Equine herpesvirus type 1 (EHV-1) can cause respiratory disease, abortions and neurological disorders in horses. The present invention is directed to a gene encoding an envelope glycoprotein of EHV-1, the glycoprotein D (gD) gene, its gene product and antibodies directed against the gD gene product. The envelope glycoproteins of herpesviruses are major targets of the immune response to herpesviral infection. Hence, another aspect of this invention is directed towards a vaccine against EHV-1 and treatment of EHV-1 infection with anti-gD antibodies.

It is now recognized that the herpesvirus of horses, referred to as equine rhinopneumonitis (also called equine abortion virus, or EHV-1) is not a single herpesvirus but two genetically and antigenically distinct viruses, sometimes designated as subtypes 1 and 2 of EHV-1. EHV-1 subtype 1 (often called simple EHV-1) causes respiratory disease, spontaneous abortion in pregnant mares and occasionally, paralysis in horses. EHV-1 subtype 2 (also referred to as EHV-4) causes respiratory disease and only occasionally, abortions. The present invention is directed to a glycoprotein isolated from subtype 1 EHV-1 (hereafter referred to as EHV-1 in accordance with the International. Committee on Taxonomy of Viruses at Edmonton, Canada in 1987).

Outbursts of EHV-1 infections in horses frequently occur in areas of concentrated horse breeding, particularly during the winter months. The incubation period of EHV-1 is from 2 to 10 days. Initial symptoms of infection include high fever for 1 to 7 days and discharge from the nostrils. White cell counts are generally depressed during the first few days

of fever and may take a week or 10 days to recover. Diarrhea and enteritis, edema of the legs and tendovaginitiε are not common in uncomplicated cases but do occur in complicated cases. All symptoms are worsened by forced exercise or work; recovery is complete in 1 to 2 weeks unless complications develop.

Reinfection may occur at intervals of 4 to 5 months or longer. These subsequent infections are usually asymptomatic and generally do not result in complications in adult horses. However, the disease ^" has been known to breakout annually in young horses on farms where no new horses have been introduced, suggesting that adult horses can act as carriers. EHV-1 infection in young horses is often associated with weaning and assembling in winter quarter.

Infected mares may have no overt signs of infection at first, with the incubation time between nasal inoculation and abortion varying from 3 weeks to 4 months. The virus spreads readily by direct contact, fomites and aerosolized secretions. It may spread from one abortive mare to others, but evidence indicates that almost all mares on a farm are infected 1 to 4 months before abortion; hence, infection spreads rapidly, probably by aerosolized secretions or direct contact. Some foals infected prenatally reach full term and are born alive, but abortion is the normal outcome of EHV-1 infection in pregnant mares. The herpesviruses are a family of structurally similar viruses. They have a double-stranded DNA genome characterized by short and long unique sequences of DNA (U and U _τ respectively) , and inverted repeats of DNA sequence which flank the unique sequences. The IT region of DNA is capable of inverting in orientation, giving rise tc :. je prototype and inverted arrangements of the EHV-1 genome. ^• All herpesviruses replicate within the nucleus of a host cell, and several

5 members of the herpes family, if not all, are capable of becoming latent after establishing a primary infection and the initiating recurring, sometimes acute, infections.

Herpesviruses are not only similar in their gross morphology, but also at the molecular level. For example, general antisera against Herpes Simplex Virus type 1 (HSV-1) and EHV-1 have been used to demonstrate some minimal crossreaction between these viruses by complement fixation, gel diffusion, immunofluorescence and immunoprecipitation. However, HSV-1 has less than 5% DNA sequence identity with ₀ EHV-1 and specific antibodies to each virus do not cross-neutralize the other ( udwig et al., 1971, Virology 45: 534-537). Despite this, the genome of EHV-1 appears to be functionally colinear with the genomes of HSV, pseudorabies virus (PRV) and varicella-zoster virus, as determined by 5 molecular hybridization experiments (Davison et al., 1983, J. Gen. Virol. 6_4: 1927-1942). Analysis of the organization and function of the EHV-1 genome is therefore not only relevant foi elucidating the mechanisms underlying EHV-1 infection, but also may identify key features of herpesvirus genomes by comparative _Q molecular biology..

A number of major structural proteins have been identified in EHV-1 viriσns, typically by protein gel electrophoresis and through the use of antibodies directed against the intact EHV-1 virion. However, interest has 5 centered on the structural glycoproteins due to their roles in the infectious process and their ability to invoke an immune response. In addition to several minor glycoproteins, eight high abundance glycoproteins have been identified in the envelope of purified EHV-1 virions. These glycoproteins have 0 molecular masses of 200, 125, 95, 90, 68, 63, 45, and 41 kilodaltons (Perdue et al., 1974, Virology 59: 201-216; Turtinen et al., 1981, Am. J. Vet. Res. 42: 2099-2104), and

5

are generally distinguished as glycoproteins by use of gp followed by the numbers 2, 10, 13, 14, 17, 18, 21, and 22a, respectively. Little is known about the antigenic or moleculai structure of most of these glycoproteins. However the genes for six of these proteins have been mapped on the EHV-1 genome ( p2, gplO, gpl3, gpl4, gpl7/18 and gp21/22a; Allen et al, 1987, J. Virol. !_-. 2454-2461). All but gp 17/18 map within the long unique (U _r ) region of the EHV-1 genome.

Two of these six glycoproteins have been identified as homologs of glycoproteins known in other herpesviruses, based on map position: gpl3 corresponds to gC of HSV (and glll of PRV) and gpl4 corresponds to gB of HSV (and gll of PRV) (Allen et al. 1987, supra) . The nucleotide sequences of EHV-1 gpl3 and gpl4 have been determined and the translated amino acid sequences of both have revealed significant homology to the corresponding HSV glycoproteins (Allen et al. , 1988, J. Virol 6_2: 2850-2858; Whalley et al. , 1989, J, Gen. Virol. 7_0: 383-394). The HSV gB glycoprotein, with extensive amino. acid sequence identity to EHV-1 gpl4, is required for virus entry and cell fusion and has been shown to invoke circulating antibodies as well as cell-mediated immune response. Because of its structural similarity the gpl4 protein may have a similar role.

A genomic library of EHV-1 DNA exists together with a physical, restriction map of the EHV-1 genome (Henry et al. , 1.981, Virology 115: 97-114). Identification and characterization of EHV-1 glycoproteins by. analysis of the DNA in the unique short (U ) region of the EHV-1 genome has led to the discovery of a new EHV-1 glycoprotein (glycoprotein D) . There is a long standing need for safe, effective, long-acting, vaccines against EHV-1 infection. A number of EHV- ^' l vaccines are currently available (e.g. U.S. Patent 4,110,433 to Purdy; U.S. Patent 4,083,958 to Bryan ) , but are

derived from live viruses. In addition, the EHV-1 vaccines currently available are generally acknowledged as being inadequate in spectrum and duration of protection (Doll, 1961, J. Am. Vet. Med. Assoc. 35J9: 1324-1330; Bryans, 1976. Jn Equine Infectious Diseases IV, Proceedings of the Fourth International Conference on Equine Infectious Diseases, T.J. Bryans and H. Gerber, eds., Princeton: Veterinary Publications: 83-92; Burrows, et al., 1984, Veterin. Rec. 11 : 369-374; and Stokes et al., 1989, J. Gen. Virol. 7_0: 1173-1183). Thus, the present discovery provides new vaccines for EHV-1 protection having significant advantages over those of the prior art, since the use of live or attenuated viruses is eliminated.

The present invention is directed to isolated DNA encoding equine herpesvirus type 1, glycoprotein D (gD), which maps within the unique short (U ) region of the EHV-1 genome (map units 0.865-0.884). The (gD) polypeptide is encoded by an open reading frame at nucleotides 511-1836, 661-1836, 679-1836, or 682-1836, as shown in Fig. 2. The gD polypeptide appears to have a cleaved signal sequence which yields a polypeptide encoded by nucleotides 739-1836, depicted in Fig. 2. The gl ¹ polypeptides, and antigenic peptides thereof, are useful ir. vaccines against EHV-1 and for the production of antibodies directed against the gD polypeptide.

Another aspect of the present invention provides an isolated nucleic acid encoding an EHV-1 gD polypeptide as described above, or a fragment thereof, and replicable expression vectors containing these nucleic acids.

A still further aspect of this invention is directed to transformed hosts such as prokaryotic microorganisms and

c cultured eukaryotic cells containing the replicable expression vectors encoding EHV-1 gD polypeptides.

Another aspect of this invention provides isolated, EHV-1 gD protein and antigenic EHV-1 gD peptides, especially ii recombinant form. A further aspect of this invention provides a vaccine composition for immunization against EHV-1 containing a gD polypeptide or antigenic portions thereof and a pharmaceutically acceptable carrier.

A still further aspect of this invention provides polyclonal and monoclonal antibodies directed against the EHV-1 glycoprotein D, hybridoma cell lines producing these monoclonal antibodies and methods of using these antibodies to detect EHV-1.

Yet another aspect of this invention is directed a method of treatment or prevention of EHV-1 infection via the antibodies directed against the EHV-1 gD protein, including treatment by passive immunization.

In the accompanying drawings, Fig. 1 depicts the genomic map location of the EHV-1 gD gene. (a) The structure of the viral genome is shown with the unique long (U ) region and unique short (U ) segment depicted with the solid lines and the internal repeat (IR) and terminal repeat (TR) segments represented by closed boxes. (b) The Ba HI restriction map of the prototype arrangement of the EHV-1 genome is shown, and restriction sites are indicated with arrows. The 5.2 Kbp BamHI M fragment maps entirely within the U _c at map position 0.869-0.884 in the prototype arrangement and at map position 0.865-0.872 in the inverted arrangement. (c) Expanded restriction map of the BamHI M fragment showing the positions of restriction cleavage sites employed in subcloning.

Restriction sites are labeled as follows: B, BamHI; K, Kpnl;

X, Xbal. The position of the pSZ-4 open reading frame encodin< gD polypeptides is indicated by the arrow.

Fig. 2 depicts the nucleotide sequence of the 2.2 Kb] BamHI/Kpnl pSZ-4 clone and the amino acid sequence of the gD open reading frame (ORF). Numbers at the end of each row note the distance of the nucleotide (upper number) from the Kpnl recognition sequence and the distance of the amino acid residue (lower number) from the first possible initiation methionine in this ORF. Features in the sequence are shown with the following symbols: o, CAAT box; x, TATA box; #, HSV ICP4 consensus binding site homolog; §, putative cis-regulatory AT-rich region; *, polyadenylation signal; +, GT-rich region following the polyadenylation signal. Strongly hydrophobic amino acids are underlined with one solid line, and potential glycosylation sites for the addition of N-linked * oligosaccharides (N-X-S/T; Hubbard and Ivatt, 1981, Ann. Rev. Biochem. ^0_: 555-583) are underlined with dashed lines.

Fig. 3. Alignment of the amino acid sequences of the EHV-1 gD ORF with HSV-1 gD (McGeoch et al., 1985, J. Mol. Biol. 181: 1-13) and PRV gp50 (Petrovskis et al., 1986, J. Virol. _9: 216-223), using the FASTP algorithm of Lipman and Pearson (1985, Science 227: 1435-1440). Amino acid identity is denoted by vertical bars between the sequences. Matches between PRV and HSV residues are noted below the HSV sequence. The asterisks (*) indicate the conserved cyεteine residues. The position of the signal peptide cleavage site for HSV gD-1 (Eisenberg et al. , 1984, J. Virol. _49: 265-268) and the for EHV-1 are denoted by arrows.

Fig. 4. Hydropathicity analysis of the amino acid sequence of EHV-1 gD. The hydrophobicity and hydrophilicity characteristics of the EHV-1 amino acid . juence were determined using the Kyte and Doolittle (_t82, J. Mol. Biol. 157: 105-132) algorithm and a 15 amino acid window. The

δ vertical axis represents a relative hydrophobic score where values above -5 (midpoint value indicated by dashed line) are hydrophobic. The horizontal axis represents the amino acid number of the EHV-1 gD translated sequence.

The present invention provides the EHV-1 gD gene, encoded by the open reading frame (ORF) indicated by the nucleotide sequence in Fig. 2 (nucleotides 511-1836). The EHV-1 gD gene lies within the unique short (U ) region of the EHV-l genome,, and is identified by sequencing a 2.2 kilobase (kb) fragment of DNA lying within this region. This 2.2 kb fragment, obtained from a libary of EHV-l genomic DNA (Henry et al. supra.), is located within the U region as determined by restriction mapping (Henry et al. , supra). No other EHV-l glycoproteins mapping within the U region have heretofore been fully characterized by sequence analysis. DNA sequence analysis of this 2.2 Kb BamHI/Kpnl fragment revealed an ORF whose translated sequence exhibits significant homology to glycoprotein D (gD) of herpes simplex virus (HSV) types 1 and 2 and to pseudorabies virus (PRV) glycoprotein 50, the gD equivalent (Fig. 2 and Fig. 3). The ORF of EHV-l gD gene is capable of encoding several polypeptides all of which are contemplated by the present invention. If translation initiates at the first in-frame ATG at nucleotides 511-513 depicted in Fig. 2, a gD polypeptide having 442 amino acids and ^" a calculated molecular weight of '49,904 is produced. Likewise, if translation initiates at the second, third or fourth in-frame ATG's (nucleotides 661-663, 679-681 or 682-684. respectively) then gD polypeptides having 393, 387 or 386 amino acids, respectively, are produced. Cleavage of a predicted signal sequence yields a mature EHV-l gD polypeptide encoded by amino acids 77-442 (367 amino acids). 7- _* ι skilled artisan can

7 identify and isolate a DNA encoding any of these gD polypeptides.

The fourth in-frame ATG at nucleotides ^■ 682-684 serve.' as a preferred initiation codon for several reasons: 1) the sequences neighboring this ATG comply most favorably with the sequence motif of an initiation codon according to Kozak's rules (Kozak, 1980, Cell 22 .' 7-8; Kozak, 1983, Microbiol. Rev. 47: 1045; Kozak 1986, Cell 44: 283-292); 2) residues following this methionine have the most likely signal sequence based on hydropathicity and a probable signal sequence cleavage site after residue number 19; 3) this gD polypeptide is 386 amino acids (a.a.) in size (43,206 molecular weight) which compares favorably to the size of the gD polypeptides of HSV-1 (394 a.a. ) , HSV-2 (394 a.a. ) , and PRV (402 a.a. ) ; .and 4) ^■ the relative positions of the 5' regulatory elements, namely the CAAT sequence at 502-505 and the TATA box at 561-564, suggest that transcription is initiated downstream of the first ATG. The nucleotide sequence of the gD gene reveals a complete transcriptional unit including CAAT and TATA elements and signals for polyadenylation. The gD polypeptide exhibits features typical of a transmembrane protein: a hydrophobic N-terminal signal sequence followed by a probable signal sequence cleavage site, four potential N-linked glycosylation sites, and a hydrophobic membrane-spanning domain near the carboxyl terminus followed by a charged membrane anchor sequence. Hence, the present invention provides a complete DNA sequence for the entife EHV-l gD gene and allows identification of the EHV-l gD polypeptides encoded in the identified ORF.

The present invention provides the EHV-l gD gene on a 2.2 kb BamHI/Kpnl fragment cloned into pUC19, to create pSZ-4. Another aspect of the present invention provides replicable expression vectors allowing regulated expression of a EHV-l gD polypeptide. Replicable expression vectors as

described herein are generally DNA molecules engineered for 1 controlled expression of a desired gene, especially high level expression where it is desirable to produce large quantities o a particular gene product, or polypeptide. The vectors encode promoters and other sequences to control expression of the gen 5 being expressed, and an origin of replication which is operabl in the contemplated host. Preferably the vectors are plasmids, bacteriophages, cosmids or viruses. Any expression vector comprising RNA is also contemplated.

* Sequence elements capable of effecting expression of

1 ₀ a gene product include promoters, enhancer elements, transcription termination signals and polyadenylation sites. Promoters are DNA sequence elements for controlling gene expression, in particular, they specify transcription initiation sites. Prokaryotic promoters that are useful

15 include the lac promoter, the trp promoter, and P. and P promoters of lambda and the T7 polymerase promoter. Eukaryotic promoters are especially useful in the invention and include promoters of viral origin, such as the baculovirus polyhedrin promoter, the vaccinia virus hemagglutinin (HA) promoter, SV40

20 late promoter, the Moloney Leukemia Virus LTR, and the Murine Sarcoma Virus (MSV) LTR. Yeast promoters and any promoters or variations of promoters designed to control gene expression, including genetically-engineered promoters are also contemplated. Control of gene expression includes the ability

25 to regulate a gene both positively and negatively (i.e., turning gene expression on or off) to obtain the desired level of expression.

One skilled in the art has available many choices of replicable expression vectors, compatible hosts and well-known

30 methods for making and using the vectors. Recombinant DNA methods are found in any of the myriad of standard laboratory manuals on genetic engineering (see for example Sambrook et

35

// al. , 1989, Molecular Cloning: A Laboratory Approach, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

The replicable expression vectors of the present invention can be made by ligating part or all of the EHV-l gD coding region in the proper orientation to the promoter and other sequence elements being used to control gene expression. For example, a DNA fragment encoding gD nucleotides 511-1839 (depicted in Fig. 2) may be operably linked, by ligation, downstream of a promoter, thereby allowing expression of a 442 amino acid gD polypeptide. Similarly, ligation of DNA fragments encoding gD nucleotides 661-1839, 679-1839, or 682-1839 downstream of a promoter allows gD polypeptides of 393 amino acids, 387 amino acids or 386 amino acids to be expressed. This juxtapositioning of promoter and other sequence elements with gD polypeptide coding region allows the production of large amounts of the gD polypeptide useful, not only as a vaccine against EHV-l infection, but also for anti-gD antibody production and for analysis of the function of gD during EHV-l infection. Preferred vectors of the present invention are derived from eukaryotic sources. Expression vectors that function in tissue culture cells are especially useful, but yeast vectors are also contemplated. These vectors include yeast plasmids and minichromosomes, retrovirus vectors, BPV (bovine papilloma virus) vectors, vaccinia virus vectors, baculovirus vectors, SV40 based vectors and other viral vectors. Baculovirus vectors and retrovirus vectors (e.g., murine leukemia viral vectors) and preferred. Tissue culture cells that are used with eukaryotic replicable expression vectors include S. frugiperda cells, VERO cells, MRC-5 cells, SCV-1 cells, COS-1 cells, NIH3T3 cells, mouse L cells, HeLa

s2, cells and such other cultured cell lines known to one skilled in the art.

The present invention also contemplates prokaryotic vectors that are suitable as cloning vectors or as expression vectors for EHV-l gD polypeptides, including bacterial and bacteriophage vectors that can transform such hosts as E. col. B. subtilis, Streptomyces sps. and other microorganisms. Many of these vectors are based on pBR322 including pUC19 and pGEM-7Zf (commercially available from Promega, Madison, WI ) an are well known in the art. Bacteriophage vectors that are use in the invention include lambda and M13.

In one embodiment the EHV-l gD gene is inserted into a lambda gtll expression vector (Sambrook e_t al. , 1989, Molecular Cloning: A Laboratory Manual Vol. 2, Cold Spring Harbor Laboratory Press: 12.1-12.44). Lambda gtll is constructed to allow insertion of foreign DNA into the structural gene for beta-galactosidase, thereby producing a beta-galactosidase-foreign-protein fusion protein, under the control of the lac promoter. Such a fusion protein is easily isolated, for example, by using commercially available anti-beta-galactosidase antibodies. The gD-beta-galactosidase fusion protein can then be used to generate antibodies against the EHV-l gD protein. As an alternative prokaryotic expressio system, the pKK223-3 expression vector, can provide high level of EHV-l gD expression in E. coli. This vector contains the strong trp-lac (tac) promoter which is IPTG inducible. (deBoer et al. , 1983, Proc. Natl. Acad. Sci. USA). A major advantage of the pKK223-3 expression vector is that an intact EHV-l gD polypeptide is expressed rather than a beta-galactosidase fusion protein. In another preferred embodiment, the EHV-l gD protei of the present invention is expressed in a baculovirus expression system. This system provides baculovirus expressio

/2> vectors into which EHV-l gD DNA encoding an EHV-l gD polypeptide can be inserted downstream of a strongly transcribed promoter. When cultured in insect cells, the recombinant baculovirus can provide stable expression of high levels of extracellular or intracellular polypeptide. Baculovirus expression vectors and their use are reviewed in Luckow et al. (1988, Bio./ Technology 6 : 47-55). A particular advantage of this system is its similarity to higher eukaryotes with regard to protein modification, processing and transport. Thus, recombinant-derived eukaryotic proteins will be processed and glycosylated in a manner important for obtaining a native protein conformation and, hence, maximal biological activity.

A further aspect of the present invention is directed to an isolated EHV-l gD polypeptide, especially a recombinant gD polypeptide. A gD polypeptide can be obtained from virally-infected cultured cells, from virally-infected animals or from microorganisms or cells transformed with an expression vector encoding a gD polypeptide. A process of preparing a recombinant EHV-l gD polypeptide includes cultivating the microorganism or cell transformed with an EHV-l gD recombinant nucleic acid for a time and under conditions sufficient to produce a gD polypeptide and then recovering the gD polypeptide. Purification of a gD polypeptide is achieved by conventional purification techniques such as ammonium sulfate precipitation, column chromatography, affinity chromatography and the like. During purification, the gD polypeptide is identified by SDS-polyacrylamide gel electrophoreεis, or by standard immunodetection techniques, such as immunoblσtting or immunoprcipitation.

Antibodies can be used to purify an EHV-l gD polypeptide. Antibodies are highly spec ^ic and are especially useful for isolating specific antigens (r. teinε) that represent only minor components of complex mixtures such as

cell lysates. The lambda gtll expression system described above provides a fusion protein of beta-galactosidase and EHV- gD proteins. This fusion protein can be purified by passage o a cell lysate containing the fusion protein over an anti-beta-galactosidase im uno-affinity column. The anti-beta-galactosidase antibodies bound to the column matrix bind the fusion protein. Any impurities can be washed off the column .and the fusion protein can be eluted by changes in pH, or by use of detergents, chaotropic agents or organic solvents. Im unoaffinity purification techniques are well known in the art (see, for example, Harlowe, et al. , 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Labortory Press: 511-552). The purified EHV-l gD fusion protein can be used to obtain antibodies specific for the EHV-l gD protein. These anti-EHV-1 gD antibodies in turn allow immunoaffinity purification of the non-fusion EHV-l gD protein or peptides thereof.

Another embodiment of the present invention provides polyclonal antibodies directed against the EHV-l protein or peptides encoding a portion of an EHV-l polypeptide. The "antibodies are useful for passive immunization of animals infected with EHV-l, and for the purification of EHV-i gD polypeptides. Antibodies can be generated by using an entire EHV-l gD polypeptide as an antigen or by using short peptides encoding a portion of an EHV-l gD polypeptide, as antigens. Computer analysis of the gD polypeptide amino acid sequence was used to identify the following peptide sequences as being strongly antigenic epitopes for all gD polypeptides; subsequent analysis has confirmed this. ^' Peptide 1 is located at amino acid residues 80-98, while Peptide 2 is located at amino acid residues 343-361 as depicted in Fig. 2. ^" jeptide 2 has an additional cysteine residue at its carbcvr _* terminus to allow coupling to a protein carrier.

The following peptides are preferred immunogens for generating antibodies:

Peptide 1 (EHV-l gD nucleotides 748-804):

NH ₂ -Cys-Glu-Lys-Ala-Lys-Arg-Ala-Val-Arg-Gly-Arg-Gln-Asρ- Arg-

Pro-Lys-Glu-Phe-Pro-COOH Peptide 2 (EHV-l gD nucleotides 1537-1593):

NH ₂ -Glu-Ile-Thr-Gln-Asn-Lys-Thr-Asp-Pro-Lys-Pro-Gly-Gln-A la- Asp-Pro-Lys-Pro-Asn-cys-COOH

Polyclonal antibodies directed against an EHV-l gD polypeptide or antigenic peptide thereof are prepared by injection of a suitable animal with an immunogenic amount of the peptide or antigenic component, collecting serum from the animal, and testing sera for the desired reactivity. If necessary, specific sera can be isolated by any of the known immunσadsorbent techniques. Detailed protocols for antibody production are provided in Harlow, E. et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Press, NY, 1988.

Another embodiment of the present invention provides monoclonal antibodies. Monoclonal antibodies are preferred because large quantities of antibodies, all of similar reactivity, are produced. The preparation of hybridoma cell lines for monoclonal antibody-production is done by fusing an immortal cell line with antibody-producing lymphocytes from an immunized animal. This can be done by techniques which are well known to those who are skilled in the art. (See, foi example, Harlow, E. and Lane, D. , Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988; or Douillard, J. Y. and Hoffman, T. , "Basic Facts About Hybridomas", in Compendium of Immunology Vol. II, L. Schwartz (Ed.), 1981.)

Unlike the preparation of polyclonal sera, the choice of animal for monoclonal antibody preparation is dependent on the availability of appropriate immortal cell lines capable of fusing with the antibody-producing lymphocytes derived from the

immunized animal. Mouse and rat have been the animals of choice for hybridoma technology and are preferably used. For the purpose of making the monoclonal antibodies of the present invention, the animal of choice may be injected with from abou 0.01 mg to about 20 mg of purified EHV-l gD antigen. Typicall the antigen is emulsified in an adjuvant to stimulate general immune responses. Boosting injections are generally also required. Lymphocytes can be obtained by removing the spleen or lymph nodes of immunized animals in a sterile fashion, and are fused ¹ to immortalized cells. A number of immortalized cell lines suitable for fusion have been developed, and the choice of any particular line is directed by any one of a number of criteria such as speed, uniformity of growth characteristics, deficiency of its metabolism for a component of the growth medium, and potential for good fusion frequency. Intraspecies hybrids, particularly between like strains, work better than interspecies fusions. Several cell lines are available, including mutant selected for the loss of ability to create myeloma immunoglobulin. Included among these are the following mouse myeloma lines: X63-Ag 8.653, MPC..-X45-6TG, P3 NSl/l-Ag4-l, P3-X63-Agl4 (all BALB/C derived), Y3*Agl.2.3 (rat), and U266 (human). X63-Ag8.653 cells are preferred.

The fused cell colonies are tested for the presence of antibodies that recognize EHV-l gD polypeptides. Detection of monoclonal antibodies can be performed using an assay where the antigen is bound to a solid support and allowed to react to hybridoma supernatanbs containing the putative antibodies. The presence of antibodies may be detected by "sandwich" techniques using a variety of indicators. Most of the common methods are sufficiently sensitive for use in the range of antibody concentrations secreted during hybrid growth.

Cloning of hybrid cells can be carried out after 20-25 days of cell growth in selected medium. Cloning can be

performed by cell limiting dilution in fluid phase or by directly selecting single cells growing in semi-solid agarose. For limiting dilution, cell suspensions are diluted serially t yield a statistical probability of having only one cell per well. For the agarose techniques, hybrids are seeded in a semisolid upper layer, over a lower layer containing feeder cells. The colonies from the upper layer may be picked up and eventually transferred to wells.

Antibody-secreting hybrid cells can be grown in various tissue culture flasks, yielding supernatants with variable concentrations of antibodies. In order to obtain higher concentrations, hybrid cells may be tranεferred into animalε to obtain inflammatory asciteε. Antibody-containing ascites can be harvested 8-12 days after intraperitoneal injection. The ascites contain a higher concentration of antibodies but include both monoclonals and immunogiubulins from the inflammatory asciteε. Antibody purification may be achieved by, for ^* example, affinity chromatography.

The present invention is alεo directed to the ^• detection of EHV-l infection by immunological techniques using antibodieε directed against EHV-l gD polypeptides. A method is provided for diagnosing EHV-l infection by contacting a blood or serum sample of an individual to be tested with an antibody directed against an EHv-1 gD polypeptide, or an antigenic fragment thereof, for a time and under conditions sufficient o form an antigen-antibody complexing and detecting a resultant antigen-antibody complex.

The presence of gD polypeptides in horse's blood sample, and therefore the virus, can be detected utilizing antibodies prepared as above, either monoclonal or polyclonal, in virtually any type of immunoassay. A method for detecting EHV-l includes contacting a test sample with an antibody directed aginst an EHV-l gD polypeptide, or an antigenic

/f peptide thereof, for a time and under conditions sufficient to form an antigen-antibody complex, and detecting the resultant antigen-antibody complex. A wide range of immunoassay techniques are available as can be seen by reference to Harlow et al. (Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988) and U.S. Patent Nos. 4,018,043 and 4,424,279. This, of course, includes both single-site and two-site, or a "sandwich" of the non-competitive types, as well as in traditional competitive binding assays. Sandwich assays are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are among the most useful and commonly used assays. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized in a solid subεtrate and the sample. to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen binary complex, a second antibody, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of a ternary complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple obεervation of the visible signal, or may ^' be quantitated by comparing with a control sample containing known amounts of antigen. Variationε on the forward assay include a simultaneouε assay, in which both the sample to be tested and the reporter conjugated antibody are first combined, incubated and then added to the unlabeled surface bound antibody. These techniques are well known to those skilled in the art, and the possibly of minor

/ ^« 7 variations will be readily apparent. As used herein, "sandwic assay" is intended to encompass all variations on the basic two-site technique.

EHV-l gD polypeptides may also be detected by a competitive binding assay in which a limiting amount of antibody specific for the gD polypeptides is combined with specified volumes of samples containing an unknown amount of the gD polypeptides and a solution containing a known amount of the detectably labeled, e.g. radio-labeled, gD polypeptides. Labeled and unlabeled molecules then compete for the available binding sites on the antibody. Phase separation of the free and antibody-bound molecules allows measurement of the amount of label present in each phase, thus indicating the amount of antigen or hapten in the sample being tested. Numerous variations on this general competitive binding assay, are known. to the skilled artisan and are contemplated by the present invention.

In any of the known immunoassays, for practical purposes, one of the antibodies or the antigen may be bound to a solid phase, and a second molecule, either the second antibody in a sandwich assay, or, in a competitive assay, the known amount of antigen, may bear a detectable label or reporter molecule in order to allow detection of an antibody-antigen reaction. When two antibodies are employed, as in the sandwich assay, it is only necessary that one of the antibodies be specific for an EHV-l gD polypeptide or its antigenic components. The following description provides the methodology for performing a typical forward sandwich assay; however, the general techniques are to be understood as being applicable to any of the contemplated immunoassays. In the typical forward sandwic assay, a first antibody having specificity for the EHV--_ gD protein or its antigenic components is either covalently or passively bound to

^■ O . a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid support may be in the form of tubes, beads, discs or microplate, or any other surface suitable for conducting an immunoassay. The binding processes are. well-known in the art and generally consist of cross-linking, covalently binding or physically adsorbing the molecule to the insoluble carrier. Following binding, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated at 25°C (or other suitable temperature) for a period of time sufficient to allow binding of any antigen. The incubation period will vary but will generally be in the range of about 2-40 minutes. Following the incubation period, the antibody-antigen solid ^* phase is washed and dried and incubated with a second antibody also specific for the gD protein or an antigenic region thereof. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten. By "reporter molecule", as used in the present specification and claims, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either - qualitative or quantitative. the most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehye or periodate. Aε will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Comιr_c.*. ^* y used enzymes include horseradish peroxidase, glucose oxidase,

β-galactosidase and alkaline phosphates, among others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the correspondin enzyme, of a detectable color change. For example, p-nitrophenyl phosphate is suitable for use with alkaline phosphatase conjugates; for peroxidaεe conjugates,

1,2-phenylenediamine, 5-aminosalicyclic acid, or tolidine are commonly used. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labeled antibody is added to the first antibody-antigen complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the ternary complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophoto etrically, to give an indication of the amount of hapten which was present in the sample.

Alternately, fluorescent compounds, such as fluorescein and rϊiodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody absorbs the light energy, inducing a state of excitability in the molecule, followed by emission of the light at a characteristic color, visually detectable w*ith a light microscope. As above, the fluorescent labeled antibody is allowed to bind to the first antibody-antigen complex. After washing off the unbound reagent, the remaining ternary complex is then exposed to the light of the appropriate wavelength, the fluorescence observed indicates the presence of the antigen of interest. Immunofluorescence techniques are both very well establiεhed in

the art. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules, can also be employed. It is readily apparent to the skilled technician how to vary the procedure to suit the required purpose. In another embodiment, the antibodies directed against EHV-l gD polypeptides are incorporated into a kit for the detection of EHV-l infection. Such a kit may encompasε any of the detection systems contemplated and described herein, and may employ either polyclonal or monoclonal antibodies directed against EHV-l gD polypeptides or antigenic regions thereof.

Both antibodies complexed to a solid surface described above or soluble antibodies are contemplated for use in a detection kit. The kit can be compartmentalized and includes at least one container containing primary anti-EHV-1 gD antibodies and another container containing secondary antibodies covalently bond to a reporter molecule, such that the secondary antibodies are capable of detecting the first antibodies or are themselves directed against EHV-l gD. For example, one contemplated kit is compartmentalized and haε the following components: the first container contains killed EHV-l virus or EHV-l gD polypeptides as a solution, or bound to a solid surface, to act as a standard or positive control, the second container containε anti-EHV-1-gD primary antibodieε either free in εolution or bound to a εolid surface, a third container contains a solution of secondary antibodieε covalently bound to a reporter molecule vΛiich are reactive againεt either the primary antibodieε or against a portion of a gD polypeptide not reactive with the primary antibody. A fourth or fifth container contains a substrate, or reagent, appropriate for visualization of the reporter molecule.

The subject invention therefore encompasses anti-EHV-1-gD antibodieε. EHV-l gD antibodieε are uεeful for

purification of the EHV-l gD protein as well as for the detection and study of the EHV-l virus.

Another embodiment of the present invention provides pharmaceutical compositions of the EHV-l gD protein or portion thereof, or of anti-EHV-1 gD antibodies. One important embodiment of the present invention provides the purified EHV-l gD protein or peptide portions thereof as a vaccine against EHV-l. The vaccine includes an immunogenic amount of an EHV-l gD polypeptide or an antigenic peptide thereof and pharmaceutically acceptable inert ingredients commonly used in vaccinations. The effective dosage of this vaccine is about 0.5 μg to about 2000 mg of antigen per kilogram of body weight. Boosting regiments may be required and the dosage regimen can be adjusted to provide optimal immunization. The vaccination of a mare prior to breeding and again during her pregnancy may prevent abortions caused by EHV-l. Other horses can be vaccinated, for example, about once a year. Foalε can be vaccinated εhortly after birth. Vaccinations can be administered parenterally or extra-parenterally to the mucosal εurfaceε of the body. The intramuscular route of innoculation is preferred.

Another embodiment of the present invention contemplates the treatment or prevention of EHV-l infection by passive immunization with anti-EHV-1 gD antibodies or horse anti-EHV-1 gD anti-serum. Pharmaceutical compositions for passive immunization include an amount of anti-EHV-1 gD antibody or antiserum effective in the treatment of EHV-l infection, and particularly, effective in preventing abortion in EHV-l infected pregnant mares. The dosage of anti-EHV-1 gD anti-serum may be from about 0.01 microliters to about 0.1 milliliters per kilogram of body weight. The dosage of anti-EHV-1-gD antibodies depends upon the efficacy and titer of the antibodies but may be from about 0.5 μg to about 2000 mg

antibody protein per kilogram of body weight. The doεage regimen can be adjusted to provide the optimum therapeutic response. For example, if a horse is exposed to EHV-l or if a horse (particularly a pregnant mare) becomes infected with EHV-l several divided does can be administered daily or a single daily dose can be proportionally reduced or increased a: indicated by the exigencies of the therapeutic situation. The active compounds for vaccination or passive immunization may be administered in a convenient manner such as by intraveneous (where water soluble), intramuscular, subcutaneouε, intranasal, or intradermal routes. Intramuscular administration is a preferred method of administration but other methods are also contemplated by the present invention. In order to administer EHV-l gD protein or anti-EHV-1 gD antibodies by other than parenteral administration, they should be coated, or administered with, a material to prevent inactivation. For example, EHV-l gD protein or anti-EHV-1 gD antibodies may be administered in an adjuvant, co-administered with enzyme inhibitors or administered in lipoεomeε. Adjuvants contemplated herein include resorcinolε, non-ionic εurfactantε such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.

The active compounds may alεo be administered parenterally or intraperitoneally. Disperεions can also be prepared in glycerol, liquid polyethylene glycolε, and mixtures thereof, and in oilε. Under ordinary conditions of εtorage and uεe, theεe preparationε can contain a preεervative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluable) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or disperεion. In all caεeε the form uεt be sterile and must be fluid to the

extent that easy syringability exists. It must be stable unde. the conditions of manufacture and storage and must be preεerve< against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or disperεio. medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agentε, for example, parabenε, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases it may be preferable to include isotonic agents, for example, sugarε or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, disperεionε are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from thoεe enumerated above. In the caεe of εterile powderε for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and the freeze-drying technique which yield a powder of the active ingredient plus any dditional desired ingredient form previously sterile-filteio solution thereof.

It is especially advantageous to formulate parentera compositions in dosage unit form for ease of administration an* uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to the treated; each unit containing a predetermined quantity of the active material calculated to produce the desired therapeutic effect in asεociation with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly depend on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding εuch active material for the treatment of diεeaεe.

The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore discloεed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expreεεed in proportions, the active compound is generally preεent in from about 10 μg to about 2000 mg/ml of carrier. In the caεe of compoεitionε containing supplementary active ingredients, the dosageε are determined by reference to the uεual doεe and manner of administration of the said ingredients.

As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and adεorption delaying agentε, and the like. The use of such media gentε for pharmaceutical active substaπceε iε well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplate . Supplementary active ingredients can also be incorporated into the compositionε.

Hence, the above pharmaceutical compositions provide therapeutically administrable forms of an anti-EHV-1 vaccine, and anti-EHV-1-gD antibodies. The vaccine is useful for long-term prevention of EHV-l infection while the anti-sera an antibodies may be used for short-term prevention and treatment of EHV-l infection.

The Examples serve to further illustrate the invention.

EXAMPLE 1 Viral and bacterial strains

EHV-l (Kentucky A strain) was propogated in mouse L- cells (O'Callaghan et al. , 1968, Virology 6: 104-114; Perdue et al., 1974, Virology ^9: 210-216). Extracellular virionε purified from infectious supernatants were used as the εource of viral DNA (Cohen et al. , 1975, Virology 6^8 _. : 561-565). All plasmidε were maintained in Escherichia coli strain DH5α [F~80dlacZ M15 (lacZYA-argF)U169 recAl endAl hsdR17 (r ~ m, ^' )

___, -i . r supE442~ thi-1 gyrAl relAl; Bethesda Research Laboratories, Gaithersburg, MD] or strain XLl-Blue [recAl lac ^" endAl gyrA96 thi hsdR17 supE44 relAl (F'proAB lacIQ lacZ M15 TnlO); Stratagene, La Jolla, CA] Cell culture

L-M strain mouse fibroblasts were grown and subcultured regularly in suspenεion culture in YELP medium

(yeast extract, lactalbumin hydrolyzate, peptone) supplemented with 3% fetal bovine serum (Microbiological Asεociateε Incorporated, Bethesda, Maryland) as deεcribed previously (O'Callaghan et al., 1968, J. Virol. 2 : 793-804 and O'Callaghan et al. 1968, Virology 36 _. = 104-114). When radioactive precurεorε were employed, the cells were transferred to LP media (no yeast extract) supplemented with dialyzed calf serum. Virus propagation and aεεay - The Kentucky A strain of equine herpeε viruε type 1, formerly known aε eq&ine abortion virus (EAV) and pasεaged fro itε hamεter host to L cells in 1962 (Randall et al. , 1962, Proc. Soc. Exp. Biol. Med. 110: 487-489), was employed. This viruε (EHV-1L) has been passaged regularly in both suspension and monolayer cultures of L-M cells. For propagation of virus, cells either grown to confluency in monolayer cultures in 250-ml disposable plastic tisεue culture flaεkε (Falcon

*ϊ

Products, Cockeysville, Maryland) or maintained as log phase suspension cultures in 500-ml or 1000-ml Erlenmeyer flasks wer employed. Infection was accomplished at a multiplicity of 5-1 plaque-forming units (PFU) per cell in a small volume of mediu

( (2200 X 10 cell/ml); attachment was carried out for 2-2.5 hr at 37°

The virus was assayed by using a modification of the plague method of Garabedian et al. (1967, Proc. Soc. Exp. Biol. Med. 126: 568-571). Briefly, 0.1 ml aliquots of appropriate dilutions of the virus sample were added to monolayers of L-M cells in plastic petri disheε (60 X 15 mm; Falcon Products,

Cockeyεville, Maryland), which were then incubated at 37° for 2 hr in 5% C0 ₂ atmosphere to permit virus attachment. After this incubation, each monolayer was overlaid with 5 ml of chilled (4 ^β ) YELP medium containing 1.5% methyl cellulose 4000 cps (Fisher Chemical Company, Fair Lawn, New Jersey), and the plates then were incubated at 37° for 4-5 days. The methyl cellulose overlay was removed by standing the plates at 4° for 30 min. and washing with ice cold phosphate-buffered saline (PBS; 0.02 M sodium phosphate, 0.14 M sodium chloride, pH 7.4!. The monolayers then were fixed with 97% methyl alcohol (2 min.) and were stained with a 0.5% aqueous solution of ethylene blue. Plagues could be counted macroscopically and were 0,75 to 1 mm in diameter. Microεcopic examination εhowed plaques tc be areas of cell-clearing due to formation of large syncytia; syncytia with as many aε 100 or more nuclei were observed. Neutralization of. EHV-l Infectivity by Antibodies

Two assays are available to ascertain whether antibodies or antisera have the ability to neutralize EHV-l infectivity. First, a plaque reduction assay is available which quantitates EHV-l neutralization and monitors its requirement for complement. This assay is time consuming, requiring approximately 6 days (O'Callaghan, et al., 1983. In

2,0

Herpesviruses, B. Roiz an, ed. ; Series 2 of Comprensive Virology, H. Frankel-Conrat and R.R. Wagner, edε. Plenum

Publishing Corp., N.Y.: 215-318). A εecond assay is used fo rapid analysis. This assay monitors EHV-l encoded thymidine kinase (Tk) activity in LTK cells to indicate neutralization. The ability of an antibody to prevent EHV-l infection is measured by ability to inhibit induction of the viral Tk activity within the cell. This assay iε deεcribed in detail below.

Dilutions of anti-EHV-1 gD antibodies or antisera are incubated with infectious EHV-l (3 pFu/cell) for 1 hour at 37°C and then added to monolayers of permissive LTK cells. At 14 hr post infection, cell extracts are prepared and asεayed for EHV-l Tk activity by a rapid assay method (Wolcott, et al., 1989, Anal. Biochem.. 178: 30-40). This method has been shown to detect and quantitate EHV-l neutralizing antibodies with a sensitivity greater than or equal to the plaque reduction assay. However, ^'* this asεay requireε only one day to complete, in contraεt to 6 dayε for the plaque reduction assay. Vaccination of hamsters, and challenge with EHV-l Suckling LSH Syrian hamsterε are separated into groups of 10. One group receiveε a single inoculation of EHV-l vaccine (i.e. purified EHV-l-gD protein or peptides derived therefrom). Inoculation may be by variouε routeε, εuch as intramuεcular, subcutaneous, or intraperitoneal. Another group receives a similar innoculation of EHV-l vaccine as well as a booster on day 14. Another control group receiveε no vaccine. Additional groups, are used if the effectiveness of varying amounts of vaccine is to be tested. Sera iε collected from all hamsters at various intervals after inoculation and tested for neutralizing antibody in cell culture assayε and for total anti-EHV-1 antibody by ELISA aεsay. All of the hamsters then receive a challenge of a known 50% lethal dose of a

hamster-adapted Kentucky strain of EHV-l by the intraperitonea route 14 days after the last immunization. Survivors are counted at 7 days and 14 days after challenge with EHV-l viruε

Livers can be examined histopathologically for typical EHV-l cytopathology. The efficacy of a EHV-l vaccine is tested in horses in a similar manner.

Testing for Passive Immunization

This procedure is similar to the vaccination test described above. Hamsters are tested first, and then horses.

Different groups of animals receive either no anti-EHV-1-gD anti-serum or one or more injections of anti-serum. After 14 days all animals are challenged with EHV-l virus, and the number of survivors are counted 1 week and 2 weeks after viral challenge.

Cloning and DNA sequencing The cloned 5.2 kbp BamHI M fragment containing the majority of Us sequences (Henry et al., 1981, Virology 115:

97-114; O'Callaghan et al. , 1981. In Developments in Molecular Virology, Vol. 1 Herpeεviruε RNA, Y. Becker, ed.: 387-418) was used as the source of DNA for cloning and sequencing. Digestion of the BamHI M fragment with Kpnl and Xbal yielded a 0.9 kbp BamHI/Xbal fragment, a 1.3 kb Xbal frac-.ent, a 1.0 Xbal/Kpnl fragment, and a 2.2 kb BamHI/Kpnl fragment; these restriction endonuclease digestion products were ligated into the plasmid vectors pUC19 or pGEM-7Zf (Promega, Madison, WI) to generate the clones pCF, pCF2, pCF3, and pSZ-4, respectively (Fig. 1).

The 2.2 kb BamHI/Kpnl subclone in pUCl9, designated pSZ-4 and located at the left terminus of the BamHI M fragment, was selected for DNA sequence analysis. A nested set of deletions was generated for both strand ccording to the method of Henikoff (1984, Gene 2j3: 351-..-i) using the Erase-a-Base system (Promega, Madison, WI) . After religation,

. 3-1 the deleted plasmid DNA was used to transform competent Escherichia coli strain XLl-Blue (Stratagene, La Jolla, CA) . Deletion clones were selected and sequenced using the dideoxy chain termination method (Sanger et al. , 1977, Proc. Natl.

Acad. Sci. USA 7_4: 5463-5467) as modified for use with T7 DNA polymerase (Pharmacia, Uppsala, Sweden) and 35S-dATP (Grundy e al., 1989, Virology 172: 223-236). 7-Deaza-dGTP was used in all sequencing reactions to reduce compression artifactε.

Sequencing reactions were analyzed in 6% polyacrylamide wedge gels. Internal sequencing primers ^' were syntheεized and used to fill in gaps in the sequence. Both strands were completely sequenced. DNA sequence analysis

DNA sequences were compiled using the programε of Staden (1980, Nuc. Acid. Res. _V2 : 505-519) and Eugene and Sam (Lark Sequencing Technologies). Searches of the Genbank and NBRF databases for sequences homologous to those of EHV-l were performed with the FASTN/P programs of Lipman and Pearεon (1985, Science 2_27: 1435-1440) using the IBI Pustell sequence analysis software. Analyses of DNA and protein sequence data were performed with the PC/GENE software package (Intelligenetics, Inc., Mountain View, CA) .

EXAMPLE 2 1 DNA SEQUENCE ANALYSIS OF CLONE pSZ-4

The EHV-l gD gene was identified by DNA sequence analysis of clone pSZ-4. The position of the pSZ-4 clone on the EHV-l genome (map units 0.865-0.872 and 0.869-0.884) is ₅ shown in Fig. 1. pSZ-4 was generated by Kpnl digestion of the 5.2 kbp BamHI M clone, a viral restriction fragment previously localized to the unique short (U ) segment of the viral genome by Southern blot analysis (Henry et al., 1981, supra. ) . Sequence analysis of both strands of pSZ-4 (Fig. 2) revealed a

1 ₀ total of 2,229 nucleotides with a base composition of 49.6% G+C. Analysis of the sequence for posεible protein coding regionε revealed one long open reading frame (ORF) which commenced with an ATG codon at nucleotides 511-513 (from-the Kpnl site) and extended 1,325 bases to a stop codon (TAA) at

15 nucleotides 1837-1839. In addition to the start codon at 511-513, there are four in-frame ATG codons located at nucleotides 661-663, 679-681, 682-684, and 706-708. The start codon at 682-684 is the most favorable for initiation of translation according to Kozak's "leaky-scanning" model (Kozak,

20 1980, Cell 22: 7-8; Kozak, 1983, Microbiol. Rev. 47: 1045; and Kozak, 1986, Cell 4_4: 283-292S). The local nucleotide sequence of this initiation codon, TTATGATGG, shows alignment at positions -3 and +1, critical residueε in Kozak'ε conεenεus motif, CC(A/G)CCATG(G) . Initiation of protein εyntheεiε from

25 the first start codon (nucleotides 511-513) results in a primary translation pfoduct of 442 amino acids with a predicted molecular weight of 49,904; however, initiation from the fourth start codon (nucleotides 682-684) produces a polypeptide of 386 amino acids having a predicted molecular weight (M ) of 43,206,

30 a size comparable to those of HSV-1 gD (394 amino acids; M

43,344; Watson et al., 1982, Science 218: 381-384; McGeoch et al., 1985, J. Mol. Biol. 181: 1-13), HSV-2 gD (394 amino

35

acids; Watson 1983, Gene 26_: 307-312) and PRV gp50 (402 amino acids; M 44,500; Petrovskis et al. , 1986, J. Virol. 5_9:

216-223). Furthermore, analysis of the translated sequence for a possible signal sequence revealed that residues following the fourth initiation methionine are most likely to serve as a signal sequence (see below).

Potential siqnals for the promotion and termination ^" of transcription were identified within sequences flanking the gD ORF and include: (1) TATA motifs at nucleotides 328-331 and 561-564, (Corden et al. , 1980, Science 209: 1406-1414) (2) CAAT box homologs at nucleotides 243-246 and 504-507, (Benoiεt et al., 1980, Nuc. Acid. Reε. : 127-142;1 Efstratiadis et.m 1980, Cell 21:. 653-668; Jones and Yamamoto, 1985, Cell _ 2 '. 559-572) (3) a polyadenylation consensus seguence (AATATA) 12 nucleotides downstream of the termination of translation (nucleotides 1848-1853) (Proudfoot and Brownlee, 1976, Nature 263_: 211-214; Nordstrom et al., 1985, Proc. Natl. Acad. Sci. USA 8 : 1094-1098), and (4) a GT-rich region at 1871-1876 (Birnεtiel et al., 1985, Cell 41,: 349-359). In addition, there are potential tranεcriptional regulatory εequenceε located in close proximity to the second TATA box. An AT-rich motif (572-578) was identified eight nucleotides downstream of the TATA box.

si

EXAMPLE 3 IDENTIFICATION OF A EHV-l GENE PRODUCT AS

A MEMBER OF THE gD FAMILY OF HERPESVIRUS GLYCOPROTEINS To determine the relationship of the EHV-l gD ORF gene product to other proteins, the translated sequence was compared to polypeptide sequences in the NBRF protein database using the amino acid ho ology algorithm, FASTP (Lip an and Pearson, 1985, Science 227: 1435-1440). Three protein sequences in the database displayed significant homology with the EHV-l sequence and were HSV-1 gD (Watson et al., 1982, supra; McGeoch et al, supra), HSV-2 gD (Watson, 1983, supra) and the PRV gD homolog, gp50 (Petrovskiε et al., supra). The results of this analysis (Fig. 3) revealed that the EHV-l gD equivalent iε 21% homologous with PRV gp50 and 18% homologous with HSV-1 gD and HSV-2 gD. A comparison of the sequences using the PAM 250 matrix (Dayhoff et al. , 1978. In Atlas of Protein Sequence and structure, M.O. Dayhoff, ed. : 345-362) which permits alignment of conservative amino acid substitutions, indicated that a sequence similarity between the EHV-l translated εequence and each of the three herpesvirus glycoproteinε may be aε high aε 32%. The EHV-l translated sequence contains 12 cysteine residueε (amino acids 17, 35, 77, 80, 138, 176, 188, 197, 259, 273, 421, and 431), while P V gp5C and HSV-1 gD and HSV-2 gD each contain seven cysteine residueε (Watson et al., 1982, supra.; Watεon, 1983, εupra.; McGeoch et al., 1985, supra.; Petrovskis et al., 1986, εupra.). The cyεteine residues at positionε 17 and 35 may not be part of the tranεlated sequence if initiation of translation were to occur after the first start codon in the EHV-l gD ORF. In the case of HSV-1 gD, six cysteine residues were judged to be essential for the proper conformation of the glycoprotein (Wilcox et al., 1988, J. Virol. 62: 1941-19747). Thus, these data indicate that the EHV-l gD ORF (nucleotides 511-1839 depicted in Fig.

2), contained within clone pSZ-4, codes for a herpes viral polypeptide of the gD family.

3?

EXAMPLE 4 ANALYSIS OF THE EHV-l gD POLYPEPTIDE SEQUENCE

Analysis of the translated sequence revealed characteristics typical of a transme brane glycoprotein. Hydropathicity plots using the algorithm of Kyte and Doolittle (1982, J. Mol. Biol. 157: 105-132) were used to identify hydrophobic domains within the EHV-l gD amino acid sequence. Two strongly hydrophobic regions at the amino terminus spanning residues 1-21 and 58-76 (as depicted in Fig. 2) were candidates for a signal sequence (Fig. 4). Further analysiε of these regions uεing the weight matrix algorithm of von Heijne (1986, Nuc. Acid. Res. 14 : 4683-4690) identified the second hydrophobic domain as the most likely sequence for a signal peptide. The signal sequence begins at the fourth methionine at amino acid position 58 in the translated sequence followed by the sequence AGR. The hydrophobic core begins at amino acid residue 62 and contains 15 hydrophobic amino acids, LVFAMAIAILSWLS. The signal sequence cleavage site is predicted to occur after εerine reεidue 76. The predicted signal sequence is 19 residues in length, and occurs in a position similar to that of HSV-1 gD (Fig. 4), and the mature polypeptide iε 367 amino acids in length. Near the carboxy1 terminus is a region (residueε 394-422) enriched for hydrophobic amino acidε and thuε it may function as transmembrane anchor domain. Further analysiε of this region using the method of Klein et al. (1985, Biochem. Biophys. Acta 815: 468-476) revealed residueε 406-422 aε poεεible membrane spanning amino acidε. Aεsuming the primary tranεlation product is processed to remove the signal peptide and that the polypeptide is anchored in the membrane, residueε 77-406 are exposed on the surface of mature virion: "ir infected cell membranes. The exposed region contains : - r consensus sequence motifs for the addition of N-linked oligosaccharides aε

. 32 indicated in Fig. 2 (N-X-S/T; Hubbard and Ivatt, 1981, Ann. Rev. Biochem. .5_0: 555-583). The putative carboxyl-terminal cytoplasmic domain spans residues 423-442, is enriched for hydrophilic amino acids, and posseεseε a net poεitive charge o

2.

EXAMPLE 5 PRODUCTION AND PURIFICATION OF A EHV-l gP-BETA-

GALACTOSIPASE FUSION PROTEIN

Expression of EHV-l gP as a beta galactosidase fusion protein. The EHV-l gD protein is expressed as a fusion protei with beta-galactosidase using a lambda gtll expression vector (Sambrook, et al. , 1989, Molecular Clonging: A Laboratory Manual Vol. 2 Cold Spring Harbor Laboratory Press: 12.1-12.44). Fragments ^' containing the EHV-l gP open reading frame, or portions thereof, are fused in-frame with the beta-galactosidase open reading frame. This is accomplished by use of restriction enzyme digestion and exonuclease treatment to generate the appropriate EHV gP PNA fragment, _. followed by ligation to appropriately cut lambda gtll PNA. Theεe recombinant PNA molecules are packaged into lambda phage heads in vitro, and the resultant phage are used to transfect E. coli KM392 cells in the presence of 5-bromo-4-chloro-3-indσlyl beta-P galactoside (X-gal). Under theεe conditionε, phage harboring EHV-l gP inεertε appear aε clear plaqueε (Benton and Paviε, 1977, Science 196:180) . Alternatively the phage are plated on the E. coli hoεt Y1090 εince this strain contains: 1) geneε for the lac repreεsor which prevents lacZ-directed gene expresεion until it iε derepreεsed by the addition of IPTG (isopropyl-beta P thiogalactopyranoside) , 2) a deficiency in the Ion protease which prevents marked degradation of the EHV-l fusion protein, and 7) supF to suppress the phage amber mutation for cell lysis [S100]. The recombinants are amplifed in E. coli KM392 carrying the plasmid pMC9 which expresses the lac repressor (Ca os et al. 1983, Proc. Natl. Acad. Sci. USA 0:1266). For both recombinant hosts [KM392 (pMc9) or Y1090], plaque formation iε initiated in the absence of any lacZ-directed expression to ensure that fusion proteins toxic

io to the host, do not inhibit the growth of any particular membe of the library.

Purification of EHV-l Polypeptide/Galactosidase Fuεion Protein

Since the lambda gtll expression vector system produces a fusion protein consisting of both beta-galactosidaε and EHV-l epitopeε, these characteristics are used to purify the EHV-l gP polypeptide by employing anti-beta galactosidase antibody linked to Sepharose 4-B in an immunoaffinity column. Briefly, rabbit anti-beta galactosidase (IgG fraction; United States Biochemical Corporation, Cleveland, OH) is covalently linked to Sepharose 4B using the cyanogen bromide activation method. Anti-beta galactosidaεe (1-30 mg/ml) iε dialyzed into an appropriate buffer and centrifuged (100,000 xG to remove aggregateε). The CNBr-activated Sepharose iε filtered, washed, and hydrated with ice cold 0.1 mM HCl. A weighed amount of activated Sepharose 4B (density - 1.0) is added to an equal volume of antibody solution (10 mg/ml) and stirred overnight at 4°C. Glycine (0.05 M) is added to saturate any remaining reactive group on the Sepharose 4B.

An EHV-l gP fuεion protein iε obtained from E. coli Y1090 infected with recombinant gtll. LacZ directed expression of the fuεion protein iε induced in the preεence of IPTG, and a lytic infection is initiated. The lysate from thiε infection iε used for a batch purification of the fusion polypeptide. Briefly, the antibody-Sepharose complex iε mixed with the lyεate εolution and the εlurry iε applied to a column. The column iε washed 10 times with waεh buffer (0.01 M Triε, pH 8.0 0.14 M NaCl, 0.025% NaN,) , and the purified fuεion protien iε eluted with high.pH triethanolamine.

An EHV-l gP fusion protein, purified by this method, is of sufficient purity for production of anti-EHV-1 gD antibodies.

<*/ >

EXAMPLE 6 EXPRESSION OF EHV-l gP IN AN pKK223-3

PROKARYOTIC EXPRESSION SYSTEM The pKK223-3 vector contains the strong trp-lac (tac promoter first described by deBoer et al. (1983, Proc. Natl. Acad. Sci. USA). The tac promoter contains the -35 region of the trp promoter and the -10 region, operator, and the ribosome binding site of the lac UV-5 promoter. In a lac i ^y host such as E. coli JM105, the tac promoter is repressed and may be derepressed at the appropriate time by the addition of IPTG. The tac promoter is followed by a polylinker derived from pUC8 (Vieria and Messing, 1982, Gene 1/7: 259-268) containing unique EcoRI, BamHI, Sail, PstI , Smal, and Hindlll restriction enzyme sites which facilitate the positioning of geneε behind the promoter and ribosomal binding site. The polylinker is followed by a PNA segment containing the strong rrnB ribosomal RNA transcription terminators which have been previously characterized (Brosius et al., 1981, J. Mol. Biol. 148 : 107 ,- and Brosius et al., 1981, Plaεmid 6_:112). The pσεition of the •terminators stabilizes the host-vector εystem, presumably by inhibiting the overexpression of detrimental RNA εpecieε or proteinε by the εtrong tac promoter contained on the vector (Gentz et al., 1981, Proc. Nat. Acad. Sci USA 7_8:4936; Brosius, 1981, supra, ) . The remainder of the plasmid consiεtε of pBR32Z εequenceε. Expreεεion of EHV-l gP polypeptideε within a pKK233-3 vector iε induced by introduction of IPTG to the culture ediu.T. when uεing E. coli JM105 aε host cells. An EHV-l gP polypeptide is detected by comparison of the electrophoretic patterns of proteins derived from JM105 cells having the gP expression vector, with the pattern from cells that do not. SPS polyacylamide gels stained with an appropriate protein stain, such as Coomassie Blue, may be used for these

comparisonε, or, Weεtern blot analyεiε uεing anti-EHV-1 gD antibodies to detect a gD polypeptide, may be employed (see fo example Harlow, et al. , 1988, Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press) .

The gD polypeptide(s) expressed in a pKK233-3 expression vector system are purified by preparative SDS gel electrophoresis, or by immunoaffinity chromatography with an anti-EHV-1 gD antibody in a manner similar to that described in Example 5.

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EXAMPLE 7 EXPRESSION OF EHV-l gD IN A BACULOVIRUS

EXPRESSION SYSTEM To obtain regulated expression of an EHV-l gD polypeptide, the coding region of the EHV-l gD gene is placed downstream of a strong baculovirus promoter, the polyhedrin promoter, within a transfer vector. The transfer vector is a plasmid which has been genetically engineered to contain baculovirus DNA flanking the polyhedrin gene, as well as convenient restriction enzyme recognition sites adjacent to the strong polyhedrin promoter. Transfer vectors are cσ-transfected with baculovirus DNA to allow homologouε recombination between the baculovirus DNA within the transfer vector and the genome of the baculovirus. Such homologouε recombination replaces the polyhedrin coding region in the baculovirus genome with the EHV-l gD coding region. This replacement causes the polyhedrin gene product to be lost and gives rise to an occlusion negative viral phenotype. Hence, baculoviruses which have incorporated the EHV-l coding region are recognized by an occlusion negative phenotype. Techniques for diεtinguiεhing thiε phenotype, aε well aε for manipulating transfer vectors, and recombinant baculoviruseε are provided in Summers et al. (1987) A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedure, Texas Agricultural Experiment Station, Bulletin No. -1555. To place the EHV-l gD coding region into a transfer vector, pSZ-4 DNA is digested with the appropriate restriction emzymes and the DNA fragment encoding EHV-l gD amino acidε 1-386 (or any portion thereof) is isolated. The transfer vector, pAc373, is digested with a restriction enzyme that cuts just downstream of the polyhedrin promot- . After ligation (T4 DNA ligase, New England Biolabs) of the iregment encoding the EHV-l gP coding region to the linearized transfer vector,

recombinants are selected for ampicillin resiεtance and reεtriction mapped to identify those having the correct structure.

The recombinant EHV-l gP tranfer vector is co-transfected with baculovirus AcMNPV PNA, into S. frugiperda Sf9 cells by the method described in Summers et al. The plaque; are screened for an occlusion negative phenotype, and several recombinant (occulsion-negative) baculovirus clones are separately plaque purified three times to ensure that the EHV-l gP recombinant clones are homogeneous.

EXAMPLE 8 GENERATION OF POLYCLONAL ANTIBOPIES

To prepare polyclonal antibodies directed againεt EHV-l gP polypeptides, any of a variety of antigens are used, such as a purified EHV-l gP fusion protein, a purified EHV-l g: polypeptide or a synthetic peptide encoding a portion of a gD polypeptide.

Synthetic peptides with the following amino acid sequences were made for immunization into rabbits:

1) Amino acids 80-98 (as depicted in Fig. 2): NH _? -Cyε-Glu-Lys-Ala-Lys-Arg-Ala-Val-Arg-Gly-Arg-Gln-Aεp -Arg- Pro-Lyε-GIu-Phe-Pro-COOH

2) Amino acids 343-361 (as depicted in Fig. 2): NH ₂ -Glu-Ile-Thr-Gln-Asn-Lys-Thr-Aεp-Pro-Lys-Pro-G.ly-Gln -Ala- Asp-Pro-Lys-Pro-Asn-Cys-COOH These EHV-l gP peptides were identified as strongly antigenic epitopes for all EHV-l gP polypeptides by computer analysiε of the EHV-l gP polypeptide sequence. Peptide 2 has an additional cysteine residue on itε carboxy terminuε to facilitate coupling to a carrier protein. New Zealand white female rabbitε were immunized by εubdermal injection with lOOμl of Freund'ε complete adjuvant containing 0.1-1 mg of oligopeptide in multiple locations along the back. The rabbits were first shaved on both sides of the back for easy subdermal injection. Typically rabbits were boosted with similar amounts of antigen at 10-40 days intervals following the primary* injection, until the serum was poεitive for gD reactivity at a dilution of greater than 10 -4 when aεsayed by ELISA, immunoblotting, and immunoprocipitation analysis.

EXAMPLE 9 PREPARATION OF HORSE ANTI-EHV-1-gD

ANTISERA Horses are immunized with the entire EHV-l gD protein, or portions thereof (i.e. the peptides of Example 8) or an EHV-l gD fusion protein, to create an antisera reactive against EHV-l. This antisera is useful for treatment or prevention of EHV-l infection. The procedure employed is analogous to that for making polyclonal antibodies (Example 8). ^* To enhance the immune reaction the antigen iε placed in an adjuvant before immunization. An adult horεe iε injected with 50 μg to 100 mg of an gD antigen. Immunization iε intramuscular, intradermal or subcutaneous. Multiple εiteε of injection are generally used with subcutaneous or intradermal immunization to stimulate the immune responεe. After primary immunization the animal is boosted about every 2-6 weeks. Horse serum is drawn after one or more boosting injections and tested for high anti-EHV-1-gD antibody titerε by the viruε neutralization aεsay, and for the ability to alleviate EHV-l infection, firεt in hamεterε and then in horses.

EXAMPLE 10 MONOCLONAL ANTIBODY PRODUCTION

Monoclonal antibodies are prepared in accordance witl the techniques developed by Kohler and Mulskin (Eur. J.

Immunol. 6:511-519, 1976) and Harlow et al. (Antibodieε: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988).

Balb/c mice are immunized subdermally with 100 ul of Freund's complete adjuvant containing 0.1-1 mg of am EHV-l gD antigen such as a purified EHV-l gD polypeptide or fusion protein, or the conjugated or non-conjugated EHV-l peptides described in Example 8. Two weeks after the initial injection, the mice are boosted with the appropriate antigen by intravenous and intraperitoneal injection of about 100 ug of antigen in phosphate buffered saline (PBS).

Five dayε after the laεt injection and after confirmation of the preεence of antibody in mouse εera, the mice are sacrificed and their spleenε removed. Spleen cells are obtained by gentle disruption of the spleen in a 7 ml

Dounce homogenizer in 3.5-4 ml PBS. The cells are then pelleted at 1200 rpm in a PR6 centriguge for 6 minutes at room temperature. The supernatant is removed into a suction flask, and the cells are resuεpended in 15 ml 0.83% NH„C1. The cells are again pelleted by centrifuatiσn for 8 minutes, at 1200 rpm at room temperature, then the supernatant is withdrawn into a suction flask cells resuspended in 20 ml PBS. The following solutionε are prepared for use in the εubεequent cell fuεion:

Hypoxanthine (H) , 680 mg/100 ml H ₂ 0; add 204 drops cone. H2-.SO4.; heat to dissolve

Aminopterin (A), 46.4 mg/100 ml H ₂ 0; add 2 drops 1.0 N NaOH to dissolve

Thymidine (T), 775 mg/100 ml H ₂ 0; add 45 mg glycine PEG-DME--melt PEG at 42°C, then add 1 ml DME (at

^• i

37°C); adjuεt pH with 1.0 N NaOH to 7.6 1 DMEM—to 500 ml DME add 37.5 ml a- horεe serum;

37.5 ml FCS, 10.0 ml L-glutamine, 0.2 ml garamycin, 2X HAT-DME—to 200 ml DME add 25.0 ml a- horse serum, 25.0 ml FCS, 4.0 ml L-glutamine, 0.2 , garamycin, ₅ 0.8 ml H, and 0.8 ml A, and 0.8 ml T (2X HT-DME omits A) Cloning Agar-350 mg unwashed Difco agar in 25 ml H-,0, autoclaved Cloning Medium—to 25 ml 2X DME, add 35 ml filtered, 0 condition DMEM, 7 ml a- horεe serum, 7 ml FCS, 1ml

L-glutamine, .1 ml garamycin. Two 30 ml flasks of X63-Ag8.653 myeloma cells are added to centrifuge tubes and εpun down at 1200 rpm for 8 minuteε at room temperature. The spleen cells are resuεpended 5 in 20 ml PBS. From each εuεpension, .01 ml is removed and added to 0.1 ml 0.4% trypan blue and 0.3 ml PBS for cell counting. The volume of each εuεpenεion iε adjuεted εo aε to obtain a spleen cell to X63-Ag8.653 cell ratio of 10:1, and the suspensions are then mixed. The mixture is pelleted at 120Q 0 rpm for 8 minutes at room temperatue and all but about 0.1 ml of supernatant removed. The cells are then resuspended in the remaining liquid and added to 1.3 ml of 1:1 PEG-DME solution, pH 7.6. Every minute the volume of the solution is doubled with DME until the final volume iε 25 ml. 25 The cellε are again pelleted, the εupernatant decanted, and the cellε reεuspended in enough 50% 2X HAT-DME/50% condition DMEM (the supernatant retained from the X63-Ag8.653 cellε above) to yield a final concentration of about 3.5 x 10 spleen cellε. The cellε are distributed into a 3096-well flat-bottom icrotiter plate (TC-96; Flow

Laboratories), at 0.1 ml/well. The plate iε incubated at 37°C in humidified air/C0 ₂ until visible colonies appear, usually

35

about 10-12 days. The contents of the well is tranferred to 0.5 ml of HAT-DME/conditioned DME in a TC-24 plate (Flow Laboratories). When healthy cell growth appears (about 2-5 days), about .35 ml medium is removed and tested for antibody production by enzyme-linked im unosorbent assay (ELISA), i munoprecipitation of EHV-l gD polypeptides, or Western blot analysis. When cells producing the antibodies of interest are growing well, one drop of each culture is transferred into 1.0 ml DMEM in a TC-24.

To clone the hybrid cells, 25ml of melted agar and 76 ml of cloning medium is combined, and 5 ml is pipetted into 60 mm petri dish and left to solidify. Cells from DMEM cultures are diluted in 50% DMEM/50% conditioned DMEM, at 10 ^_1 or 10 ^~2 dilutions depending on cell growth. Into sterile tubes is placed 0.1 ml of each of the two dilutions, and to- each is added 0.9 ml of cloning medium/agar mixture. This iε mixed well and poured over the εurface of the agar underlay. After εolidification the plateε are incubated at 37°C in a CO,, incubator until colonies are visible with the naked eye, typically about 7-10 days. Colonies are then picked and tranεferred .1 ml of DMEM/conditioned DMEM in a TC-99 plate and incubated at 37°C in a C0_ incubator. After the culture is acidic (usually 1-4 days), transfer is made to .05 ml DMEM in

TC-24 plate. When the growth is 50% confluent, the medium iε removed and tested for antibody p ^' roduction aε previouεly. Thoεe clones producing EHV-l gD specific antibodieε are moved

2 into 5 ml DMEM in 25 cm flaεkε. Cloned cellε are then frozen or injected into mice for aεcites production.