Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
2000-04-07
2002-09-17
Stucker, Jeffrey (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S205100, C424S233100, C435S069100, C435S235100, C435S320100
Reexamination Certificate
active
06451319
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to viral vectors for vaccination of animals. In particular, the present invention pertains to viral vectors having insertion sites for the introduction of foreign DNA.
BACKGROUND OF THE INVENTION
The adenoviruses cause enteric or respiratory infection in humans as well as in domestic and laboratory animals.
Inserting genes into adenoviruses has been accomplished. In the human adenovirus (HuAd) genome there are two important regions: E1 and E3 in which foreign genes can be inserted to generate recombinant adenoviruses.
This application of genetic engineering has resulted in several attempts to prepare adenovirus expression systems for obtaining vaccines. Examples of such research include the disclosure of U.S. Pat. No. 4,510,245 of an adenovirus major late promoter for expression in a yeast host; U.S. Pat. No. 4,920,209 of a live recombinant adenovirus type 7 with a gene coding for hepatitis-B surface antigen; European patent No. 389,286 of a non-defective human adenovirus 5 recombinant expression system in human cells; and published International application No. WO 91/11525 of live non-pathogenic immunogenic viable canine adenovirus in a cell.
However, because they are more suitable for entering a host cell, an indigenous adenovirus vector would be better suited for use as a live recombinant virus vaccine in different animal species compared to an adenovirus of human origin. For example, bovine adenovirus-based expression vectors have been reported for bovine adenovirus 3 (BAV-3) (see U.S. Pat. No. 5,820,868).
Bovine adenoviruses (BAVs) comprise at least nine serotypes divided into two subgroups. These subgroups have been characterized based on enzyme-linked immunoassays (ELISA), serologic studies with immunofluorescence assays, virus-neutralization tests, immunoelectron microscopy and by their host specificity and clinical syndromes. Subgroup 1 viruses include BAV 1, 2, 3 and 9 and grow relatively well in established bovine cells compared to subgroup 2 viruses which include BAV 4, 5, 6, 7 and 8.
BAV-3 was first isolated in 1965 and is the best characterized of the BAV genotypes and contains a genome of approximately 35 kilobases. The locations of hexon and proteinase genes in the BAV-3 genome have been identified and sequenced.
Genes of the bovine adenovirus 1 (BAV-1) genome have also been identified and sequenced. However, the location and sequences of other genes such as certain early gene regions in the BAV genome have not been reported.
The continued identification of suitable viruses and gene insertion sites are valuable for the development of new vaccines. The selection of (i) a suitable virus and (ii) the particular portion of the genome to use as an insertion site for creating a vector for foreign gene expression, however, pose a significant challenge. In particular, the insertion site must be non-essential for the viable replication of the virus, as well as its operation in tissue culture and in vivo. Moreover, the insertion site must be capable of accepting new genetic material, while ensuring that the virus continues to replicate.
What is needed is the identification of novel viruses and gene insertion sites for the creation of new viral vectors.
SUMMARY OF THE INVENTION
In one embodiment, the present invention provides recombinant viruses. While not limited to a particular use, these recombinant viruses can be used to generate vaccines.
While not limited to a particular virus, in one embodiment the present invention provides a recombinant virus comprising a foreign DNA sequence inserted into the E4 gene region of a bovine adenovirus. In a preferred embodiment, the insertion is to a non-essential site. In another embodiment, the present invention provides a recombinant virus comprising a foreign DNA sequence inserted into the E3 gene region of a bovine adenovirus 1. In a preferred embodiment, the insertion is to a non-essential site.
While not limited to its ability to replicate, in a preferred embodiment, the recombinant virus is replication competent. Likewise, while not limited to the foreign DNA to be inserted, in a preferred embodiment, the foreign DNA encodes a polypeptide and is from a virus or bacteria selected from the group consisting of bovine rotavirus, bovine coronavirus, bovine herpes virus type 1, bovine respiratory syncytial virus, bovine para influenza virus type 3 (BPI-3), bovine diarrhea virus, bovine rhinotracheitis virus, bovine parainfluenza type 3 virus,
Pasteurella haemolytica, Pasteurella multocida
and/or
Haemophilus somnus.
In another preferred embodiment, the foreign DNA encodes a cytokine. In a further preferred embodiment, the polypeptide comprises more than ten amino acids and is antigenic. Finally, in a particularly preferred embodiment, the foreign DNA sequence is under the control of a promoter located upstream of the foreign DNA sequence.
The present invention also contemplates mutant viruses. While not limited to a particular mutant virus, in one embodiment, the mutant virus comprises a deletion of at least a portion of the E4 gene region of a bovine adenovirus. In a preferred embodiment, the deletion is of a non-essential site. In another embodiment, the virus comprises a deletion of at least a portion of the E3 gene region of a bovine adenovirus 1. In a preferred embodiment, the mutant virus is replication competent. In a further preferred embodiment, at least one open reading frame of the relevant gene region of the bovine adenovirus is completely deleted.
In yet another embodiment, the present invention provides a method for preparing a recombinant virus comprising inserting at least one foreign gene or gene fragment that encodes at least one antigen into the genome of a virus wherein said gene or gene fragment has been inserted into the early gene region 4 of a bovine adenovirus or inserted into the early gene region 3 of bovine adenovirus 1. In a preferred embodiment, the method includes the insertion of at least a part of the genome of a virus into a bacterial plasmid, transforming said bacteria with said plasmid, and incubating said bacteria at approximately 25° C.
In another embodiment, the present invention provides vaccines. While not limited to a particular vaccine, in one embodiment, the vaccines comprise the recombinant viruses described above.
The present invention also contemplates methods of vaccination, including, but not limited to, the introduction of the above-described vaccines to an animal.
Definitions The term, “animal” refers to organisms in the animal kingdom. Thus, this term includes humans, as well as other organisms. Preferably, the term refers to vertebrates. More preferably, the term refers to bovine animals.
A “vector” is a replicon, such as a plasmid, phage, cosmid or virus, to which another DNA sequence may be attached so as to bring about the expression of the attached DNA sequence.
For purposes of this invention, a “host cell” is a cell used to propagate a vector and its insert. Infecting the cell can be accomplished by methods well known to those skilled in the art, for example, as set forth in Transfection of BAV-1 DNA below.
A DNA “coding sequence” is a DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, procaryotic sequences, cDNA from eucaryotic mRNA, genomic DNA sequences from eucaryotic (e.g., mammalian) DNA, viral DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence can be located 3′ to the coding sequence.
A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase or an auxiliary protein and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is in
Chiang Christina H.
Cochran Mark D.
Schering-Plough Veterinary Corporation
Stucker Jeffrey
Winkler Ulrike
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