Chemistry: molecular biology and microbiology – Virus or bacteriophage – except for viral vector or...
Reexamination Certificate
1998-08-20
2002-04-02
Mosher, Mary E. (Department: 1648)
Chemistry: molecular biology and microbiology
Virus or bacteriophage, except for viral vector or...
C435S320100, C424S199100
Reexamination Certificate
active
06365393
ABSTRACT:
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
Büttner Mathias
Rziha Hans-Joachim
Schmeer Norbert
Strube Walter
Akorli Godfried R.
Bayer Aktiengesellschaft
Gil Joseph C.
Mosher Mary E.
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