Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1999-01-29
2001-09-04
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S229100, C435S235100, C435S236000, C435S320100, C435S005000, C435S007100, C435S007400
Reexamination Certificate
active
06284251
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with recombinant bovine herpesvirus vaccines and corresponding methods. More particularly, the present invention is preferably concerned with the construction of an infectious recombinant bovine herpesvirus type 1 (BHV-1) having deleted portion of the native glycoprotein E (gE) coding region and a functional &bgr;-galactosidase gene (&bgr;-gal) inserted therein at the gE locus. The deletion of the native gE coding region attenuates the virus and serves as a genotypic or immunological marker which differentiates gE-deleted recombinant virus from wild type virus infection. Additionally, the insertion of the &bgr;-gal gene provides for a phenotypic method of assaying for the presence of gE-deleted recombinant virus infection by expressing &bgr;-gal activity in host cells.
2. Description of the Prior Art
Bovine herpesvirus type 1 (BHV-1), also known as infectious bovine rhinotracheitis virus (IBRV), is associated with a variety of clinical diseases including rhinotracheitis, conjunctivitis, genital infections, and occasionally abortion, enteritis, encephalitis, and generalized systemic infections in cattle. The genome of BHV-1 consists of a linear dsDNA molecule of about 140 kb. It is composed of a unique long (U
L
) region and a unique short (U
S
) region that are flanked by internal and terminal inverted repeat sequences (I
R
and T
R
, respectively). The BHV-1 genome encodes for approximately 70 proteins (Misra et al., Proteins Specified Bovine Herpesvirus 1 (Infectious Bovine Rhinotracheitis, 40 J. Virol. 367-378 (1981)). Like several other animal herpesviruses, the BHV-1 genome encodes for the glycoprotein (g) gE gene. The BHV-1 gE gene sequence, which codes for 575 amino acid (aa) residues, has been reported for two different strains (Leung—Taek, P. et al., The Complete DNA Sequence and the Genetic Organization of the Short Unique Region (U
S
) of the Bovine Herpesvirus Type 1 Strain (ST strain), 199 Virology 409-421 (1994); Rebordosa, X. et al., Mapping, Cloning and Sequencing of a Glycoprotein—Encoding Gene From Bovine Herpesvirus Type 1 Homologous to the gE Gene From HSV-1, 149 Gene 203-209 (1994)). The predicted gE amino acids contain stretches of hydrophobic amino acids at the N terminus (putative signal sequence) and near the C terminus (transmembrane sequence), which is typical of class 1 integral membrane proteins. The BHV-1 gE, and its homologs in other herpesviruses, have been shown to be dispensable for in vitro replication, but deletion of the entire gE coding sequence of the pseudorabies virus (PRV) genome is responsible for both the reduced virulence of the live vaccine strains Norden and Bartha (Petrovskis, E. A. et al., Deletion in Vaccine Strains of pseudorabies Virus and Their Effect on Synthesis of Glycoprotein gp 63, 60 J. Virol. 1166-1169 (1986) and the alteration of neuroinvasiveness (Card, J. P. et al., Pseudorabies Virus Envelope Glycoprotein gI Influences Both Neurotropism and Virulence During Infection of the Rat Visual System, 66 J. Virol. 3032-3041 (1992)). Thus, expression of the gE gene is required for full pathogenic potential of viruses in animals but is not required for growth in tissue culture (Kritas et al., Invasion and Spread of Single Glycoprotein Deleted Mutants of Aujeszky's Disease Virus (ADV) in the Trigeminal Nervous Pathway of Pigs After Intranasal Inoculation, 50 Vet. Micobiol. 323-334 (1994); Kritas et al., Role of Envelope Glycoproteins gI, gp63 and gIII in the Invasion and Spread of Aujeszky 's Disease Virus in the Olfactory Nervous Pathway of the Pig, 75 J. General Virol. 2319-2327 (1994)).
Recently, gE gene-deleted mutants of PRV and IBR have been of interest with respect to their usefulness as differential marker vaccines. Currently, a gE-deleted marker vaccine is being used for the eradication of IBR in Europe. However, this gE-deleted vaccine strain in Europe lacks a &bgr;-gal marker which allows for in situ histochemical detection methods for the detection of &bgr;-gal enzyme activity and in situ histochemical methods or immunoblotting methods for the detection of &bgr;-gal protein. The coding region of &bgr;-gal also serves as a genotypic marker of the recombinant virus. The virus can be easily detected by Southern blot hybridization and PCR tests can also determine the genetic purity of the vaccine virus from the wild type. Therefore, what is needed is an avirulent gE-deleted IBRV strain which contains a suitable phenotypic/histochemical/genotypic &bgr;-gal marker.
SUMMARY OF THE INVENTION
A BHV-1 recombinant virus (gE&Dgr;3.1IBR&bgr;) has been constructed in which gE open reading frames (ORF's) comprising a portion of the gE gene-coding sequences have been deleted and a chimeric reporter/marker gene has been inserted in their place. The inserted &bgr;-galactosidase (&bgr;-gal) gene plays no regulatory role in the replication of the virus but does serve as a phenotypic marker for gE&Dgr;3.1IBR&bgr; virus.
To construct this recombinant BHV-1, the BHV-1 gE gene-coding region and the flanking upstream and downstream sequences were cloned. To create a deletion in the gE gene-coding region, the above cloned DNA was digested with suitable enzymes to release the amino two-thirds of that region and ligated to the &bgr;-gal gene. The resulting plasmid DNA was cotransfected with DNA from full-length, wild type IBR virus strain Cooper into MDBK cells. Recombinant viruses expressing &bgr;-gal (blue plaques) were plaque purified and assayed further by blot hybridization for genetic characterization and by immunoblotting for reactivity against BHV-1 gE peptidespecific rabbit polyclonal antibody. One recombinant virus, gE&Dgr;3.1IBR&bgr;, was characterized in vitro for its growth properties and in vivo in calves for its pathogenic properties. The ability of the recombinant virus to induce BHV-1 neutralizing antibodies in infected calves was investigated by plaque reduction tests.
The regulation and expression of the chimeric &bgr;-gal gene are unique to this recombinant BHV-1 virus in two ways. The first unique aspect of this recombinant virus is that the &bgr;-gal gene is regulated by a strong human cytomegalovirus immediate early (HCMV-IE) promoter (not by a BHV-1-derived regulatory sequence). The second unique aspect is that the gene is expressed as a BHV-1-encoded gene at both early and late phases of infection. The in vitro and in vivo properties of this gE-deleted recombinant virus were analyzed by comparing it with the parental IBRV strain Cooper.
In tissue culture experiments, gE&Dgr;3.1IBR&bgr; virus grew to a lower titer than the wild type (parent strain Cooper) at early times post-infection, but at late times postinfection, the recombinant virus grew to an almost equal titer to the wild type strain. The recombinant virus usually developed significantly smaller plaques compared to the parental wild type strain Cooper. This could have been due to the lack of cell-to-cell spread of virus and is consistent with the results for other herpesviruses.
In animal experiments, gE&Dgr;3.1IBR&bgr;-infected calves shed approximately 100-fold less virus compared to the parental strain Cooper infected calves throughout the duration of virus shedding. The duration of this virus shedding was also two days shorter for calves infected by gE&Dgr;3.1IBR&bgr;. Whereas the calves infected with gE&Dgr;3.1IBR&bgr; virus remained healthy, the calves infected with parental strain Cooper showed typical IBR symptoms and lesions. Results of serum neutralization indicated that both the wild type and gE-deleted IBRV infected calves induced comparable BHV1 neutralizing antibody. It has been previously reported that thymidine kinase (TK) gene-deleted IBRV grew, both in vitro and in vivo, with a significantly lower titer (Chowdhury, S. I., Construction and Characterization of an Attenuated Bovine Herpesvirus Type 1 (BHV-1) Recombinant Virus, 52 Vet. Microbiol. 13-23 (1996)). Taken together, these results indicate that even though the recombinant gE
Hovey Williams Timmons & Collins
Kansas State University Research Foundation
Mosher Mary E.
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