Recombinant papilloma virus L1

• US 6,013,262 A
• Filed: 01/16/1997
• Issued: 01/11/2000
• Est. Priority Date: 05/17/1994
• Status: Expired due to Term

• Current Assignee: The University of Queensland
• Original Assignee: The University of Queensland
• Inventors: Zhou, Jian, Frazer, Ian
• Primary Examiner(s): Mosher, Mary E.
• Assistant Examiner(s): SALIMI, ALI REZA
• Application Number:
  US08/737,336
• Time in Patent Office: 1,090 Days
• Field of Search: 435/69.1, 435/5, 435/7.1, 435/69.3, 435/252.3, 435/325.1, 435/320.1, 435/235.1, 435/253.33, 530/350, 530/403, 536/23.72, 424/204.1
• US Class Current: 424/204.1
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 16/084 Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 2710/20022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2710/20023 Virus like particles [VLP]
• CPC Class Codes: Y10S 977/917 Vaccine
• CPC Class Codes: Y10S 977/918 Immunological
• CPC Class Codes: Y10S 977/92 Detection of biochemical

Abstract

This invention relates to a recombinant papilloma virus L1 protein which can elicit an immune response which recognises papilloma virus VLP including L1 protein and can form extracellularly a multimeric structure or VLP wherein the multimeric structure comprises a plurality of recombinant papilloma virus L1 proteins. This invention also includes the use of the recombinant papilloma virus L1 protein to detect the presence of papilloma virus and can form the basis of a vaccine for prophylactic and therapeutic use.

Claims

1. A recombinant papilloma virus L1 protein which has an amino acid sequence shown in FIGS. 1(a)-1(c) (SEQ ID NO:

6).

2. A multimeric structure comprising a plurality of recombinant papilloma virus L1 proteins, wherein each of said recombinant papilloma virus L1 proteins is a recombinant papilloma virus L1 protein as claimed in claim 1.

3. A multimeric structure as claimed in claim 2, which is able to induce an immune response that recognizes papilloma virus VLP.

4. A multimeric structure as claimed in claim 2, wherein the multimeric structure is a pentamer.

5. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein according to claim 1;

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein.

6. A method as claimed in claim 5, wherein the nucleotide sequence encoding said papilloma L1 virus protein is inserted into pTrcHiSB in a correct reading frame with respect to expression of the papilloma virus L1 protein;

and wherein said papilloma virus L1 protein is produced by a strain of E. coli.

32. A vaccine comprising one or more multimeric structures as claimed in any one of claims 2, 3 or 4, together with a pharmaceutically acceptable vaccine adjuvant.

7. A recombinant papilloma virus L1 protein which has an N terminal amino acid sequence including:

MetArgGlySerHisHisHisHisHisHisGlyMetAlaSerMetThrGlyGlyGlnMetGlyArgAspLeuTyrAspAspAspAspLysAsp (SEQ ID NO;

1).

8. A multimeric structure comprising a plurality of recombinant papilloma virus L1 proteins, wherein each of said recombinant papilloma virus L1 proteins is a recombinant papilloma virus L1 protein as claimed in claim 7.

9. A multimeric structure as claimed in claim 8, which is able to induce an immune response that recognizes papilloma virus VLP.

10. A multimeric structure as claimed in claim 8, wherein the multimeric structure is a pentamer.

11. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein according to claim 7;

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein.

12. A method as claimed in claim 11, wherein the nucleotide sequence encoding said papilloma virus L1 protein is inserted into pTrcHisB in a correct reading frame with respect to expression of the papilloma virus L1 protein;

and wherein said papilloma virus L1 protein is produced by a strain of E. coli.

31. A vaccine comprising one or more multimeric structures as claimed in any one of claims 8, 9 or 10, together with a pharmaceutically acceptable vaccine adjuvant.

13. A multimeric structure comprising a plurality of recombinant papilloma virus L1 proteins, wherein each of said recombinant papilloma virus L1 proteins has an N terminal amino acid sequence including (His)₆.

14. A multimeric structure as claimed in claim 13 which is able to induce an immune response that recognises papilloma virus VLP.

15. A multimeric structure as claimed in claim 13 wherein the multimeric structure is a pentamer.

16. The multimeric structure of claim 13, wherein said recombinant papilloma virus L1 proteins are bacterially-expressed.

30. A vaccine, comprising one or more multimeric structures as claimed in any one of claims 13, 14 or 15, together with a pharmaceutically acceptable vaccine adjuvant.

17. A recombinant DNA molecule having a nucleotide sequence selected from any one of the following:

(a) A nucleotide sequence encoding a recombinant papilloma virus L1 protein and having a 5'"'"' nucleotide sequence including;

ATGCGGGGTTCTCATCATCATCATCATCATGGTATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTACGACGATGACGATAAGGAT (SEQ ID NO;

2);

(b) A nucleotide sequence that is complementary to the sequence in (a); and

(c) A nucleotide sequence that is a synonymous sequence to the sequence in (a).

18. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein comprising an N terminal amino acid sequence including (His)₆ ;

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein,

wherein said recombinant DNA molecule is the recombinant DNA molecule as claimed in claim 17.

19. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein comprising an N terminal amino acid sequence including;

MetArgGlySerHisHisHisHisHisHisGlyMetAlaSerMetThrGlyGlyGlnMetGlyArgAspLeuTyrAspAspAspAspLysAsp (SEQ ID NO;

1);

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein

wherein said recombinant DNA molecule is the recombinant DNA molecule as claimed in claim 17.

20. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein comprising an amino acid sequence as shown in FIGS. 1(a)-1(c) (SEQ ID NO;

6);

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein,

wherein said recombinant DNA molecule is the recombinant DNA molecule as claimed in claim 17.

21. A recombinant DNA molecule encoding a recombinant papilloma virus L1 protein and having a 5'"'"' nucleotide sequence which includes six repeats of the trinucleotide sequence CAT wherein the recombinant DNA molecule has a nucleotide sequence as shown in FIGS. 1(a)-1(c) (SEQ ID NO:

5).

22. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein comprising an N terminal amino acid sequence including (His)₆ ;

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein,

wherein said recombinant DNA molecule is the recombinant DNA molecule as claimed in claim 21.

23. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein comprising an N terminal amino acid sequence including;

MetArgGlySerHisHisHisHisHisHisGlyMetAlaSerMetThrGlyGlyGlnMetGlyArgAspLeuTyrAspAspAspAspLysAsp (SEQ ID NO;

1);

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein

wherein said recombinant DNA molecule is the recombinant DNA molecule as claimed in claim 21.

24. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein comprising an amino acid sequence as shown in FIGS. 1(a)-1(c) (SEQ ID NO;

6);

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein,

wherein said recombinant DNA molecule is the recombinant DNA molecule as claimed in claim 21.

25. A method for preparing a multimeric structure, comprising:

(i) expressing in a bacterium a recombinant DNA molecule which encodes a recombinant papilloma virus L1 protein which has an N terminal amino acid sequence including (His)₆ ;

(ii) substantially purifying said recombinant papilloma virus L1 protein; and

(iii) forming said multimeric structure extracellularly from a plurality of molecules of said papilloma virus L1 protein.

26. The method of claim 25, wherein said DNA molecule comprises a 5'"'"' nucleotide sequence having six repeats of the trinucleotide sequence CAT.

27. The method of claim 26, wherein said N terminal amino acid sequence encoding by said DNA comprises:

MetArgGlySerHisHisHisHisHisHisGlyMetAlaSerMetThrGlyGlyGlnMetGlyArgAspLeuTyrAspAspAspAspLysAsp (SEQ ID NO;

1).

28. The method of claim 26, wherein said N terminal amino acid sequence encoded by said DNA comprises an amino acid sequence as shown in FIGS. 1(a)-1(c) (SEQ ID NO:

6).

29. A method as claimed in claim 25, wherein the nucleotide sequence encoding said papilloma virus L1 protein is inserted into pTrcHisB in a correct reading frame with respect to expression of the papilloma virus L1 protein;

and wherein said papilloma virus L1 protein is produced by a strain of E. coli.