Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1999-04-09
2001-10-09
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S229100, C435S320100, C435S235100, C435S069100, C435S069300, C536S023720
Reexamination Certificate
active
06299882
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 gene insertion sites for the introduction of foreign DNA.
BACKGROUND
Marek's disease is a lymphoproliferative disease of chickens caused by Marek's disease virus (MDV). MDV, a naturally occurring herpesvirus, infects bursa-derived and thymus-derived lymphocytes in chickens, and may subsequently induce a lymphoma of thymus-derived lymphocytes. MDV is a designation of a family of avian herpesviruses. For example, MDV1 is a virulent strain of herpesvirus in chickens, MDV2 is a naturally attenuated herpesvirus strain in chickens, and MDV3 is a nonpathogenic herpesvirus of turkey.
Since Marek's disease is contagious, the virus has become an important pathogen of chickens, particularly in an environment of large scale breeding such as in the poultry industry. Currently, Marek's disease is controlled by vaccination of embryos at 17-19 days of incubation, or one day old chicks.
The application of recombinant DNA techniques to animal viruses in general has a recent history. The first viruses to be engineered have been those with the smallest genomes. For example, in the case of the papovaviruses, because these viruses are so small and cannot accommodate much extra DNA, their use in genetic engineering has been as defective replicons. Thus, foreign DNA sequence expression from these viruses requires a wild-type helper virus and is limited to cell culture systems. On the other hand, for adenoviruses, there is a small amount of nonessential DNA that can be replaced by foreign sequences. This technique has also been applied to portions of the herpesvirus genome in an avian herpesvirus (see U.S. Pat. No. 5,853,733 to Cochran et al).
The cases of deletion or insertion of genes into herpesviruses demonstrate that it is possible to genetically engineer herpesvirus genomes by recombinant DNA techniques. In the past, the methods that have been used to insert genes involve homologous recombination between the viral DNA cloned in plasmids and purified viral DNA transfected into the same animal cell. However, the extent to which one can generalize the location of the deletion and the sites for insertion of foreign DNA sequences is not known from these previous studies.
The identification of suitable DNA sequence insertions sites in avian herpesviruses 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 DNA sequence 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
The present invention provides mutant and recombinant herpesviruses comprising a foreign DNA sequence inserted into a site in the herpesvirus genome. In one embodiment, the site is non-essential for viral replication. In a preferred embodiment, the foreign DNA sequence is capable of being expressed in a host cell infected with the recombinant herpesvirus and its expression. In a particularly preferred embodiment, the foreign DNA sequence is also under control of a promoter located upstream of the foreign DNA sequence.
The present invention is not limited to particular sites for insertion or deletion. In one embodiment, the deletion and/or insertion is in the UL54.5 open reading frame of a Marek's disease virus. In another embodiment, the deletion and/or insertion is in the UL43 open reading frame of a Marek's disease virus. In a preferred embodiment, the insertion is in the genome of Marek's disease virus type 1.
While not limited to particular types of DNA inserted, in one embodiment of the present invention the foreign DNA sequence inserted into the herpesvirus genome encodes a polypeptide. Preferably, the polypeptide is immunogenic to the animal into which the recombinant herpesvirus is introduced. Preferably, this immunogenic polypeptide is a linear polymer of more than 10 amino acids linked by peptide bonds which stimulates the animal to produce antibodies. In a preferred embodiment, the foreign DNA sequence also encodes a detectable marker. Preferably, the detectable marker is
E. coli
B-galactosidase.
In preferred embodiments, the recombinant herpesvirus contains a foreign DNA sequence encoding an immunogenic polypeptide from chicken anemia virus (CAV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV), Newcastle disease virus (NDV), infectious iaryngotracneitis virus (ILTV), or infectious bronchitis virus (IBV), fragments thereof and/or substantially homologous sequences. In another preferred embodiment, the foreign DNA encodes a cytokine. The present invention also contemplates recombinant herpesviruses having more than one foreign DNA sequence encoding an antigen or antigens.
When the foreign DNA sequence of the recombinant herpesvirus of the present invention encodes an immunogenic polypeptide from infectious bursal disease virus (IBDV), it is preferred that the immunogenic polypeptide is IBDV VP2, VP3 or VP4 protein, fragments thereof and/or substantially homologous sequences. When the foreign DNA sequence encodes an immunogenic polypeptide from MDV. Preferably, the immunogenic polypeptide is MDV glycoprotein B (gB), glycoprotein D (gD), or glycoprotein A (gA) fragments thereof and/or substantially homologous sequences.
When the foreign DNA sequence encodes an immunogenic polypeptide from Newcastle disease virus (NDV), it is preferred that the immunogenic polypeptide is NDV fusion (F) protein or NDV hemagglutinin-neuraminidase (HN), fragments thereof and/or substantially homologous sequences.
When the foreign DNA sequence encodes an immunogenic polypeptide from infectious laryngotracheitis virus (ILTV), it is preferred that the immunogenic polypeptide is ILTV glycoprotein “B” (gB), ILTV glycoprotein D (gD), or ILTV glycoprotein I (gI), fragments thereof and/or substantially homologous sequences.
When the foreign DNA sequence encodes an immunogenic polypeptide from infectious bronchitis virus (IBV), it is preferred that the immunogenic polypeptide is IBV spike protein, IBV matrix protein, nucleocapsid protein, fragments thereof and/or substantially homologous sequences.
The expression of the inserted foreign DNA sequence can be under control of a promoter located upstream of the foreign DNA sequence. Preferably, the promoter is a herpesvirus promoter. More preferably, the promoter is selected from a group consisting of pseudorabies virus (PRV) gX promoter, MDV gB promoter, MDV gA promoter, MDV gD promoter, ILTV gB promoter, ILTV gD promoter, ITLV gI promoter, human cytomegalovirus virus (HCMV) immediate early promoter, and/or substantially homologous sequences.
The present invention further provides for a homology vector for producing a recombinant herpesvirus by inserting a foreign DNA sequence into the herpesvirus genome. In one embodiment, the homology vector comprises a double-stranded DNA molecule consisting essentially of a double-stranded foreign DNA sequence, with at one end of the foreign DNA sequence, double-stranded DNA homologous to the genomic DNA located at one side of a non-essential site of the herpesvirus genome, and at the other end of the foreign DNA sequence, double-stranded DNA homologous to the herpesvirus genomic DNA sequence located at the other side of the same site. In such an embodiment, the double-stranded DNA can be homologous to a DNA sequence present within a 3212 base pair Sac I to Bgl II subfragment contained within the Bam HI “B” genomic fragment of a Marek's disease virus type 1. Preferably, a DNA seq
Salimi Ali R.
Salkeld Pamela G.
Schering Corporation
Zaradic Sandy S.
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