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  • C12N2710/16761—Methods of inactivation or attenuation

Definitions

• Herpesvirus Type-1 Viruses Harboring Mutations Within the Immediate Early Gene
• This invention relates to mutant Equine herpesvirus type-1 (EHV-l) isolates, and in particular, EHV-l isolates carrying mutations in the sole immediate-early (IE) gene of the viral genome.
• the present invention further relates to vaccine compositions and methods of treating EHV-l infections. Methods of determining the pathogenicity of an EHV-l virus are also provided.
• Equine herpesvirus type-1 (EHV-l) is a major pathogen in horses. In infected animals, it is usually associated with upper respiratory tract infections, but may also cause neurological sequelae. EHV-l infection in pregnant mares causes abortigenic disease and has an important 'economic impact on the worldwide equine industry.
• EHV-l Although the horse is the natural host of the equine herpesviruses, a variety of animals and tissue culture systems can be used to propagate the viruses.
• Experimental animals for EHV-l include Syrian hamsters, baby hamsters, chick embryos, baby and adult mice, and kittens.
• Primary tissue culture systems used to propagate EHV-l include cells from a variety of equine tissues such as fetal lung, dermis, spleen and kidney, as well as cells from domestic cats, dogs, hamsters, rabbits, mice, sheep and swine.
• permanent tissue culture systems commonly -used to cultivate EHV-l include HeLa, Vero, CV-1, rabbit kidney (RK) , mouse L-M and equine Edmin337 cells.
• Natural respiratory EHV-l infection of the horse only results in a short-lived humoral response and does not confer long-term protection against subsequent infection.
• a number of vaccines have been developed to combat EHV-l infections, among them inactivated vaccines which mostly contain both EHV-l and EHV-4 (Pneumabort KTM, ResequinTM, PrestigeTM and DuvaxynTM) , modified live vaccines (RhinomuneTM or PrevaccinolTM) and subunit vaccines (Cavalon IRTM) .
• EHV vaccines cause undesirable side effects, and most do not afford acceptable levels of protection. There is a need for safe and effective vaccines against EHV-l infection.
• the present invention is directed to mutant equine herpesvirus type-1 (EHV-l) isolates carrying one or more mutations in the immediate-early (IE) gene of the viral genome .
• EHV-l equine herpesvirus type-1
• the present invention provides EHV-l isolates carrying in the IE gene of the viral genome, at least one of the mutations listed in Table 1.
• the present invention provides replication-competent EHV-l isolates carrying one or more mutations in the IE gene.
• the mutation in the IE gene does not significantly interfere with the structure and/or function of any of the four domains of the IE protein, TAD (aa 1-89) , SRT (aa 181-220), DBD (aa 422-597) and NLS (aa 963-970), which are essential for viral replication.
• the present invention provides replication-competent EHV-l isolates which carry a mutation in the IE gene that involves a substitution of an amino acid residue within TAD, SRT, DBD or NLS .
• the substitution involves an exchange of amino acids within an exchange group, i.e., amino acids that resemble each other with respect to their overall impact on protein structure.
• the substitution is one of D24N, D20N, F15D, L12E or E34Q.
• Particularly preferred mutations include insertions or deletions of one or more, preferably, at least three, more preferably at least five, amino acid residues within amino acid 90-180, 221-421, 598-962, or 971-1487.
• Most preferred mutations include d644/824 (deletion of aa 644-824) , nl029 (nonsense mutation at 1029 and deletion of aa 1030-1487) , nl411 (nonsense mutation at 1411 and deletion of aa 1411- 1487) , in628 (insertion at 1411) and inl411 (insertion at 1411) .
• the present invention provides replication-competent EHV-l isolates which carry one or more mutations in the IE gene and which have reduced virulence or no virulence, also referred herein as "nonpathogenic" EHV-l isolates.
• Non-pathogenic, replication competent EHV-l isolates can be of an EHV-l strain such as KyA, KyD, Ab4 , Abl, RacLll, RacH and RacM wherein one or more mutations have been introduced into the IE gene of the viral genome.
• Preferred mutant EHV-l isolates are KyA mutant isolates.
• the present invention is further directed to immunogenic compositions which include one or more of the non- pathogenic, replication-competent mutant EHV-l isolates described herein.
• the present invention further provides methods of stimulating an immune response against EHV-l in a horse subject by administering to the horse an immunogenic composition of the present invention.
• present invention is directed to live attenuated vaccine compositions which include one or more of the non-pathogenic, replication-competent mutant EHV-l isolates described herein.
• the present invention provides methods of treating EHV-l infections in a horse by administering to the horse subject, a therapeutically effective amount of a non-pathogenic, replication-competent mutant EHV-l isolate of the present invention.
• the present invention provides methods for determining the pathogenicity of an EHV-l strain present in a subject- previously vaccinated with a nonpathogenic EHV-l isolate of the present invention. The determination is achieved by carrying out assays which distinguish the wild type EHV-l and the non-pathogenic EHV-l isolate previously administered to the subject.
• Figure 1 depicts the structure of the EHV-l genome and location of the immediate-early gene.
• a schematic of the EHV-l genome is shown at the top.
• the lower portion of the figure depicts the functional domains of the IE protein (1,487 amino acids) : the transactivation domain (TAD, aa 3-89) , a serine rich tract (SRT, aa 181-220) , the DNA-binding domain (DBD, aa 422-597) , nuclear localization signal (NLS, aa 963- 970) .
• TAD transactivation domain
• SRT serine rich tract
• DBD DNA-binding domain
• NDS nuclear localization signal
• Figure 2 depicts the recombination system to generate mutations in the IE gene.
• Construct pIECassette contains the 5' portion of the IE gene. Unique Nc ⁇ l and Nael restriction sites facilitate the cloning of sequences encoding mutagenized TADs spanning amino acids 3-89.
• Construct pBR322IE contains the entire IE ORF. EcoRV/Barri ⁇ X fragments from various pIECassette constructs containing mutagenized TADs were easily cloned into pBR322IE to reconstitute an ORF that encodes a mutant form of the IE protein.
• ICE infected-cell extracts derived from RK-13 cells infected with EHV-l KyA (Lanel) , KyAd644/824 (Lane 2), KyAnl411 (Lane 3), or KyAInl411 (Lane 4) at an MOI of 10.
• ICE were subjected to SDS-PAGE, and the proteins were blotted to nitrocellulose and were stained with the polyclonal anti -IE peptide antibody.
• the 200-kDa band representing the IE protein was clearly detected in Lanes 1, 3, and 4.
• the 175- kDa band derived from KyAd644/824-infected cells is shown in Lane 2.
• Lane 1 shows the 200-kDa IE protein band detected in nuclear extracts of RK-13 cells infected with EHV- 1 KyA.
• Lane 2 shows the 138 kDa IE protein band detected in nuclear extracts of RK-13 cells infected with KyAnl029.
• Figure 4 depicts the growth analyses of selected IE mutant viruses.
• RK-13 cells were infected with either wild- type EHV-l KyA or selected EHV-l IE mutant viruses at an MOI of 1 and incubated for 1 h at 37°C in 5% C0 2 to permit virus attachment. After attachment, the cells were washed three times with Eagle's without FBS to remove unattached virus and were incubated at 37°C in 5% C0 2 . At the times indicated after the attachment period, virus titers in the culture supernatant were determined by plaque assay using RK-13 cells.
• Figure 5A-5C depict the analysis of the CTL activity of the lymphocytes isolated from CBA mice immunized with wild type EHV-l or EHV-l containing mutations in the IE gene.
• EHV-l equine herpesvirus type-1 isolates carrying one or more mutations in the immediate-early (IE) gene of the viral genome .
• EHV-l has a linear, double-stranded DNA genome, characterized by short and long unique sequences (U s and U respectively) , and inverted repeats which flank the unique short sequences.
• the entire genome of EHV-l, strain Ab4 has been sequenced and shown to be 150,223 bp in size and contain 80 potential open reading frames (ORFs) .
• the IE gene is the sole immediate-early gene of EHV- 1 and is present in both inverted repeats of the viral genome.
• the open reading frame (ORF) of the IE gene (SEQ ID NO: 1) is transcribed to a 6.0-kb spliced mRNA that gives rise to both structurally and antigenically-related protein species (Caughman et al . Virology 163:563-571, 1988) .
• the predominant IE protein species of 1,487 amino acids (SEQ ID NO: 2) is comprised of discrete, functional domains ( Figure 1) .
• a potent transcriptional activation domain (TAD) maps within the first 89 amino acids.
• a serine rich tract may contain a site(s) for phosphorylation.
• the DNA-binding function lies within amino acid residues 422-597.
• Amino acids spanning 963-970 are necessary for nuclear localization of the protein.
• the IE protein is essential for viral growth in cell culture and is required for both early and late gene expression during the course of a productive infection (Smith et al. J. Virol. 66:936-945, 1992) . Following IE polypeptide synthesis, approximately 45 early transcripts can be detected. Three of these early proteins serve as regulatory proteins and are designated EICP22, EICP27 and EICP0.
• EHV-l isolates carrying mutations in the IE gene can be generated by a recombination system provided herein.
• mutations in the IE gene can be generated by employing any of the myriad recombinant cloning techniques, e.g., those described in Current Protocols in Molecular Cloning (Ausubel et al .
• a mutant IE nucleotide sequence carrying one or more mutations is then placed on a recombination vector appropriate for transfection and transfected into an appropriate host cell, e.g., RK-13 cells.
• Transfected host cells are then infected with a null EHV-l virus devoid of the IE gene . Mutant viruses are thus generated by homologous recombination between the genome of the null virus and the recombination vector containing the mutant IE gene.
• Null EHV-l viruses for use in this recombination system can be generated using any EHV-l strain, e.g., KyA, KyD, Ab4 , Abl, RacLll, RacH and RacM.
• A.deposit of the EHV-l KyA strain was made with the American Tissue Type Culture, 10801 University Boulevard., Manassas, VA 20110-2209, on July 20, 2000 (ATCC deposit # PTA-2253) .
• Null virus of strain KyA, i.e., KyA ⁇ lE has been generated as described by Garko- Buczynski et al . (Virology 248: 83-94, 1998) .
• mutant EHV-l isolates carrying identical or different mutations in the two copies of the IE gene can be generated.
• the mutant EHV-l isolates of the present invention carry identical mutation (s) in both copies of the IE gene in the viral genome.
• One embodiment of the present invention provides EHV-l isolates carrying in the IE gene of the viral genome, at least one of the mutations listed in Table 1.
• Nonsense Mutations n627 aa 628-148 . 7 deleted Deletion of NLS and C-term n951 aa 952-1487 deleted Deletion of NLS and C-tenn nl029 aa 1030 -1487 deleted Deletion of C-term nl411 aa 1412 - 1487 deleted Deletion of C-term
• the present invention provides mutant EHV-l isolates that are replication-competent .
• replication competence refers to the ability of a viral isolate to propagate in a host cell in the absence of a complementing IE protein expressed in trans .
• the replication competence of a mutant viral isolate can be determined by a number of assays, e.g., a plaque assay using non-complementing cells (cells that do not express IE protein) .
• a plaque assay using non-complementing cells cells that do not express IE protein
• cells of a monolayer are infected with a mutant isolate of interest and are subsequently overlaid with 2% agarose mixed in a 1:1 ratio with appropriate growth medium.
• Host cells which can be employed for this purpose include cells from a variety of equine tissues such as fetal lung, dermis, spleen and kidney, as well as cells from domestic cats, dogs, hamsters, rabbits, mice, sheep and swine.
• cells commonly used to cultivate EHV-l in tissue culture are used, including HeLa, Vero, CV-1, rabbit kidney (RK) , mouse L-M and equine Edmin337 cells.
• RK-13 cells are used in the assay.
• a mutant viral isolate is "replication competent" if such mutant isolate can form plaques on the non-complementing cells, even if the plaques may be of a smaller size than those formed by wild type EHV-l.
• Replication-incompetent cells can only form plaques in complementing cells where the IE protein is expressed in trans e.g., IE13.1 cells.
• the IE protein four domains of the IE protein, TAD (aa 1-89) , SRT (aa 181-220) , DBD (aa 422- 597) and NLS (aa 963-970) , are essential for viral replication.
• mutations in the IE gene that significantly interfere with the structure and/or function of these four domains likely produce replication incompetent viral isolates.
• a deletion of five or more contiguous amino acid residues within any of these four domains of the IE protein is likely disruptive to the function of such domain, and the resulting mutant virus is likely to be replication-incompetent.
• substitution of one or more amino acid residues to residues similar in size and/or hydrophobicity can be less disruptive to the structure and/or function of the respective domain and thus, the resulting mutant virus can still be replication-competent.
• one embodiment of the present invention provides replication-competent EHV-l isolates harboring at least one mutation in the IE gene, wherein the mutation is a substitution at a residue anywhere within TAD (aa 1-89) , SRT (aa 181-220) , DBD (aa 422-597) or NLS (aa 963-970) and wherein the mutation does not substantially disrupt the function of the IE protein in viral replication.
• a preferred substitution according, to the present invention can be between amino acids within an exchange group, i.e., amino acids that resemble each other with respect to their overall impact on protein structures.
• aromatics Phe, Tyr and Typ form an exchange group
• the positively charged residues Lys , Arg and His form an exchange group
• the large aliphatic non-polar residues Val, Leu and lie form an exchange group which also contains the slightly polar Met and Cys .
• All small residues Ser, Thr, Asp, Asn, Gly, Ala, as well as Glu, Gin and Pro are also within an exchange group.
• the substitution is one of D24N, D20N, F15D, L12E or E34Q.
• the mutant EHV-l isolates carrying one or more of these subtitutions are of a strain selected from KyA, KyD, Abl, Ab4 , RacLll, RacM or RacH.
• Particularly preferred mutant EHV-l isolates are KyAD24N, KyAD20N, KyAF15D, KyAL12E and KyAE34Q.
• the replication-competent mutant EHV-l isolates harbor a mutation that is localized outside of any of the four domains described above and does not cause any substitution, deletion or insertion within any of the TAD (aa 1-89) , SRT (aa 181-220), DBD (aa 422-597) and NLS (aa 963-970) domains.
• Particularly preferred mutations include insertions or deletions of one or more, preferably, at least three, more preferably at least five, amino acid residues within the region of amino acid 90-180, 221-421, 598-962, or 971-1487.
• Most preferred mutations include d644/824 (deletion 'of aa 644-824) , nl029 (nonsense mutation at 1029 and deletion of aa 1030-1487) , nl411 (non-sense mutation at 1411, and thus deletion of aa 1411-1487) , in628 (insertion at 1411) and inl411 (insertion at 1411) .
• the mutant EHV-l isolates carrying one or more of these mutations are preferably of a strain selected from KyA, KyD, Abl, Ab4 , RacLll, RacM or RacH, and more preferably, KyA.
• the present invention provides replication-competent mutant EHV-l isolates that have reduced virulence or no virulence, also referred herein as "non-pathogenic" or “attenuated” EHV-l isolates.
• virulence or "pathogenicity” as used herein refers to the capacity of a strain of EHV-l to induce EHV-1-related diseases in horses, e.g., infection in the respiratory tracts, spontaneous abortions as well as neurological diseases. Infections caused by pathogenic EHV-l are typically characterized by fever, profuse nasal discharge and congestion of the nasal mucosa. Accordingly, a “non- pathogenic” or “attenuated” strain of EHV-l, or an ⁇ HV-1 strain with “reduced virulence” as used herein is a strain having a substantially reduced capacity, as compared to a pathogenic EHV-l, in inducing the development of EHV-related clinical diseases.
• the pathogenicity of an EHV-l isolate can be conveniently determined in mouse models.
• Clinical signs of EHV-l infection in mice include, e.g., ruffled fur, loss of body weight, labored breathing, lethargy and huddling, as described by, e.g., Colle et al . Virus Res . 43: 111-124 (1996) and Zhang et al. Virus Res . 56: 11-24 (1998) .
• the degree of infection can also be assessed by isolating the viruses from the lungs of infected mice, plating the viruses on RK-13 cell monolayers and determining the number of plaques formed, as described in the Examples that follow.
• the non-pathogenic mutant EHV-l isolates of the present invention can be generated by introducing a mutant IE gene into null viruses of a non-pathogenic EHV-l strain via the recombinant system described herein. Any of those naturally non-pathogenic EHV-l strains or EHV-l strains that are made non-pathogenic by laboratory procedures can be used for generating null-viruses, e.g., KyA, KyD, Ab4 , Abl, RacLll, RacH and RacM. Null viruses of any of these non-pathogenic EHV-l strains can be generated, e.g., by following the procedure described by Garko-Buczynski et al . (Virology 248: 83-94, 1998) .
• a preferred EHV-l strain for use in generating the mutant isolates of the present invention is the EHV-l KyA strain, a deposit of which was made with the American Tissue Type Culture, 10801 University Boulevard., Manassas, VA 20110-2209, on July 20, 2000 (ATCC deposit # PTA-2253) .
• preferred non-pathogenic, replication competent mutant EHV-l isolates of the present invention include KyA isolates carrying at least one mutation in the IE gene, wherein the mutation is a substitution at a residue anywhere within TAD (aa 1-89) , SRT (aa 181-220) , DBD (aa 422- 597) or NLS (aa 963-970) and wherein the mutation does not substantially disrupt the function of the IE protein in viral replication.
• Examples of such mutant EHV-l isolates are KyAD24N, KyAD20N, KyAF15D, KyAL12E and KyAE34Q.
• Non-pathogenic, replication competent mutant EHV-l isolates of the present invention include KyA isolates carrying at least one mutation in the IE gene, wherein the mutation is localized outside of any of the four domains described above and does not cause any substitution, deletion or insertion within any of the TAD (aa 1-89), SRT (aa 181-220), DBD (aa 422-597) and NLS (aa 963-970) domains.
• Examples of such mutant EHV-l isolates are KyAd644/824, KyAnl029, KyAnl411, KyAin628 and KyAinl411 .
• the nonpathogenic, replication-competent mutant EHV-l isolates of the present invention as described hereinabove are included in immunogenic compositions .
• immunogenic is meant the capacity of an EHV-l isolate in provoking a immune response in a horse subject, either a cellular immune response mediated primarily by cytotoxic T-cells, or a humoral immune response mediated primarily by helper T-cells which in turn activate B-cells leading to antibody production.
• the immunogenic compositions of the present invention include at least one, i.e., one or more of the non- pathogenic replication competent mutant EHV-l isolates described hereinabove.
• Preferred mutant EHV-l isolates to be included in the immunogenic compositions include, e.g., an EHV-l isolate harboring one or more of the mutations D24N, D20N, F15D, L12E or E34Q in the IE gene, or an EHV-l isolate harboring one or more mutations within aa 90-180, 221-421,
• mutant EHV-l isolates for use in an immunogenic composition of the present invention are of a non-pathogenic EHV-l strain such as KyA, KyD, Ab4 , Abl, RacLll, RacH and RacM.
• Particularly preferred mutant EHV-l isolates for use in an immunogenic composition include KyAD24N, KyAD20N, KyAF15D, KyAL12E, KyAE34Q, KyAd644/824, KyAnl411 and KyAinl411.
• the immunogenic compositions of the present invention can also include additional active ingredient such as other immunogenic compositions against EHV-l, e.g., those described in U.S. Patent 5,707,629 ("Immunogenic composition against equine herpesvirus type 1") and U.S. Patent 5,795,578 ("Vaccine against equine herpesvirus type 1), or immunogenic compositions against EHV-4.
• additional active ingredient such as other immunogenic compositions against EHV-l, e.g., those described in U.S. Patent 5,707,629 ("Immunogenic composition against equine herpesvirus type 1") and U.S. Patent 5,795,578 ("Vaccine against equine herpesvirus type 1), or immunogenic compositions against EHV-4.
• the immunogenic compositions of the present invention can include one or more pharmaceutically- acceptable carriers.
• a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, adjuvant, vaccine stabilizing agents, antibacterial and antifungal agents, isotonic agents such as sugar and sodium chloride, adsorption delaying- agents, and the like.
• the use of such media gents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the immunogenic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• the immunogenic compositions of the present invention can made in forms suitable for injectable use, e.g., in the form of sterile aqueous solutions or dispersions, or can be made in lyophilized forms using vacuum-drying and freeze-drying techniques. Lyophilized vaccine compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or and HEPES, with or without adjuvant. In all cases the form of the immunogenic compositions must be sterile.
• a stabilizing solution e.g., saline or and HEPES
• the immunogenic compositions of the present invention can be administered to a horse to induce an immune response against EHV-l. Accordingly, another embodiment of the present invention provides methods of stimulating an immune response against EHV-l in a horse subject by administering an effective amount of any one of the above- described immunogenic compositions of the present invention.
• the amount of an immunogenic composition to be adminisitered to be "effective" in inducing an immune response may depend on the immunogenicity of the particular EHV-l isolate used in the immunogenic composition.
• the immunogenicity of an EHV-l isolate i.e., the type and extent of an immune response induced by a viral isolate can be conveniently assessed in mice, which are generally accepted as models representative of horse subjects.
• a variety of techniques may be used for analyzing the immune responses induced in mice by a viral isolate. For example, one skilled in the art can determine whether a viral isolate induces a cell-mediated immune response by, e.g., detecting the presence of responding CTLs in the mouse spleen or other lymphoid tissues. One skilled in the art can also readily determine whether a viral isolate stimulates a humoral immune response by, e.g., detecting the neutralizing titer of EHV-l specific antibody in the serum or the presence of antibody secreting cells in the serum. These techniques are well described in the art, e.g., Coligan et al . Current Protocols in Immunology, John Wiley & Sons Inc. (1994) .
• an immunogenic composition of the present invention can be administered to a horse subject via intravenous, intraperitoneal, intramuscular, or intramucosal (e.g. nasal or respiratory spray or injection) routes, or by other forms of parenteral administration.
• An immunogenic composition can also be administered via an implant or orally.
• Another embodiment of the present invention is directed to live attenuated vaccine compositions.
• the vaccine compositions of the present invention include one or more of the non-pathogenic, replication-competent mutant EHV-l isolates as described hereinabove .
• a pathogenic strain of EHV-l leads to the production of pathogenic virions in the infected subjects and the EHV-l related diseases.
• a non-pathogenic EHV-l strain of the present invention generally replicates to an extent to sufficient to protect the subject against challenge by a virulent or pathogenic EHV-l strain.
• the term "vaccine” as used herein refers to a composition which prevents or reduces the risk of infection or which ameliorates the symptoms of infection.
• the protective effects of a vaccine composition against a pathogen are normally achieved by stimulating an immune response in the subject which may involve either or both of cell-mediated or humoral immune response.
• the strength and duration of the immune responses induced by an EHV-l isolate can be taken into consideration in determining the amount of such isolate that should be included in a vaccine composition, as well as the vaccination schedules.
• abolished or reduced incidences of EHV-l infection, amelioration of the symptoms, or accelerated elimination of the viruses from the infected subjects are indicative of the protective effects of a vaccine composition.
• the mutant EHV-l isolate for use in a vaccine composition is of a non-pathogenic EHV-l strain such as KyA, KyD, Ab4, Abl, RacLll, RacH and RacM.
• Particularly preferred EHV-l isolates for use in a vaccine composition include KyAD24N, KyAD20N, KyAF15D, KyAL12E and KyAE34Q.
• the mutant EHV-l isolate for use in a vaccine composition is of a non-pathogenic EHV-l strain such as KyA, KyD, Ab4, Abl, RacLll, RacH and RacM.
• Particularly preferred EHV-l isolates for use in a vaccine composition include KyAd644/824, KyAnl411 and KyAinl411.
• the vaccine compositions of the present invention can also include additional active ingredient such as other immunogenic compositions against EHV-l, e.g., those described in U.S. Patent 5,707,629 ("Immunogenic composition against equine herpesvirus type 1") and U.S. Patent 5,795,578 ("Vaccine against equine herpesvirus type 1), or immunogenic compositions against EHV-4.
• additional active ingredient such as other immunogenic compositions against EHV-l, e.g., those described in U.S. Patent 5,707,629 ("Immunogenic composition against equine herpesvirus type 1") and U.S. Patent 5,795,578 ("Vaccine against equine herpesvirus type 1), or immunogenic compositions against EHV-4.
• the vaccine compositions of the present invention can include one or more pharmaceutically-acceptable carriers as described hereinabove.
• the vaccine compositions of the present invention can made in forms suitable for injectable use, e.g., in the form of sterile aqueous solutions or dispersions, or can be made in lyophilized forms using vacuum-drying and freeze- drying techniques. Lyophilized vaccine compositions are typically maintained at about 4°C, and can be reconstituted in a stabilizing solution, e.g., saline or and HEPES, with or without adjuvant. In all cases the form of the vaccine compositions must be sterile.
• the above vaccine compositions of the present invention are used in treating EHV-l infections. Accordingly, the present invention provides methods of treating EHV-l infections in a horse by administering to the horse subject, a therapeutically effective amount of a non-pathogenic EHV-l isolate of the present invention.
• treating is meant preventing or reducing the risk of infection by a pathogenic strain of EHV-l, ameliorating the symptoms of an EHV-l infection, or accelerating the recovery from an EHV-l infection.
• a vaccine composition of the present invention can be administered to a horse subject via intravenous, intraperitoneal, intramuscular, or intramucosal (e.g. nasal or respiratory spray or injection) routes, or by other forms of parenteral administration.
• a vaccine composition can also be administered via an implant or orally.
• 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.
• Foals can be vaccinated shortly after birth.
• Non-pathogenic, replication competent mutant EHV-l isolates of the present invention can also have immune protective effects against infections caused by equine herpesvirus type 4 (EHV-4) .
• EHV-4 equine herpesvirus type 4
• the use of the mutant EHV-l isolates of the present invention in immunogenic or vaccine compositions for preventing or inhibiting EHV-4 infections is also contemplated by the present invention.
• the vaccine compositions of the present invention have the additional feature that the non-pathogenic EHV-l isolate included therein is generally discernable from wild type EHV-l strains in terms of the composition and size of the IE protein expressed, or the sensitivity of growth to temperature. This feature is useful, e.g., in determining whether a subject tested positive for EHV-l in certain laboratory tests carries a pathogenic EHV-l or a previously inoculated non-pathogenic EHV-l.
• the present invention provides methods of determining the pathogenicity of an EHV-l strain present in a subject previously vaccinated with a non-pathogenic EHV-l isolate of the present invention.
• the methods are premised on a distinction between the wild type EHV-l and the non-pathogenic EHV-l isolate previously administered to the subject, the choice of assays for making the distinction depends on the nature of the mutation of the non-pathogenic EHV-l isolate.
• EHV-l having d644/824, nl029 or nl411, assays based on detection of IE proteins or antibodies in serum against IE proteins
• infected cells or tissues can be isolated from the subject.
• Intracellular extracts can be made from such cells or tissues and can be subjected to, e.g., Western Blot analysis, as described in the Examples hereinbelow.
• the observation of an IE protein of a lower Mw is indicative of the non-pathogenicity of the EHV-l strain present in the subject.
• an antibody specific for the deleted portion of amino acid residues can be used in Western Blot, and absence of an IE protein band is also indicative of the non-pathogenicity of the EHV-l strain present in the subject. Additionally, the absence of antibodies in the serum against the deleted or truncated portion of the IE protein, may also be an indication of the non-pathogenicity of the virus in the subject. The presence or absence of antibodies in the serum of the subject can be determined by using a peptide corresponding to the deleted portion in an appropriate immunoassay, e.g., ELISPOT.
• an appropriate immunoassay e.g., ELISPOT.
• temperature shift assays can also be used, as described in the Examples hereinbelow.
• such assays can be applied in distinguishing wild type EHV-l from, e.g., EHV isolates having d644/824, nl029, nl411, inl411, D24N, L12E, F15D or E34Q, and in particular, nl029 and nl411.
• KyAnl411 and KyA1029 fail to grow at 39 C.
• KyA carrying d644/824, inl411, D24N, L12E, F15D or E34Q has a reduced capacity to grow as compared to wild type KyA.
• the determination of the pathogenicity can also be achieved by using nucleic acid-based assays to screen for mutations in the IE gene of the viruses isolated from the subject.
• nucleic acid-based assays include Southern or Northern blot analysis, PCR, and sequencing.
• a mutation in the IE gene may result in a reduced expression of another EHV-l protein, e.g., EICPO or gD .
• EICPO EHV-l protein
• gD another EHV-l protein
• nl029 causes significant reduction in the mRNA levels of both EICPO and gD .
• detection of the reduced expression of such other proteins, either at the mRNA level or the protein level is indicative of the non-pathogenicity of the virus in the subject. Any variations of the foregoing assays are also encompassed by the present invention.
• Mouse LM cells were propagated in suspension culture with YELP medium (Eagle's minimum essential medium [EMEM] supplemented with yeast extract, lactalbumin hydrolysate, peptone) containing 0.12% methylcellulose- 12 , 100 ⁇ g/ml streptomycin, 100 U/ml penicillin, and 5% fetal bovine serum (FBS) .
• EMEM Eagle's minimum essential medium
• Rabbit kidney cells (RK-13) were maintained in complete EMEM supplemented with 100 ⁇ g/ml streptomycin, 100 U/ml penicillin, nonessential amino acids and 5% FBS. Infectious virus was measured by a plaque assay as described by Perdue et al . Virology 59: 201-216 (1974) .
• IE13.1 cells express the IE protein in trans and both copies of the IE gene on the viral genome are replaced with LacZ sequences from E. coli .
• Generation and characterization of IE13.1 cells as well as the IE knock-out virus KyA ⁇ lE were described by Garko-Buczynski et al . Virology 248: 83-94 (1998) .
• Plasmids A recombination system was established ( Figures 1-2) so that a panel of mutant viruses could be generated by homologous recombination between the KyA ⁇ lE viral genome and various recombination vectors derived from plasmid pBR322IE.
• pBR322 was digested with EcoRV and PvuII, and the resulting fragment was re-ligated to generate plasmid pBR322E/P.
• An 8.0-kb JVdel fragment containing the entire IE ORF from nt -215 to nt +8140 of the IE gene was cloned into the sole Ndel site of pBR322E/P to generate pBR322IE.
• a second vector, pIECassette was generated by cloning an EcoRV/Ba EI fragment that harbors nt - 257 to +1266 of the IE ORF into the ScoRV/BamHI sites of pBR322.
• a "domain swap" strategy was used to generate recombination plasmids containing mutant IE forms with nonsense, insertion, point, or deletion mutations.
• Recombination plasmids pBR322nl411, pBR322d644/824 , pBR322In628, and pBR322nl029 were generated by digesting pBR322IE with BamHI and PvuII and replacing this fragment with mutagenized BamHI -PvuII IE ORF sequences derived from mutants described by Smith et al . J. Virol. 69: 3857-3862 (1995).
• Plasmids pBR322dl78/627 and pBR322d552/897 were generated by digesting pBR322IE with iVcoI and PvuII and replacing this fragment with mutagenized Ncol -PvuII IE ORF sequences derived from mutants described by Buczynski et al . , Virus Res . 65:
• Recombination plasmid pBR322 ⁇ SRTl was generated by digestion of pSVIE with BspEI (nt 1523 to nt 1733) followed by fill-in with Klenow to generate blunt-ends and insertion of an in-frame 10-mer EcoRl linker.
• plasmid pSVIE was first digested with .Hindi11 and BamHI, and the resultant HindiII-BamHI fragment was cloned into the Hindlll -BamHI sites of pUCIO to generate plasmid pUIE .
• pUIE was digested with iVael and self-ligated to generate plasmid pUIE ⁇ SRT2.
• Plasmid pUIE ⁇ SRT2 was digested with JWCOI, and the resultant fragment was cloned into the .Ncol site of pBR322IE.
• PCR mutagenesis (Stratagene) was performed utilizing primers harboring the mutation of choice (Oligo' s Etc. Wilsonville, OR; Integrated DNA Technologies, Inc., Coralville, IA) .
• Mutagenized TADs were cloned into the pCR-Blunt vector (Invitrogen, Carlsbad, CA) and were sequenced in their entirety to confirm the presence of the desired mutation.
• Mutagenized TADs were cloned into unique _WcoI-_WaeI sites of the vector pIECassette.
• EcoRV and BamHI fragments housing various mutagenized TADs were then cloned into the ScoRV-Ba HI sites of the pBR322IE vector, thereby generating a reconstituted IE ORF with the mutation of choice.
• the recombination vectors containing nonsense, insertion, point, or deletion mutations in the IE gene were used to generate mutant viruses that allowed domains of the IE protein essential for virus replication to be identified.
• RK-13 cells were transfected with various recombination vectors containing a mutant IE gene, followed by infection with the null virus KyA ⁇ lE. Mutant viruses were thus generated by homologous recombination between the KyA ⁇ lE viral genome and various recombination vectors containing mutant IE genes .
• the recombination vector of choice was transfected into RK-13 cells as follows.
• RK-13 cells were plated at a density of 2.5 x 10 s cells per 25cm 2 in complete EMEM and permitted to attach overnight at 37°C in 5% C0 2 .
• the following day, RK-13 monolayers were subjected to liposome-mediated DNA transfection using Lipofectin reagent (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. More specifically, liposomes were prepared by mixing 20 ⁇ l Lipofectin reagent with 230 ⁇ l serum-free EMEM and incubating the mixture at room temperature for 45 min.
• IE13.1 cells were superinfected at an MOI of 10 with KyA ⁇ lE.
• CPE cytopathic effects
• IE13.1 cells were overlaid with 2% agarose mixed in a 1:1 ratio with 2X EMEM supplemented with 6% FBS and 1 mM BluoGal (Life Technologies) to distinguish between white and blue plaques.
• White plaques were picked and subjected to five rounds of plaque purification on IE13.1 cells to ensure the removal of residual KyA ⁇ lE.
• Viruses were isolated from culture supernatants by ultracentrifugation in an SW50.1 rotor at 28,000 rpm for 2 h at 4°C. Supernatants were aspirated, and the remaining pellet was resuspended in 50 ⁇ l of RIPA buffer (150mM NaCI, 50mM Tris-HCl [pH 8.0], 0.1% SDS, 0.5% deoxycholate, 1% NP40) and incubated at room temperature for 1 h with occasional mixing. Resuspended pellets served as templates for PCR analyses.
• RIPA buffer 150mM NaCI, 50mM Tris-HCl [pH 8.0], 0.1% SDS, 0.5% deoxycholate, 1% NP40
• IE-specific DNA fragments were amplified using an IE forward primer (5' CCTTCCCTTCTCGGTCTT3 ' (SEQ ID NO: 3); nt 913-930) and an IE reverse primer (5' CTCCACCCCGAACATGTT3 ' (SEQ ID NO: 4); nt 1155-1138) .
• the lacZ-specific DNA fragments were amplified using the IE forward primer (above) and a lacZ-specific primer (5'GGGTAACGCCAGGGTTTT3' (SEQ ID NO : 5)) derived from the 5' coding region of the E. coli lacZ gene.
• the mutations were confirmed by sequence analyses using Promega's fmol Sequencing System (Madison, WI) . In the case of the mutant viruses, it was found that both copies of the bacterial lacZ ORF of KyA ⁇ lE parent virus were replaced with the mutated IE ORF derived from the recombination vectors.
• Nuclear extracts of EHV-l infected cells were prepared by a modification of the procedures described by Paterson et al . (Nuc. Acids. Res. 16:11005-11025, 1988) .
• RK- 13 cells were infected with wild-type EHV-l or mutant EHV-l at an MOI of 10.
• Six h post-infection, cells were scraped into PBS, pelleted, and resuspended into 4 volumes of buffer A (10 mM HEPES [pH 7.0], 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, 0.5% NP40, and 0.5 mM TPCK) .
• Total protein in cell or nuclear extracts was determined by use of the bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL) .
• BCA bicinchoninic acid
• Protein samples of infected cell extracts were boiled for 5 min with an equal volume of 2X Laemmli sample buffer (10% 2-mercaptoethanol, 4% SDS, 20% glycerol, 120 mM Tris-HCl [pH 6.8], 0.001% bromphenol blue) . Proteins were separated through a 4% stacking gel and an 8% resolving gel and were electrophoretically-transferred to a nitrocellulose membrane (Schleicher &. Schuell, Inc., Keene, NH) at 100V for 1 h at 4°C.
• BCA bicinchoninic acid
• the membrane was blocked for 1 h at room temperature in TBST buffer (100 mM Tris-HCl [pH 7.5], 0.9% NaCI, 0.1% Tween 20) containing 1% gelatin (Sigma) .
• TBST buffer 100 mM Tris-HCl [pH 7.5], 0.9% NaCI, 0.1% Tween 20
• the nitrocellulose membrane was then incubated with a polyclonal anti-IE peptide-specific antiserum (1:1000 dilution), followed by incubation with goat anti-rabbit antibody (Sigma; 1:30,000 dilution) conjugated to alkaline phosphatase.
• This anti-IE peptide antibody was generated to a peptide spanning amino acids 425 to 445 of the IE protein and had been demonstrated to be highly reactive to the IE protein in immunofluorescence (IF) and Western blot analyses (Smith et al . , Virology 202: 760-770, 1994). Each antibody was incubated in TBST containing 0.1% gelatin for 2 h with shaking, followed by three washes with TBST containing 0.1% gelatin for 15 min with shaking.
• the reactive proteins were visualized by incubating the membrane in NBT buffer (100 mM Tris-HCl [pH 9.5], 100 mM NaCI, 5.0 mM MgCl 2 , 0.33 mg/ml nitroblue tetrazolium [NBT, Gibco-BRL], and 0.165 mg/ml 5- bromo-4-chloro-3-indolyl phosphate [BCIP] Gibco-BRL) .
• NBT buffer 100 mM Tris-HCl [pH 9.5], 100 mM NaCI, 5.0 mM MgCl 2 , 0.33 mg/ml nitroblue tetrazolium [NBT, Gibco-BRL], and 0.165 mg/ml 5- bromo-4-chloro-3-indolyl phosphate [BCIP] Gibco-BRL
• the mutant IE protein was synthesized in infected RK-13 cells. Representative data for four of the IE mutant viruses are shown in Figure 3. In contrast to the wild type IE protein which migrates on SDS Page as a band at about 190-200 kDa,
• KyAd644/824 produced an IE protein that migrates at about 180 kDa.
• Mutant KyAnl029 harbors an IE ORF that lacks sequences encoding the 458 carboxyl terminal resides and thus, its IE gene product migrates at about 138 kDa.
• the IE ORF of the KyA ⁇ SRT2 virus lacks sequences encoding amino acids 88-243 and thus its predicted IE protein would migrate at approximately 179 kDa.
• An IE gene product of this size was detectable but at very low levels. This indicated that only very low titers of this mutant could be obtained.
• the IE protein encoded by the KyA ⁇ SRT2 virus could be very unstable.
• RK-13 cells and IE13.1 cells were infected with mutant viruses at a range of dilutions. Infected cells were overlaid with 2% agarose mixed in a 1:1 ratio with 2X EMEM supplemented with 6% FBS. Plaque formation was observed.
• KyA ⁇ SRTl and KyA ⁇ SRT2 delete the entire SRT region of the IE protein, although the deletion within the IE ORF of KyA ⁇ SRT2 (residues 88-243) is in very close proximity to the IE TAD and may affect the function and/or conformation of this critical domain.
• KyAd644/824 contains a deletion within region 3 of the IE protein and demonstrated no defect in growth on RK-13 cells, indicating that this region contains no domains essential for the IE protein to transactivate early genes required for virus replication.
• mutant EHV-l that contain nonsense mutations at amino acids 627 and 951 were not able to replicate on RK-13 cells (both of these mutants lack the nuclear localization signal) .
• Nonsense mutations at amino acids 1411 (KyAnl411) and 1029 (KyAnl029) had no effect on the growth of these viruses on non-complementing cells.
• plaques generated by KyAnl029 were larger than those produced by wild-type KyA, and the appearance of plaques generated by KyAnl029 was delayed as compared to the wild-type KyA virus.
• insertion mutations within the IE ORF of KyAIn628 and KyAInl411 had no deleterious effect on the growth or the phenotype of these mutants on RK-13 cells.
• Wild-type EHV-l or mutant viruses were serially diluted and plated in triplicate onto RK-13 cells. Plates were incubated at 33°C, 37°C, or 39°C for five days and fixed using 10% buffered formalin (Sigma, St. Louis, MO). Following staining with methylene blue, plaques were enumerated to determine plating efficiency at the various temperatures.
• Wild-type EHV-l replicated efficiently at both 33°C and 37°C, but virus yield was reduced by approximately 70% at 39°C. Additionally, the plaque size of wild-type
• EHV-l was significantly reduced at 39°C to an extent that the very small plaque size made plating efficiency difficult to calculate.
• Mutant viruses were divided into two groups based on their ability or inability to grow at 39°C (Table 3) .
• Viruses KyAnl029, KyAIn628, KyAnl411 and KyA ⁇ SRT2 were categorized into Group 1 based on a severe impairment of growth in RK-13 cells at 39°C as compared to growth observed for wild-type KyA. Both KyAnl029 and KyAnl411 displayed impaired growth at both at 37°C ( Figure 4) and at 39°C (Table 3) .
• Viruses categorized into Group 2 (Table 3) , with the exception of KyAInl411, grew at 39°C, although some impairment of growth was observed. KyAInl411 was able to grow at 33°C, 37°C and 39°C to levels that appeared to be comparable to those of wild-type EHV-l.
• Viruses were divided into two groups (I and II) based on their on their ability or inability to grow at 39°C as compared to wild-type EHV-l. Growth at 39°C scored as comparable to wildtype EHV-l. corresponds to titers that range from 10 4 -10 "s . ++ corresponds to titers that range from 10 "3 -10 "4 .
• RPA ribonuclease protection assays
• RPA the RIBOQUANT RNASE PROTECTION ASSAY SYSTEM (Pharmingen, San Diego, CA) was utilized as per the manufacturer's instructions. Approximately 20 ⁇ g of total RNA was used for each reaction of the RPA.
• Probes for RPA analyses were generated using Promega's (Madison, WI) RIBOPROBE IN VITRO TRANSCRIPTION SYSTEM.
• IE probes plasmid pG3IE was digested with BamHI to generate run-off transcripts.
• Plasmid pG3IE was constructed by cloning an Nhel/Dral fragment containing the entire 4,773-bp IE ORF into the Xbal/Sma.1 sites of plasmid pGEM-3Z (Promega, Madison, WI) . Digested pG3IE plasmid was purified by phenol-chloroform extraction and ethanol precipitation, and approximately 1 ⁇ g of plasmid was used for riboprobe generation as per the manufacturer's instructions. For ElCPO-specific probes, plasmid pGEMICPOK (Bowles et al . J. Virol. 71:4904-4914, 1997; Bowles ' et al . , J. Virol.
• plasmid p72D392 (Zhang et al . Virus Res. 56:11-24, 1998) was transcribed directly using the Promega's RIBOPROBE IN VITRO TRANSCRIPTION SYSTEM. mRNA levels were quantitated by using the MOLECULAR DYNAMICS PHOSPHOIMAGERY SYSTEM (Sunnyvale, CA) .
• EICPO 99% 42% 3% gD b 7% 5% "Values shown are depicted as the precent mRNA detected as compared to those obtained for wild-type EHV-l IE, EICPO, and gD mRNA expression.
• b gD is a late gene and no gD transcript was detected at immediate-early times in cells infected with wild-type virus or any of the mutant viruses.
• mice Female CBA (H-2k) mice, 3 to 6 weeks of age, were obtained from Harlan Sprague Dawley, Indianapolis, Ind., or Jackson Laboratory Bar Harbor, Maine. Mice were maintained in the Animal Resource Facility of the Louisiana State University Medical Center, Shreveport, in cages equipped with filter tops. All mice were rested for a minimum of 1 week prior to use .
• mice were anesthetized with Halothane (Sigma Chemical Co., St. Louis, Mo.) and inoculated intranasally (i.n.) with 2 x 10 s PFU of EHV-l KyA or a mutant virus in a volume of 50 ⁇ l .
• Control mice received 50 ⁇ l of culture medium alone .
• mice were monitored daily for development of clinical signs of EHV-l infection such as ruffled fur, loss of body weight, labored breathing, lethargy and huddling, as described by, e.g., Colle et al . Virus Res. 43: 111-124 (1996) and Zhang et al . Virus Res . 56: 11-24 (1998) . No clinical disease was observed with mice infected with any of the four mutant viruses tested.
• lymphocytes were isolated from the mediastinal lymph nodes (MLN) 5 days postinoculation, and a single-cell suspension was obtained by pressing the lymphoid tissues through a 60-gauge wire mesh screen.
• the lymphocytes were washed and cultured (10 7 cells per well) for 3 days at 37°C and 5% C0 2 in 12-well flat-bottom plates (Corning Inc., Corning, N.Y.) in complete RPMI 1640 (Sigma) containing 5% FCS, 20 ⁇ M - mercaptoethanol, 20 mM HEPES, 2 mM L-glutamine and antibiotics.
• Cytolytic activity was assessed in a standard 4-h sl Cr release assay in 96-well V-bottom plates (Nunc, Denmark) at a range of effector-to-target ratios against 10 4 51 Cr-labeled, infected or uninfected target LM cells.
• mice were infected as described above and maintained for 2-26 weeks. Spleen tissues were pressed through a 60-gauge wire mesh screen, and the cell suspension was subjected to a brief exposure at at 37°C to Tris-buffered 0.83 NH 4 C1 to lyse erythrocytes . The resulting lymphocytes were then cultured (10 7 cells per well) in complete RPMI 1640 at 37°C and 5% C0 2 , for 5 days in 12-well flat-bottom plates in the presence of 3 x 10 5 mitomycin C-treated stimulator cells, as described by Jennings et al . Cell. Immunol. 133: 234-252 (1991) .
• the stimulator cells were L-M mouse fibroblasts cells that had been infected with EHV-l KyA at a multiplicity of infection of 10 for 18 h to allow the expression of late viral gene products. Cytolytic activity was assessed in a standard 4-h 51 Cr release assay in 96-well V-bottom plates (Nunc, Denmark) against 10 4 51 Cr-labeled, infected or uninfected LM cells.
• Serum samples from immunized and control mice were assayed for the presence of neutralizing antibody (nAb) . Briefly, after the sera were heated to 56°C for 1 h to inactivate complement, serial, two-fold serum dilutions (1:20 to 1:1028) were incubated with 3 x 10 3 pfu EHV-l KyA for 1 h at 37°C. Each sample was assayed in triplicate for infectious virus by plaque assay on RK-13 cell monolayers. The nAb titer was determined as the reciprocal of the highest dilution of serum resulting in a 50% reduction in pfu. Preimmune serum was employed as a control.
• nAb neutralizing antibody
• ASC EHV-Specific Antibody Secreting Cells
• mice On specified days postchallenge, the mice were terminated, and the lungs, MLN, and CLN were removed, and the spectrum of ASC was determined by ELISPOT assay.
• tissue was fragmented with sicssors and then pressed through a 60 -gauge screen. After centrifugation, the cells were resuspended in 10 ml collagenase type
• ELISPOT assay Briefly, nitrocellulose-based microtiter 96-well plates (Millititer-HA, Millipore Corporation, Bedford, MA) were coated with EHV-l KyA- infected cell lysate (10-200 ⁇ g/ml) in phosphate-buffered saline (PBS) and incubated in humidified chambers at 4°C overnight, or at 37°C for 2 h. Coated plates were stored at 4°C until use. For each experimental determination, the coating buffer (PBS) was decanted and the plates were washed three times by rinsing with PBS.
• PBS phosphate-buffered saline
• lymphocytes were diluted to the appropriate concentration (10 3 to 10 s cells/well) in DMEM-5, were added to the coated plates.
• Lipopolysaccharide LPS, 5 ⁇ g/well; Calbiochem-Novabiochem, La Jolla, CA was added as a nonspecific activator of B cells.
• a control plate of uninfected cells was also used for the assays and was treated in the same manner as the plates of EHV-l infected cells.
• the cells were incubated for 3 to 4 h at 37°C in 5% C0 2 and then were discarded by washing with PBS containing 0.005% Tween 20 (PBST) .
• PBS containing 0.005% Tween 20 PBST
• One hundred microliters of alkaline phosphatase-conjugated antibody was added to each well, and the plates were incubated for 2 to 3 h at room temperature in humidified chambers.
• EHV-1-specific ASC were visualized by adding 5-bromo-4chloro-3-indolyphosphate p- toluidine salt (BCIP, Gibco-BRL) and Nitroblue tetrazolium chloride (NBT, Gibco-BRL) as substrate, according to the manufacturer's instructions, for 5 to 30 min until the blue color developed.
• BCIP 5-bromo-4chloro-3-indolyphosphate p- toluidine salt
• NBT Nitroblue tetrazolium chloride
• mice were lightly anesthetized with Halothane (Halocarbon Laboratories, River Edge, NJ) and then inoculated intranasally with a pathogenic EHV-l strain at 2 x 10 6 pfu diluted into PBS (50 ⁇ l total volume/mouse) .
• Mice were monitored daily for the development of clinical signs of EHV-l infection such as ruffled fur, loss of body weight, labored breathing, lethargy and huddling.
• the level of virus in the lungs of challenged mice were determined. Lungs from the challenged animals were isolated at days 2, 5 and 10 post-challenge. Virus was recovered from mouse lung tissue as described previously by Awan, et al . J. Gen. Virol. 71: 1131-1140
• Tissues were homogenized in 1 ml Eagles minimal essential medium using a 2 ml Wheaton Dounce homogenizer. Each sample was then sonicated in an Ultrasonic processor
• SEQ ID NO: 1 Equine herpesvirus type 1 immediate- early gene, GENEBANK Accession number J04366
• SEQ ID NO: 2 (Equine herpesvirus type 1 immediate-early protein

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