2, 3, 4, 5, 6 , Fig. 7, Fig. 8 or Fig. 9 or one of them by one or more amino acids different amino acid sequence.

Another object of the invention is a DNA coding for a peptide of a papillomavirus main capsid protein, the DNA being the base sequence of FIG. 1, FIG. 2, FIG. 3, FIG. 4, FIG. 5, FIG. 6, FIG 7, 8 or 9, or a base sequence different from one or more base pairs.

1 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL231 at the DSM (German Collection of Microorganisms and Cell Cultures) under DSM 1 1405 on February 13, 1 997.

FIG. 2 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL250 at the DSM under DSM 1 1 406 on 1 February 3, 1 997.

FIG. 3 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. That DNA was deposited as plasmid DL253 at DSM under DSM 1 1407 on Feb. 3, 1 997.

FIG. 4 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL267 with the DSM under DSM 1 1 408 on February 3, 1 997.

FIG. 5 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was plasmid DL284 at DSM under DSM 1 1409 on the 3rd of February. 1 997 deposited.

FIG. 6 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL285 with the DSM under DSM 1 1 41 0 on 1 February 3, 997.

FIG. 7 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL287 at DSM under DSM 1 141 1 on February 13, 1 997.

FIG. 8 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide from L1 of a papilloma virus. This DNA was deposited as plasmid DL297 at the DSM under DSM 1 141 2 on 1 February 3, 1 997.

FIG. 9 shows the base sequence and the amino acid sequence derived therefrom of a DNA coding for a peptide of L1 of a papilloma virus. This DNA was deposited as plasmid DL332 at the DSM under DSM 1 141 3 on February 1, 1 997. The above DNA was compared to the DNA of known papilloma viruses. Sequence homology studies were carried out. A homology that is less than 90% shows a DNA according to the invention as a new HP virus. The DNAs according to the invention have the following sequence homologies with known papilloma viruses:

DNA of Fig. 1 77% to HP virus 5c

DNA of Fig. 2 80% to HP virus 9

DNA of Figure 3 76% to HP virus 38

DNA of Fig. 4 79% to HP virus 38

DNA of Fig. 5 74% to HP virus 20

DNA of Fig. 6 77% to HP virus 9

DNA of Fig. 7 73% to HP virus 20

DNA of Fig. 8 87% to HP virus 5b

DNA of Fig. 9 78% to HP virus 5b

According to the invention, the above DNA can be present in a vector or expression vector. Examples of such are known to the person skilled in the art. In the case of an expression vector for E. coli, these are e.g. pGEMEX, pUC derivatives, pGEM-T and pGEX-2T. For expression in yeast e.g. to call pYl OO and Ycpad l, while for expression in animal cells e.g. pKCR, pEF-BOS, cDM8 and pCEV4 must be specified.

The person skilled in the art knows suitable cells in order to express the above DNA present in an expression vector. Examples of such cells include the E. coli strains HB101, DH1, x1776, JM101, JM 109 and XU-Blue, the yeast strain Saccharomyces cerevisiae and the animal cells L, NH-3T3, FM3A, CHO, COS, Vero, and Hey.

The person skilled in the art knows how the above DNA has to be inserted into an expression vector. He is also aware that the above DNA in Connection with a DNA coding for another protein or peptide can be inserted so that the above DNA can be expressed in the form of a fusion protein.

Another object of the invention is a papilloma virus genome comprising the above DNA. The term "papilloma virus genome" also includes an incomplete genome, i.e. Fragments of a papilloma virus genome comprising the above DNA. This can e.g. a DNA coding for L1 or a part thereof.

A common method can be used to provide the above papillomavirus genome. A method comprising the following method steps is favorable:

(a) isolation of the total DNA from a biopsy of epithelial neoplasm,

(b) hybridizing the total DNA of (a) with the above DNA, thereby detecting a papillomavirus genome contained in the total DNA of (a), and

(c) Cloning of the total DNA from (a) containing the papillomavirus genome in a vector, and optionally subcloning of the clone obtained, all process steps originating from conventional DNA recombination technology.

With regard to the isolation, hybridization and cloning of Zeil DNA, reference is also made to Sambrook et al., Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory (1,989).

The term "epithelial neoplasm" encompasses any neoplasms of epithelial tissue in humans and animals. Examples of such neoplasms are warts, condylomas in the genital area and carcinomas of the skin. The latter are lying preferably used to isolate the above papillomavirus genome.

The term "vector" includes any vector suitable for cloning chromosomal or extrachromosomal DNA. Examples of such vectors are cosmids such as pWE1 5 and Super Cos1, and phages such as Λ-phages, e.g. ΛZAP Expressvector, ΛZAPII Vector and Λgt10 Vector. In the present case, Λ phages are preferably used. The above vectors are known and are available from Stratagene.

Papillomavirus genomes according to the invention can be integrated in chromosomal DNA or extrachromosomal. Methods are known to the person skilled in the art to clarify this. He also knows how to find the optimal restriction enzymes for cloning the papillomavirus genomes. It will be based on genomes of known papilloma viruses. In particular, the person skilled in the art will observe the aforementioned HP viruses accordingly.

The provision of a papilloma virus genome designated DL231 -G is described by way of example. For this purpose, the total DNA is isolated from a biopsy of a squamous cell carcinoma, cleaved with BamHI and electrophoretically separated in an agarose gel. The agarose gel is then subjected to a blotting process, whereby the DNA is transferred to a nitrocellulose membrane. This is used in a hybridization process in which the DNA from FIG. 1, possibly in combination with a DNA from HP virus 5c, is used as the labeled sample. Hybridization with the papilloma virus DNA present in the total DNA is obtained.

Furthermore, the above total DNA cleaved with BamHI is cloned in a Λ phage. The corresponding clones, ie the clones containing the papillomavirus DNA, are identified by hybridization with the DNA from FIG. 1, possibly in combination with a DNA from the HP virus 5c. The insert of these clones is then subjected to a further cloning in a plasmid vector, whereby a clone is obtained which contains the papillomavirus genome DL231 -G. The genome is confirmed by sequencing.

Analogously, further papillomavirus genomes are provided. They are named according to the DNAs used for their preparation, with: DL250-G, DL253-G, DL267-G, DL284-G, DL285-G, DL287-G, DL297-G or DL332-G.

Another object of the invention is a protein encoded by the above papillomavirus genome. Such a protein is e.g. a major capsid protein (L1) or a minor capsid protein (L2). The above protein is produced in a conventional manner. The production of L1 or L2 of the papilloma virus genome DL231 -G is described as an example. The HP virus 5c, which is related to the DNA of FIG. 1, is used for this purpose. The complete sequence and the position of individual DNA regions coding for proteins are known from this. These DNAs are identified on the papillomavirus genome DL231 -G by parallel restriction cleavages of both genomes and subsequent hybridization with different fragments relating to L1 or L2 coding DNA. They are confirmed by sequencing. The DNA coding for L1 is called DL231 -G-L1 DNA and the DNA coding for L2 is called DL231 -G-L2 DNA.

Furthermore, the DNA coding for L1 or L2 is inserted into an expression vector. Examples of such for E. coli, yeast and animal cells are mentioned above. In particular, reference is made to the vector pGEX-2T for expression in E. coli (cf. Kirnbauer, R. et al., Supra). After insertion of the DL231 -G-L1 - or DL231 -G-L2-DNA, pGEX-2T-DL231 -G-L1 or pGEX-2T-DL1 7-G-L2 is obtained. After transformation of E. coli, these expression vectors express a glutathione S-transferase-L1 or glutathione S-transferase-L2 fusion protein. These proteins are purified in the usual way.

For a further expression of the above coding L1 or L2 DNA, the Bacculovirus or vaccinia virus system called. Expression vectors that can be used for this are, for example, pEV mod. and pSynwtVI ^" for the bacculovirus system (cf. Kirnbauer, R. et al., supra). For the vaccinia virus system, vectors with the vaccinia virus are in particular" early "(p7. 5 k) - or" late "( Psynth, pl 1 K) promoter (cf. Hagensee, M., E. et al., Journal of Virology (1 993), pages 31 5-322), in the present case the bacculovirus system is preferred DNA encoding L1 or L2 in pEV mod. PEVmod.-DL231 -G-L1 or pEVmod.-DL231 -G-L2 is obtained.

The former expression vector alone or both expression vectors together lead to the formation of virus-like particles after infection of SF-9 insect cells. In the former case such a particle comprises an L1 protein, while in the latter case it contains an L1 protein as well as an L1 protein.

A virus-like particle of the latter case is also obtained by inserting the above DL231 -G-L1 and DL231 -G-L2 DNAs together into the expression vector pSynwtVI ^" and the obtained pSynwtVI ^" DL231 -G-L1 / L2 for infection of SF-9 insect cells is used. The above virus-like particles are cleaned in the usual way. They also represent an object of the invention.

Another object of the invention is an antibody directed against an above protein or virus-like particle. Such is produced in the usual way. It is described by way of example for the production of an antibody which is directed against a virus-like particle comprising L1 of DL231 -G. For this purpose, the virus-like particle BALB / c mice is injected subcutaneously. This injection is repeated every 3 weeks. About 2 weeks after the last injection, the serum containing the antibody is isolated and tested in the usual way.

In a preferred embodiment, the antibody is a monoclonal antibody. After the fourth injection above, the mice are produced Spleen cells removed and fused with myeloma cells in the usual way. The further cloning is also carried out according to known methods.

The present invention makes it possible to detect papilloma viruses, in particular in carcinomas of the skin. For this purpose, the DNA according to the invention can be used as such or encompassed by a further DNA. The latter can also be a papilloma virus gome or part of it.

The present invention also enables the provision of previously unknown papilloma viruses. These are found particularly in carcinomas of the skin. Furthermore, the invention provides proteins and virus-like particles which are due to these papillomaviruses. Antibodies are also provided which are directed against these proteins or particles.

The present invention thus makes it possible to take diagnostic and therapeutic measures for papillomavirus diseases. In addition, it provides the opportunity to build a vaccine against papillomavirus infections. The present invention thus represents a breakthrough in the field of papilloma virus research.

The invention is illustrated by the examples.

Example 1: Identification of the papilloma virus genome DL231 -G

The total DNA is isolated from a biopsy of Verruca vulgaris. 10 yg of this DNA are cleaved with the BamHI restriction enzyme and electrophoresed in a 0.5% agarose gel. At the same time, 10μg of the above DNA, which has not been cleaved, are also separated. The agarose gel is subjected to a blotting process, whereby the DNA from the agarose gel is transferred to a nitrocellulose membrane. This is used in a hybridization process in which the above 1 in combination with HP virus 5c DNA is used as the p ³² -labeled sample. Hybridization with the blotted DNA is obtained.

The above methods are known to those skilled in the field of DNA recombination technology. In addition, reference is made to Sambrook et al., Supra.

Example 2: Cloning of the papilloma virus genome DL231 -G

The biopsy DNA obtained from Example 1 is cleaved with the restriction enzyme BamHI. The fragments obtained are used in a ligase reaction in which the BamHI-cleaved and dephosphorylated vector ΛZAP Express is also present. The recombinant DNA molecules obtained in this way are packaged in bacteriophages and used to infect bacteria. The ZAP Express Vector Kit offered by Stratagene is used for these process steps. The phage plaques obtained are then subjected to a hybridization process in which the p ³² -labeled DNA from FIG. 1 used in Example 1 is used in combination with p ³² -labeled HP virus 5c DNA. Hybridization with corresponding phage plaques is obtained. The BamHI fragments of DL231 -G are isolated from these and used together with a BamHI-cleaved, dephosphorylated plasmid vector, pBluescript, in a further ligase reaction. The recombinant DNA molecules obtained are used to transform bacteria, E. coli XL1-Blue. A bacterial clone containing the papillomavirus genome DL231 -G is identified by restriction cleavage or hybridization with the above DNA samples. The plasmid of this bacterial clone is designated pBlue-DL231 -G.

Claims

claims

DNA coding for a peptide of a papillomavirus main capsid protein, the peptide having the amino acid sequence of FIGS. 1, 2, 3, 4, 5, 6, 7, 8 or FIG. 9 or an amino acid sequence different from one or more amino acids, and the DNA being obtainable by the following method steps:

(a) isolation of the total DNA from a biopsy of epithelial neoplasm,

(b) Hybridization of the total DNA of (a) with a DNA from FIGS. 1, 2, 3, 4, 5, 6, 7, 8 or 9, whereby a papilloma virus genome contained in the total DNA of (a) is detected, and

(c) cloning the total DNA of (a) containing the papillomavirus genome in a vector and sequencing the clone.

DNA according to claim 1, characterized in that the DNA coding for the peptide of the papillomavirus main capsid protein has the base sequence of Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6. Fig. 7, FIG. 8 or FIG. 9 or a base sequence different from one or more base pairs, and the DNA being obtainable by the following method steps:

(a) isolation of the total DNA from a biopsy of epithelial neoplasm,

(b) Hybridization of the total DNA from (a) with a DNA from FIG. 1, FIG.

2, 3, 4, 5, 6, 7, 8 or 9, whereby a papilloma virus genome contained in the total DNA of (a) is detected, and (c) cloning the total DNA of (a) containing the papillomavirus genome in a vector and sequencing the clone.

3. DNA according to claim 1 or 2, characterized in that the DNA comprises a papilloma virus genome.

4. Protein encoded by the papilloma virus genome according to claim 3.

5. Protein according to claim 4, characterized in that the papillomavirus main capsid protein is present as a virus-like particle.

6. Protein according to claim 5, characterized in that the virus-like particle also contains a papilloma virus secondary capsid protein.

7. Expression vector comprising a DNA coding for the protein according to claim 4.

8. transformant containing the expression vector according to claim 7.

9. A method for producing the protein of claim 4, comprising culturing the transformant of claim 8 under suitable conditions.

10. Antibody directed against the protein according to any one of claims 4-6.

1 1. Use of the DNA according to one of claims 1 -3 as a reagent for diagnosis.

1 2. Use of the protein according to any one of claims 4-6 as a reagent for diagnosis, therapy and / or vaccination.

1 3. Use according to claim 1 1 or 1 2, wherein the diagnosis of papilloma virus infections or diseases.

4. Use according to claim 1 2, wherein the therapy and / or vaccination relates to papillomavirus infections or diseases.