Technical Field

The present invention relates generally to the prevention of disease in cattle. More particularly, the instant invention is directed to the recombinant production of certain bovine herpesvirus type 1 (BHV-l) antigens especially a truncated BHV-l giv antigen, for use in vaccines to protect cattle against bovine herpesvirus type 1 infection.

Backgro nd Bovine herpesvirus type l (BHV-l) is an economi¬ cally significant pathogen of cattle. BHV-l, which is also known as infectious bovine rhinotracheitis virus, causes severe respiratory infections, conjunctivitis, vulvovaginitis, abortions, encephalitis, and generalized systemic infections. If an animal recovers from a primary infection, the virus remains in the host in a latent state. Reactivation of the virus can be provoked by certain endogenous or exogenous physical modifications in the animal, or experimentally by treatment of the animal with glucocorticoids like dexamethasone.

In an effort to control BHV-1 infections, killed virus and attenuated live-virus vaccines have been developed. While these vaccines appear to induce some level of protection in cattle, the level of immunity is well below that necessary to afford complete OL?

SUBSTITUTESHEET

near-co plete protection. For example, the vaccines do not always prevent the establishment of a latent infection by a virulent field strain of BHV-l. Furthermore, the safety of the live-virus vaccines has been questioned. It has been shown recently that two live BHV-1 vaccine strains can be reactivated by the use of dexamethasone, indicating that at least some BHV-1 vaccines can themselves establish a latent infection. See, e.g., Gerber et al. (1978) Am. J. Vet. Res. 3.9:753-760; Jericho et al. (1983) Can. J. Com. Med. 17:133-139; Pastoret et al. (1980) Infect. Immun. 29_:483-488. Subunit vaccines, i.e. vaccines including select proteins separated from the whole virus _r afford a method for overcoming the problems inherent in the use of live and attenuated virus vaccines.

Several polypeptides of BHV-1 have now been studied. Mlsra et al. (1981) J. Virol. 40:367-378, reports on the partial characterization of a number of BHV-1 polypeptides and their immunoprecipitation with antiseru . van Drunen Littel-van den Hurk et al. (1984) Virology 135:466-479 and van Drunen Littel-van den Hurk et al. (1985) Virology 144:216-227 are directed to monoclonal antibodies developed against BHV-1 glycoproteins, and the ability of the monoclonal antibodies to neutralize virus and participate in antibody-dependent complement-mediated lysis in vitro. See also Collins et al. (1984) J. Virol. J£:403-409; Okazaki et al. (1986) Virology 150:260-264. van Drunen Littel-van den Hurk et al. (1985) Virology 111:204-215 is directed to the purification of BHV-1 glycoproteins by immunoadsorbent chromatography and the production of antiserum in rabbits, van Drunen Littel-van den Hurk et al. (1986) J. Clin. Microbiol. 22.:274-282 is directed to in vitro immunoreactivity of purified BHV-1 glycoproteins and bovine antiserum. Okazaki et al. (1987) Arch. Virol.

£2:17-26 pertains to in vitro studies of the reactivities of monoclonal antibodies against BHV-1 glycoproteins with infected cells. Babiuk et al. (1987) Virology 159:57-66 relates to the purification of gl, gill and gIV from virus infected cell lysates. This reference also discloses that gl of BHV-1 corresponds to gB of herpes simplex virus (HSV) ; gill corresponds to gC; and gIV corresponds to gD. Purified gl, gill and gIV have been shown to induce high levels of neutralizing antibody in cattle and participate in antibody dependent cell cytotoxicity of BHV-l cells. The purified glycoproteins were also shown to protect cattle from disease. Babiuk et al. (1987) Virology 159:57-66. van Drunen Littel-van den Hurk et al. (1990) Vaccine 8.:358-368 confirmed the protectivity of gl, gill and gIV and studied the epitope specificity of the immune response to the glycoprotein vaccines. Hughes et al. (1988) Arch. Virol. 103:47-60 identified three neutralizing antigenic domains on gIV. Mayfield et al. (1983) J. Virol. 47:259-264 discloses the cloning of a BHV-1 strain and a restriction map. Fitzpatrick et al. (1989) virology 173:46-57. describe the nucleotide sequence of gill. Pachl et al. (1987) J. Virol, jϋ:315-325 describe the recombinant expression of a glycoprotein from the human pathogen HSV- 1. There was no demonstration, however, that the re¬ combinant polypeptide from the human virus was, in fact, protective in a human host. See also PCT Pub. No. WO88/02634; U.S. Patent NOS. 4,661,349; 4,642,333.

Fitzpatrick et al. (1988) J. Virol. 62:4239-4248 describe the expression of gl and gill in urine LMTK- cells. The transfected cells were shown to stimulate the production of neutralizing antibodies in mice. Fitzpatrick et al. (1990) describe the expression of deleted, truncated and hybrid forms of gl and gill in murine LMTK-cells and epitope mapping of the same. Tikoo

et al. (1990) J. Virol. 6 5132-5142 disclose the mapping, cloning and sequencing of BHV-1 gIV, as well as the expression of gIV in bovine cells, van Drunen Littel-van den Hurk et al. (1989) J. Virol. 6_3:2159-2168 disclose the expression of gl and gill in a vaccinia virus vector. The recombinant vectors elicited a neutralizing antibody response in cattle immunized with the same, van Drunen Littel-van den Hurk et al. (Jan. 1991) J. Virol. 6^:263-271 describe the expression of gIV by recombinant baculovirus. This disclosure was based in part on the present invention. Cattle immunized with recombinant gIV raised neutralizing antibodies thereto.

U.S. patent 5,151,267 issued September 29, 1992 discloses vaccines comprising at least one recombinant antigenic determinant of BHV-1 gl, BHVl-glll and/or BHV-l gIV and the preparation thereof. Babiuk et al. Viroloσv 159:57-66 (1987) also describes the protection of cattle with individual viral BHV-1 glycoproteins.

Disclosure of the Invention

It has been discovered that recombinant subunit vaccines, based on selected BHV-1 glycoproteins, will protect cattle from disease. These vaccines are particularly useful in protecting cattle from the shipping fever complex syndrome which often includes infection by BHV-1. Surprising, these subunit vaccines are substantially more protective than prior art killed virus and attenuated live-virus vaccines. The recombinant subunit vaccines do not suppress the immunological response to other components often found in ultivalent attenuated shipping fever viral vaccines. Further, the recombinant subunit vaccines of the present invention also eliminate the risk of infection from the live-virus vaccines. It has also been discovered that recombinant BHV-l polypeptides maintain the proper

epitopes necessary to protect immunized animals from * disease. Both nonglycosylated polypeptides, and

,- polypeptides glycosylated by heteroiogous host organisms,

=* effectively elicit antibodies that neutralize virus

5 infectivity and induce complement-mediated cell lysis. Based on these discoveries, the present invention can take several embodiments.

The present invention is directed to a vaccine composition comprising a pharmaceutically acceptable

10 vehicle plus a recombinant subunit antigen comprising a truncated bovine herpesvirus type 1 (BHV-1) gIV glycoprotein, optionally with at least one additional recombinant subunit antigen comprising a neutralizing epitope of glycoprotein selected from the group

15 consisting of BHV-1 gl, BHV-1 gill and a second BHV-1 gIV protein. Production of a truncated protein was by secretion. An example of a plasmid containing a truncated BHV-1 gIV is pG4HUNEO having the ATCC accession number 69076.

20 In other embodiments, the subject invention is directed to nucleotide sequences encoding proteins substantially homologous and functionally equivalent to truncated BHV-1 gIV.

In yet other embodiments the subject invention is

25 directed to DNA constructs comprising an expression cassette comprised of: (a) a DNA coding sequence for a polypeptide containing at least one neutralizing epitope of a truncated BHV-1 gIV; and (b) control sequences that are operably linked to the coding sequence whereby the

30 coding sequence can be transcribed and translated in a host cell, and at least one of the control sequences is heteroiogous to the coding sequence.

In another embodiment, the present invention is directed to a host cell stably transformed by the above

35 DNA constructs.

In still another embodiment, the subject invention is directed to methods of producing recombinant truncated BHV-1 gIV polypeptides comprising:

(a) providing a population of the above host cells; and

(b) growing the population of cells in a growth media under conditions whereby the polypeptide encoded by the expression cassette is expressed and secreted; and optionally (c) recovering the secreted truncated BHV-l gIV polypeptide from the growth media.

In another embodiment, the instant invention is directed to methods of treating or preventing BHV-l infection in a bovine host comprising administering to the bovine host a therapeutically effective amount of a vaccine compositions described above.

These and other embodiments of the present invention will readily be apparent to those of skill in the art from the following disclosure.

Brief Description of the Figures

Figure 1 is the nucleotide sequence and deduced amino acid sequence from a viral clone of gIV. The gene encodes a peptide 417 amino acids long. The right-angle arrow marks the position of the mature gIV sequence. Putative transmembrane sequences are underlined.

Figure 2 shows the construction of the vaccinia expression vector pW-1.

Figure 3 depicts the construction of the vaccinia expression vector pWSL-1.

Figure 4 depicts the vaccinia expression vector pW-1/gIV with the full length gIV gene cloned into the Bglll cloning site of pWSL-1.

Figure 5 shows the vaccinia expression vector pW- 1/gIVt which includes a modified BHV-l gIV gene inserted into the Bglll cloning site of pWSL-1.

Figure 6 shows various BHV-1 genomic library clones.

Figure 7 depicts the £. coli expression plasmid pgpll complete, carrying the entire gib coding sequence plus DNA encoding the first eight amino acids of glc. See Example V. Figure 8 shows the £. coli expression plasmid pBHC150Δ, carrying a deletion mutant of the BHV-1 gill gene. See Example V.

Figure 9 shows the cloning strategy and the construction of pBHDsib. See Example V. Figure 10 depicts the jg. coli expression plasmid pBHDsib, containing a gIV gene encoding the mature protein. See Example V.

Figure 11 depicts the adenovirus transfer vector, pAdBM5. * indicates the gene insertion site. See Example VI.

Figure 12 shows the adenovirus vector, pAdBM5.gIV, which includes the gene coding for full-length gIV. See Example VI.

Figure 13 shows the effects of immunization with truncated BHV-1 glVA protein (-A-) , as compared to glVA (-■-) and a placebo (-o-) in virus shedding in animals challenged with BHV-1.

Figure 14 shows the daily number of sick calves when treated with BHV-IV protein, truncated BHV-IV protein and a placebo.

Figure 15. Structure of BHV-1 gIV deletions and truncations. The amino acid sequence of gIV is depicted schematically, at the top of the figure, with signal sequence . transmembrane anchor sequence ( jff ) / cysteine residues (S) and potential N-linked

glycosylation sites (t) . Deleted or truncated forms of gIV are shown below the diagram of intact gIV with the deleted regions indicated by solid line (■■■) . The name given to each mutant protein is indicated on the right. Figure 16. Predicted secondary structure of BHV-l gIV. The deduced amino acid sequence of gIV was analysed for alpha-helix, beta sheet, and beta turn probabilities using a version of algorithm of Chou and Fasman. Segments of alpha-helix, are looped, segments of beta sheets are zig-zagged, and beta turns are indicated as bends. Cysteine residues (C) and potential N-linked glycosylation sites (N) are indicated. The approximate location of the mapped epitopes is indicated by arrows whereas the region of the gIV required for proper processing and transport marked by bracket.

Figures 17(a) and (b) show the effects of immunization with truncated BHV-l gIV vaccine.

Detailed Description The practice of the present invention will employ, unless otherwise indicated, conventional virology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al.. Molecular Cloning; A Laboratory Manual (2nd Edition, 1989); Maniatis et al.. Molecular Cloning: A Laboratory Manual (1982) ; DNA Cloning: A Practical Approach. vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985);

Transcription and Translation (B. Hames & S. Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed. , 1986) ; Perbal, A Practical Guide to Molecular Cloning (1984) .

All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

A. Definitions

The following terminology will be used in ac¬ cordance with the definitions set out below in describing the present invention.

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, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bound at the 3' terminus by the translation start codon (ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eucaryotic promoters will often, but

now always, contain "TATA" boxes and "CAT" boxes. Procaryotic promoters contain Shine-D lgar o sequences in addition to the -10 and -35 consensus sequences.

DNA "control sequences" refers collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, enhancers, and the like, which collectively provide for the transcription and translation of a coding sequence in a host cell.

A coding sequence is "operably linked to" or "under the control of" control sequences in a cell when RNA polymerase will bind the promoter sequence and transcribe the coding sequence into mRNA, which is then translated into the polypeptide encoded by the coding sequence.

A "host cell" is a cell which has been transformed, or is capable of transformation, by an exogenous DNA sequence. A cell has been "transformed" by exogenous DNA when such exogenous DNA has been introduced inside the cell membrane. Exogenous DNA may or may not be integrated (covalently linked) to chromosomal DNA making up the genome of the cell. In procaryotes and yeasts, for example, the exogenous DNA may be maintained on an episomal element, such as a plasmid. With respect to eucaryotic cells, a stably transformed cell is one in which the exogenous DNA has become integrated into the chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eucaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the exogenous DNA. Episomal replication is also possible in mammalian cells e.g.

plasmids containing the EBV ori and EBNA gene, SV40 plasmids in COS cells.

A "clone" is a population of cells derived from a single cell or common ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations.

Two DNA or polypeptide sequences are "substantially homologous" when at least about 85% (preferably at least about 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra: DNA Cloning, vols I & II, supra; Nucleic Acid Hybridization. supra.

The term "functionally equivalent" intends that the amino acid sequence of the subject protein is one that will elicit an immunological response, as defined below, equivalent to the specified BHV-1 immunogenic polypeptide.

A "heteroiogous" region of a DNA construct is an identifiable segment of DNA within or attached to another DNA molecule that is not found in association with the other molecule in nature. Thus, when the heteroiogous region encodes a viral gene, the gene will usually be flanked by DNA that does not flank the viral gene in the genome of the source virus or virus-infected cells.

Another example of the heteroiogous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene) . Allelic variation or

naturally occurring mutational events do not give rise to a heteroiogous region of DNA, as used herein.

A composition containing molecule A is "substan¬ tially free of" molecule B when at least about 75% by weight of the total of A + B in the composition is molecule A. Preferably, molecule A comprises at least about 90% by weight of the total of A + B in the composi¬ tion, more preferably at least about 99% by weight.

"Bovine host" refers to cattle of any breed for which it may be desirable to immunize against BHV-l infection, whether or not the bovine host is already infected or latently infected by BHV-1. A bovine host can be of any age. Thus, the term encompasses calves as well as adult cattle. "Native" proteins or polypeptides refer to proteins or polypeptides recovered from BHV-1 virus or BHV-1-infected cells. Thus, the term "native BHV-l polypeptide" would include naturally occurring BHV-1 proteins and fragments thereof. "Non-native" polypeptides refer to polypeptides that have been produced by recombinant DNA methods or by direct synthesis. "Recombinant" polypeptides refer to polypeptides produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.

An "antigen" refers to a molecule containing one or more epitopes that will stimulate a host's immune system to make a humoral and/or cellular antigen-specific response. The term is also used interchangeably with "immunogen."

By "subunit antigen" is meant an antigen entity separate and discrete from a whole virus (live or killed) . Thus, an antigen contained in a cell free extract would constitute a "subunit antigen" as would a substantially purified antigen.

A "hapten" is a molecule containing one or more epitopes that does not stimulate a host's immune system to make a humoral or cellular response unless linked to a carrier. The term "epitope" refers to the site on an antigen or hapten to which a specific antibody molecule binds. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." An "immunological response" to a composition or vaccine is the development in the host of a cellular and/ or antibody-mediated immune responses to the composition or vaccine of interest. Usually, such a response consists of the subject producing antibodies, B cells, helper T cells, suppressor T cells, and/or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.

The terms "immunogenic polypeptide" and ••immunogenic amino acid sequence" refer to a polypeptide or amino acid sequence, respectively, which elicit antibodies that neutralize viral infectivity, and/or mediate antibody-complement or antibody dependent cell cytotoxicity to provide protection of an immunized host. An "immunogenic polypeptide" as used herein, includes the full length (or near full length) sequence of the desired BHV-1 glycoprotein or an immunogenic fragment thereof. By "immunogenic fragment" is meant a fragment of a polypeptide which includes one or more epitopes and thus elicits antibodies that neutralize viral infectivity, and/or mediates antibody-complement or antibody dependent cell cytotoxicity to provide protection of an immunized host. Such fragments will usually be at least about 5 amino acids in length, and preferably at least about 10 to 15 amino acids in length. There is no critical upper limit to the length of the fragment, which could comprise nearly the full length of the protein sequence, or even a

fusion protein comprising fragments of two or more of the BHV-1 subunit antigens.

The term "treatment" as used herein refers to either (i) the prevention of infection or reinfection (prophylaxis) , or (ii) the reduction or elimination of symptoms of BHV-l (therapy) .

B. General Methods

Bovine herpesvirus type l, or BHV-l, is a well-known and well-characterized virus, of which many strains are known. See, e.g., Gibbs et al. (1977) Vet. Bull. (London) 17:317-343. BHV-1, also known as infectious bovine rhinotracheitis virus, is similar in structure to other herpesviruses, and possesses a linear double-stranded DNA genome of approximately 140 kilobase pairs. BHV-l can remain latent in infected animals, probably in trigeminal or sacral ganglia, and, as discussed above, can be reactivated with relative ease. The antigen of the present invention is a truncated BHV-1 gIV and optionally used with at least one additional subunit antigen comprising a antigenic determinant selected from the group of BHV-1 glycoproteins consisting of gl, gill, and a second gIV. These optional antigens are disclosed in U.S. patent 5,151,267 issued September 29, 1992.

The truncated gIV sequence need only encode a "polypeptide neutralizing epitope"; i.e., an epitope which elicits antibodies that neutralize virus infectivity, and/or mediates antibody-complement or antibody dependent cell cytotoxicity to provide protec¬ tion of an immunized host. See, e.g., Babiuk et al. (1975) Infect. Immun. 12:958-963; Babiuk et al. (1987), supra.

In general, the polypeptide subunit antigens will usually be at least about 5 amino acids in length in

order to encode an epitope, and preferably at least about 10-15 amino acids in length. There is no critical upper limit to the length of the subunit antigen, which could comprise the entire viral glycoprotein sequence, or even a fusion protein comprising the sequences of two or more of the viral glycoproteins.

The truncated and optional antigens of the present invention are recombinant polypeptides. These recombinant antigens can take the form of partial glycoprotein sequences, full-length viral protein sequences, or even fusion proteins (e.g., with an appropriate leader for the recombinant host, or with another subunit antigen sequence for BHV-1 or another pathogen) . The antigen, even though carrying epitopes derived from glycoproteins, does not require glycosylation. The generation of truncated BHV-1 gIV gene constructs was for directing secretion of gIV for the purposes of 1) increasing gIV yields and 2) producing gIV in relatively pure form by simply harvesting from the growth media.

Particularly preferred epitopes for screening the antigens will be those epitopes that are exposed on the cell membrane of infected host cells, as well as those epitopes encompassing the attachment site on intact virions. Such epitopes can be defined by monoclonal antibody analysis. See, e.g., van Drunen Littel-van den Hurk et al. (1985) Virology 111:216-227; Okazaki et al. (1987) , supra.

In the vaccines of the present invention, it will sometimes be preferable to have more than one polypeptide neutralizing epitope in the antigen(s) . Furthermore, it may also be desirable to include polypeptide neutralizing epitopes from more than one glycoprotein in the antigen(s) . In its simplest form, this can be achieved by employing a polypeptide encoding a truncated BHV-1 gIV

alone or optionally with the partial or complete sequence of a glycoprotein (usually encompassing more than one neutralizing epitope) , or by employing a combination of polypeptides encoding the sequences of two or three of the viral glycoproteins (e.g. gl, gill and gIV) .

The antigens of the present invention, may be conjugated to a vaccine carrier. Such carriers are well known in the art, such as bovine serum albumin (BSA) , hu¬ man serum albumin (HSA) and keyhole limpet hemocyanin (KLH) . A particularly preferred carrier protein, rotavirus VP6, is disclosed in U.S. patent No. 5,071,651, the disclosure of which is hereby incorporated by reference in its entirety. The BHV-1 neutralizing epitopes of the present invention may also be incorporated within particle-forming viral polypeptides as a fusion protein, as described in U.S. Patent No. 4,722,840 and EPO Pub. No. 174,759. Alternatively, the BHV-1 antigens of the present invention can be incorporated into a foreign virus (e.g., vaccinia or adenovirus) as is known in the art and described more fully below in the examples.

The antigen(s) are formulated into a vaccine composition comprising a pharmaceutically acceptable vehicle and, optionally, an adjuvant. Such formulations are well within the skill of the art. In general, the formulations will be particularly adapted for intra¬ muscular injection, since intravenous injection is usually not practical for large-scale application to domestic animals. Alternatively, the vaccines are given orally or intranasally, and the subunits formulated with a suitable oral carrier. It may be preferred to administer the vaccines of the present invention orally to raise mucosal immunity, as well as intramuscularly for systemic immunity. A pharmaceutically acceptable vehicle, suitable for parenteral injection, is usually

nontoxic and nontherapeutic. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and Hank's solution. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Parenteral vehicles may also take the form of suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. The vehicle will also usually contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and tris buffer, while examples of preservatives include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard formula¬ tions will either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a nonliquid formulation, the vehicle may comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline could be added prior to administration. Various adjuvants are known in the art which can also be employed in the vaccine formulations of the present invention; e.g., Freund's adjuvant, Avridine, aluminum salts [A1(0H)_, A1P0 ₄ , A1_(S0 ₄ ) ₈ ], Ca_(P0 ₄ ) ₂ , saponin, DDA, Pluronics, oil-in-water emulsions (contain- ing, e.g., Avridine, dextran sulphate, Emulsigen PLUS or vitamin E) and water-in-oil emulsions (containing, e.g., polysorbate 80) . The selection of the appropriate adjuvant and its concentration in the vaccine composition is within the skill of the art. Many protocols for administering the vaccine composition of the present invention to animals are within the skill of the art. The preferred route of administration is parenteral, particularly intramuscular. The concentration of the antigen(s) in the vaccine composition is selected so that an effective dose is

presented in the bovine host to elicit antibodies to the polypeptide neutralizing epitopes. Within wide limits, the dosage is not believed to be critical. Typically, the vaccine composition is administered in a manner which will deliver between about 1 to about 1,000 μg of the subunit antigen in a convenient volume of vehicle (e.g., about 1-10 ml) . Preferably, the dosage in a single immunization will deliver from about 1 to about 500 μg of subunit antigen, more preferably about 5-10 μg to about 100-200 μg (e.g., 10-100 μg) . It may also be preferred, although optional, to administer a second, booster immunization to the animal several weeks to several months after the initial immunization. To insure sustained high levels of protection against disease, it may be helpful to readminister a booster immunization to the animals on a periodic basis.

The antigen can be produced from protein recovered from virus or virus-infected cells, or cells expressing the recombinant glycoproteins. For example, purified virus or virus-infected cells can be disrupted or lysed and subjected to immunoadsorbent chromatography to purify gl, gill or gIV. See, e.g., van Drunen Littel-van den Hurk et al. (1985) Virology 144:204-215. The production of monoclonal antibodies is within the skill of the art. See, e.g., van Drunen Littel-van den Hurk et al. (1984), supra: Okazaki et al. (1987) , supra. Briefly, a mammal, such as a mouse, is immunized with either purified virus or the purified viral glycoprotein of interest (e.g., SDS-PAGE purified) and antibody-producing B lymphocytes recovered. Typically, these B lymphocytes are then fused with a continuous cell line to produce an immortal antibody-producing cell line; i.e., a hybridoma, trioma, etc. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B-lymphocytes with oncogenic DNA, or

transfection with Epstein-Barr virus. See, e.g., M. Schreier et al. (1980) Hybridoma Techniques; Ha merling et al. (1981) Monoclonal Antibodies and T-Cell Hybridomas; Kennett et al. (1980) Monoclonal Antibodies; see also U.S. Patent Nos. 4,341,761; 4,399,121;

4,427,783; 4,444,887; 4,451,570; 4,466,917; 4,472,500; 4,491,632; 4,493,890. Native BHV-1 proteins which are immunopurified can be used in their entirety as subunit antigens, or fragments of the entire proteins containing the neutralizing epitopes can be employed as subunit antigens.

Non-native BHV-1 polypeptides can be produced by a number of methods. For example, oligopeptides containing neutralizing epitopes can be prepared synthetically by known techniques. See, e.g., U.S. Patent No. 4,735,896. It is preferred, however, to prepare the non-native polypeptide subunit antigens by recombinant DNA methods.

Recombinant polypeptide subunit antigens are produced according to the present invention by construct- ing an expression cassette and transforming a host cell therewith to provide a cell line or culture capable of expressing the subunit antigen which is encoded within the expression cassette. The first step in constructing the expression cassette is to obtain a coding sequence for the glycoprotein or glycoprotein epitopes of interest. Thus, coding sequences can either be prepared directly by synthetic methods based on the disclosed sequence (or equivalent sequences encoding the same amino acids) , or by using the disclosed sequence to design oligonucleotide probes to clone coding sequence using known techniques. See, e.g., Mayfield et al. (1983), supra. The coding sequence can be comprised entirely of BHV-1 glycoprotein-encoding sequences, or such glycoprotein sequences can be fused to other sequences (e.g., leader sequences) so that a fusion protein is en-

coded. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397. Synthetic coding sequences will also allow for the convenient construction of coding sequences which express BHV-1 glycoprotein analogs or "muteins". Alternatively, coding sequences for muteins can be prepared by site-directed mutagenesis of native BHV-1 nucleotide sequences. The techniques of site-directed mutagenesis are known in the art.

Once an appropriate coding sequence for the antigen has been prepared or isolated, it can be cloned into any suitable vector or replicon. Numerous cloning vectors or replicons are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which can be transformed include various bacteriophage lambda vectors fE. coli) , pBR322 fE. coli) , pACYC171 (E. coli) , pKT230 (gram-negative bacteria) , pGV1106 (gram-negative bacteria) , pLAFRl (gram-negative bacteria) , pME290 fnon-E. coli gram-negative bacteria) , pHVl4 fE. coli and Bacillis subtilis) , pBD9 (Bacillis) , pIJ61 (Str ptomyces) , pUC6 (Strepto γces) , actinophage dC31 (Streptomyces) , YIp5 (Saccharomyces) , YCpl9 (Saccharomvces) , 2-micron plasmid (Saccharomyces) , and bovine papillo a virus (mammalian cells) . See, generally, DNA Cloning, vols I & II, supra: Maniatis et al., supra; Perbal, supra.

To complete construction of expression cassettes, the coding sequence as described above for the antigens is then operably linked to control sequences (e.g., a promoter, etc.), so that the DNA sequence encoding the subunit antigen is transcribed into messenger RNA in the host cell transformed by the expression cassette. It is within the skill of the art to operably link the subunit antigen coding sequence to appropriate control sequences

in order to bring about transcription and translation. In general, the coding sequence will be downstream from the promoter sequence and any expression regulatory regions, such as enhancers or operator sequence. If the subunit antigen coding sequence is linked to a heteroiogous coding sequence or start codon, then it is important to place the subunit antigen coding sequence in reading frame with the latter. If the intended expression host is procaryotic, then it will also be necessary to include a ribosome binding site among the upstream control sequences. Downstream operably linked control sequences will usually comprise a transcription termination sequence, and a polyadenylation signal (for mammalian expression hosts) . When the intended expression host is a procaryotic or yeast cell, the promoter and other control sequences will necessarily be heteroiogous to the subunit antigen coding sequence. If the selected expression host cell is a mammalian cell, the control sequences can be homologous BHV-1 sequences, or preferably heteroiogous mammalian control sequences. The expression cassette can be constructed, for example, as a discrete molecular entity flanked by convenient restriction sites, or it can be constructed by inserting the coding sequence into a previously constructed expression vector with an appropriate insertion site.

A number of procaryotic expression vectors are known in the art. See, e.g., U.S. Patent Nos. 4,440,859; 4,436,815; 4,431,740; 4,431,739; 4,428,941; 4,425,437; 4,418,149; 4,411,994; 4,366,246; 4,342,832; see also U.K. Publication Nos. GB2,121,054; GB2,008,123; GB2,007,675; and European Publication No. 103,395. The preferred procaryotic expression vectors are those for E. coli. Other preferred expression vectors are those for use in eucaryotic systems. Yeast expression vectors are also

known in the art. See, e.g., U.S. Patent Nos. 4,446,235; 4,443,539; 4,430,428; see also European Publication Nos. 103,409; 100,561; 96,491.

One of the preferred expression hosts of the present invention are mammalian cells. Various cell lines and expression vectors are known in the art. Examples of appropriate mammalian expression hosts include kidney cell lines (e.g., CV-1 monkey kidney cell lines), fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines) , Chinese hamster ovary (CHO) cells, HeLa cells, mouse NIH/3T3 and/or LMTK~ cells. It is also known to express heteroiogous proteins in myeloma cell lines employing immunoglobulin promoters. See, e.g, Banerjee et al. (1983) Cell 3_3:729-740; U.S. Patent No. 4,663,281. The selection of a mammalian cell line is not critical, and is within the skill of the art. Various mammalian expression vectors employing viral promoters (e.g., SV40 early region promoter, Rous sarcoma virus, LTR promoter, etc.) are also well known in the art. See, e.g., Pachl et al. (1987), supra; Gorman et al. (1982) Proc. Natl. Acad. Sci. USA 79.:6777-6781; Southern et al. (1982) J. Mol. App. Genet. 1:327-341; PCT Publication No. WO87/02062.

Preferred eucaryotic expression vectors are those employing the vaccinia virus, the SV40 virus, or the Rous sarcoma virus, which are also well known in the art. See, e.g., Mackett et al. (1984) J. Virol. .49:857; DNA Cloning, vol. II, pp. 191-211, supra; PCT Publication No. WO86/07593; Chakrabarty et al. (1985) Mol. Cell. Biol. 5:3403.

Another preferred embodiment of the present invention is the expression of recombinant BHV-1 polypeptides in insect cells using viral vectors, such as baculovirus. For example, high levels of expression have been achieved with vectors based on Autograoha

californica nuclear polyhedrosis virus (AcNPV) in Spodoptera frugiperda cells. See, e.g., Smith et al. (1983) J. Virol. 16:584-593; U.S. Pat. App. Serial No. 07/092,120, supra; Canadian Pat. App. Serial No. 545,803, supra.

Generally, a host (cell) which has been stably transformed by an expression cassette for the subunit antigen is selected to produce the recombinant polypeptide. A stably transformed host is one wherein the expression cassette has integrated into the host cell's chromosome. In the case of bacteria, yeast or mammalian expression hosts or the like, it may be preferred*to select expression hosts which maintain the cassette on a non-integrating episomal element, such as a plasmid. The subunit antigen is produced by growing host cells transformed by the expression cassette under conditions which cause the expression of biologically active subunit antigen polypeptide. The appropriate conditions to bring about expression are well known in the art, and will depend primarily on the expression system and host selected. The subunit antigen polypeptide may be isolated from the host cells and puri¬ fied. If the expression system secretes the subunit antigen, then the polypeptide can be purified directly from the growth media. If antigen is not secreted, however, it may be necessary to disrupt the host cells and purify the subunit antigen polypeptide from the cellular lysate. Various purification techniques, such as HPLC and immunoaffinity chromatography, are known, and the selection of the appropriate purification and re¬ covery method is within the skill of the art.

It is known that BHV-1 can be immunosuppressive. This could interfere with the effectiveness of other bacterial or viral vaccines administered concurrently with, or within a few days of BHV-1 vaccine. However, it

has been discovered that vaccination with BHV-l gIV and/or truncated BHV-1 gIV does not have an immunosuppressive effect on other bacterial or viral vaccines. Accordingly, subunit gIV can be used in conjunction with other vaccines without fear of causing reduced immune responses to them. Any bovine bacterial or viral vaccine reducing or preventing infection can be the second vaccine. Such materials are well known in the art and include but are not limited to vaccines against Pasteurella haemolytica. Haemophilus somnus. parainfluenza virus, coronavirus, rotavirus, adenovirus, bovine respiratory syncytial virus, bovine diarrhea virus and the like. Thus, a wide variety of co- infections can be co-treated. Accordingly, the invention also includes a method for co-treating or preventing a BHV-1 infection and a second infection in a bovine host which comprises administering to the bovine host a therapeutically effective amount of (1) a vaccine composition of BHV-glV and (2) a vaccine against the second infection and compositions which comprise (1) and (2) .

An alternative route of administration involves gene therapy or nucleic acid immunization. Thus, nucleotide sequences (and accompanying regulatory elements) encoding the subject proteins can be administered directly to a subject for in vivo transcription and translation thereof. Alternatively, gene transfer can be accomplished by transfecting the subject's cells or tissues ex. vivo and reintroducing the transformed material into the host. DNA can be directly introduced into the host organism, i.e., by injection (see. International Publication No. WO90/11092; and Wolff et al., Science (1990) 247:1465-1468) . Liposome-mediated gene transfer can also be accomplished using known

ethods. See e.g., Hazinski et al.. Am. J. Respir. Cell Mol. Biol. (1991) 1:206-209; Brigham et al. , Am. J. Med. Sci. (1989) 298:278-281; Canonico et al., Clin. Res. (1991) 29_:219A; ^a ™* Nabel et al.. Science (1990) 249:1285-1288. Targeting agents, such as antibodies directed against surface antigens expressed on specific cell types, can be covalently conjugated to the liposomal surface so that the nucleic acid can be delivered to specific tissues and cells susceptible to BHV-1 infection.

Diagnostic Assays for BHV-1 Antibodies The recombinant antigens of BHV-1 gl, gill and/or gIV can be used as substrate reagents in immunόassays to identify antibodies to BHV-1 gl, gill and/or gIV in a sample, e.g. , blood, from a bovine host as one means of determining if the bovine host is infected with BHV-1 and to determine the concentration of the antibodies in the sample. The immunoassays in which the recombinant antigens of BHV-1 gl, gill and/or gIV can be used include, but are not limited to, radioimmunoassay, competition immunoprecipitation, enzyme-linked immunoadsorbent assay, immunofluorescence assay and the like. Detection is convenient, rapid, sensitive and specific. The recombinant antigens of BHV-1 gl, gill and/or gIV are used in assay compositions in a concentration sufficient to form a detectable complex with the antibodies. The BHV-1 antigens can be mixed with or attached to a suitable matrix (support) or carrier, such as a latex particle or plastic microtiter plate or the like. They can also be conjugated with an enzyme, dye, radio-labelled or the like, depending upon what immunological method is used. The details of

conducting various types of immunoassays is well known in the art and also described in Tyssen, P., "Laboratory Techniques in Biochemistry and Molecular Biology, Practice and Theory of Enzyme Immunoassays" (Ed. R.H. Burton and P.H. van Knippenberg, the disclosures of which are incorporated by reference. Accordingly, the invention includes a method for determining the presence or absence of or concentration of antibodies for BHV-l in a sample by employing an immunoassay, the immunoassay characterized by using recombinant antigenic BHV-1 gl, gill and/or gIV reactive with BHV-1 antibodies as a reagent in the immunoassay, whereby a complex of the BHV-1 antibodies and the recombinant antigenic BHV-1 gl, gill and/or gIV is formed, and determining the presence or absence of or concentration of the complex formed as indicative of the presence or absence of or concentration of the antibodies.

Described below are examples of the present invention which are provided only for illustrative purposes. The examples are not intended to limit the scope of the present invention in any way, as numerous embodiments within the scope of the claims will be appar¬ ent to those of ordinary skill in the art in light of the present disclosure. Those of ordinary skill in the art are presumed to be familiar with (or to have ready access to) the references cited in the application, and the dis¬ closures thereof are incorporated by reference herein.

C. Experimental

EXAMPLES

I

This example demonstrates the protection of cattle immunized with subunit vaccines made from purified BHV-l glycoproteins.

I.A. Materials and Methods

I.A.I. Virus and Bacteria

Strains P8-2 and 108 of BHV-l were propagated in

Georgia bovine kidney cells as described previously. Babiuk et al. (1975) Infect. Immun. 12:958-963. For virus challenge of animals strain 108 was used, whereas for glycoprotein isolation the P8-2 strain was used.

A culture of Pasteurella haemolvtica (biotype A, serotype 1) was prepared as described previously. Bielefeldt Ohmann et al. (1985) J. Infect. Dis.

151:937-947. In each case, the bacterial challenge was in the log phase of growth and had a titer of 1 to 2 x

10 ⁹ CFU/ml.

I.A.2. Monoclonal Antibodies and Immunoadsorbent

Purificatipn

Monoclonal antibodies against gl, gill, and giv were produced as described previously, van Drunen Littel-van den Hurk et al. (1984), supra. Clones 1E11-1F6, 1D6-G11 and 1G6-2D9 which recognize gl, gill, and gIV, respectively, were selected to prepare immunoadsorbent columns.

Purification of IgG fractions of monoclonal antibodies was carried out using protein A-Sepharose CL-4B (Pharmacia Montreal, Quebec) . L'ltalien in Method

of Protein Microcharacterization. pp. 279-314 (J.E. Shively ed. 1986) . Monoclonal IgG was eluted from the protein A-Sepharose column with 50 mM triethylamine and was dialyzed thoroughly against 0.1 M HEPES, pH 7.5 (coupling buffer: CB) . The purified IgG was linked to activated Affigel-10 (Bio-Rad Laboratories, Mississauga, Ontario) at 5 mg protein/ml gel, according to the manufacturer's instructions.

Glycoproteins gl, gill, and gIV were purified from virus-infected cell lysate as previously described (van Drunen Littel-van den Hurk et al. (1985) Virology 144:216-227) . Twenty-four hours postinfection, at a m.o.i. of 1, cultures were harvested and centrifuged at 1000 rpm to obtain infected cell pellets. Cells were resuspended in 1% Nonidet-P40 (NP-40) and 1% sodium deoxycholate (DOC) in 0.10 M Tris-hydrochloride, 0.15 M NaCl, (pH 7.5) and used as starting material for purification.

Immunoadsorbent columns with specificities for gl, gill, and gIV, respectively, were prepared. After passage of the sample over the column in sample applica¬ tion buffer, the column was exchanged with 1 vol of fresh sample application buffer prior to washing with 2 vol of wash buffer [100 mM Tris, 500 mM NaCl, 1% NP-40 (pH 7.5)]. The wash buffer was displaced from the column with 2 vol of water prior to elution of the specifically bound antigen with 50 mM triethylamine. The eluted fractions were monitored by removing 5-50 μl collected fraction and performing a nonquantitative Bradford assay. Those fractions that contained protein were then directly concentrated for further analysis. The column was reequilibrated in sample application buffer for reuse or stored in sample application buffer plus 0.02% thimerosal. Columns prepared, used, and stored in this way have retained significant activity for almost a year.

I.A.3. Immunization and Pathogen Challenge

Purified glycoproteins were formulated with Avridine (N,N-dioctadecy1-N,N-bis)

(2-hydroxylethylpropanediamine) as follows: 150 mg of Avridine was dissolved in 1 ml of absolute EtOH and then combined with 90 μl Tween 80 by thorough mixing. Next, 4.7 ml of Intralipid were combined with Avridine/EtOH and thoroughly mixed by vortexing. 4.0 mis of biological buffer, e.g. Hanks' buffered salt solution or PBS were added to the solution to complete the adjuvant preparation. The vaccine was prepared by mixing equal volumes of antigen and adjuvant solutions such that each animal received a dose of 100 μg of glycoprotein + 15 mg of Avridine in a 2 ml volume. Groups of five animals each were immunized intramuscularly with the above preparations. Twenty-one days later animals were boosted and then challenged 3 weeks after booster immunization. Control unvaccinated calves were immunized with Avridine (adjuvant alone) . A further control group was immunized with a commercial killed virus vaccine (Triangle 3, Fort Dodge Laboratories, Iowa) as recommended by the manufacturer. Blood samples were taken from animals at 10-day intervals for assessment of antibody responses. Following immunization, animals were transported into an isolation pen and examined clinically, and rectal temperatures were recorded and blood samples were col¬ lected for various immunological assays to establish baseline immunological activity. The calves were then individually exposed to an aerosol of BHV-1, followed 4 days later with E _Λ . haemolytica. In each case, the aerosol was generated by a DeVilbiss Nebulizer, Model 65 (DeVilbiss, Barry, Ontario, Canada) . Treatment was for 4 min in the case of the virus and 5 min with P.

haemolytica as described previously. Bielefeldt Ohmann et al. (1985) , supra.

I.A.4. SDS-PAGE. Western Blot. ELISA and ADCC Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was carried out in 7.5% dis¬ continuous slab gels under reducing conditions, as described previously (van Drunen Littel-van den Hurk et al. (1984) , supra; Laemmli (1970) Nature (London) 227:680-685..

The Western blotting technique was performed as described previously (van Drunen Littel-van den Hurk et al. (1984) , supra) . After electrophoresis, virus lysates were electrophoretically transferred to nitrocellulose sheets. Subsequently, the instructions for use of the

Bio-Rad (Mississaugo, Ontario) immunoblot assay kit were followed.

In order to determine the antibody responses of cattle immunized with purified glycoproteins, the ELISA was performed essentially as described previously (van Drunen Littel-van den Hurk et al. (1984) , supra) . However, affinity-purified, peroxidase-conjugated rabbit anti-bovine IgG (Zymed) at a dilution of 1:3000 was used as the detecting antibody. The neutralization titers of the bovine sera were determined as described previously (Babiuk et al. (1975) , supra) . To determine complement-enhanced neutralization, guinea pig serum (1:40 final dilution) was added to the virus-antibody mixture. The titers were expressed as the reciprocal of the highest dilution of antibody which caused a 50% reduction of plaques relative to the virus control.

ADCC assays were performed in microtiter plates as described previously (Babiuk et al. (1975) , supra) . The ratio of effector cells (polymorphonuclear cells) to

target cells (BHV-1-infected, 51_ _r _ _labeled ___, _ _{ells) was}

50:1. Controls consisted of BHV-l-infected GBK target cells plus anti-BHV-l serum or targets with polymorphonuclear cells in the absence of antibody.

I.A.6. Clinical Evaluation and Necropsy

The clinical evaluations were performed at the same time each day by two independent investigators who were uninformed about the specific treatments of the individual animals. The parameters evaluated included depression, appetite, fever, conjunctivitis, rhinitis, mouth-breathing, tracheitis, and pneumonia. In each case a score of 0 was assigned to healthy animals. Clinical scores of 1-4 were assigned to sick animals for each individual parameter as follows: 4, severe; 3, marked; 2, moderate; 1, mild. Total clinical scores for each animal are the sums of scores for each parameter.

Postmortem examinations were done on animals that died or were euthanized during the experiments. The nasal passages, larynx, trachea, and lungs were examined and photographed. Viral and bacterial lesions were recorded. The extent of pneumonia was assessed by a numerical method developed by Thomson et al. (1975) Canad. J. Comp. Med. 3_£:194-207. The pneumonic lesions in each lung lobe (except for the accessory lobe) were graded from 0 to 5 according to the amount of tissue involved. Total scores for seven lung lobes ranged from 0 to a theoretical maximum of 35 if the entire lung was affected.

I.A.7. Leukocyte Function

To study post-BHV-1-challenge leukocyte function, venous blood was collected into syringes containing citrate dextrose. The blood was centrifuged at 1000 g

for 20 min, the buffy coat was collected, and the peripheral blood mononuclear leukocytes (PBL) were further purified on Ficoll-Hypaque as described previously. Bielefeldt Ohmann et al. (1985) , supra. The polymorphonuclear neutrophils (PMN) were isolated from the original pellet by lysis of the erythrocytes as described previously. The viability of both PBLs and PMNs was greater than 99% as determined by trypan blue exclusion. (i) Functional Analysis of PBL. Lectin-driven lymphocyte proliferation was assayed as described previ¬ ously. Id. Briefly, 1 x 10 PBL were added into quadruplicate wells of a flat-bottomed microtiter plate (Nunc, Roskilde DK) in a final volume of 200 μl of RPMI 1640 plus 5% fetal bovine serum, 50 mM HEPES, and 25 mg gentamycin (all media components are from Grand Island Biological Co., Grand Island, NY). Lectins, phytohemagglutinin (PHA) , and concanavalin A (Con A, Calbiochem, La Jolla, CA) were added to the cultures. The cultures were incubated for 72 hr and labeled with fmethyl- 3HIthymidine (H_-Tdr) (Amersham Co., Oakville,

Ontario) during the last 16-18 hr of incubation. The amount of radioactivity incorporated by PBLs was quantitated by liquid scintillation counting. (ii) Functional Analysis of PMNs. Chemotaxis of PMNs was measured using microchemotaxis chambers. Gee et al. (1983) Proc. Natl. Acad. Sci. USA 80:7215-7218. Briefly, 25 μl of the chemoattractant was added to the bottom wells of the chemotaxis chamber, whereas the top chamber wells contained 45 μl of PMNs. The chemotaxis chambers were incubated for 2 hr in a humidified C0 ₂ atmosphere at 37°C. After incubation, the membranes were removed and nonmigrating cells were scraped from the upper surface. Membranes were fixed, stained with Giemsa, and examined microscopically for the presence of

migrating cells. Cell counts are presented as the mean counts of three representative high-power microscope fields.

Luminol-enhanced chemiluminescence was measured by the method of Abramson et al. (1982). Briefly, 2 x lθ ⁷ cells were added to vials containing 5 ml of Hank's bal¬ anced salt solution, 400 μl of opsonized zymosan, and 20 μl of luminol. Immediately upon addition of the cells, the reaction was followed over time using a Packard Picolite 6500 Luminometer (United Technologies Packard,

7 Downers Grove, IL) . Results are plotted as CPM/10 cells at the peak of the response which occurs at 45 min.

Superoxide anion generation and release were measured by the superoxide dismutase (SOD) inhibitable reduction of ferracytochrome C as described previously. Johnson et al. (1978) J. Exp. Med. H£:115-127. All samples were assayed in duplicate and in suspension in a final volume of 1 ml. The samples were incubated for 45 min at 37 ^β C. The reaction was terminated by transferring 1 ml aliquots to an ice bath followed by centrifugation. The cytochrome c. reduction was monitored on a spectrophotometer at 550 nm. The OD value was then converted to nm 0_/cell.

II

This example demonstrates the production of non-native BHV-1 subunit antigens in recombinant vaccinia virus vectors.

II . A. Recombinant Production of oIV in a Vaccinia Vector

II.A.1. Construction of the pWSL-l Insertion Vector

The pWSL-1 insertion vector was constructed as depicted in Figures 2 and 3. Vaccinia virus vectors are known, such as VAC-I and VAC-III deposited as ATCC VR-2223 and VR-2224, respectively July 22, 1988.

Specifically, the pW-l expression vector was derived from the pGS-20 plasmid (van Drunen Littel-van den Hurk et al. (1989) J. Virol. 62:2159-2168). pGS-20 was digested with Xhol and Aval, treated with DNA poll Klenow fragment to blunt the ends and then ligated. This process resulted in the deletion of a 2,200 bp fragment and the production of pW-l (Figure 2) . pWSL-1 was then constructed from elements of pW- 1 and a series of four synthetic oligonucleotides representing an adenine rich region, a spacer and a consensus sequence from the vaccinia virus late gene promoter. (This sequence was based on a sequence described by Davison and Moss (1989) J. Mol. Biol. 210:771-784.. A 3,080 bp EcoRI-Pstl fragment and a 1,700 bp Xmal-Pstl fragment were isolated from restriction enzyme digests of pW-l. The fragments were then ligated together with the four oligonucleotides to form pWSL-1. The pWSL-1 has two unique cloning sites; Bglll or Smal (Figure 3) .

II.A.2. Insertion of the Full-Lenoth BHV-1 gIV Gene into pWSfr-l The procedures for gene insertion and recombinant vaccinia virus recovery were the same as that described for pGS-20. (See, van Drunen Littel-van den Hurk et al. (1989) J. Virol. 62:2159-2168.)

The BHV-l gIV sequence shown in Figure 1 was digested with Mael restriction endonuclease. The resulting Mael sites at sequence positions 42 and 1344 were converted to Bglll sites using commercially obtained

oligonucleotide linkers (Pharmacia) . The resulting Bglll adapted BHV-1 gIV gene was then cloned into the Bglll cloning site of pWSL-1 to yield the expression vector pW-l/glV (Figure 4).

II. .3. Insertion of a Truncated BHV-1 gIV Gene into PWSL-1 The gene for BHV-1 gIV was modified to incorporate a stop codon in the reading frame immediately preceding the putative membrane spanning region of the mature protein (Figure 1) . This modification of the gene results in the early termination of translation and secretion of the truncated protein. This system eliminates the requirement for extensive downstream processing that is associated with the production of antigens that are associated with membranes, and causes an up to tenfold increase in product yield.

The Bglll adapted gene for BHV-1 gIV was partially digested with SacII. The SacII site at position 1154 (Figure 1) was then converted into a Xhol site using commercially obtained oligonucleotide linkers (Pharmacia) . A second synthetic oligonucleotide linker was then inserted at the Xhol site to yield an in-frame stop codon. The resulting modified BHV-l gIV gene was then cloned into the Bglll cloning site of pWSL-1 to yield the expression vector pW-1/gIVt (Figure 5) .

II.A.4. Purification of Recombinantlv Expressed gIV

BSC-1 cells were cultured in MEM containing 10% fetal bovine serum. Confluent monolayers were infected with the recombinant vaccinia, expressing either full- length or truncated and secreted BHV-1 gIV at a multiplicity of 0.1. 72 hours after infection or at the appearance of total cytopathic effect, the recombinant gIV was harvested.

For full-length gIV, the cells were scraped from the surface of the culture flasks into the growth media, which was then centrifuged (lOOOg for 20 min) and the cell pellet collected. The cells were disrupted with detergent and further processed as previously described. Truncated gIV was collected by harvesting the media from the culture flasks. Cell debris was removed by centrifugation at lOOOg for 20 min. The clarified media was frozen at -70°C until processing. After thawing, the media was filtered through a 0.45 micron filter. The detergents, Nonidet P40 and Na Deoxycholate, were then added to the filtrate to final concentrations of 0.1%. The truncated gIV was then purified by affinity chromatography through BHV-l gIV specific columns, as has been described previously.

Ill

This example demonstrates the production of non- native BHV-1 subunit antigens using a Baculovirus system.

III.A. Materials and Methods

III.A.l. Cells. Viruses and Antibodies s_. frugiperda (SF9) cells were grown and maintained in TNM-FH medium (GIBCO) containing 10% fetal bovine serum as described by Summers and Smith (Summers, M.D. , and Smith, G.E., "A manual of methods for baculovirus vectors and insect cell culture procedures" Texas Agricultural Experiment Station bulletin no. 1555 (1987) . Texas Agricultural Experiment Station, College Station, Tex.) . Virus stocks of the wild type AcNPV and recombinant baculoviruses were prepared in SF9 cells as described by Summers and Smith (supra) . Monoclonal antibodies specific for gIV were developed and

characterized by van Drunen Littel-van den Hurk et al. (1987) Virology 160:465-472: and Hughes et al. (1988) Arch. Virol. 103:47-60. The gIV-specific monoclonal antibody mixture used for identification of recombinant glV consisted of equivalent amounts of 136 (epitope la) , 9D6 (epitope lb) , 3E7 (epitope II) , 10C2 (epitope Ilia) , 4C1 (epitope Illb) , 2C8 (epitope IIIc) , 3C1 (epitope Hid) , and 3D9S (epitope IV) .

III.A.2. Insertion of BHV-1 Glycoproteins into the AcMNPV Transfer VectQr The gIV glycoprotein gene was isolated from a subclone of pSD9 as a 1.3 kb Mael fragment (Tikoo, S.K. et al. (1990) J. Virol. 6 :5132-5142, which was blunt end repaired and inserted into the plasmid pRSV cat

(Fitzpatrick, D.R. et al. (1988) J. Virol. 62:4239-4248). The 1.3 kb Bglll fragment was then subcloned into the BamHl site of the baculovirus transfer vector pVL941 (Luckow, V.A. and Summers, M.D. (1989) Virology 170:31- 39) . Plasmid DNA was prepared by alkaline lysis and cesium chloride gradient centrifugation by standard methods. After transformation of £. coli JM105, colonies appearing on LB agar containing 100 μg of ampicillin per ml were inoculated into L broth containing ampicillin (50 μg/ml) and incubated at 37 ^β C overnight with vigorous shaking. Small scale preparations of plasmid from each colony were prepared, and the presence of the gIV gene was confirmed by digestion with endonucleases Aval and EcoRV. A single clone was identified containing the gIV gene in the desired orientation and designated pVLglV. Clone pVLglV was inoculated into 500 ml. of L broth containing ampicillin; after 24 h at 37°C, the plasmid was prepared by alkaline lysis and further purified by equilibrium centrifugation on cscl.

A modified BHV-1 gIV gene, as described in Example II.A.3, that produces a truncated form of gIV, was digested with Bglll, the fragment isolated and subcloned into the BamHl site of the baculovirus transfer vector pVL941. This construct was named pVLglVt.

The gl glycoprotein gene contained in plasmid pgB complete, described in Example II.B.l of U.S. patent 5,151,267, was digested with Bglll and BamHl and inserted into the BamHl site of the baculovirus transfer vector pVL941.

The gill glycoprotein gene contained in plasmid pll3Rl Bgl 3.0, as described in Example II.B.l of U.S. patent 5,151,267, was transferred to the BamHl site of baculovirus transfer vector pVL9 1 as an EcoRI + BamHl subfragment. This construct was designated pVLglll.

III.A.3. Transfection and Selection of Recombinant Viruses After two cycles of ethanol precipitation, purified plasmid was mixed with an equal amount of A.. californica viral DNA and used to transfect subconfluent monolayers of SF9 cells as outlined by Summers and Smith (supra) . After incubation at 28°C for 5 days, the supernatant was serially diluted and inoculated onto confluent monolayers of SF9 cells. After 1 h, an overlay consisting of TNM-FH medium containing 6% fetal bovine serum and 1.5% low-gelling-temperature agarose was added, and the plates were incubated at 28°C for 5 days. Recombinant baculoviruses were identified by plaque hybridization essentially as outlined by Summers and

Smith (supra) . The polyhedrin-negative recombinants were plaque purified three to four times on SF9 cells to remove contaminating wild-type baculoviruses.

III.A.4. Preparation of Cell Lvsates

To analyze expression of recombinant gIV confluent monolayers of SF9 cells on 35 mm petri dishes were infected with individual polyhedrin-negative recombinants at a multiplicity of infection of 5 and incubated for 48 h at 28 ^Q C. The cells were scraped into phosphate- buffered saline (PBS) , pelleted at 150 x g for 1 min, and suspended in 50 μl of RIPA buffer (0.02 M Tris hydrochloride [pH 8.0], 0.15 M NaCl, 1% sodium deoxycholate, 1% Nonidet P-40, 10 mM EDTA, 10 mM phenylmethylsulfonyl fluoride) . The postnuclear supernatant was collected, combined with reducing electrophoresis sample buffer, and boiled for 2 min.

IV

This example demonstrates the production of non- native BHV-1 subunit antigens in J_, coli vectors.

IV.A. Cloning and Expression of the BHV-1 gl Gene in E. coli A BHV-1 genome library of BHV-1 strain Cooper as represented in 12 HindiII fragments cloned in to pBR322, was screened according to the methods of Southern, using probes corresponding to HSV-l gB and the pseudorabies virus (PRV) gB gene counterpart. A single clone, pSD106, was found to bind to the probes.

In order to specify the actual coding sequences of the BHV-1 gl gene, a detailed restriction map was constructed for pSD106. The application of the techniques of Southern using separate probes specific for the HSV-1 gB amino and carboxy-termini, located the BHV-1 gl gene within a Kpnl - Sail sub-fragment of pSD106 (pSD106 Kpnl-Sall; Figure 6) .

It is not expected that foreign viral proteins produced in Ej_ coli would be folded into a configuration that mimics the protein in the native virus particle. Therefore, the most active immunogen obtained from bacterially expressed genes is likely to be composed of a contiguous stretch of amino acids rather than one which requires the positioning of two or more contiguous sequences of amino acids (i.e., tertiary structure). An additional limitation is that E^. coli will not process pre gl to gib + glc. Thus, it was hypothesized that the optimal formulation of bacterially expressed gl would be composed of gib and glc, expressed individually and then utilized either separately or in combination.

To construct a clone expressing the amino terminus of mature gib + glc, the pSD106 Kpnl - Sail fragment was cut with Apal and the lengths of the Apal fragments randomized by treatment with Bal31 exonuclease. Any asymmetric fragment\ends produced by imprecise Bal31 activity were blunted by Klenow fill-in reactions. The Apal digested plasmid was then cut with Xmal and the population of fragments carrying sequences encoding the amino terminus of gib were isolated from LMP agarose gels as a 600-520 bp smear. This fragment sub-population was then ligated to the Hpal - Xmal sites of polink 26. Polink 26 was a modified form of pBR328 and contained a synthetic oligonucleotide inserted into the EcoRI and Sail sites. The oligonucleotide contained restriction enzyme sites in the following order: EcoRI, Xbal, Bglll, Ball, Hpal, Clal, Hindlll, Kpnl, BamHl, Smal, Sphl, Sad, Xhol, Sail.

An insert from the clone bank of the amino terminus of gl was isolated by Bglll and Smal digestion of pBHB5' and transferred to the Bglll plus Smal sites of the £-. coli expression vector pHK414. The resulting clone was named pRed30. To complete the construction of

the gib coding sequence, SmaG was isolated as a 775 bp Xmal fragment from pSdl06 Kpnl - Sail and was ligated to the Xmal site of pRed30 to produce pgpll: _/ 3gal. This plasmid was transformed into the E^ coli strain NF1829, produced a gpll:3-galactosidase fusion protein of approximately 175k (gpll is another designation of the gib peptide) . An unfused gib peptide was made by the "gpll complete" clone, "pgpll complete" (Figure 7) was constructed by transferring the 1340 bp Bglll - BamHl fragment from pgpll:/3gal to the S _J . coli expression plasmid pGH346. The 1340 bp fragment carries the entire gib coding sequence plus DNA encoding the first eight amino acids of gib. In NF1829, "pgpll complete" produces a 60k protein which specifically reacts to BHV-l gib polyclonal sera and gib specific monoclonal antibodies.

IV.B. Cloning and Expression of BHV-1 gill gene in E. coli The BHV-1 genomic library was screened by the techniques of Southern using probes specific for the coding sequence of the HSV-1 gC and the PRV gC homolog (PrV gill) . One of the Hindlll clones, designated pSD113 (Figure 6) , hybridized to both the probes. A restriction endonuclease map of the pSD113 plasmid was prepared in order to locate the region within the Hindlll fragment which specifically encoded the BHV-1 gill gene. By using a probe specific for the PRV gC gene homolog, the BHV-1 gill gene was located within an approximately 2500 bp EcoRI - Bglll subfragment of pSD113. An £_. coli clone expressing the carboxy-terminal two-thirds of the BHV-1 gill gene was made in the following way. The pSD113 EcoRI - Bgll subfragment of pSD113 was digested with Xmal and inserted into the Xmal site of the plasmid pJS413 to form pBHC3'. The pSD113 EcoRI - Bglll subfragment of pSD113 was digested with

Pvul and the Pvul asymmetric ends were treated with Klenow enzyme to blunt them. The Pvul digested plasmid was then cut with Sad, and the 1600 bp Pvul - Sad fragment containing the carboxy-terminus of the gill was purified from LMP agarose gels. This fragment was ligated to Smal plus Sad sites of plasmid ptac413 to produce pll3Pvu/413. The extraneous sequences 3' to the stop codon were removed by replacing the 900 bp Aval - Sad fragment from pll3Pvu/413 with the 2220 bp Aval - Sad fragment from BHC3'. The new plasmid "pll3 Pvu end" carried 900 bp of BHV-l gill gene carboxy-terminus in frame to the reconstituted _/ S-galactosidase gene. The E. coli strain NF1829 was transformed with this plasmid.

Upon induction with lactose, these cells produced approximately 5% of the total protein as a gill related protein. The fusion product was collected as aggregates and solubillzed in 10 mMol urea, pH 9.0. The solubilized protein was dialyzed against 10 mMol triethylamine, pH 9.0 to remove urea and then concentrated by Amicon filtration to a final concentration of 2 mgs/ml of BHV-l protein. The concentrated gill fusion protein was formulated with Freunds' Complete Adjuvant and injected intramuscularly to rabbits in 1 mg doses. The antisera raised in these rabbits was used to immunoprecipitate a BHV-l infected cell extract and was found to specifically react with the gill glycoprotein.

In an attempt to express the full-length BHV-1 gill gene in J_. coli. the expressor clone "113Pvu end" was extended to include all gill coding sequences up to the Ncol site ("gill complete") . This full-length clone failed to produce significant amounts of gill protein. The simple removal of the signal sequence and/or addition of preferred E^. coli codons upstream of the gill sequences failed to improve expression. In order to produce the largest EL. coli gill expressor clone

possible, and to avoid the apparent problem in expressing the amino terminal gene sequences, a library of gill clones was generated with randomized amino termini. The E. coli plasmid "gill complete", which does not express significant amounts of gill protein, was digested with Ncol and then treated with Bal3l for varying lengths of time. Ncol cut at the ATG of the gill gene and Bal31 treatment removed nucleotides starting from the Ncol cut ends to varying extents. The population of random sized fragments were treated with Klenow enzyme to blunt asymmetric ends and were then digested with BamHl. The population of Ncol - Bal31 - BamHl fragments ranging in size from 1530 bp to 1250 bp were isolated from LMP agarose and ligated to the Smal - BamHl sites of pJS413. Ej. coli NF1829 was transformed with this ligation mixture and plated to MacKonkey's Agar. Putative expressor clones were picked as red colonies. The positive colonies were then tested for protein production by lactose induction and protein analysis by SDS-PAGE. Three clones were found to express gill as a β- galactosidase fusion protein. The clones were found to differ in the amount of amino terminal sequence removed by the Bal31 treatment: losing 150, 250, and 350 bp, respectively. The gill gene inserts were excised by Bglll - BamHl digestion of the Bal31 expressor plasmids and then purified from LMP agarose gels. Each gill carrying fragment was then ligated into the Bglll - BamHl sites of the Ε . coli expressor plasmid GH435. pGH435 carries stop codons in each of the three possible reading frames immediately 3' to the BamHl insertion site. Therefore, expression of any insert at the Bglll and BamHl sites would generate a nonfused peptide. The largest Bal31 clone, called pBHC150Δ (Figure 8), which had a deletion of approximately 150 bp at the amino terminus of gill, makes a peptide of approximately 53K

upon lactose induction. This plasmid is carried in the E. coli strain W3110F'Iq.

IV.C. Expression of Full-Length Mature BHV-1 gIV in E. coli

The BHV-1 genomic library clone pSD98 (Figure 6) was identified as carrying the gIV gene, in addition to several other putative BHV-1 genes. Restriction enzyme mapping of pSD98 mapped the gIV protein encoding sequences to a Xmall - Xhol fragment of this plasmid. pSD98 was digested with Xmal and Xhol, the fragment isolated and inserted into the plasmid polink 26 to produce p98XmaI - Xhol.

The construction of the gIV gene containing £-. coli expression vector is depicted in Figure 9. The signal sequence does not appear in the mature gIV protein and does not contribute to immunogenicity of the glycoprotein. Therefore, the signal sequence was removed by making a synthetic oligonucleotide corresponding to the coding sequence of the first amino acid of the mature BHV-l gIV gene (i.e.. Leu) and extending to the Sail site 88 bp downstream from the start (ATG) of the gene (see Figure l) . An engineered Hindlll asymmetric overhang was added immediately 5' to the Leu codon and a Xmal asymmetric end was added immediately 3' to the Sail site (see Figure 9) . The Hindlll and Xmal overhangs permitted ligation of the oligonucleotide into the Hindlll plus Xmal sites of the J,. coli expression vector pHK414. The resultant plasmid was called BHDsyn43 and is carried in the J . coli strain MC1066.

The amino-terminus of the gIV clone BHDsyn43 was extended by ligating the 590 bp BamHl - Sail fragment from p98Xma-XhoI to the Sail plus BamHl sites of the BHDsyn43. This ligation produced pBHD5' and carries the first 620 bps of the coding sequence for mature BHV-1

glV. The pBHD5' was also maintained in the J_. coli strain MC1066.

An Ej. coli clone expressing full length mature BHV-1 gIV was made by first transferring the gIV insert from pBHD5' carried in a 630 bp Bglll - BamHl fragment, to the Bglll - BamHl sites of the J_. coli expression plasmid GH432. The carboxy terminal half of the gIV gene was then added by ligating the 640 bp Xmal fragment from pSD98 to the Xmal site in the new construction BHD5'/432. The plasmid pSD98 was part of the original BHV-1 genomic library BHV-1 strain Cooper (Figure 6) . The library takes the form of 12 Hindlll clones of the viral genome inserted into the pBR322. Plasmid pSD98 was identified as part of the gIV gene partially by its location in the "S" region of the BHV-1 genome, that corresponds to the location of the gD of other herpesviruses, e.g., HSV-1 and 2, PRV and EHV-1, and reactivity in Southern blots using a probe corresponding to the PRV gIV gene homologue. The final BHV-1 gIV Z _Λ . coli expressive plasmid is called pBHDsib (Figure 10) and was transformed in the E. coli strain W31104'Iq. Upon induction with lactose (2% final concentration) , pBHDsib made a 58K protein which represented approximately 10% of the total protein produced by the clone. In a western blot assay, the 58K protein reacted specifically with anti-BHV-1 hyperimmune serum and the BHV-1 gIV specific monoclonal antibody 3D9.

An aggregate preparation from pBHDsib was solubilized in 50 mMol /3-mercaptoethanol, 0.5% SDS pH 8.0. Doses containing 150 μg of the 58K solubilized protein were prepared in Freunds' Complete Adjuvant and administered to rabbits by intramuscular injection. Sera raised in these animals reacted specifically with a glycoprotein from BHV-1 infected cell extracts indistinguishable from gIV. When assessed for BHV-1

2

-46-

virus neutralizing activity in vitro. the rabbit sera was found to have a plaque reduction titre of 1:128.

IV.D. Purification of Recombinant BHV-1 Glycoproteins

E. coli W3110F'Iq transfected with pBHDsib was cultured in L broth supplemented with ampicillin (50 μg/ml) . Late log growth phase cultures were induced with either 2% lactose or 2 mM IPTG. Five hours after induction, the cultures were harvested, the cells pelleted by centrifugation (2000g for 20 min) and then mechanically disrupted. The aggregates were subsequently dispersed by treatment with 6M GuHCl and then dialyzed to reduce the GuHCl concentration to 2M. The other BHV-l glycoproteins expressed in J*. coli are similarly purified.

The following example illustrates the production of non-native BHV-l subunit antigens in recombinant adenovirus vectors.

V.A. Expression of BHV-1 gIV Using Adenovirus Vectors In one form, this expression system is based on human adenovirus serotype 5. The El and E3 regions of the virus have been deleted to facilitate the accommodation of large gene inserts into the viral genome. 293 cells, human kidney cells transformed with human adenovirus type 5 DNA, express the viral El proteins constitutively. As a result, the combination of vectors based on human adenovirus type 5 and 293 cells form an ideal expression system.

A transfer vector, pAdBM5 (Figure 11) , was developed in order to insert foreign gene sequences into

human adenovirus serotype 5. The vector contains the adenovirus major late promoter (Ad2MLP) , enhancer sequences, and polyadenylation sequences, flanked by the adenovirus serotype 5 El flanking sequences. A unique BamHl site is located downstream of the MLP and is used for the insertion of foreign genes.

For the expression of either full-length BHV-l gIV or truncated BHV-1 gIV, the appropriate gene constructs were described above, were digested with Bglll and inserted into the dephosphorylated BamHl site of the pAdBM5. Figure 12 depicts pAdBM5.gIV, the adenovirus vector including the gene encoding full-length gIV. The plasmid DNA was digested with Clal, mixed with purified human adenovirus serotype 5 DNA and was used to transfect 293 cells using the calcium phosphate technique. The transfected cells were plated out and incubated at 37°C until a cytopathic effect developed. Supernatant from these cultures was then removed and used to reinfect 293 cells. Recombinant virions expressing BHV-l gIV were identified, selected and then further purified by plaque assay.

V.B. Purification of Recombinant Expressed BHV-1 gIV 293 cells were cultured in MEM containing 10% fetal bovine serum and supplemented with IX vitamins and minerals. Confluent monolayers were infected with the recombinant adenovirus expressing BHV-l gIV at a multiplicity of 0.1. 24 hours after infection or at the appearance of total cytopathic effect, recombinant gIV was harvested.

For full-length gIV, the cells were scraped from the surface of the culture flasks into the growth media, which was then centrifuged (1000 g for 20 min) and the cell pellet collected. The cells were disrupted with detergent and further processed as previously described.

Truncated gIV was collected by harvesting the media from the culture flasks. Cell debris was removed by centrifugation at lOOOg for 20 min. The clarified media was frozen at -70°C until processing. The truncated gIV was purified by affinity chromatography through BHV-1 gIV specific columns, as has been described previously.

VI

This example illustrates the efficacy of recombinantly produced BHV-1 subunit antigens.

VI-A. Materials and Methods

VI.A.1. Cells and Viruses

E. coli cells were cultured in L Broth. Madin Darby bovine kidney cells (MDBK) cells, BSC-1 cells, and 293 cells were cultured in Eagle's minimal essential medium (MEM; GIBCO laboratories. Grand Island, N.Y. , USA) , supplemented with 10% fetal bovine serum (FBS; GIBCO) . Spodoptera frugjperda (SF9) cells were grown and maintained in TNM-FH medium (GIBCO) containing 10% FBS. Strains P8-2 and 108 of BHV-1 were propagated in MDBK cells as described previously (Babiuk et al. (1975)

Infect.Immun. 12:958). Virus recovered from nasal swabs was quantified by plaque titration on MDBK cells in microtiter plates with an antibody overlay as previously described (Rouse et al. (1974) J.Immunol. 112:1391). Vaccinia virus (WR strain) and recombinant vaccinia virus were propagated in BSC-1 cells as described in Example II. Human adenovirus type-5 and recombinant adenovirus were grown in 293 cells as described in Example V. Virus stocks of the baculovirus AcNPV and recombinant virus were prepared in SF9 cells as described by Summers and

Smith (Summers et al., "A manual of methods for baculovirus vectors and insect cell culture procedures. Texas Agricultural Experiment Station Research bulletin no. 1987, 1555, Texas Agricultural Experiment Station, College Station, Tex) .

VI.A.2. Recombinant Expression of gIV

BHV-1 gIV was recombinantly expressed in E. coli. baculovirus, SF9 cells, adenovirus, vaccinia virus and mammalian cells, as explained in the above examples.

VI.A.3. Preparation of Immunoadsorbent Columns

The IgG fraction of the gIV-specific monoclonal antibody 3DlGb was prepared from ascites fluid, using a protein A-Sepharose CL-4B (Pharmacia, Montreal, Quebec, Canada) column. The purified IgG was dialyzed thoroughly against 0.1 M HEPES, pH 7.5 and linked to activated Affigel-10 (BioRad Laboratories, Mississauga, Ontario, Canada) at 5 mg protein per ml gel according to the manufacturer's instructions. An immunoadsorbent column was packed for each of four different gIV species.

vitftt4t Purificatipn of glycoproteins Glycoprotein gIV was purified from BHV-1 (strain P8-2) infected MDBK cells, recombinant AcNPV infected SF9 cells, recombinant adenovirus infected 293 cells or recombinant vaccinia virus infected BSC-l cells. Cell lysates were prepared from the virus infected cells, essentially as described previously (Van Drunen Littel- van den Hurk et al. (1985) Virology 111:204-215). Briefly, the cells were harvested, centrifuged at 1000 rpm and resuspended in 10 mM Tris-hydrochloride, 150 mM NaCl, pH 7.5 containing 1% Nonidet P40 (NP40) and 1% sodium deoxycholate. After the cell lysates had cycled

three times through the respective gIV-specific monoclonal antibody columns, the columns were washed with one volume of sample application buffer and two volumes of wash buffer (10 mM Tris-hydrochloride, 500mM MaCl, 0.1% NP40, pH 7.5). Specifically bound antigen was eluted with 50 mM diethylamine, pH 11.5, immediately neutralized with 1 M Tris-hydrochloride, pH 7, and concentrated on an Amicon YM30 membrane. The columns were re-equilibrated in sample application buffer for re- use or stored in phosphate-buffered saline (PBS) , containing 0.02% sodium azide. The protein content was determined with the BioRad protein determination kit. The purity was assessed by pdlyacrylamide gel electrophoresis alone and in combination with western blotting. Finally, the purified proteins were applied to appropriate cells to test for residual input virus.

VI.A.5. Polvacrvlamide Gel Electrophoresis

Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) was carried out in 10% discontinuous gels under reducing conditions as described previously (Laemmli, U.K. (1970) Nature 227:680-685).

VI.A.6. Immunization Groups of eight calves each were immunized intramuscularly with 25 ug of purified gIV, produced by BHV-1, baculovirus, adenovirus or vaccinia virus, or 100 ug of gIV produced in £ _* . coli. The glycoproteins were combined with the adjuvant Avridine as described previously (Babiuk et al. (1987) Virology 159:57-66.. A control group was vaccinated with Avridine only. Twenty- one days later the animals were boosted and then challenged with BHV-1 fourteen days after the booster immunization. Blood samples were taken from the animals at the time of the first immunization, booster

immunization, and challenge, as well as ten days after challenge for assessment of antibody responses.

VI.A.7. Experimental Challenge Fourteen days after the second immunization, animals were transported into an isolation pen and examined clinically. Their weights and rectal temperatures were determined and recorded. Blood samples and nasal swabs were collected to establish baseline values. The calves were then individually exposed to an aerosol of 10 ⁷ pfu per ml of BHV-1 strain 108, which was generated by a DeVilbis Nebulizer, model 65 (DeVilbis, Barrie, Ontario, Canada) . The duration of the treatment was 4 min per calf.

VI.A.8. Clinical Evaluation

The clinical evaluations were performed at the same time each day by two independent investigators who were uninformed about the specific treatments of the individual animals. The parameters evaluated included depression, appetite, fever, conjunctivitis, rhinitis, mouth-breathing and tracheitis. In each case, a score of 0 was assigned to healthy animals. Clinical scores of 1- 4 were assigned to sick animals as follows: 4, severe; 3, marked; 2, moderate; 1, mild. Temperatures were taken every day and nasal swabs were collected every other day and processed the same day. Blood samples were collected ten days after challenge.

VI.A.9. Enzyme-linked Immunosorbent Assay (ELISA)

In order to determine the gIV-specific antibody responses of the calves, the ELISA was performed essentially as previously described (Van Drunen Littel- van den Hurk (1984) Virology 135:466) . Polystyrene microtiter plates (Immulon 2, Dynatech Laboratories Inc.,

Alexandria, VA, USA) were coated with 0.05 ug purified gIV per well and incubated with serially diluted bovine sera. Affinity-purified horseradish peroxidase (HRPO)- conjugated rabbit anti-bovine IgG (Kirkegaard _ Perry Laboratories, Gaithersburg, Maryland, USA), at a dilution of 1:5000, was used as the detecting antibody. Antibody isotypes were determined in an indirect ELISA using giv- coated plates and isotype-specific monoclonal antibodies (provided by Dr. K. Nielsen, Agriculture Canada, Animal Diseases Research Institute, Nepean) . Affinity-purified HRPO-conjugated goat anti-mouse IgG (Boehringer-Mannheim, Dorval, Quebec, Canada) at a dilution of 1:10,000 was used as the detecting antibody.

VI.A.10. Competitive Antibody Binding Assay fCBA. The CBA was based on the ELISA modified as previously described (Van Drunen Littel-van den Hurk et al. (1985) Virology 144:216-227.. Briefly, gIV coated plates were incubated with serially diluted competitor antibodies from the gIV-immunized calves. After a 3 hour incubation at 37°C, the plates were washed and incubated with HRPO-conjugated monoclonal antibodies specific for eight different epitopes on gIV (Van Drunen Littel-van den Hurk (1984) Virology 135:466: and Hughes et al. (1988) Arch. Virol. 103:47) . After a 1 hour incubation at 37°C, the plates were washed again and developed. The percentage competition was calculated using the formula [100x(A-B)/A] where A is absorbance in absence of competitor antibody and B is absorbance in the presence of competitor monospecific antibody.

VT.A.ll. Neutralization Test

The neutralization titers of the bovine sera were determined as described previously (Babiuk, L.A. et al. (1975) Infect.Immun. 12:958). The titers were expressed

as the reciprocal of the highest dilution of antibody that caused a 50% reduction of plaques relative to the virus control. Neutralization titers were also determined for the nasal swabs of the immunized animals and calculated in the same manner.

VLB. Results

VI.B.l. Purification of Authentic and Recombinant gIV Authentic and recombinant gIVs were purified on gIV-specific monoclonal antibody columns. All of the recombinant gIV glycoproteins were produced at higher levels than the authentic gIV from BHV-1 (Table 1) .

Table 1

Source of σiv 'Yieldl tug/ 10 ⁶ cells.

BHV-1 1 - 2.5 Baculovirus 15 - 35

Adenovirus 3.5 - 8.5

Vaccinia virus 2.5 - 5.5

£-. coli ^b 500 - 1000

* The yields of gIV from mammalian cells were determined with the BioRad protein determination kit. ^b The yields of gIV from J _s . coli are expressed in ug per liter and represent values obtained on bench scale before optimalization.

The purity of the glycoprotein preparations was assessed by SDS-PAGE. All of the recombinant forms of gIV bound specifically to the columns. The apparent molecular weights of authentic gIV and gIV from vaccinia virus and adenovirus were identical, indicating that processing and

glycosylation of authentic gIV in MDBK cells and recombinant gIV in BSC-1 or 293 cells are very similar. Recombinant gIV from baculovirus, however, had an apparent molecular weight of 63 kDa, which is lower than that of the 71 kDa authentic form. In addition to the 63 kDa species, four bands of lower apparent molecular weight were observed. These bands were consistently seen both in pure and in crude preparations of gIV from baculovirus. Recombinant gIV from £_. coli had an apparent molecular weight of 54 kDa, which corresponds to the molecular weight of the unglycosylated form of gIV (Van Drunen Littel-van den Hurk (1986) Virology ≤_:401- 410) . As about 50% of the total protein preparation from E. coli consisted of gIV, this recombinant protein was not further purified. The gIV from £,. coli was not dimerized at all, whereas the gIV from baculovirus showed a much reduced degree of dimerization as compared to authentic gIV.

VLB.2. Immune Responses to Authentic and Recombinant

_____

In order to determine whether the different forms of recombinant gIV have the same protective capacity as authentic gIV, they were evaluated in a BHV-1 challenge experiment as described above. The level and the specificity of the total antibody response following immunization was determined in an ELISA using authentic gIV, gl or gill as the antigens. After one immunization, high levels of gIV-specific antibodies were found in the sera of all immunized animals. The antibody titers increased following the booster immunization. There was no significant difference between the antibody titers induced by gIV from BHV-1, baculovirus, adenovirus or vaccinia virus. However, the antibody titers generated by gIV from £_. coli were 5-fold higher after the booster

immunization. None of the animals reacted with gl or gill, showing the specificity of the immune response. In no case did the placebo-vaccinated animals produce any immune response. In order to predict the effectiveness of the glycoprotein-specific antibodies to prevent infection, the serum neutralizing antibody titers were determined. After one immunization, gIV from BHV-1, baculovirus, adenovirus and vaccinia virus induced reasonably good levels of neutralizing antibodies, which increased to very high levels following the booster immunization. Again, there was essentially no difference between the immune responses to these four forms of gIV. In contrast, there was a significant difference in the neutralizing antibody response to gIV from S _J . coli. Even after two immunizations, the neutralizing antibody titer induced by this form of gIV was lower than the level induced by one immunization of any of the other forms of gIV. The contribution of antibody isotypes to the immune response was investigated by indirect ELISA. The IgGl titers were higher than the IgG2 titers throughout the period before challenge. The IgGl titers reached peak values after two immunizations and then started to plateau and decrease after challenge. The IgG2 titers were lower initially, but generally continued to increase after challenge. The IgM titers were much lower than the IgGl or IgG2 titers throughout the duration of the experiment. The antibody isotypes were generally similar between the groups immunized with the different forms of recombinant gIV. However, the IgM levels induced by gIV from £j. coli were significantly higher than those induced by the other forms of gIV. The IgGl response, however, was slower in this group.

VI.B.3. Epitope Specificity of the Immune Response to Authentic and Recombinant gIV

Recombinant gIV from E^. coli induced a lower level of neutralizing antibodies to BHV-1 than the other recombinant gIVs, although the total antibody response was equivalent or higher. In order to determine which of the neutralizing epitopes on gIV were recognized, the sera from all immunized animals were tested with respect to epitope specificity. Seven neutralizing epitopes (epitopes la, lb, II, Ilia, IHb, IIIc, and Hid) and one non-neutralizing epitope (epitope IV) have been mapped on gIV (Hughes et al. (1988) Arch. Virol. 102:47). All epitopes on gIV were recognized by animals immunized with gIV from BHV-1, baculovirus, adenovirus, or vaccinia virus; blocking varied between 30 and 95%, depending on the epitope. These values correlate well with previously reported values between 30 and 85% (Van Drunen Littel-van den Hurk (1990) Vaccine 2:358-368). However, the neutralizing epitopes on gIV were either not at all (la, lb, II, Ilia, and IHb) , or poorly (IIIc and Hid) recognized by animals immunized with gIV from J . coli. The only epitope recognized well by these animals was the non-neutralizing epitope IV. These results indicate that the neutralizing epitopes on gIV, most of which are conformation-dependent (Hughes et al. (1988) Arch. Virol. 103:47) . are present on gIV from baculovirus, adenovirus and vaccinia virus, but not on gIV from Ej. coli.

VLB.4. Protection from Challenge with BHV-1 All animals were challenged with an aerosol of

BHV-1. Prior to challenge, all animals were healthy and they had a normal rectal temperature. However, within 24 h post infection, the animals in the placebo-immunized group started to exhibit a sharp rise in temperature. The temperatures continued to increase until three days

post challenge, whereafter they declined again. There was no significant increase in temperature in the glV- vaccinated groups, although the animals immunized with gIV from E coli did experience some elevated temperatures during the first two days after infection. In addition to the temperature responses the calves were clinically evaluated for signs of respiratory disease. The clinical illness scores correlated well with the temperature responses. The animals in the placebo- immunized group showed signs of clinical illness from day 1 until day 7 post challenge, whereas the groups immunized with gIV from BHV-1, baculovirus, adenovirus or vaccinia virus experienced no illness at all. The group that received gIV from J_. coli showed mild disease for three days after infection. A further non-subjective assessment of morbidity is the extent of weight loss of animals challenged with BHV-1. The weight loss observed in the placebo-immunized group is a reflection of the anorexia as a result of the morbidity due to viral challenge. In contrast to the placebo-immunized group, gIV immunized animals experienced minimal or no weight loss during the 8 days following challenge.

VLB.5. Induction of Mucosal Immunity With the exception of the group immunized with gIV from £_. coli. all animals vaccinated with authentic or recombinant gIV were fully protected from disease, when challenged with BHV-1. To determine whether they were also protected from viral infection, the extent of virus shedding from the nasal passages was assessed.

Essentially no virus was recovered from the nasal swabs of animals vaccinated with gIV from BHV-1, baculovirus, adenovirus or vaccinia virus. One animal in each of the groups vaccinated with gIV from baculovirus and adenovirus shed virus for one day. In contrast, all

animals immunized with gIV from Ej. coli or placebo shed virus for 7 to 9 days post challenge. These data indicated that intramuscular immunization with a subunit vaccine induced mucosal immunity in the nasal passages, thereby preventing viral infection. In addition, the extent of the mucosal immunity appeared to correlate with the level of the neutralizing antibodies in the serum. To confirm the presence of a mucosal immune response in the nasal passages, the antibody titers in the nasal swabs were determined. On the challenge day, groups vaccinated with gIV from BHV-1, baculovirus, adenovirus, or vaccinia virus had mean neutralizing antibody titers between 25 and 65. The gIV-specific ELISA titers correlated well with the neutralizing antibody titers. The group immunized with gIV from Ij_. coli did not have any neutralizing antibodies in the nasal secretions, although the total gIV-specific antibody levels were as high as in the other groups. No gl-or gill-specific antibodies were found in the nasal secretions (data not shown) . These data correlate well with the serum antibody levels.

VII This example illustrates the production of a BHV-1 gl by recombinant baculovirus vectors.

VILA. Materials and methods

VII.A.l. Cells, viruses and antibodies - Madin Darby bovine kidney (MDBK) cells were cultured in Eagle's minimal essential medium (Grand Island Biological Co. , Grand Island, N.Y.) supplemented with 10% fetal bovine seruma (FBS) (Gibco) . Virus stocks of BHV-l strain Cooper were grown in MDBK cells as previously described (Babiuk et al., 1975 Infect. Immun. 12:958-963). Spodoptera frugioerda (SF9) cells were grown and

aintained in TNM-FH medium (GIBCO) containing 10% FBS according to the procedures described by Summers and Smith (1987 Texas Agricultural Experimental Station Bulletin No. 1555, College Station, TX) . Virus stocks of wild-type AcNPV and recombinant virus were prepared in SF9 cells as described by Summers and Smith (1987 supra). Monoclonal antibodies specific for gl were developed and characterized by van Drunen Littel-van den Hurk et al. (1984 Virology 135:466-479) . The gl-specific monoclonal antibody mixture used for identification of recombinant gl consisted of equivalent amounts of 1B10 (epitope I) , 3F3 (epitope II) , 1E11 (epitope HI) , 1F8 (epitope IVa) , 5G2 (epitope IVb) , 3G11 (epitope IVb) , 5G11 (epitope IVc) , 6G11 (epitope IVc) , 1F10 (epitope V) and 2C5 (epitope V) .

TI _τ A-2. Insertion of BHV-1 gl DNA into the transfer vector - A cassette of the gl glycoprotein gene has been prepared in plasmid pSV2Neo as previously described (Fitzpatrick et al., (1988), J. Virol., 62:4239-4248. The plasmid was digested with restriction endonuclease Bglll and the fragment representing the gl gene was purified by agarose gel electrophoresis and ligated into the BamHl site of baculovirus transfer vector pVL941. After transformation of £. coli strain JM105, colonies appearing on L agar containing 100 μg/ml ampicillin were inoculated to L broth containing ampicillin and incubated at 37°C overnight with vigorous shaking. Small scale preparations of plasmid from each colony were prepared and the presence of the gl gene was confirmed by digestion with endonucleases Aval and EcoRV. A single clone was identified containing the gl gene in the desired orientation and designated pVlgB. Clone pVlgB was inoculated into 500 ml L broth containing ampicillin and after 24 h at 37°C, plasmid was prepared by alkaline

lysis and further purified by equilibrium centriguation on CsCl.

VILA.3. Transfection and selection of recombinant viruses - After two cycles of ethanol precipitation, purified plasmid was mixed with an equal amount of A., californica viral DNA and used to transfect subconfluent monolayers of SF9 cells as outlined by Summers and Smith (1987) supra. Recombinant baculoviruses were identified by plaque hybridization essentially as outlined by Summers and Smith (1987) supra. The polyhedrin-negative recombinants were plaque- purified 3 to 4 times on SF9 cells to remove contaminating wild-type baculovirus.

V_I _r At4t Preparation of cell lvsates - To analyze expression of recombinant gl, confluent monolayers of SF9 cells on 35 mm petri dishes were infected with individual polyhedrin-negative recombinants at a moi of 5 and incubated for 48 h at 28 ^β C. The cells were scraped into PBS, pelleted at 150xg for 1 min, and resuspended in 50 μl of RIPA buffer (0.02 M Tris-hydrochloride [pH 8.0], 0.15M NaCl, 1% 10 mM phenylmethylsulfonylfluoride .[PMSF]) . The postnuclear supernatant was collected and 5 μl was combined with reducing electrophoresis sample buffer and boiled for 2 min for analysis by SDS-PAGE and immunoblotting. To determine approximate yields of recombinant gl, SF9 cells in monolayers or suspension cultures were infected with recombinant virus at a moi of 1. The cells were harvested at various times post infection, washed with PBS and resuspended in RIPA buffer at lxlO ⁷ cells/ml for analysis by ELISA. Equivalent samples from uninfected cells and/or cells infected with the parental virus were always included as controls.

VII.A.5 Analysis of carbohydrates - Proteins were digested with endoglycosidase H or glycopeptidase F as described by Ronin et al. (1987), Biochemistry. 26:5848- 5853. Infected cells were collected by centrifugation and 2x10 ^s cells were resuspended in 10 μl of appropriate enzyme incubation buffer. Digestion with glycopeptidase F (Boehringer-Mannheim, Laval, Quebec, Canada) was performed in 50 mM Trishydrochloride (pH 8.6), 25 mM EDTA, 1% Triton X100, 1% 2-mercaptoethanol, 0.2% SDS and ι.5 U of enzyme. Digestion with endo H (Boehringer- Mannheim) was performed in 0.1 M sodium acetate (pH 5), 0.15 M sodium chloride, 1% Triton X100, 1% 2- mercaptoethanol, 0.2% SDS, and 1.5 mU of enzyme. The cells were incubated for 18 h at 37°C. Proteins were precipitated by adding 1 ml of ice-cold acetone and centrifugation. They were subjected to SDS-PAGE followed by immunoblot analysis. Tunicamycin was added to recombinant AcNPV-infected SF9 cells or BHV-1 infected MDDBK cells at the time of infection from a stock solution of 1 mg/ml in ethanol. SF9 cells were harvested at 48 h post infection and MDBK cells were harvested at 24 h post infection.

VILA.6 SDS-PAGE. Immunoblot and ELISA - Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS- PAGE) was performed in 8.5% or 10% polyacrylamide discontinuous gels as previously described (van Drunen Littel-van den Hurk et al., 1984), supra. Electrophoresis was carried out under reducing conditions. Protein bands were visualized by staining with coo assie brilliant blue. In order to identify recombinant gl, produced by baculovirus, an immunoblot assay was performed as previously described (van Drunen Littel-van den Hurk et al., 1984), supra. Briefly, after electrophoresis, cell lysates were electrophoretically

transferred to nitrocellulose sheets. Subsequently, the instructions for use of the Bio-Rad (Mississauga, Ontario) immunoblot assay kit were followed. One gl- positive recombinant baculovirus, named Bac-gl, was amplified by growth on SF9 cells. The supernatants from this infection were stored at 4°C and used in all subsequent experiments.

Sandwich and indirect ELISA's were used to determine the yields of glycoprotein gl in recombinant baculovirus-infected SF9 cells. In the sandwich assay, microtiter plates were coated with the IgG fraction of bovine hyperimmune serum as the capture antibody and then incubated with lysates from recombinant virus - infected and control cells, or affinity-purified standard gl. In the indirect assay, the cell lysates and glycoproteins were directly adsorbed to the microtiter plates. Mixtures of gl-specific monoclonal antibodies, followed by horseradish peroxidase (HRPO)-conjugated goat anti- mouse IgG (Boehringer-Mannheim) were used for detection as previously described (van Drunen Littel-van den Hurk et al. _r 1984) supra) . The reaction was visualized using 0.8 mg/ml of 5-aminosalicylic acid and 0.006% H ₂ 0 ₂ as described.

VILA.7. Immunofluorescence and flow cytrometrv - The expression of glycoprotein gl in recombinant baculovirus- infected SF9 cells was determined at 24, 48 and 72 h post infection. Briefly, cells were washed in PBS and cytospin smears were prepared and fixed in methanol. They were incubated for 30 min at 37 ^β C with a 1:100 dilution of a gl-specific monoclonal antibody mixture and washed in PBS and ddH ₂ 0. They were stained with fluorescein isothiocyanate-conjugated (FITC) rabbit anti- mouse IgG (Boehringer-Mannheim) for 30 min at 37 ^β C and washed again before being mounted in PBS-glycerol for

examination. For surface staining and flow-cytometric analysis, cells were suspended in PBS containing 0.2% gelatin and 0.03% NaN ₃ (PBSG) at 4xl0 ⁷ cells/ml. They were plated in microtiter plates at 2x10° cells per well and incubated with serial dilutions of monoclonal antibody mixtures for 30 min on ice. Subsequently, they were washed in PBSG and then incubated with FITC rabbit anti-mouse IgG for 30 min at 4°C. After washing, the cells were fixed in 2% formaldehyde and analyzed with an EPICS CS (Coulter Electronics Ltd.) flow cytometer as described elsewhere (Campos et al. (1989), Cell. Immunol.. 120:259-269. The percentage of positive cells was calculated using the immuno-program (Coulter Electronics Ltd., MDAPS system) for the analysis of immunofluorescence histograms.

VILA.8. Cell fusion assay - Monolayers of SF9 cells in 24-well tissue culture plates were infected with recombinant virus at a moi of 5-10 PFU per cell. At 36 h post infection, the medium was replaced with TNM-FH medium, adjusted to a pH ranging from 5.0 to 6.5 Syncytia formation was monitored under a phase contrast microscope (Zeiss Model IM35; magnification 200x) . Monospecific and monoclonal antibodies were added at a dilution of 1:100 at the time of pH shift.

V__.A.9 _. Immunization of cattle - Glycoprotein gl was purified by immunoadsorbant chromatography from Bac-gl infected SF9 cells or BHV-1 infected MDBK cells as described in detail previously (van Drunen-Littel van den Hurk and Babiuk (1985), Virology. 144:204-215. Groups of eight animals each were immunized with 10 μg of affinity- purified recombinant or authentic gl in Emulsigeri ¹ * PLUS at a ratio of 7:3 (vol/vol) as outlined by the manufacturer (MVP Laboratories, Ralston, Ne.). The

animals were injected intramuscularly and they received a booster immunization 28 days later. They were bled at the times of immunization and two weeks after the second immunization for assessment of antibody responses. The antibody response to gl in the vaccinated animals was assayed in an immunoblot assay with purified BHV-l as the antigen, as described previously (van Drunen Littel-van den Hurk et al. (1990), Vaccine. 2:358-368.

VII.B Results

VII.B.l Production of recombinant gl glycoprotein in SF9 cells - Recombinants containing the gl gene inserts were tested for their ability to produce BHV-1 glycoprotein I after infection of SF9 cells. All of the gl recombinants directed the synthesis of a polypeptide with an apparent molecular weight of 116 kDa, which was visible on a coomassie brilliant blue stained gel at 48h post infection. This protein was missing in uninfected cells and cells infected with the parental baculovirus. In order to confirm the identify of this glycoprotein, immunoblot analyses were performed on Bac-gl infected SF9 cells and BHV-1 infected MDBK cells. A gl-specific monoclonal antibody mixture that recognized the 130k, 74k and 55k components of authentic gl in BHV-l infected MDBK cells, reacted with three polypeptides with apparent molecular weights of 116 kDa, 63 kDa, and 52 kDa in Bac-gl infected SF9 cells. This indicates that terminal glycosylation of gl has not occurred in the recombinant virus-infected SF9 cells. Recombinant gl was cleaved in infected SF9 cells, but not with the same efficiency as authentic gl. No reaction was observed between the gl- specific monoclonal antibodies and SF9 cells infected with the parental baculovirus.

VI-ιBt_τ Processing of σl in mammalian and insect cells - To further analyze the observed difference in molecular weight of the recombinant and authentic gl, Bac-gl infected SF9 cells and BHV-1 infected MDBK cells were treated with tunicamycin, an inhibitor of N-linked glycosylation. In these cells only one polypeptide with an apparent molecular weight of 105k was observed, which corresponds to the previously identified polypeptide backbone of authentic gl (van Drunen Littel-van den Hurk and Babiuk, (1986) J. Virol.. 52; 401-410. This experiment proved that the reduced molecular weight of gl expressed in insect cells was due to incomplete glycosylation. To compare the type of carbohydrate attached to recombinant and authentic gIV, both glyco¬ proteins were subjected to digestion with endo H or endo F. Digestion with endo H resulted in a slight decrease in apparent molecular weight of authentic gla and glc, but had no effect on gib, which confirms previous studies (van Drunen Littel-van den Hurk et al., (1986) supra. The greater portion of recombinant gla and glc was sensitive to endo H, showing the presence of high-mannose type oligosaccharides. However, the recombinant gib was not sensitive to endo H, indicating that these oligosaccharides are trimmed. All of the recombinant and authentic forms of gl were endo F sensitive, showing precursor molecules with similar apparent molecular weights in BHV-1 and Bac-gl infected cells.

Authentic and recombinant gl are both cleaved during processing to the mature polypeptide. However, the cleavage process is incomplete in mammalian cells and even less efficient in insect cells. It has been proposed that Arg-Arg-Ala-Arg-Arg sequence (501-505) , which occurs in the region of non-similarity with HSV-l, may be the processing site for PRV gll (Robbins et al., (1987) J. Virol.. £1:2691-2701 and BHV-1 gl (Whitbeck et

al., (1988) J. Virol.. £2:3319-3327. To confirm the position of the cleavage site of authentic as well as recombinant gl, we sequenced the N-terminus of the glc glycoprotein from infected MDBK and SF9 cells. This analysis confirmed that the first 12 N-terminal amino acids of authentic and recombinant glc correspond to positions 506-517. Since recombinant gl was cleaved at the same site as authentic gl, the reduced cleavage efficiency is probably due to the presence of relatively low amounts of enzyme in baculovirus-infected cells, as compared to the large amounts of gl produced in these cells. N-terminal sequencing of the gib glycoprotein demonstrated that the signal is cleaved in MDBK and SF9 cells and that the amino terminal residue of authentic as well as recombinant gl is Arg-68.

VII.B.3 Kinetics and level of expression of the recombinant gl glycoprotein - The amount of gl synthesized in recombinant baculovirus-infected SF9 cells was quantitated by ELISA, standardized with affinity- purified recombinant gl. SF9 cells grown as monolayers in 35 mm petri dishes were infected with Bac-gl at a moi of 5 _f and aliquots of 1x10* cells were harvested at various times post infection. Cell lysates were prepared and the level of expression of recombinant gl was tested in the ELISA. Immunoreactive gl could be detected as early as 24 h after infection and maximal expression was observed between 36 and 48 h, whereafter a slight decrease in measurable glycoprotein occurred. This decline presumably reflected cell lysis and sub-sequent degradation of the glycoprotein. This analysis showed that, at maximal levels of expression, 30 μg of gl were produced per 10* cells. In order to analyze the possibility to produce recombinant gl at a larger scale, SF9 cells were grown in suspension cultures and infected

with the recombinant baculovirus at a moi of 1. In addition to yield by ELISA, the viability of the cells and percentage of infected cells were determined. Flow cytometric analysis showed an increase in percentage of infected cells (y-axis) as well as total protein yield (x-axis) over time. The percentage of infected cells increased gradually, reaching peak levels of 85% at 72 h after infection, when the viability of the cells was down to 25%. The viability of the cells was too low for flow cytometric analysis beyond this time point. Analysis by ELISA demonstrated that up to 35 μg of gl were produced per 10 ⁶ cells. This demonstrated the feasibility of growing the recombinant baculovirus on a larger scale and yet obtain good yields of the glycoprotein.

V.Itg-4 Intracellular localization of recombinant gl in

SF9 cells - The intracellular distribution of the recombinant gl glycoprotein was examined by an indirect immunofluorescence assay. At 48 h post infection, recombinant gl was primarily localized in the perinuclear membranes of the infected SF9 cells. To determine whether the recombinant gl was present on the surface of infected cells, immunofluorescence analysis was carried out on unfixed cells. Localization of gl was demonstrated by bright fluorescence on the surface.

Wild-type AcNPV-infected control cells did not show any fluorescence with the gl-specific monoclonal antibody panel (not shown) .

VII.B.5. Fusogenic properties of recombinant gl in insect cells - It has been shown previously that one of the functional characteristics of gl is its ability to induce cell fusion in absence of other viral proteins (Fitzpatrick et al., (1988) supra; (1990) J. Gen. Virol.. 21:1215-1219. To determine whether this functional

property was retained in the recombinant protein, SF9 cells were infected with Bac-gl. Fusion of the insect cells was not evident under standard culture conditions, but after a shift to pH 5.4, fusion was apparent in Bac-gl infected SF9 cells within two hours. The syncytia formation observed in these cells continued to increase over 8 h of observation. Inclusion of gl-specific rabbit serum or a mixture of gl-specific monoclonal antibodies completely inhibited fusion by gl as set forth in Table 2.

Table 2. Inhibition of fusion activity mediated bv gl expressed in baculovirus

Treatment* Fusion activity (%) ^b

TNM-FH, pH 5.4 80

Trypsin 80

Normal Rabs 80 gl-specific Rabs 0 Control Mabs 80 gl-specific Mabs mixture 0

1B10 mab (I) 5

3F3 Mab (II) 80

1E11 Mab (III) 80 1F8 Mab (IVa) 80

5G2 Mab (IVb) 10

5G11 Mab (IVC) 60

1F10 Mab (V) 80

a. Cell fusion was induced at 36h post infection by replacing the cell culture medium with TNM-FH pH 5.4. At the time of pH shift a final dilution of 1:100 of Rabs (rabbit serum) or Mabs (monoclonal antibodies) was added to the medium. Treatment

with 20 μg trypsin was carried out for 10 min, just before pH shift at 36h. b The cells were counted 8h after the pH shift.

The percentage of fused cells was calculated on a total of 400 cells and rounded to the nearest decimal.

When individual monoclonal antibodies were included in the media, fusion was almost completely inhibited by the monoclonal antibodies 1B10 (epitope I) and 5G2 (epitope IVb) and partially inhibited by 5G11 (epitope IVc) . Inclusion of trypsin at the time of pH shift did not affect the fusion activity.

VII.B.6. Antigenic and immunogenic properties of gl expressed in SF9 cells - The antigenic properties of recombinant gl were evaluated using a panel of gl- specific monoclonal antibodies. The epitopes recognized by these monoclonal antibodies have been identified and characterized previously (van Drunen Littel-van den Hurk et al. (1985) supra; Fitzpatrick et al. (1990) Virology. 176:145-157. Reactivity of all of these monoclonal antibodies in an ELISA (Table 3b) indicated that all of the epitopes identified on the authentic glycoprotein were also present on the recombinant gl glycoprotein.

The reaction between the monoclonal antibodies and two carbohydrate-dependent epitopes IVa and IVc; (van Drunen Littel-van den Hurk et al., 1990b) was weaker on recombinant gl than on authentic gl, which is in agreement with lack of terminal glycosylation of gl in Sf9 cells. Epitopes I, II, and III, however, appeared to be more reactive on recombinant gl than on its authentic counterpart.

To compare the immunogenicity of recombinant and authentic gl, calves were immunized with affinity-

purified glycoprotein from recombinant Bac-gl-infected Sf9 cells or BHV-1 infected MDBK cells. Two immunizations of recombinant or authentic gl in Emulsigen PLUS elicited antibodies that were reactive with gl from BHV-1 in an immunoblot assay. The antibody titers induced by recombinant and authentic gl were very similar (Table 3a) .

Table 3a Immune Response to Authentic and Recombinant gl

* Animals received two intramuscular immunizations of authentic gl, recombinant gl, or PBS (placebo) in Emulsigen PLUS.

* ELISA titers were determined against affinity-purified gl and expressed as the reciprocal of the highest dilution resulting in a reading of two standard deviations above the negative control value.

a. Monoclonal antibodies developed by van Drunen

Littel et al. (1984) b. gl epitopes assigned by competitive binding assays (van Drunen Littel-van den Hurk al.,

1985) . c. Neutralizing titers were determined for ascites fluids in the presence of guinea pig serum as a source of complement. -, titer < 4; +/-, titer<100 +, titer > 100; ++; titer > 10,000.

(Van Drunen Littel-van den Hurk et al., (1985) supra.

Antigen titer was expressed as the reciprocal of the highest dilution of infected cells giving a reading of at least 0.05 OD (492nm) .

A 1:100 dilution corresponds to 2xl0 ⁴ cells.

The reaction between the monoclonal antibodies and two carbohydrate-dependent epitopes (IVa and IVc; van Drunen Littel-van den Hurk et al. (1990) . Gen. Virol.. 71:2053-2063) was weaker on recombinant gl than on authentic gl, which is in agreement with lack of terminal glycosylation of gl in SF9 cells. Epitopes I, II and HI, however, appeared to be more reactive on recombinant gl than on its authentic counterpart.

To study the immunogenicity of recombinant gl, cattle were immunized with 10 μg of affinity-purified glycoprotein from recombinant baculovirus-infected SF9 cells. Two immunizations of recombinant gl in Emulsigen elicited antibodies that were reactive with gl from BHV-1 in an immunoblot assay.

VIII Construction of Recombinant AcMNPV Expressing Secreted BHV-1 gIV.

VIII.A. Standard protocols, described in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures" by Summers and Smith were used to generate the above recombinant. We used the gene replacement vector, pAcYMl (see Ann. Rev. Microbiol. 12:177), to direct the insertion of the modified gIV gene (see below) into the polyhedron gene locus of AcMNPV. Therefore, gIV gene expression was directed by the polyhedron gene promoter and the recombinant virus displayed a polyhedron negative phenotype. The following procedures were used to generate the insertion plasmid required to produce a baculovirus capable of secreting gIV from infected cells. The entire gIV gene coding sequence is contained within a single 1303 bp Mae I fragment (see gIV gene sequence Figure 1 + restriction endonuclease map. Figure 15) . A Mae I site

exists approx. 45 bp upstream of the gene's initiation codon and approx. 10 bp downstream of the stop codon. This Mae I subfragment was isolated from the BHV-1 Hindlll K fragment and treated with T4 DNA polymerase to blunt the ends. The modified fragment was then inserted into the Bgl II site (also treated with T4 DNA polymerase) of a cloning vector. This procedure regenerated the Bgl II cloning sites, such that the gIV gene's coding sequence was now flanked by unique Bgl II sites. In order to make a construct that will permit gIV glycoprotein secretion, the Bgl II modified plasmid was first partially digested with Sac II and a TAB linker (TCGAGC) was added to create a unique Xho I site at the C-terminal Sac II site (located at 1115 bp in the accompanying gIV gene sequence) . The C-terminal Sac II site is located at the very 5' terminus of the sequence which encodes the gIV transmembrane sequence. The new plasmid was then digested with Xho I, blunt-end repaired with T4 DNA polymerase followed by insertion of a triphasic translation stop codon linker (CTAGCTAGCTAG) which causes premature translation termination at the 5' terminus of the gIV gene's anchor sequence. The modified gIV gene construct was excised by Bgl II digestion and then inserted into the Bam HI cloning site of pAcYMl. Proper orientation of the gIV gene was established by mapping asymmetric restriction endonuclease sites within the insert relative to unique restriction endonuclease sites in the pAcYMl backbone. This final construct was used to co-transfect SF9 cells along with purified wild- type genomic AcMNPV DNA by the prescribed procedures of Smith and Summers. (Summers et al., "A manual of methods for baculovirus vectors and insect cell culture procedures'*, Texas Agricultural Experiment Station Research bulletin no. 1987, 1555, Texas Agricultural Experiment Station, College Station, Tex.) rτυTE SHEET

VIII.B. Individual polyhedron negative plaques were isolated (see plaque purification procedures) , amplified by growth on SF9 cells and tested for expression of secreted gIV by western analysis of serum-free growth media (Ex-Cell 400, JR Scientific) collected from virus infected cells (moi 1.0) 48h post infection. Our analyses demonstrated that approximately 70% of the total gIV produced by this virus was secreted into the media.

VIILC. Groups of six animals were immunized intramuscularly at a 3 week interval with 25 μg of crude truncated gIV produced from baculovirus-infected Sf9 cells. The tglV was combined with the adjuvant Avridine as described previously (Babiuk et al., 1987). A control group was vaccinated with Avridine only. Twenty-one days later the animals were boosted and then challenged with BHV-1 fourteen days after the booster immunization. Blood samples were taken from the animals at the time of the first immunization, booster immunization and challenge, as well as ten days after challenge for assessment of antibody responses. One immunization with truncated gIV did not induce detectable levels of serum neutralizing antibodies to BHV-l. However, following the booster immunization good titers of serum neutralizing antibodies were observed in all vaccinated animals

(Table 4). After challenge with BHV-1, the animals in the placebo-immunized groups showed signs of clinical illness from day 1 until day 7 post infection (Figure 17a) , whereas the group immunized with truncated gIV experienced very little illness. All animals in the placebo group shed high titers of virus for at least 10 days, whereas in the vaccinated group only one animal shed very low levels of virus for 5 days (Figure 17b) . Results of these immunization experiments are shown in Table 4 below and in Figures 17(a) and 17(b) .

Table 4 SERUM NEUTRALIZATION TITERS: MEAN + RANGE

* Groups of 6 animals were immunized twice at a 3-week interval, with 25 μg of crude truncated IV from baculovirus in Avridine. ^b Geometric mean serum neutralizing antibody titers and ranges in parenthesis. Where no range is shown, all animals had the same titer: <2.

IX IX.A.1. Cells and viruses

Madin-Darby bovine kidney (MDBK) cells, BSC-1 cells and human thymidine kinase negative (TK ^" ) 143 cells were grown as monolayers in Eagle's minimum essential medium (MEM) (GIBCO/BRL, Mississauga, Ontario, Canada) , supplemented with 5% fetal bovine serum (FBS) (GIGCO/BRL, Mississauga, Ontario, Canada) . LMTK" cells were grown in Dulbecco's minimum essential medium (DMEM) (GIBCO/BRL, Mississauga, Ontario, Canada) supplemented with 5% FBS. The P8-2 strain of BHV-1 was propagated in MDBK cells and quantitated as described in Rouse et al. (1974) J. Immunol.. 112:1391-1398). Wild type (WR strain) and recombinant Ws were propagated in BSC-1 cells and LMTK ^* cells (Mackett et al. (1984) J. GgR. Virol.. !£:857-864) •

IX.A.2. Construction of recombinant plasmids

Restriction endonucleases, and other DNA modifying enzymes were purchased from Pharmacia (Dorval,

Quebec, Canada) and New England Biolabs (Mississauga, Ontario, Canada) , and were used as directed by the manufacturer.

Construction of the RSV1.3 and RSV1.3X plasmid: The full-length gIV gene was excised from plasmid pRSDneoglV (Tikoo et al. (1990) J. Virol.. 6 :5132-5142) as a 1.3 kilobase (kb) Bglll fragment and inserted into Bglll digested pRSV-0 (Fitzpatrick et al. (1988) J. Virol. _f 62:4239-4248) creating the pRSV1.3 plasmid. This plasmid was partially digested with SacII and a TAB linker (pTCGAGC) was added (Barany, F. , (1985) Proc. Natl. Acad. Sci. USA 22.:4202-4206) to create a unique Xhol site at the C-terminal SacII site. This plasmid was called pRSV1.3X. All subsequent deletions and truncations were constructed beginning with either of these two plasmids. a) Plasmid pSTglV: The gIV gene was subcloned from plasmid pRSDneoglV (Tikoo et al. (1990) supra) as a 1.3 kb Bglll fragment, treated with T4 DNA polymerase and ligated to Smal digested pGS20 (Mackett et al. (1984) supra) . b) Plasmid pSTglVdl: Plasmid pRSV1.3X was digested with Xhol, blunt-end repaired with T4 DNA polymerase, followed by insertion of a triphasic stop codon Nhel Linder (pCTAGCTAGCTAG) . The DNA was then digested with Nhel and religated. The truncated gIV gene was inserted into the Smal site of pGS20 as a blunt-end repaired Bglll fragment. c) Plasmids pSTgIVd2 and pSTglVdδ: Plasmid pRSVl.3 was partially digested with Narl and a triphasic stop codon Hpal linker (pd[TTAAGTTAACTTAA]) was inserted after treating the Narl digested plasmid with T4 DNA polymerase. The DNA was finally digested with Hpal and religated. The insertion of the linker at one of the two Narl sites in the gIV gene was confirmed by restriction

endonuclease mapping. These two truncations were cloned separately into the Smal site of pGS20 as a blunt end repaired Bglll fragment, creating plasmid pSTgIVd2 (Hpal linker insertion at 3' Narl site) and plasmid pSTglVdδ (Hpal linker insertion at 5' Narl site). d) Plasmid pSTgIVd3: Plasmid pRSV1.3 was partially digested with Sail, blunt-end repaired with T4 DNA polymerase followed by ligation with a triphasic stop codon Nhel linker (pCTAGCTAGCTAG) . The DNA was digested with Nhel and religated. The truncated gene was inserted into the Smal site of pGS20 as a blunt-end repaired Bglll fragment. e) Plasmid pSTgIVd4: Plasmid pRSV1.3 was digested with Smal, the large fragment purified and then ligated with a triphasic stop codon Nhel linker

(pCTAGCTAGCTAG) . The DNA was digested with Nhel and religated. The truncated gene was inserted into the Smal site of pGS20 as a blunt-end repaired Bglll fragment. f) Plasmid pSTglVdδ: Plasmid pRSV1.3X was digested to completion with Drain and Xhol. The large fragment was purified,, blunt-end repaired with T4 DNA polymerase and ligated. The partial gene deletion was inserted into the Smal site of pGS20 as a blunt-end repaired Bglll fragment. g) Plasmid pSTgIVd7: Plasmid pRSV1.3 was digested to completion with Sail and the large fragment was purified and religated. The partial gene deletion was inserted into the Smal site of pGSlO as a blunt-end repaired Bglll fragment. h) Plasmid pSTglVdβ: Plasmid pRSV1.3X was digested with Xhol and treated with mung bean nuclease to create blunt ends. Then DNA was partially digested with Xmal, treated with Klenow enzyme and the large fragment was then purified and religated. The partial gene

deletion was inserted into the Smal site of pGS20 as a blunt-end repaired Bglll fragment. i) Plasmid pSTgIVd9: Plasmid pRSV1.3 was partially digested with Sail followed by complete digestion with Xhol. The large fragment was purified, blunt-end repaired and then religated. The partial gene deletion was inserted into the Smal site of pGS20 as a blunt-end repaired Bgll fragment.

IX.A.3. Isolation of recombinant vaccinia viruses The desired recombinant Ws were made by homologous recombination as previously described (Mackett et al. (1985) "DNA Cloning: A Practical Approach," pp. 191-211, Ed. by D.M. Clover, Oxford: IRL press) . A newly confluent monolayer (75 cm ² ) of BSC-1 cells was infected with wild-type W (WR strain) at a multiplicity of infection of 0.05 PFU/cell. At 4 hrs post infection the cells were collected by mild trypsinization, washed three times with Hepes buffer (pH 7.1) and adjusted to a concentration of 1-2X10* cells/ml in Hepes buffer (pH 7.1). Approximately 10 μg of cesium chloride gradient purified linearized plasmid DNA was mixed with 750 μl of the infected cell suspension and placed on ice in an electroporation cuvette for 10 min before and immediately after electroporation at 200 volts and 500 μFD using a Bio-Rad Gene Pulser. The cells were then diluted in MEM containing 10% FBS and incubated at 37 ^β C. After 2-3 days, to permit virus replication, transfected cells and supernatants were collected, frozen and thawed twice and sonicated for 20 sec to release virus. Putative recombinants were selected by plating the sonicated supernatants on TK ^" 143 cells and overlaying with 1% agarose in growth medium containing 5-bromo-2'- deoxyuridine (25 μg/ml) . After 3 days, the TK ^" plaques were visualized by staining the monolayer with neutral

red, picked individually and grown on BSC-1 cells to amplify virus. Recombinant W were selected by screening the TK ^" plaques for gIV expression by immunocytochemistry before replaquing and making viral stocks in LMTK ^" cells.

IX.A.4. Polyclonal and monoclonal antibody gIV specific monoclonal antibody (MAb) production and characterization, in particular their reactivity with native or denatured gIV, neutralizing activity, and grouping based on competition binding assays have been described (Hughes et al. (1988) Arch. Virol.. 103:47-60 and van Drunen Littel-van den Hurk et al. (1990) J. Gen. Virol.. 21:2053-2063). Before use, MAb ascites fluids were clarified and filtered. Monospecific polyclonal gIV specific antisera produced in rabbits have been described (Hughes et al. (1988) supra) .

IX.A.5. Protein expression

For immunoprecipitation, LMTK" cells were infected at a multiplicity of infection (MOI) of 5. At 10 hrs post infection, the cells were washed and incubated in cysteine-methionine free DMEM for 90 min before labeling with [ ³⁵ S] cysteine-methionine (100 μCi/ml) . After 4-8 hrs of labeling, the cells and/or medium was harvested. In pulse-chase experiments, cells were labelled at 10 hrs post infection with 150 μCi of [ ^M S] methionine-cysteine for 15 min. Depending on the specific experiment, either the cells were harvested immediately or the label was removed and cells were incubated for different time periods in DMEM containing an excess of cold methionine (chase) . Proteins were immunoprecipitated from the medium or from the infected cells, lysed with modified RIPA buffer and analyzed by SDS-PAGE as previously described (van Drunen Littel-van den Hurk (1990) supra1.

IX.A.6. Enzyme treatments

Immunoprecipitated proteins were eluted in 20 μl of 0.5% SDS by boiling for 3 min. The eluted proteins were digested with 20 mU of endo H in 0.125M sodium citrate pH 5.5, 0.1M 2-mercaptoethanol, 0.5mM phenylmethylsulphonyl fluoride and 0.1% SDS. For analysis by SDS-PAGE, the digested proteins were precipitated with ice-cold acetone, resuspended in electrophoresis sample buffer and boiled for 3 min before analysis (van Drunen Littel-van den Hurk (1990) supra..

IX.A.7. Immunoperoxidase staining

LMTK ^" cells grown in 4 well Lab-Tek chamber slides were infected with the appropriate recombinant W at an MOI of 5. After 16 hrs of incubation the cells were fixed with 3% paraformaldehyde for 15 minutes at 4°C (surface staining) and stained by immunoperoxidase staining procedure as previously described (Fitzpatrick et al. (1988) supra) .

RESULTS IX.B.

To examine the structure and function of different domains of gIV, the complete open reading frame and the mutated forms (internal deletions or truncations) of gIV gene (Fig. 15) were cloned into the W expression vector pGS20 (Fig. 2) to generate recombinant Ws designated here as STglV expressing wild-type gIV, and STglVdl to STgIVd9 expressing mutant proteins dl to d9 respectively.

IX.B.l. Characterization of proteins made by recombinant WS

To examine the product of the wild type gIV gene, LMTK" cells were infected with recombinant W STglV and metabolically labelled with [ ³⁵ S] cysteine-methionine. For comparison with authentic gIV, MDBK cells were infected with BHV-l and labelled similarly with [ ³⁵ S] cysteine-methionine. The radiolabelled proteins were immunoprecipitated with rabbit anti-glV antiserum and analyzed by SDS-PAGE under reducing conditions.

Radioimmunoprecipitation of recombinant pSTglV W infected cells revealed a major protein band of approximately 71 kDa molecular weight which comigrated with the authentic gIV protein produced in BHV-l infected cells. No similar band was observed in uninfected cells or cells infected with wild-type W. This suggests that recombinant gIV produced in LMTK ^* cells was post- translationally modified in a manner similar to authentic gIV. The proteins produced by the recombinants carrying deleted or truncated forms of gIV were also analyzed by SDS-PAGE under reducing conditions. The mutant forms of gIV protein dl-d9 were detected as single bands at approximately the expected molecular weights, except d7 which migrated more slowly than expected. This aberrant mobility of d7 protein appears to be due to the addition of 0-1inked oligosaccharides (Tikoo et al. unpublished data) .

IX.B.2. Antigenic structure of oIV proteins To examine the antigenic properties of wild type gIV, radiolabelled protein was immunoprecipitated from W STglV infected cell lysates with gIV specific MAbs (Hughes et al. (1988) supra and van Drunen Littel- van Den Hurk et al. (1984) Virology. 12__:466-479) and analyzed by SDS-PAGE under reducing conditions. In

addition to the recognition of the recombinant gIV by MAbs directed against continuous epitopes lb (MAb 9D6) , IV (MAb 3D9S) and Ilia (MAb 10C2) , the protein was also recognized by MAbs directed against discontinuous epitopes lb (MAb 136), II (MAb 3E7) , IIIc (MAb 2C8) , Hid (MAb 3C1) and IHb (MAb 4C1) . This suggests that the antigenic structure of gIV produced in W stglV infected cells is similar to gIV produced by BHV-1 infected cells. In order to locate the antigenic sites on the gIV glycoprotein, the mutated proteins were similarly immunoprecipitated from recombinant infected cell lysates with individual MAbs and analyzed by SDS-PAGE under reducing conditions. The results are as follows: a) A truncated form of gIV (AAs 1-355) , expressed by recombinant W STglVdl, which lacks 62 amino acids at the carboxy terminus including the transmembrane anchor sequence, reacted with all of the gIV specific MAbs recognizing both continuous and discontinuous epitopes. b) A truncated form of gIV (AAs 1-320) , expressed by recombinant W STgIVd2, which lacks 97 amino acids at the carboxy terminus, reacted with all of the MAbs, except 3D9S (which recognized a continuous epitope) and 136 (which recognized a discontinuous epitope) . The reactivity of MAbs 2C8 and 4C1 to this protein was also reduced. c) A truncated form of gIV (AAs 1-244) , expressed by recombinant W StgIVd3, which lacks 173 amino acids at the carboxy terminus reacted only with MAb 9D6, which recognizes a continuous epitope. d) A truncated form of gIV (AAs 1-216) , expressed by recombinant W STgIVd4, which lacks 201 amino acids at the carboxy terminus, also reacted only

^' with MAb 9D6.

e) A truncated form of gIV (AAS 1-164), expressed by recombinant W STgIVd5, which lacks 253 amino acids at the carboxy-terminus, did not react with any of the MAb. f) A deleted form of gIV expressed by recombinant W STgIV6, which lacks 265 AAs from residue 90 to 354 in the extracellular domain of gIV, also did not react with any of the MAbs. g) A deleted form of gIV expressed by recombinant W STgIVd7, which lacks 213 residues from AAs 32-244 in the extracellular region of gIV, reacted only with MAb 3D9S which recognize a continuous epitope. h) A deleted form of gIV expressed by recombinant pSTglVdδ, which lacks 139 AAs from residue 218-355 in the extracellular region of gIV, reacted only with MAb 9D6. i) A deleted from of gIV expressed by recombinant W STgIVd9, which lacks 112 residues from AAs 245-355 in the extracellular region of gIV reacted only with MAb 9D6.

These observations suggest that binding sites for MAb 9D6 and 3D9 lie between amino acid 164-216 and amino acid 320-355, respectively. In addition amino acids 244-320 are important for the formation of discontinuous epitopes recognized by MAbs 2C8 and 4C1, whereas amino acids 320-355 are critical for the formation of discontinuous epitope recognized by MAb 136.

IX.B.3. Secretion of truncated gIV proteins. In order to determine whether truncated forms of gIV were efficiently secreted into the medium, LMTK ^" cells infected with recombinant Ws were labeled with [ ³⁵ S] cysteine-methionine for 4-8 hrs beginning 10 hrs after infection. Cell culture supernatants were immunoprecipitated with rabbit anti-giv polyclonal

antiserum. Proteins expressed by recombinant W STglVdl and W STgIVd2 were detected in the medium where as proteins truncated at or upstream of amino acid 244 (W STgIVd3 to W STgIVd5) were never detected in the medium. This suggests that amino acid 244-320 are required for efficient secretion of the truncated gIV molecules and confirmed our previous observation concerning the location of transmembrane anchor domain between amino acids 361 to 389 (Tikoo et al. (1990) supra) .

IX.C.l.

A number of strategies have been used to locate the antigenic sites of a viral glycoprotein. Since the induction of protective humoral immune response is dependent on the conformation of gIV, the approach of expressing deleted and truncated forms of gIV in mammalian cells by recombinant vaccinia viruses has allowed the mapping of the binding sites of different MAbs in gIV and study the effect of these mutations on the native structure of the glycoprotein (Fig. 16) . A similar approach has been used to localize the functional domains of HSV-1 glycoprotein D (Cohen et al. (1988) J. Virol. 62:1932-1940) .

To confirm the validity of this approach, insertion of the full-length gIV gene into vaccinia virus showed that gIV expressed by recombinant W STglV had an antigenic profile indistinguishable from authentic gIV synthesized after viral infection (Hughes et al. (1988) and van Drunen Littel-van den Hurk et al. (1986) supra) . These results confirm and extend the observations previously reported for recombinant gIV expressed in transfected bovine cells (Tikoo et al. (1990) supra) .

Previously, four antigenic domains of gIV were identified using a panel of MAbs (Hughes et al. (1988) and van Drunen Littel-van den Hurk et al. (1986) supra) .

Domain I consists of two epitopes; epitope la is a continuous epitope recognized by MAb 9D6 and epitope lb is a discontinuous epitope recognized by MAb 136. The present results indicate that epitope la is located between residue 164-216 and a portion of epitope lb is located between residue 320-355. The second portion of epitope lb is located upstream of residue 245, perhaps upstream of residue 216 but downstream of residue 31. This assumption is based on the fact that recombinant W STgIVd7 which expresses a mutant protein devoid of residues 32-244 is not recognized by MAb 136. In addition, competitive bidding experiments indicate that either these two epitopes share common amino acids or that they lie in close proximity to one another (Hughes et al. (1988) supra).

Domain II of gIV is represented by a discontinuous epitope which is recognized by MAB 3E7. This epitope is located upstream of residue 320 and at least a portion of the epitope is located upstream of residue 245. This is based on two observations. First, if the binding site is composed entirely of residues between 245-320, the protein expressed by recombinant w STgIVd7 should be recognized by MAB 3E7. Second, the epitope is destroyed by the addition of reducing agents, and all of the cysteine residues that could possibly contribute to disulphide binding are located between residue 74 to 214.

Domain HI is represented by four epitopes, three of which have been shown to be discontinuous (Hughes et al. (1988) supra) . Analysis of the mutant proteins expressed by recombinant W STgIVd2, W STgIVd7 and W STgIVd9 indicate that the binding site for MAb 10C2, which recognizes the continuous epitope Ilia, lies in close proximity to amino acids 244 and 245. The epitopes IHB, IIIC and Hid recognized by conformation

dependent MAbs 4C1, 2C8 and 3C1 respectively, are located between amino acid 19 to 320.

Domain IV is represented by a continuous epitope recognized by a non-neutralizing MAb 3D9S. This epitope was mapped between residues 320-355.

Formation of discontinuous epitopes depends on certain tertiary structures of gIV which in part involve disulphide bonds. The observation that the MAbs recognizing discontinuous epitopes (destroyed by reducing agent) react with residues 1-355 suggests that this polypeptide maintains its normal disulphide bonding pattern. Six of the seven gIV cysteine residues located within residues 75 to 213 probably play a role in the structure of these discontinuous epitopes. Interestingly, these six cysteine residues are readily aligned in all gIV homologs thus far identified (Tikoo et al. (1990) supra) . All six cysteines are involved in intramolecular disulphide bond formation in HSV-1 gD (Wilcox et al. (1988) J. Virol. 62:1941-1947) and are suggested to be important for the structure and function of the protein (Long et al. (1990) J. Virol. 64:5542- 5552) . The cysteine at residue 376 is within the transmembrane domain of gIV and is not involved in the formation of these epitopes, indicating that this cysteine is not involved in intramolecular disulphide bonding in gIV required for attaining the proper tertiary structure. A similar observation has been made previously for the HSV-1 gD glycoprotein (Wilcox et al. (1988) supra) . Earlier studies have shown the presence of both

N-linked and O-linked oligosaccharides in gIV (van Drunen Littel-van den Hurk et al. (1986) supra) . In BHV-1 infected cells, N-linked oligosaccharides are processed from high mannose oligosaccharides present on precursor gIV (pglV) to complex oligosaccharides of mature gIV

which is transported to the surface of the infected cell and also incorporated into the virion envelope (Marshall et al. (1986) J. Virol. 22:745-753 and van Drunen Littel- van den Hurk (1986) supra). At 24 hrs postinfection, most of the protein is found in the mature form.

Essentially similar transport and processing kinetics were observed for recombinant gIV produced by W STglV indicating that W is an acceptable vector for expressing BHV-1 glycoproteins. Processing and transport of a viral glycoprotein through the exocytic pathway is dependent on its conformational and structural signals, which may include the location of N-linked glycosylation sites, position of cysteine residues forming disulphide bonds that promote the juxtaposition of residues on the molecule, and amino acid residues required for membrane insertion, anchoring, local folding of monomers and formation of oligomers (Guan et al. (1985) Cell 42:489- 496; Kreis et al. (1986) Cell 16.:929-937; Rose et al. (1988) Ann. Rev. Cell Biol. 1:257-288 and Wilcox et al. (1988) supra) . Alterations of any of these signals may affect processing, and/or transport of a glycoprotein. The results of this study indicate that the extent of processing of the genetically engineered gIV mutant proteins correlated with the transport of the proteins to the cell surface/media. However, a loss in the ability to form discontinuous epitopes was not associated with the loss of transport of the mutant protein to the cell surface/media. All mutant proteins containing amino acids 245-320 (dl, d2, d7) were processed from precursor to product, contained endo H resistance oligosaccharides and were located on the surface of the cell or secreted into the medium when the transmembrane anchor sequence was also been deleted. These results suggest that these proteins retained signals necessary for the proper

folding, processing and consequently transport of the protein to the cell surface. In contrast, all mutants lacking amino acids 245-320 (d3, d4, d5, d6, d8 , d9) failed to be processed from precursor to product form and were not transported to the cell surface or secreted in the medium. In addition, virtually all of the oligosaccharides were of the high-mannose form indicating that these mutant proteins are retained in the endoplasmic reticulum. It is in this organellese that membrane-bound and secretory proteins acquire high- mannose oligosaccharides, fold and in many cases oligomerize (Rose et al. (1988) supra) . Both misfolded and unassembled subunits are retained in the endoplasmic reticulum and prevented from further transport by interactions with resident cellular proteins (Rose et al. (1988) supra) . The altered processing and transport of the mutants lacking amino acid 245-320 could be due to misfolding of the proteins, however, we could not detect protein aggregation (data not shown) , as has been observed with the other misfolded proteins (Wilcox et al. (1988) supra) . Alternatively, a block in transport could be due to the absence of required signals residing in residues 245-320. Preliminary studies indicate that the 0-linked oligosaccharides are attached to serine/threonine located in this region (Tikoo et al. unpublished data) . It is possible that the absence of either amino acid sequence or protein modifications present in this region may be responsible for the observed effects.

X.A. Animal trials were conducted with full-length gIV (glVA) and truncated gIV (TglVA) and results analyzed as described previously in VII above.

X.B. Results - Analysis of the serum samples obtained from the vaccinated animals showed that both the full- length gIV (gIV) and the truncated gIV (TglV) , prepared as in XI above, produced strong immune responses as measured by ELISA and plaque reduction assays as set forth in Tables 5 and 6 below. Significantly nasal secretions also contain neutralizing antibody, as set forth in Table 6 below. Clinical examination indicated that gIV and TglV significantly reduced virus shedding and sick-days. The placebo animals in every case succumbed to BHV-1 infection as indicated by conventional virus-shedding (Figure 13) and prolonged sickness (Figure 14).

Table 5 - ELISA Assay

αglV ELISA Titer

Group No. Dose

*.

Table 6

Neutralization Titers

55

Group No. Dose Days in nasal secretions I vo

Placebo 1 gIV 2 TglV 3

XI

XLA. BHV-1 DNA Immunization Section

XI. .1 Reagent Preparation

Plasmid DNA was produced by cultivating E. coli. strain HblOl, transformed with pRSO, Fitzpatrick, D.R. et al. (1988) Journal of Virology _r 62. 4239-4248, and pRSglV, Tikoo, S.K. et al. (1990) Journal of Virology. 64. 5132-5142. Plasmid DNA was extracted and purified on CsCl gradients followed by dialysis against distilled water and ethanol precipitation. Purified plasmid was solubilized in 0.85% saline.

XI.A.1.2. Vaccination

Plasmid DNA was presented by intramuscular injection of four sites in the hind quarters of hereford calves. Volume of the injections was 2 ml with plasmid at a concentration of 62.5 μg/ml. Venous blood was drawn on regular intervals the presence of BHV-1 gIV-specific IgG antibody was determined by ELISA, western blots and neutralization assays.

XI.B. Results A gIV gene-specific immune response has been demonstrated by Western blots and IgG ELISA titres of 3200 have been attained. Plaque-reduction assays indicate serum neutralizing titres as high as 48 (a titre of 16 correlates highly with protection from disease) .

XII Use of recombinant BHV-1 proteins for diagnostic purposes.

XII.A. The recombinant BHV-1 gl, gill or gIV can be used as antigens in standard immunological assays, for example ELISA tests, to indicate the presence of antibodies to BHV-1. In this manner, the immunological status of an animal can be assessed with respect to present infection or predisposition to BHV-1.

The recombinant gl, gill or gIV proteins were diluted in sodium carbonate buffer pH 9.6 and used to coat the wells of an ELISA plate at concentrations ranging from 0.1 to lμg of protein/well. After incubating for a minimum of 1 hour, the plates were washed and dilutions of animal sera added to the plate in a serial fashion. The processing of the ELISA proceeded as described in example X.

XII.B. The developed ELISA plate indicated that animals infected by BHV-1 had detectable levels of antisera specific for gl, gill or gIV and any one of these recombinant BHV-1 proteins would be suitable for use in the diagnosis of BHV-l infection.

DEPOSIT OF BIOLOGICAL MATERIALS The following materials have been deposited or will be deposited with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. during the pendancy of this application as necessary. These deposits will be maintained under the terms of the Budapest Treaty on the deposit of microorganisms. The nucleotide sequences of the deposited materials are incorporated by reference herein, as well as the sequences of polypeptides encoded thereby. In the event of any discrepancy between a sequence expressly disclosed herein and a deposited sequence, the deposited sequence is controlling. The deposit of the sequence is not the grant of a license to make, use or sell any of the deposited materials. Material Accession Number Deposit Date 1E11-1F6 HB 9774 July 22, 1988

1D6-G11 HB 9775 July 22, 1988

1G6-2D9 HB 9776 July 22, 1988 pW-l/gl December 9, 1992 pW-1/gIII December 9, 1992 pG4HUNEO ATCC 69076 September 29, 1992

VAC-I VR 2223 July 22, 1988

VAC-III VR 2224 July 22, 1988 While the present invention has been illustrated above by certain specific embodiments, it is not intended that the specific examples should limit the scope of the invention as described in the appended claims.