Title:  Parapoxviruses containing foreign DNA, their production and their use in vaccines

United States Patent:  6,365,393

Assignee:  Bayer Aktiengesellschaft (Leverkusen, DE)

Filed:  August 20, 1998

PCT NO:  PCT/EP97/00729

102(e) Date:  August 20, 1998

PCT PUB. Date:  September 4, 1997

Foreign Application Priority Data:  Feb 28, 1996[DE] (196 07 458); Sep 26, 1996[DE] (196 39 601)

The present invention relates to recombinantly prepared parapoxviruses which carry, in their genomes, deletions or insertions in the form of foreign hereditary information and contain hereditary information, to the preparation of such constructs and to their use in vaccines.

Description of the Invention

The present invention relates to recombinant parapoxviruses, to their preparation, and to vaccines and immunomodulators which contain them.

The novel, recombinantly altered parapoxviruses carry deletions and/or insertions in their genome. The deletion of segments of the genome of the parapoxviruses and/or the insertion of foreign DNA can lead to the reduction or loss of their pathogenicity (attenuation). Hereditary information from pathogens or biologically active substances is incorporated into the genome of the parapoxviruses by means of insertions. This foreign hereditary information is, as a constituent of the recombinant parapoxviruses, expressed, for example, in cell cultures, tissues or in intact organisms.

The recombinant parapoxviruses which have been prepared in accordance with the invention are employed, for example, in vaccines or immunomodulators. Expression of the foreign DNA in the genome of the parapoxviruses elicits, for example in a vaccinated individual, a defensive reaction against the pathogens which are represented by the foreign hereditary information. The non-specific resistances of the vaccinated individual can also be stimulated. (In that which follows, the term parapoxviruses is abbreviated to PPV).

PPV can themselves have an immunomodulatory effect since they stimulate non-pathogen-specific immune reactions in the organism. Thus, preparations of parapoxviruses are, for example, successfully employed in veterinary medicine for increasing general resistance.

While vaccines which have a pathogen-specific effect require several days to weeks, depending on the antigen, for establishing protection, they then provide long protection which lasts for months to years.

Consequently, vaccines which are prepared on the basis of recombinant parapoxviruses can be employed as biological products for the improved control of infectious diseases since they build up a long-lasting pathogen-specific immunity in the organism and also induce a non-pathogen-specific protection which sets in very rapidly.

The combination of the immunostimulatory properties of the PPV and the expression of foreign antigens which induce a homologous and/or heterologous pathogen-specific protection is novel. This permits the preparation of products which both mediate a rapid-onset, broad non-pathogen-specific protection against infections and also provide a long-lasting, pathogen-specific protection against infection.

The family of the vertebrate poxviruses (Chordopoxvirinae) is subdivided into individual, independent genera. The present invention relates to the genus of the PPV, which differ both structurally and genetically from the other poxviruses. The PPV are divided into three different species (Lit. #1):

Parapoxvirus ovis (also termed ecthyma contagiosum virus, contagious pustular dermatitis virus or orf virus), which is regarded as the prototype of the genus,

Parapoxvirus bovis 1 (also termed bovine papular stomatitis virus or stomatitis papulosa virus) and

Parapoxvirus bovis 2 (also termed udderpoxvirus, paravaccinia virus, pseudocowpox virus or milker's nodule virus).

Parapoxvirus representatives which have been isolated from camels, red deer, chamois, seals and sealions have also been described. Whether these viruses are autonomous species within the parapoxvirus genus or whether they are isolates of the above-described species has still not been finally clarified.

Infections with PPV can elicit local diseases in both animals and man (zoonotic pathogens). Lit. #1 provides an overview of the syndromes which have so far been described. Prophylactic measures, such as vaccines, can be used to control the diseases. However, the activity of the vaccines which have thus far been obtainable, and which have been developed exclusively on the basis of Parapoxvirus ovis, is unsatisfactory (Lit. #2).

The invention relates to using PPV as a vector for foreign genetic information which is expressed.

Vectors based on avipox, racoonpox, capripox, swinepox or vaccinia virus have already been described as vectors for expressing foreign genetic information. The insights which have been gained in this connection cannot be transferred to PPV. As comparative investigations have demonstrated, there are morphological, structural and genetic differences between the individual genera of the poxviruses. Thus, serological methods can, for example, be used to differentiate the PPV from other poxvirus genera, a fact which is attributable to different protein patterns and to different hereditary information which is associated with this. For example, some representatives of the poxviruses have the ability to agglutinate erythrocytes. This activity is mediated by way of a surface protein, the so-called haemagglutinin (HA). PPV do not possess this activity.

Knowledge of the organization of the PPV genome is currently restricted to determinations of the size of the genome, the GC content of the nucleic acid, comparative restriction enzyme analyses, the cloning of individual genome fragments, and sequence analyses of part regions and the associated preliminary description of individual genes (for a review, see Lit. #1, Lit. #5, Lit. #6).

It is not currently possible to use insertion sites which are known in the case of vaccinia due to the fact that these sites are either lacking or have not been demonstrated in PPV.

Thus, attempts to identify the gene for thymidine kinase in the PPV genome and to use it as an insertion site, as in the case of the orthopoxviruses, were not successful. While Mazur and coworkers (Lit. #3) describe the identification of a segment of the PPV genome which they claim resembles the thymidine kinase gene of vaccinia virus (an orthopoxvirus), our own extensive investigations have not been able to confirm the existence of such a gene in PPV. Other authors (Lit. #1) have also not been able to find a thymidine kinase gene in PPV. The gene for HA is used as an insertion site for foreign DNA in vaccinia virus. As described above, PPV do not possess this activity.

In 1992, Robinson and Lyttle mentioned alternative insertion sites on the PPV genome (Lit. #1) without, however, providing a description or a precise characterization of these sites. There has furthermore still not been any description of the successful use of PPV as vectors.

In our own analytical investigations of the sequence of HindIII fragment I from PPV strain D1701, we found an ORF which possesses amino acid homology (36.1 to 38.3% identity; 52.8 to 58.6% similarity, GCG, Wisconsin Package 8.1, e.g. Pikup Program) with vascular endothelial growth factor (VEGF) from various mammalian species (e.g. mouse, rat, guinea pig, cow and man). Seq. ID No: 1 shows the nucleotide sequence of the gene in D1701, while Seq. ID No: 15 shows the amino acid sequence of the corresponding D1701 protein. Recently, a homologous gene was also described in PPV strains NZ2 and NZ7 (Lit. #6); however the function of this gene is not known. Other poxviruses, e.g. orthopoxviruses, are not known to have a corresponding gene. In the remainder of the text, this gene is termed VEGF gene.

Our sequence analysis of HindIII fragment I of D1701 led to the identification of another ORF which possesses homology with orthopoxvirus protein kinase genes and is known in vaccinia as F10L. The identity with the vaccinia F10L gene is 51% while the similarity is 70%. In the remainder of the application, this gene is termed PK gene. Seq. ID No: 2, No: 9 and No: 13 show versions of the nucleotide sequence of the gene in D1701, while Seq. ID No: 14 shows the amino acid sequence of the corresponding D1701 protein.

An additional ORF was found which overlaps the 3' end of the PK gene and the 5' end of the VEGF gene. Homology investigations showed that there was low identity (28%) and low similarity (51%) with the F9L gene in vaccinia. Seq. ID No: 5 and No: 10 show versions of the nucleotide sequence of the gene in D1701. In the remainder of the text, this gene is termed the F9L gene.

A further ORF, which, due to its similarity to a gene in PPV NZ2 (identity 76%, similarity 83%), is termed ORF3, was found within the ITR region. Seq. ID No: 4 shows the nucleotide sequence of the gene in D1701. In the remainder of the text, this gene is termed ORF3 gene.

The present invention relates to

1. Recombinantly prepared PPV having insertions and/or deletions.

2. Recombinantly prepared PPV having insertions and/or deletions in genome segments which are not required for virus multiplication.

3. Recombinantly prepared PPV having insertions and/or deletions in genome segments which are required for virus multiplication.

4. Recombinantly prepared PPV which contain insertions and/or deletions in the regions of HindIII fragment I from D1701 which are not expressed.

5. Recombinantly prepared PPV which contain insertions and/or deletions in the regions of HindIII fragment I from D1701 which are expressed.

6. Recombinantly prepared PPV according to 1 to 5, in which insertions and/or deletions are located in D1701 HindIII fragment I or in the DNA from other PPV which corresponds to this fragment.

7. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the VEGF gene or adjoining this region.

8. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the PK gene or adjoining this region.

9. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the ITR segment or adjoining this region.

10. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the HD1R gene or adjoining this region.

11. Recombinantly prepared PPV which contain insertions and/or deletions in the region of the F9L gene or adjoining this region.

12. Recombinantly prepared PPV which contain insertions and/or deletions in the region, or in the vicinity, of the gene which encodes the 10 kDa protein.

13. Recombinantly prepared PPV which contain insertions and/or deletions in the region of EcoRI fragment E from D1701, in which the gene encoding the 10 kDa protein is located.

14. Plasmid which contains HindIII fragment I from D1701 or DNA from other PPV which corresponds to this fragment.

15. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in regions which are required for virus replication.

16. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in regions which are not required for virus replication.

17. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in the regions which are not required for virus replication and which are not expressed.

18. Plasmid which contains HindIII fragment I from D1701 and which, in this fragment, contains deletions and/or insertions in the regions which are not required for virus multiplication and which lie in regions which are expressed.

19. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the VEGF gene of this fragment.

20. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the PK gene of this fragment.

21. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the ITR segment of this fragment.

22. Plasmid which contains HindIII fragment I from D1701 and which contains deletions and/or insertions in, or adjacent to, the HD1R gene and/or the F9L gene.

23. Plasmid which contains EcoRI fragment E from D1701 and which contains deletions and/or insertions in, or adjacent to, the gene which encodes the 10 kDa protein.

24. Plasmid which contains part of HindIII fragment I from D1701 in which deletions and/or insertions in accordance with 14 to 23 are present.

25. Plasmid according to 14 to 24, in which the DNA fragment from D1701 is replaced with a DNA from other PPV which corresponds to this fragment.

26. Plasmid according to 14 to 25, which either contains the whole of HindIII fragment I or only a part of it.

27. D1701 HindIII fragment I, or parts thereof, or fragments from other PPV which correspond to this fragment, having the sequence according to sequence listing ID No: 8 or No: 12.

28. DNA segment or parts of D1701 HindIII fragment I, or the segment from other PPV which corresponds to this segment, or parts thereof, which encodes VEGF protein in accordance with sequence listing ID No: 1.

29. DNA segment or parts of D1701 HindIII fragment I, or the segment from other PPV which corresponds to this segment or parts thereof, which encodes PK protein according to sequence listing ID No: 2, No: 9 or No: 13.

30. DNA segment, or parts thereof, for the HD1R gene having the sequence according to sequence listing ID No: 3 of PPV.

31. DNA segment, or parts thereof, for F9L having the sequence according to sequence listing ID No: 5 or ID No: 10 of PPV.

32. DNA segment, or parts thereof, for the ITR region having the sequence according to sequence listing ID No: 4 of PPV.

33. Gene products which have been prepared on the basis of the sequences of the DNA segments according to 27 to 32.

34. Recombinantly prepared PPV according to 1 to 13 which contain, as insertions, foreign DNA which encodes immunogenic constituents from other pathogens.

35. Recombinantly prepared PPV according to 1 to 13 and 34 which contain, as insertions, foreign DNA which encodes cytokines.

36. Process for preparing the viruses according to 1 to 13, 34 and 35, characterized in that the plasmids according to 14 to 26 are recombined with PPV in cells in the manner known per se and selected for the desired viruses.

37. Process for preparing the plasmids according to 23, characterized in that

1. a suitable PPV strain is selected,

2. its genome is purified,

3. the purified genome is treated with restriction enzymes,

4. the resulting fragments are inserted into plasmids, and

5. selection is carried out for the plasmids which contain the gene which encodes the 10 kDa protein, and

6. where appropriate, insertions and/or deletions are introduced into the gene encoding the 10 kDa protein,

7. the fragments described under 4 (above) can, where appropriate, also be prepared using alternative methods such as polymerase chain reaction (PCR) or oligonucleotide synthesis.

38. Process for preparing the plasmids according to 14 to 22 and 24 to 26, characterized in that

1. a suitable PPV strain is selected,

2. its genome is purified,

3. the purified genome is treated with restriction enzymes,

4. the resulting fragments are inserted into plasmids, and

5. selection is carried out for the plasmids which contain HindIII fragment I or fragments or constituents which correspond to this fragment,

6. and, where appropriate, insertions and/or deletions are introduced into these fragments in the resulting plasmids.

7. the fragments described under 4 (above) can, where appropriate, also be prepared using alternative methods such as polymerase chain reaction (PCR) or oligonucleotide synthesis.

39. Process for preparing D1701 HindIII fragment I or EcoRI fragment E, which encodes the 10 kDa protein, or the region from other PPV which corresponds to this fragment or segment, or parts thereof, characterized in that

1. a suitable PPV strain is selected,

2. its genome is purified,

3. the purified genome is treated with restriction enzymes,

4. and the desired fragments or segments are selected, or

5. where appropriate, the resulting fragments of the genome are initially inserted in plasmids and the plasmids containing the desired fragments are isolated, after which these plasmids are multiplied and the desired fragments are isolated from them.

6. the fragments described under 4 (above) can, where appropriate, also be prepared using alternative methods such as PCR or oligonucleotide synthesis.

40. Process for preparing the gene products according to 33, characterized in that the fragments obtainable in accordance with 39 are transferred into suitable expression systems and the genes are expressed using these systems.

41. Use of the recombinantly prepared PPV according to 1 to 13 in vaccines.

42. Use of the recombinantly prepared PPV according to 1 to 13 in products which both immunize and stimulate non-pathogen-specific immune defence.

43. Use of the recombinantly prepared PPV in immunomodulators which stimulate non-pathogen-specific immune defence.

44. Use of the recombinantly prepared PPV for heterologously expressing foreign DNA.

45. Use of the recombinantly prepared PPV as vectors for foreign DNA.

46. Use of the plasmids according to 14 to 16 for expressing parapox-specific genome segments.

47. Use of the plasmids according to 14 to 26 for preparing diagnostic agents.

48. Use of the genome fragments according to 27 to 32 for preparing diagnostic agents.

49. DNA segments according to sequence listing ID No: 6 (promoter of the VEGF gene).

50. Use of the DNA segment according to 49 as a promoter for expressing DNA.

The above-described genome fragments of PPV, which can be inserted into plasmids or viruses and which can be present as free DNA segments, encompass the given DNA sequences and their variants and homologs.

Claim 1 of 10 Claims

What is claimed is:

1. A recombinantly prepared parapoxvirus being derived from parapoxvirus strain D 1701 deposited under Reg. No. CNCM 1-751 and containing at least one insertion of a foreign DNA element within the Hind III fragment I of parapoxvirus strain D 1701, said Hind III fragment I having a size of about 5.6 kbp.

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