Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1996-02-26
2001-04-24
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...
C435S235100, C435S320100, C435S236000, C424S229100
Reexamination Certificate
active
06221360
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is broadly concerned with vaccines composed of recombinant bovine herpesviruses and corresponding methods. More particularly, in its preferred form, the present invention is concerned with the construction of an infectious recombinant bovine herpesvirus type 1 having a functional &bgr;-galactosidase gene inserted in and thereby inactivating the thymidine kinase gene (e.g., Gal-TK); infection of an animal with the resultant avirulent vaccine virus is detected by assaying the animal's respiratory secretions for the presence of virus having &bgr;-galactosidase activity in host cells.
2. Description of the Prior Art
Bovine herpesvirus type 1 (BHV-1), also known as infectious rhinotracheitis virus (IBR), is associated with a variety of clinical diseases including rhinotracheitis, conjunctivitis, genital infections, abortion, enteritis, encephalitis, and generalized systemic infections in cattle (Ludwig, 1983; Wyler et al., 1989). The genome of BHV-1 consists of a linear double-stranded DNA molecule about 140-kb in length and is composed of a unique long (U
L
) region and a unique short (U
S
) region. The U
s
region is flanked by an internal and a terminal inverted repeat sequence (I
R
and T
R
, respectively). The BHV-1 genome encodes approximately 70 proteins (Misra et al., 1981). Many herpesviruses, including BHV-1, encode thymidine kinase (TK) (Kit and Qavi, 1983; Weinmaster et al., 1982). The BHV-1 TK gene has been mapped (Bello et al., 1987), and the sequences of the TK genes of four BHV-1 strains have been determined. (Kit and Kit, 1987; Mittal and Field, 1989; Smith et al., 1990; Bello et al., 1992).
The TK genes of these four strains encode between 357 and 359 amino acid (aa) residues. The deduced aa sequences of these genes reveal that BHV-1 TK is composed of five protein domains (I-V). These domains have a high degree of homology to corresponding domains in TKs of other herpesviruses, suggesting that these domains have functional significance.
The TK gene is not essential for replication of BHV-1 in vitro or in vivo (Kit and Kit, 1987; Mittal and Field, 1989), but does play an important role in pathogenicity. BHV-1 and other herpesviruses having defective TK genes grow normally in vitro but are highly attenuated in vivo (Field and Wildy, 1978; Kit et al., 1985 a, b; Slater et al., 1993). Thus, the basis of several BHV-1 vaccines has been inactivation of the TK gene (Kit and Qavi, 1983; Kit and Kit, 1987; Kit and Kit, 1989).
SUMMARY OF THE INVENTION
The present invention is directed to (1) recombinant bovine herpesvirus genes having foreign genes inserted therein, (2) infectious recombinant bovine herpesviruses carrying these recombinant genes, (3) methods of producing these recombinant bovine herpesviruses, (4) methods of immunizing animals against diseases caused by bovine herpesviruses using these recombinant bovine herpesviruses as vaccines, and (5) methods of detecting infection of an animal by these recombinant bovine herpesviruses. The invention is unique and unknown in the prior art in that a foreign gene within a bovine herpesvirus gene carried by an infectious recombinant bovine herpesvirus may serve dual purposes. First, the foreign gene may inactivate a bovine herpesvirus gene required for virulence, thereby resulting in an attenuated virus suitable for a vaccine. Second, if the foreign gene is expressed, detection of the product of the foreign gene in an animal demonstrates that the animal has been infected with the recombinant bovine herpesvirus.
In the first preferred embodiment, the recombinant bovine herpesvirus gene has a foreign gene inserted therein. Advantageously, the recombinant bovine herpesvirus gene is a bovine herpesvirus type 1 gene encoding thymidine kinase, and the foreign gene is a hybrid gene having a promoter and a coding region from different organisms. Preferably, the promoter is the immediate early promoter from human cytomegalovirus, and the coding region encodes a &bgr;-galactosidase (e.g.,
Escherichia coli
&bgr;-galactosidase). This recombinant bovine herpesvirus gene is ideally the recombinant bovine herpesvirus gene carried by pGal-TK.
In the second preferred embodiment, the infectious recombinant bovine herpesvirus carries a recombinant bovine herpesvirus gene described in the first preferred embodiment. Advantageously, this recombinant bovine herpesvirus gene is at least partially inactive and produces no functional product in a host cell, and in its most preferred form encodes thymidine kinase and produces no functional thymidine kinase in a host cell. Preferably, the foreign gene inserted within the recombinant bovine herpesvirus gene is expressed and produces
Escherichia coli
&bgr;-galactosidase in a host cell. The recombinant bovine herpesvirus is ideally attenuated or avirulent (e.g., Gal-TK). Additionally, the recombinant bovine herpesvirus may have at least part of one or more glycoprotein gene(s) not essential for viral replication (e.g., the glycoprotein E gene, the glycoprotein I gene, and the glycoprotein C gene) deleted.
In the third preferred embodiment, an infectious recombinant bovine herpesvirus described in the second preferred embodiment is produced as follows: A hybrid DNA molecule composed of a cloning vector and a recombinant bovine herpesvirus gene described in the first preferred embodiment is constructed. Advantageously, the cloning vector and the hybrid DNA molecule is a plasmid (e.g., pGal-TK). Host cells (e.g., Madin-Darby bovine kidney cells) are co-transfected with this hybrid DNA molecule and with infectious DNA from a bovine herpesvirus (e.g., bovine herpesvirus type 1). The resultant recombinant bovine herpesvirus is subsequently recovered. Preferably, after co-transfection and before recovery, the recombinant bovine herpesvirus is enriched. Ideally, this enrichment consists of passage of the recombinant bovine herpesvirus through host cells in the presence of at least one chemical (e.g., thymidine arabinoside and/or 5-bromo-2′-deoxyuridine) that selects for the growth of thymidine kinase-inactivated virus. Preferably, after enrichment and before recovery, bovine herpesviruses are screened by detecting the foreign gene product. Ideally, this product is an
Escherichia coli
&bgr;-galactosidase which is detected by the hydrolysis of a &bgr;-galactosidase substrate (e.g., halogenated indolyl-&bgr;-D-galactoside). Additionally, one or more glycoprotein gene(s) not essential for viral replication (e.g., the glycoprotein E gene, the glycoprotein I gene, and the glycoprotein C gene) may be deleted from the recombinant bovine herpesvirus.
In the fourth preferred embodiment, an animal is immunized against disease caused by bovine herpesvirus by making a vaccine composed of an infectious recombinant bovine herpesvirus described in the second preferred embodiment in a pharmaceutically acceptable carrier (e.g., a cell culture medium), and administering this vaccine to the animal, ideally by spraying the vaccine into the animal's nostrils. Additionally, the recombinant bovine herpesvirus vaccine strain may have at least part of one or more glycoprotein gene(s) not essential for viral replication (e.g., the glycoprotein E gene, the glycoprotein I gene, and the glycoprotein C gene) deleted.
In the fifth preferred embodiment, an animal vaccinated as described in the fourth embodiment is tested to determine whether the animal has been infected by an infectious recombinant bovine herpesvirus described in the second preferred embodiment. Advantageously, a specimen is obtained from the vaccinated animal and is assayed for the presence of a macromolecular unit (e.g., the recombinant bovine herpesviruses themselves) produced by the vaccinated animal in response to infection. Ideally, the specimen is a respiratory secretion, and the assay comprises assaying the respiratory secretion for the presence of recombinant bovine herpesviruses that produce &bgr;-galactosidase in host cells by detecting the hydrolysis of a &bgr;-galac
Hovey Williams Timmons & Collins
Kansas State University Research Foundation
Mosher Mary E.
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