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WO1996013575A1 - Herpes-virus felin recombinant - Google Patents

Herpes-virus felin recombinant Download PDF

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Publication number
WO1996013575A1
WO1996013575A1 PCT/US1995/013975 US9513975W WO9613575A1 WO 1996013575 A1 WO1996013575 A1 WO 1996013575A1 US 9513975 W US9513975 W US 9513975W WO 9613575 A1 WO9613575 A1 WO 9613575A1
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Prior art keywords
herpes virus
fhv
virus
recombinant
feline herpes
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PCT/US1995/013975
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English (en)
Inventor
Mark D. Cochran
Michael W. Mcdonnell
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Syntro Corporation
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Publication date
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Priority to AU41971/96A priority Critical patent/AU4197196A/en
Publication of WO1996013575A1 publication Critical patent/WO1996013575A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16741—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16743—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Herpesviruses contain 100,000 to 200,000 base pairs of DNA as their genetic material having a long unique segment and a short unique segment, the short unique segment bounded by an internal repeat sequence and a terminal repeat sequence. Within a given genome, several regions have been identified that are not essential for the replication of virus in cell culture. Modifications of some of these ' regions of the DNA have been known to lower the pathogenicity of the virus, i.e., to attenuate the virus when it infects an animal species.
  • inactivation of the thymidine kinase gene of either human herpes simplex virus [1] or pseudorabies virus of swine [2] renders the human herpesvirus less pathogenic in mice and the pseudorabies virus less pathogenic in swine.
  • glycoproteins gene deletions in herpes simplex virus render the herpes simplex virus less pathogenic in mice [see 37 for review] . While, combinations of glycoprotein gene deletions in pseudorabies virus render the pseudorabies virus less pathogenic in swine [see 38 for review] .
  • Herpesviruses contain non-essential regions of DNA in various parts of the genome, and that modification of these regions can attenuate the virus, leading to a non-pathogenic strain from which a vaccine may be derived.
  • the degree of attenuation of the virus is important to the utility of the virus as a vaccine. Deletions which cause too much attenuation of the virus will result in a vaccine that fails to elicit an adequate immune response.
  • attenuating deletions are known, the appropriate combination of deletions for any herpesvirus is not readily apparent.
  • Feline herpesvirus 1 is the causative agent of feline viral rhinotracheitis, an acute upper respiratory disease in cats [7, 8] . Serological studies indicate that 50 to 70% of adult cats have detectable antibodies to the virus [9, 10] . Currently available inactivated and attenuated live virus vaccines reduce disease but do not prevent infection by FHV [11] .
  • feline herpesvirus is a member of the family herpesviridae, which are commonly known as the herpesviruses and a member of the subfamily alphaherpesvirus with a group
  • the FHV genome is comprised of approximately 134 kilobase (kb) pairs that is subdivided into a long unique segment of approximately 104 kb and a short unique segment of approximately 8 kb [13] .
  • the unique short region is bounded by inverted repeat sequences which are approximately 11 kb.
  • the thymidine kinase gene of FHV has been sequenced and an FHV virus containing a deletion of the TK gene was isolated using a drug selection technique [14] .
  • An FHV having a deletion of the TK gene and an insertion of the feline leukemia virus (FeLV) envelope (e ⁇ v) and gag genes at the TK deletion site has been constructed [36] .
  • feline herpesviruses in this invention are useful as vectors for the delivery of vaccine antigens from microorganisms causing diseases in animals other than cats or dogs and for the delivery of therapeutic agents.
  • the therapeutic agent that is delivered by a viral vector of the present invention must be a biological molecule that is a by-product of feline herpesvirus replication. This limits the therapeutic agent in the first analysis to either DNA, RNA or protein.
  • therapeutic agents from each of these classes of compounds in the form of anti- sense DNA, anti-sense RNA [18] , ribozy es [19] , suppressor tRNAs [20] , interferon-inducing double stranded RNA and numerous examples of protein therapeutics, from hormones, e.g., insulin, to lymphokines, e.g., interferons and interleukins, to natural opiates.
  • hormones e.g., insulin
  • lymphokines e.g., interferons and interleukins
  • This invention provides a recombinant feline herpes virus comprising the feline herpes virus viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein E (gE) gene.
  • gE glycoprotein E
  • This invention provides a recombinant feline herpes virus comprising the feline herpes virus viral genome which contains an insertion into the unique short region of the viral genome, wherein the insertion is in the glycoprotein E (gE) gene.
  • Homology vectors for producing a recombinant feline herpes virus and host cells are provided for.
  • This invention provides a vaccine for feline herpes virus which comprises an effective immunizing amount of the recombinant feline herpes virus and a suitable carrier.
  • a method of immunizing an animal against a human or feline pathogen is also provided for.
  • This invention provides a method of distinguishing an animal vaccinated with the feline herpes virus vaccine from an animal infected with a naturally-occuring feline herpes virus.
  • Figure 1 Restriction endonuclease map of the feline herpesvirus genome.
  • FIG. 1 Feline leukemia virus envelope epitopes
  • Bam ⁇ I and Bgrlll ends so that multiple copies of each epitope could be cloned as fusion proteins in the FHV homology vectors.
  • FIGS. 3A-3D DNA insertion in Homology Vectors 411-
  • the diagram shows the orientation of DNA fragments assembled in plasmid 411-91.01A and 411-91.01B.
  • the diagram shows the orientation of DNA fragments assembled in plasmid 411-91.01A and
  • junction A SEQ ID NO: 11
  • junction B SEQ ID NO: 12
  • junction C SEQ ID NO: 13
  • junction D SEQ ID NO: 14
  • junction E SEQ ID NO:
  • restriction endonuclease sites used to generate each fragment as well as the synthetic DNA sequences that were used to join the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar.
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction.
  • the following abbreviations are used, feline herpesvirus (FHV) , human cytomegalovirus (HCMV) , immediate early
  • IE feline leukemia virus
  • PRV pseudorabies virus
  • poly A polyadenylation site
  • Figures 4A-4B DNA insertion in Homology Vector 416-
  • the diagram shows the orientation of DNA fragments assembled in plasmid 416-80.21B.
  • the source of each fragment is described in the Materials and Methods section.
  • the sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 16) , junction B (SEQ ID NO: 17) , and junction C (SEQ ID NO: 18) .
  • junction A SEQ ID NO: 16
  • junction B SEQ ID NO: 17
  • junction C SEQ ID NO: 18
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction.
  • the following abbreviations are used, feline herpesvirus (FHV) , glycoprotein I (gl) and glycoprotein E (gE) .
  • FIGS. 5A-5C DNA insertion in Homology Vectors 416-
  • junction A SEQ ID NO: 19
  • junction B SEQ ID NO: 20
  • junction C SEQ ID NO: 21
  • junction D SEQ ID NO: 20
  • restriction endonuclease sites used to generate each fragment as well as the synthetic DNA sequences that were used to join the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar.
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction.
  • the following abbreviations are used, feline herpesvirus (FHV) , pseudorabies virus (PRV) , glycoprotein X (gX) , polyadenylation site (poly A) .
  • FIG. 6 DNA insertion at the gE deletion site of S-FHV-010.
  • the diagram shows the synthetic DNA inserted at the gE deletion site and the unique Sfil restriction endonuclease site of the recombinant S-FHV-010.
  • the sequences - 8 - located at the junctions between each fragment are shown (SEQ ID NO: 23) .
  • the restriction endonuclease sites used to generate each fragment as well as the synthetic DNA sequences that were used to join the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar.
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction.
  • the following abbreviation is used, feline herpesvirus (FHV) .
  • Figures 7A-7E DNA insertion in Homology Vectors 478-
  • FHV homology vector 416- 80.21B The sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 24) , junction B (SEQ ID NO: 25) , junction C (SEQ ID NO: 26) , junction D (SEQ ID NO: 27), junction E (SEQ ID NO:
  • junction F SEQ ID NO: 29
  • junction G SEQ ID NO: 30
  • junction H SEQ ID NO: 31
  • junction I SEQ ID NO: 32
  • junction J SEQ ID NO: 33
  • IE feline leukemia virus
  • PRV pseudorabies virus
  • gX Escherichia coli
  • E. coli herpes simplex virus type 1
  • poly A polyadenylation site
  • Figures 8A-8E DNA insertion in Homology Vectors 510-
  • FHV DNA sequences is FHV homology vector 416-
  • junction A SEQ ID NO: 34
  • junction B SEQ ID NO: 35
  • junction C SEQ ID NO: 36
  • restriction endonuclease sites used to generate each fragment as well as the synthetic DNA sequences that were used to join the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar. The following convention is used: restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction. The following abbreviations are used, feline herpesvirus (FHV) , human cytomegalovirus (HCMV) , immediate early
  • IE feline leukemia virus
  • PRV pseudorabies virus
  • gX Escherichia coli
  • E. coli herpes simplex virus type 1
  • poly A polyadenylation site
  • Figures 9A-9E DNA insertion in Homology Vectors 639-
  • junction A SEQ ID NO: 43
  • junction B SEQ ID NO: 44
  • junction C SEQ ID NO: 45
  • junction D SEQ ID NO: 45
  • junction E SEQ ID NO: 47
  • junction F SEQ ID NO: 48
  • junction G _ SEQ ID NO: 49
  • DNA sequences that were used to join - the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar.
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction.
  • the following abbreviations are used, feline herpesvirus (FHV) , human cytomegalovirus (HCMV) , immediate early
  • IE Dirofilaria immi tis
  • D. immi tis Dirofilaria immi tis
  • PRV pseudorabies virus
  • gX glycoprotein X
  • Escherichia coli Escherichia coli
  • HSV-1 herpes simplex virus type 1
  • poly A polyadenylation site
  • the diagram shows the orientation of DNA fragments assembled in plasmid 644-09.Al. The source of each fragment is described in the Materials and Methods section. The sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 50) , junction B (SEQ ID NO: 51) and junction C (SEQ ID NO: 52) .
  • junction A SEQ ID NO: 50
  • junction B SEQ ID NO: 51
  • junction C SEQ ID NO: 52
  • DNA sequences that were used to join the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar.
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during cons t ru c t ion .
  • the f o l l owi ng abbreviations are used, feline herpesvirus (FHV) , glycoprotein I (gl ) and glycoprotein E (gE) .
  • the diagram shows the orientation of DNA fragments assembled in plasmid 644-09.B2.
  • the source of each fragment is described in the Materials and Methods section.
  • the sequences located at the junctions between each fragment are shown, including junction A (SEQ ID NO: 53) , junction B (SEQ ID NO: 54) and junction C (SEQ ID NO: 55) .
  • junction A SEQ ID NO: 53
  • junction B SEQ ID NO: 54
  • junction C SEQ ID NO: 55
  • DNA sequences that were used to join the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar.
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction.
  • the following abbreviations are used, feline herpesvirus (FHV) , glycoprotein I (gl) and glycoprotein E (gE) .
  • Figures 12A-12E DNA insertion in Homology Vectors 669-
  • junction A SEQ ID NO: 56
  • junction B SEQ ID NO: 57
  • junction C SEQ ID NO: 58
  • junction D SEQ ID NO:
  • junction E SEQ ID NO: 60
  • junction F SEQ ID NO: 61
  • restriction endonuclease sites used to generate each fragment as well as the synthetic DNA sequences that were used to join the fragments are described for each junction.
  • the synthetic DNA sequences are underlined by a solid bar.
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during- construction.
  • the following abbreviations are used, feline herpesvirus (FHV) , human cytomegalovirus (HCMV) , immediate early (IE) , feline immunodeficiency virus (FIV) , pseudorabies virus (PRV) , glycoprotein X (gX) , Escherichia coli
  • herpes simplex virus type 1 HSV-1
  • polyadenylation site poly A
  • Figures 13A-13E DNA insertion in Homology Vectors 725-
  • junction E SEQ ID NO: 68
  • junction F SEQ ID NO: 69
  • junction G SEQ ID NO: 70
  • restriction endonuclease sites in brackets [] indicate the remnants of sites that were destroyed during construction.
  • feline herpesvirus FHV
  • human cytomegalovirus HCMV
  • immediate early IE
  • feline immunodeficiency virus FMV
  • PRV pseudorabies virus
  • glycoprotein X (gX) , Escherichia coli
  • E. coli herpes simplex virus type 1 (HSV-1) , thymidine kinase (TK) , polyadenylation site (poly A) .
  • HSV-1 herpes simplex virus type 1
  • TK thymidine kinase
  • poly A polyadenylation site
  • the present invention involves recombinant feline herpesviruses useful in the preparation of vaccines to protect cats from naturally-occuring infectious feline herpesvirus.
  • the present invention is also useful for expression of foreign genes from other pathogens for protection against disease.
  • Recombinant feline herpesvirus expressing foreign genes from avian or mammalian pathogens are useful as vaccines in avian or mammalian species including but not limited to chickens, turkeys, ducks, feline, canine, bovine, equine, and primate, including human.
  • the present invention provides a recombinant feline herpes virus comprising the feline herpes virus viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein E (gE) gene.
  • Said recombinant feline herpes virus contains a deletion which attenuates the virus, rendering it suitable for use as a vaccine against feline herpesvirus.
  • the feline herpes virus contains a foreign DNA sequence inserted into a non-essential region of the feline herpes virus genome.
  • the foreign DNA sequence is inserted into a unique short region of the feline herpes virus.
  • the foreign DNA sequence is inserted in the deleted gE gene.
  • the recombinant feline herpes virus is designated S-FHV-004.
  • S-FHV-004 was deposited on October 26, 1994 under ATCC Accession No. VR 2487 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.
  • the recombinant feline herpes virus is designated S-FHV-014.
  • S-FHV-014 virus was deposited on October 26, 1994 under ATCC Accession No. VR 2488 pursuant to the Budapest Treaty on the International Deposit of Microorganisms for the Purposes of Patent Procedure with the Patent Culture Depository of the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852 U.S.A.
  • a recombinant feline herpes virus is a live feline herpes virus which has been generated by the recombinant methods well known to those of skill in the art, e.g., the methods set forth in DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS in Materials and Methods, and the virus has not had genetic material essential for the replication of the feline herpes virus deleted.
  • the present invention also provides viruses in which (a) DNA corresponding to the gG or gl genes have been deleted, and (b) DNA encoding gG or gl have been altered or deleted, and a foreign DNA sequence from a pathogen of an animal has been inserted.
  • the present invention also provides viruses in which the gE gene is deleted in combination with the gG and/or gl genes.
  • a recombinant feline herpes virus which further comprises the foreign DNA sequence inserted into the unique short region, wherein the foreign DNA sequence is capable of being expressed in a recombinant feline herpes virus host cell.
  • the foreign DNA sequence is inserted into the unique short region or any non-essential region of the feline herpes virus viral genome in such a way that it is capable of being expressed in a recombinant feline herpes virus infected host cell.
  • a non-essential site of the feline herpes virus viral genome is a region of the viral genome which is not necessary for viral infection and replication.
  • non-essential sites of the feline herpes virus viral genome are preferred sites for inserting a foreign DNA sequence or gene into the virus, include but are not limited to: the glycoprotein E (gE) gene, the glycoprotein G (gG) gene, or the glycoprotein I (gl) gene.
  • Other non-essential sites are known to those skilled in the art.
  • the foreign DNA sequence which is inserted into a non-essential site in the feline herpes virus viral genome, encodes a screenable marker, such as E. coli B-galactosidase or E. coli beta- glucuronidase.
  • a "polypeptide which is a detectable marker” includes but is not limited to: the bimer, trimer and tetramer form of the polypeptide.
  • E. coli beta-galactosidase is a tetramer composed of four polypeptides or monomer sub-units.
  • the foreign DNA sequence which is inserted into a non-essential site in the feline herpes virus viral genome when introduced into the host cell, induces production of protective antibodies against a feline disease causing agent from which the antigen is derived or derivable.
  • antigenic polypeptide may be derived or derivable from the following: feline pathogen, canine pathogen, equine pathogen, bovine pathogen, avian pathogen, porcine pathogen, or human pathogen.
  • Antigenic polypeptides include, but are not limited to: infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus, infectious laryngotracheitis virus, infectious bursal disease virus VP2 protein, infectious bursal disease virus VP3 protein, infectious bursal disease virus VP4 protein, Marek's disease virus glycoprotein gB, Marek's disease virus glycoprotein gA, Marek's disease virus glycoprotein gD, Newcastle disease virus fusion (F) protein, Newcastle disease virus hemagglutinin-neuraminidase (HN) , infectious laryngotracheitis virus glycoprotein I, infectious laryngotracheitis virus glycoprotein D, infectious laryngotracheitis virus glycoprotein B, infectious bronchitis virus spike protein, or infectious bronchitis virus matrix protein.
  • infectious bronchitis virus infectious laryngotracheitis virus
  • Newcastle disease virus infectious bursal disease virus
  • infectious bursal disease virus VP2 protein
  • Such antigenic polypeptide may also be derived or derivable from avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent, Salmonella spp . , E. coli . , Pasteurella spp. , Bordetella spp. Eimeria spp. Histomonas spp. , Trichomonas spp. , poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.
  • the foreign DNA sequence or gene may be put under control of an endogenous upstream feline herpes virus promoter, or it may be put under control of a heterologous upstream promoter.
  • the heterologous upstream promoter may be derived from the HCMV IE promoter, the PRV gX promoter, or BHV-1.1 VP8 promoter.
  • the promoter is selected from a group consisting of MDV gB promoter, MDV gA promoter, MDV gD promoter, ILTV gB promoter, ILTV gD promoter, or HSV-1 alpha 4 promoter.
  • the foreign DNA sequence or genes include, but are not limited to: feline leukemic virus envelope gene, hepatitus B core antigen gene; Pseudorabies virus glycoprotein C gene; Dirofilaria immi tis 22 kD or p39 gene; feline immunodeficiency virus gag, pol, or env; or E. Coli beta-galactosidase gene .
  • the foreign DNA seuquence is a fusion protein two or more foreign DNA sequences or genes.
  • the present invention further provides a recombinant feline herpes virus comprising the feline herpes virus viral genome which contains a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycoprotein gE gene, so that upon replication, the recombinant virus produces no glycoprotein gE.
  • the present invention further provides a recombinant feline herpes virus comprising the feline herpes virus viral genome which contains a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycoprotein gl gene, so that upon replication, the recombinant virus produces no glycoprotein gl.
  • the present invention further provides a recombinant feline herpes virus comprising the feline herpes virus viral genome which contains a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycoprotein gG gene and in the glycoprotein gl gene, so that upon replication, the recombinant virus produces no glycoprotein gG and no glycoprotein gl .
  • any one the above genes will attenuate the virus, rendering it suitable to be used as a vaccine against feline herpesvirus.
  • a foreign DNA sequence may be inserted within any one of these sites in such a way that it may be expressed in a host cell which is infected which the recombinant feline herpes virus of the present invention.
  • the recombinant feline herpes virus further contains a deletion in a unique short region of the feline herpes virus genome. In another embodiment the recombinant feline herpes virus is further characterized by a deletion in a glycoprotein I (gl) gene. In another embodiment the recombinant feline herpes virus is further characterized by a deletion in a glycoprotein G (gG) gene.
  • gl glycoprotein I
  • G glycoprotein G
  • the present invention further provides a homology vector for producing a recombinant feline herpes virus by inserting a foreign DNA sequence into the feline herpes virus genome which comprises a double-stranded DNA molecule consisting of: a) double-stranded foreign DNA sequence encoding an antigenic polypeptide derived from a feline pathogen; b) at one end of the foreign DNA sequence, double-stranded feline virus genomic DNA homologous to the genomic DNA located at one side of a non-essential site of the feline herpes viral genomic DNA; c) at the other end of the foreign DNA sequence, double stranded feline herpes virus genomic DNA homologous to the genomic DNA located at the other side of the same site.
  • the antigenic polypeptide of a human pathogen is derived from human herpesvirus, herpes simplex virus-1, herpes simplex virus-2, human cytomegalovirus, Epstein-Barr virus, Varicell-Zoster virus, human herpesvirus-6, human herpesvirus-7, human influenza, human immunodeficiency virus, rabies virus, measles virus, hepatitis B virus and hepatitis C virus.
  • the antigenic polypeptide of a human pathogen may be associated with malaria or malignant tumor from the group conisting of Plasmodium falciparum, Bordetella pertusis, and malignant tumor.
  • the double stranded foreign DNA sequence in the homology vector encodes an antigenic polypeptide derived from an equine pathogen.
  • the antigenic polypeptide of an equine pathogen can derived from equine influenza virus or equine herpesvirus.
  • antigenic polypeptide examples include equine influenza virus type A/Alaska 91 neuraminidase, equine influenza virus type A/Prague 56 neuraminidase, equine influenza virus type A/Miami 63 neuraminidase, equine influenza virus type A/Kentucky 81 neuraminidaseequine herpesvirus type 1 glycoprotein B, and equine herpesvirus type 1 glycoprotein D.
  • the double stranded foreign DNA sequence of the homology vector encodes an antigenic polypeptide derived from bovine respiratory syncytial virus or bovine parainfluenza virus.
  • the antigenic polypeptide of derived from bovine respiratory syncytial virus equine pathogen can derived from equine influenza virus is bovine respiratory syncytial virus attachment protein (BRSV G) , bovine respiratory syncytial virus fusion protein (BRSV F) , bovine respiratory syncytial virus nucleocapsid protein (BRSV N) , bovine parainfluenza virus type 3 fusion protein, and the bovine parainfluenza virus type 3 hemagglutinin neuraminidase.
  • BRSV G bovine respiratory syncytial virus attachment protein
  • BRSV F bovine respiratory syncytial virus fusion protein
  • BRSV N bovine respiratory syncytial virus nucleocapsid protein
  • the double stranded foreign DNA sequence in the homology vector encodes a cytokine capable of stimulating human immune response.
  • the cytokine may be, but is not limited to, interleukin-2, interleukin-6, interleukin-12 , interferons, granulocyte-macrophage colony stimulating factors, and interleukin receptors.
  • Preferred embodiments of this invention are the homology vectors designated Homology Vector 416-80.21B, 644-09.Al, 644-09.B2, 478-17.IT, 478.10.11, 411-91.01A, 411-91.01B, 510-36.IC, 510-36.2F, 416-88.2L, 639.85.1H, 669-42.04 or 725-26.A10.
  • the present invention further provides a homology vector for producing a recombinant feline herpes virus by inserting a foreign DNA sequence into the feline herpes virus genome which comprises a double-stranded DNA molecule consisting of: a) double-stranded foreign DNA sequence encoding an antigenic polypeptide derived from a cytokine capable of stimulating an immune response; b) at one end of the foreign DNA sequence, double-stranded feline herpes virus genomic DNA homologous to the genomic DNA located at one side of a non-essential site of the feline herpes virus genomic
  • the present invention further provides a host cell infected with the recombinant feline herpes virus.
  • the host cell is a mammalian cell.
  • Other host cells are known to those skilled in the art.
  • the present invention further provides a vaccine for feline herpes virus which comprises an effective immunizing amount of the recombinant feline herpes virus and a suitable carrier.
  • the vaccine against an feline pathogen comprises an effective immunizing amount of the recombinant feline herpes virus and a suitable carrier.
  • This vaccine may contain either inactivated or live recombinant virus.
  • Suitable carriers for the recombinant virus are well known to those skilled in the art and include but are not limited to proteins, sugars, etc.
  • a suitable carrier is a physiologically balanced culture medium containing one or more stabilizing agents such as hydrolyzed proteins, lactose, etc.
  • the live vaccine is created by taking tissue culture fluids and adding stabilizing agents such as stabilizing, hydrolyzed proteins.
  • the inactivated vaccine uses tissue culture fluids directly after inactivation of the virus.
  • the present invention further provides a method of immunizing an animal against a human pathogen which comprises administering to the animal an effective immunizing dose of the feline herpes vaccine.
  • the method of immunizing an animal against an feline pathogen comprises administering to the animal an effective immunizing dose of the feline herpes vaccine.
  • the present invention further provides a multivalent vaccine for feline herpes virus and for one or more of other feline diseases which comprises an effective immunizing amount of a recombinant virus comprising the feline herpes virus viral genome which contains a deletion in the unique short region, wherein the deletion is in the glycoprotein E gene, and an insertion of a foreign gene into a non-essential site of the viral genome.
  • the foreign DNA sequence encodes an antigenic polypeptide which induces host cell production of protective antibodies against a feline disease causing agent from which the antigen is derived or derivable.
  • the invention further provides foreign RNA which encodes a polypeptide.
  • the polypeptide is antigenic in the animal.
  • this antigenic polypeptide is a linear polymer of more than 6 amino acids linked by peptide bonds which stimulates the animal to produce antibodies.
  • the present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant feline herpes virus comprising the feline herpesvirus viral genome which contains a deletion or other alteration in the unique short region of the viral genome, wherein the deletion or alteration is in the glycoprotein E gene so that upon replication, the recombinant virus produces no glycoprotein E.
  • an "effective immunizing amount" of the recombinant feline herpes virus of the present invention is within the range of IO 3 to ⁇ L0 PFU/dose.
  • the immunizing amount is IO 5 to fO PFU/dose.
  • the immunizing amount is IO 6 PFU/dose.
  • the method comprises administering to the animal an effective immunizing dose of the vaccine of the present invention.
  • the vaccine may be administered by any of the methods well known to those skilled in the art, for example, by intramuscular, subcutaneous, intraperitoneal or intravenous injection. Alternatively, the vaccine may be administered intranasally or orally.
  • the present invention also provides a method of immunizing an animal, wherein the animal is a feline, canine, ovine, bovine, caprine, swine or human.
  • this includes immunizing the animal against the virus or viruses which cause the disease or diseases feline herpesvirus.
  • the present invention further provides a method of distinguishing an animal vaccinated with a feline herpes virus from an animal infected with a naturally- occuring feline herpes virus which comprises analysing a sample of a body fluid from the animal for the presence of feline herpes virus gE and at least one other antigen normally expressed in an animal infected by a naturally-occuring feline herpes virus, determining whether the antigen and gE are present in the body fluid, the presence of the antigen and the absence of gE indicative of an animal vaccinated with the vaccine and not infected with a naturally-occuring feline herpes virus.
  • the presence of the antigen and of gE in the body fluid is determined by detecting in the body fluid antibodies specific for the antigen and for gE.
  • the present invention provides a method for testing a feline to determine whether the feline has been vaccinated with the vaccine of the present invention, particularly the embodiment which contains the recombinant feline herpes virus S-FHV-004 or S-FHV-014.
  • S-FHV-000 was obtained from the ATCC (ATCC No. 636) and S-FHV-001 was obtained from the NVSL (NVSL Challange Virus Strain SGE, Lot KS) .
  • FHV virus stock samples were prepared by infecting Crandell Feline Kidney (CRFK) cells at a multiplicity of infection of 1.0 PFU/cell in Dulbecco's Modified Eagle Medium (DMEM) containing 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin (these components were obtained from Irvine Scientific or equivalent supplier, and hereafter are referred to as complete DME medium) plus 5% fetal bovine serum. After cytopathic effect was complete, the medium and cells were harvested, aliquoted and frozen at -70°C. The titers were approximately 1 x IO 7 to 1 x 10 B PFU/ml.
  • a confluent monolayer of CRFK cells in a 25 cm 2 flask or 60 mm petri dish was infected with 100 ml of virus sample. After overnight incubation, or when the cells were showing 100% cytopathic effect, the cells were scraped into the medium. The cells and medium were centrifuged at 3000 rpm for 5 minutes in a clinical centrifuge. The medium was decanted, and the cell pellet was gently resuspended in 0.5 ml solution containing 0.5% NONIDET P-40 ® (octyl phenol ethylene oxide condensate containing an average of 9 moles of ethylene oxide per molecule) (NP-40 ® , purchased from Sigma Chemical Co., St. Louis, MO.) .
  • NONIDET P-40 ® octyl phenol ethylene oxide condensate containing an average of 9 moles of ethylene oxide per molecule
  • the sample was incubated at room temperature for 10 minutes. Ten ml of a stock solution of RNase A (Sigma Chemical Co., St. Louis, MO.) were added (stock was 10 mg/ml, boiled for 10 minutes to inactivate DNAse) . The sample was centrifuged to pellet nuclei. The DNA pellet was removed with a pasteur pipette or wooden stick and discarded. The supernatant fluid was decanted into a 1.5 ml Eppendorf tube containing 25 ml of 20% sodium dodecyl sulfate (Sigma) and 25 ml proteinase-K (10 mg/ml; Boehringer Mannheim Biochemicals, Indianapolis, IN) .
  • RNase A Sigma Chemical Co., St. Louis, MO.
  • the sample was mixed and incubated at 37°C for 30- 60 minutes. An equal volume of water-saturated phenol was added and the sample was mixed briefly. The sample was centrifuged in an Eppendorf minifuge for 5 minutes at full speed. The upper aqueous phase was removed to a new Eppendorf tube, and two volumes of absolute ethanol were added and the tube- put at -20°C for 30 minutes to precipitate nucleic acid. The sample was centrifuged in an Eppendorf minifuge for 5 minutes. The supernatant was decanted, and the pellet was air dried and rehydrated in -16 ml H 2 0. For the preparation of larger amounts of DNA, the procedure was scaled up to start with roller bottles or 175 cm 2 flasks of CRFK cells. The DNA was stored in 0.01 M tris pH 7.5, 1 mM EDTA at 4°C.
  • MOLECULAR BIOLOGICAL TECHNIQUES Techniques for the manipulation of bacteria and DNA, including such procedures as digestion with restriction endonucleases, gel electrophoresis, extraction of DNA from gels, ligation, phosphorylation with kinase, treatment with phosphatase, growth of bacterial cultures, transformation of bacteria with DNA, and other molecular biological methods are described in [22 and 23] .
  • the polymerase chain reaction (PCR) was used to introduce restriction endonuclease sites convenient for the manipulation of various DNAs [24] .
  • amplified fragments were less than 500 base pairs in size and critical regions of amplified fragments were confirmed by DNA sequencing. Except as noted, these techniques were used with minor variations.
  • T4 DNA ligase BBL
  • Ligation reactions contained various amounts of DNA (from 0.2 to 20mg) , 20mM Tris pH 7.5, lOmM MgCl 2 , lOmM dithiothreitol (DTT) , 200 mM ATP and 20 units T4 DNA ligase in 10-20 ml final reaction volume. The ligation proceeded for 3-16 hours at 15°C.
  • Sequencing was performed using the USB Sequenase Kit and 35 S-dATP (NEN) . Reactions using both the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, compressed areas were resolved on formamide gels. Templates were double- stranded plasmid subclones or single stranded M13 subclones, and primers were either made to the vector just outside the insert to be sequenced, or to previously obtained sequence. Alternatively, DNA sequencing was performed on the Applied Biosystems Automated Sequencer Model 388A per instructions of the manufacturer using Taq DNA polymerase and fluorescently-labelled dideoxynucleotides. The sequence obtained was assembled and compared using DNAStar software. Subsequent manipulation and comparison of sequences obtained was performed with Superclone and Supersee programs from Coral Software and DNAStar.
  • the method is based upon the calcium phosphate procedure of Graham and Van der eb [25] with the following modifications.
  • Virus and/or Plasmid DNA were diluted to 298 ml in 0.01 M Tris pH 7.5, ImM EDTA.
  • Forty ml 2M CaCl 2 was added followed by an equal volume of 2X HEPES buffered saline (lOg N-2-hydroxyethyl piperazine N' -2-ethanesulfonic acid (HEPES) , 16g NaCI, 0.74g KC1, 0.25g Na ⁇ PO ⁇ H , 2g dextrose per liter H 2 0 and buffered with NaOH to pH 7.4) .
  • 2X HEPES buffered saline laOg N-2-hydroxyethyl piperazine N' -2-ethanesulfonic acid (HEPES) , 16g NaCI, 0.74g KC1, 0.25g Na ⁇ PO ⁇ H , 2g dextrose
  • the mixture was then incubated on ice for 10 minutes, and then added dropwise to an 80% confluent monolayer of CRFK cells growing in a 60 mm petri dish under 5 ml of medium (DME plus 5% fetal bovine serum) .
  • the cells were incubated 4 hours at 37°C in a humidified incubator containing 5% C0 2 .
  • Media on the plates were aspirated, and cells were treated with 20% glycerol in 1XPBS (1.15g Na 2 HP0 4 , 0.2g KH 2 P0 4 , 0.8g NaCI, 0.2g KCl per liter 2 H 0) for one minute.
  • the cells were washed three times with 5 ml of 1XPBS and then fed with 5ml of medium (DME plus 5% fetal bovine serum) . The cells were incubated at 37°C as above for 3-7 days until cytopathic effect from the virus was 50-100%.
  • Virus was harvested as described above for the preparation of virus stocks. This stock was referred to as a transfection stock and was subsequently screened for recombinant virus by the
  • This method relies upon the homologous recombination between herpesvirus DNA and plasmid homology vector DNA which occurs in tissue culture cells co-transfected with these elements. From 0.1-1.0 mg of plasmid DNA containing foreign DNA flanked by appropriate herpesvirus cloned sequences (the homology vector) were mixed with approximately 0.3mg of intact herpesvirus DNA. The DNAs were diluted to 298 ml in 0.01 M Tris pH 7.5, ImM EDTA and transfected into CRFK cells according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS (see above) .
  • herpesvirus DNA did not require flanking herpesvirus DNA sequences but only required that it have restriction endonuclease sites available to cut out the foreign gene fragment from the plasmid vector.
  • a compatible restriction enzyme was used to cut herpesvirus DNA.
  • a requirement of the technique is that the restriction enzyme used to cut the herpesvirus DNA must cut at a limited number of sites.
  • the restriction enzymes Sfil in S-FHV-010 is appropriate.
  • Herpesvirus DNA is mixed with a 30-fold molar excess of plasmid DNA (typically 5mg of virus DNA to lOmg of plasmid DNA) , and the mixture is cut with the appropriate restriction enzyme.
  • the DNA mixture is phenol extracted and ethanol precipitated to remove restriction endonucleases, and ligated together according to the ligation procedure detailed above.
  • the ligated DNA mixture is then resuspended in 298 ml 0.01 M Tris pH 7.5, ImM EDTA and transfected into CRFK cells according to the DNA TRANSFECTION FOR GENERATING RECOMBINANT VIRUS (see above) .
  • PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS FROM SUBGENOMIC DNA FRAGMENTS The ability to generate herpesviruses by cotransfection of cloned overlapping subgenomic fragments is known to those skilled in the art [26, 27] . If deletions and/or insertions are engineered directly into the subgenomic fragments prior to the cotransfection, this procedure results in a high frequency of viruses containing the genomic alteration, greatly reducing the amount of screening required to purify the recombinant virus. In the first step of this procedure deletions are introduced into separate viruses via homologous recombination with enzymatic marker genes as described below.
  • the homology vector used in this step is constructed such that the enzymatic marker gene is flanked by a restriction endonuclease site that does not cut FHV in the region of the DNA to be deleted.
  • a library of overlapping subgenomic fragments, capable of regenerating wild-type virus is constructed from randomly sheared S-FHV-001 DNA.
  • subgenomic fragments are cloned from each of the individual recombinant viruses containing attenuating deletion/marker gene insertions, which were generated in the first step. In each case the subcloned fragment corresponds in size and location to one of the wild-type subgenomic fragments constructed in the second step.
  • the E.coli ⁇ -galactosidase (lacZ) marker gene When the E.coli ⁇ -galactosidase (lacZ) marker gene was incorporated into a recombinant virus the plaques containing recombinants were visualized by a simple assay. The enzymatic substrate was incorporated (300 mg/ml) into the agarose overlay during the plaque assay. For the lacZ marker gene the substrate BLUOGAL" (halogenated indolyl-?-D-galactoside, Life Technologies, Inc., Bethesda, MD) was used. Virus that expressed active marker enzyme turned blue. The blue plaques were then picked onto fresh CRFK cells, and the recombinant virus was purified by further blue plaque isolation.
  • lacZ E.coli ⁇ -galactosidase
  • a subsequent assay involved plaque purifying white plaques from a background of parental blue plaques. In both cases viruses were typically purified with three to five sequential rounds of plaque purification.
  • viruses containing genomic deletions involved the use of either homologous recombination and/or direct ligation techniques. Initially a virus was constructed via homologous recombination, in which the DNA to be deleted was replaced with a marker gene such as E. coli ⁇ - galactosidase (lacZ) . A second virus was then constructed in which the marker gene was subsequently deleted either by homologous recombination or via direct ligation.
  • lacZ E. coli ⁇ - galactosidase
  • the primary antibody was diluted to the appropriate dilution with PBS plus Blotto and incubated with the cell monolayer for 2 hours to overnight at room temperature. Unbound antibody was removed from the cells by washing four times with PBS at room temperature.
  • the appropriate secondary antibody conjugate was diluted 1:500 with PBS and incubated with the cells for 2 hours at room temperature. Unbound secondary antibody was removed by washing the cells three times with PBS at room temperature.
  • the monolayer was rinsed in color development buffer (lOOmM Tris pH 9.5/ lOOmM NaCI/ 5mM MgCl2), and incubated 10 minutes to overnight at room temperature with freshly prepared substrate solution (0.3 mg/ml Nitro Blue tetrazolium + 0.15 mg/ml 5-Bromo-4-Chloro-3-Indolyl Phosphatase in color development buffer) . the reaction was stopped by replacing the substrate solution with TE
  • ELISA ASSAY Indirect Elisa assay was performed using standard techniques as described [35] .
  • Sera were tested for FHV- and FeLV-specific antibodies by a microtiter technique.
  • Test serum was inactivated for 30 minutes at 56°C.
  • Duplicate 2-fold dilutions of test sera were made in 96-well microtitration plates with a 25 ml- pipettor.
  • Equal volumes of virus suspension containing approximately 300 PFU were added to individual wells and the serum/virus mixtures were incubated at 37°C for 1 hour.
  • 0.05 ml of a CRFK cell suspension containing approximately 4 X IO 5 cells/ml was added to each well. The presence of antibody was indicated by the formation of a complete monolayer in 48 hours.
  • the homology vector 416-80.21B was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It contains a unique Hindlll site into which foreign genes were inserted. Upstream of the unique Hi ⁇ dlll site is an approximately 1417 base pair fragment of FHV DNA which includes all of the gl gene coding sequence. Downstream of the unique Hindlll site is an approximately 5200 base pair fragment of FHV DNA which includes part of the terminal repeat sequence. A detailed description of the plasmid is given in Figures 4A-4B. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of pSP65 (Promega Corp., Madison, WI) .
  • Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment
  • the homology vector 644-09.A1 was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It contains a unique Hindlll site into which foreign genes were inserted. A detailed description of the plasmid is given in Figures lOA-lOC. It was constructed from the indicated DNA sources utlilizing standard recombinant DNA techniques [22, 23] . The plasmid vector is derived from an approximately 2958 base pair Asp7l8I restriction endonuclease fragment of a pSP18/pSP19 fusion such that the multiple cloning site is EcoRI / Sacl /Asp!
  • Fragment 1 is an approximately 1415 base pair Asp718I to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 2205 base pair Sail to Asp718I subfragment of FHV EcoRI E fragment .
  • the homology vector 644-09.B2 was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It contains a unique site into which foreign genes were inserted.
  • a detailed description of the plasmid is given in Figures 11A-11B. It was constructed from the indicated DNA sources utlilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2958 base pair Asp718I restriction endonuclease fragment of a pSP18/pSP19 fusion such that the multiple cloning site is EcoRI/SacI/Asp718I/SacI/EcoRI.
  • Fragment 1 is an approximately 1415 base pair Asp718I to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 2220 base pair Sail to Asp718I subfragment of FHV EcoRI E fragment .
  • the homology vector 478-17.IT was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli 3-galactosidase gene and a pseudorabies virus glycoprotein C-feline leukemia virus glycoprotein 70 (PRV gC-FeLV g70) envelope fusion polypeptide flanked by FHV DNA.
  • the FeLV g70 envelope epitope (FEE #1) polypeptide consists of a 14 amino acid antigenic epitope (NH 2 -MGPNLVLPDQKPPS-COOH; Ref. 21; Figure 2, SEQ ID NO: 8) .
  • IT contains eight copies of FeLV g70 envelope epitope #1 fused in frame internal to the PRV gC polypeptide.
  • the foreign DNA was inserted into a unique Sail site in the FHV homology vector 416-80.21. Upstream of the unique Sail site is an approximately 1417 base pair fragment of FHV DNA that includes all of the FHV gl gene coding sequence. Downstream of the unique Sail site is an approximately 5200 base pair fragment of FHV DNA that includes the unique short sequence and the terminal repeat sequence.
  • a detailed description of the plasmid 478-17.IT is given in Figures 7A-7E. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of pSP65.
  • Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 1154 base pair Pstl to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 3 is an approximately 943 base pair iVcol to Notl subfragment of PRV BamHI #2 [29] .
  • Fragment 4 is an approximately 408 base pair fragment of FeLV g70 envelope gene DNA (8 copies of FeLV g70 envelope epitope #1; Figure 2) .
  • Fragment -5 is an approximately 1116 base pair X ol to Ncol subfragment of PRV BamHI #2 and #9 [29] .
  • Fragment 6 is an approximately 543 base pair iVdel to Kpnl subfragment of PRV Ba ⁇ iHI #7 [29] .
  • Fragment 7 is an approximately 423 base pair Sail to BamHI subfragment of PRV BamHI #10 [29] .
  • Fragment 8 is an approximately 3388 base pair BamHI to Ball subfragment of pJF751 [28] .
  • Fragment 9 is an approximately 784 base pair Smal to Smal subfragment of HSV-1 BamHI Q [30] .
  • Fragment 10 is an approximately 221 base pair Kpnl to Sail subfragment of PRV BamHI #7 [29] .
  • Fragment 11 is an approximately 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment
  • the homology vector 478-10.11 was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli / S-galactosidase gene and a PRV gC-FeLV g70 envelope fusion polypeptide flanked by FHV DNA.
  • the FeLV g70 envelope epitope (FEE #1) polypeptide consists of a 14 amino acid antigenic epitope (NH 2 -MGPNLVLPDQKPPS-COOH; Ref. 21; Figure 2, SEQ ID NO: 8) " .
  • the homology vector 478-10.11 contains four copies of FeLV g70 envelope epitope #1 fused in frame internal to the PRV gC polypeptide. The foreign genes were inserted into a unique Sail site in the FHV homology vector 416-80.21.
  • a detailed description of the plasmid 478-10.11 is given in Figures 7A-7E. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of pSP65. Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment (of the FHV Sail B fragment.
  • Fragment 2 is an approximately 1154 base pair Pstl to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 3 is an approximately 943 base pair Wcol to Notl subfragment of PRV BamHI #2 [29] .
  • Fragment 4 is an approximately 204 base pair fragment of FeLV g70 envelope gene DNA (4 copies of FeLV g70 envelope epitope #1; Figure 2) .
  • Fragment 5 is an approximately 1116 base pair Xhol to Wcol subfragment of PRV BamHI #2 and #9 [29] .
  • Fragment 6 is an approximately 543 base pair Ndel to Kpnl subfragment of
  • Fragment 7 is an approximately 423 base pair Sail to BamHI subfragment of PRV BamHI #10
  • Fragment 8 is an approximately • 3388 base pair BamHI to Ball subfragment of pJF751 [28] .
  • Fragment 9 is an approximately 784 base pair Smal to Smal subfragment of HSV-1 BamHI Q [30] .
  • Fragment 10 is an approximately 221 base pair Kpnl to Sail subfragment of PRV BamHI #7 [29] .
  • Fragment 11 is an approximately 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment
  • the homology vector 411-91.01A was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli j ⁇ -galactosidase gene and a PRV gC-FeLV g70 envelope fusion polypeptide flanked by FHV DNA.
  • the FeLV g70 envelope epitope (FEE #1) polypeptide consists of a 14 amino acid antigenic epitope (NH 2 -MGPNLVLPDQKPPS-COOH; Ref. 21; Figure 2, SEQ ID NO: 8) .
  • the homology vector 411-91.01A contains four copies of FeLV g70 envelope epitope #1 fused in frame internal to the PRV gC polypeptide. The foreign genes were inserted into Pstl and Sail sites in the FHV homology vector 416-80.21.
  • a detailed description of the plasmid 411-91.01A is given in Figures 3A-3D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of pSP65. Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 1154 base pair Pstl to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 3 is an approximately 3006 base pair BamHI to PvuII subfragment of pJF751 [28] .
  • Fragment 4 is an approximately 204 base pair of FeLV g70 envelope gene DNA (4 copies of FeLV g70 envelope epitope #1; Figure 2) .
  • Fragment 5 is an approximately 751 base pair Wdel to Sail subfragment of PRV BamHI #7 [29] .
  • Fragment 6 is an approximately • 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment.
  • the homology vector 411-91.01B was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli lacZ-FeLV g70 envelope fusion polypeptide flanked by FHV DNA.
  • the FeLV g70 envelope epitope (FEE #1) polypeptide consists of a 14 amino acid antigenic epitope (NH 2 -MGPNLVLPDQKPPS-COOH) (Ref.
  • the FeLV g70 envelope epitope (FEE #3) polypeptide consists of a 14 amino acid antigenic epitope (NH 2 -AKLRERLKEREQLF-COOH) (Ref. 21; Figure 2, SEQ ID NO: 10) .
  • the homology vector 411-91.01A contains four copies of FEE #1, one copy of FEE #3, one copy of FEE #1 and one copy of FEE #3 fused in frame to the carboxy terminus of the E. coli ⁇ - galactosidase (lacZ) polypeptide.
  • lacZ E. coli ⁇ - galactosidase
  • the plasmid 411-91.01B is given in Figures 3A-3D. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of pSP65.
  • Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 1154 base pair Pstl to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 3 is an approximately 3006 base pair BamHI to PvuII subfragment of pJF751 [28] .
  • Fragment 4 is an approximately 357 base pair assembly of the FeLV g70 envelope gene (a concatemer of four copies of FEE #1, one copy of FEE #3, one copy of FEE #1, and one copy of FEE #3; Figure 2) .
  • Fragment 5 is an approximately 751 base pair Wdel to Sail subfragment of PRV BamHI #7 [29] .
  • Fragment 6 is an approximately 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment.
  • HOMOLOGY VECTOR 510-36.1C The homology vector 510-36. IC was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli / S-galactosidase gene and a hepatitis B core antigen-FeLV g70 envelope fusion polypeptide flanked by FHV DNA.
  • the FeLV g70 envelope epitope (FEE #1) polypeptide consists of a 14 amino acid antigenic epitope (NH 2 -MGPNLVLPDQKPPS-COOH; Ref. 21; Figure 2, SEQ ID NO: 8) .
  • the homology vector 510- 36.1C contains four copies of FeLV g70 envelope epitope #1 fused in frame at the carboxy terminus of the hepatitis B core antigen polypeptide.
  • the foreign genes were inserted into Pstl and Sail sites in the FHV homology vector 416-80.21.
  • a detailed description of the plasmid 510-36.IC is given in Figures 8A-8E. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of pSP65.
  • Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 1154 base pair Pstl to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 3 is an approximately 204 base pair fragment of FeLV g70 envelope gene DNA (4 copies of FEE #1; Figure 2) .
  • Fragment 4 is an approximately 590 base pair BamHI to Kpnl fragment of hepatitis B core antigen DNA.
  • Fragment 5 is an approximately 543 base pair Wdel to Kpnl subfragment of PRV BamHI #7
  • Fragment 6 is an approximately 423 base pair Sail to BamHI subfragment of PRV BamHI #10 [29] .
  • Fragment 7 is an approximately 3388 base pair ⁇ BamHI to Ball subfragment of pJF751 [28] .
  • Fragment 8 is an approximately 784 base pair Smal to Smal subfragment of
  • Fragment 9 is an approximately 221 base pair Kpnl to Sail subfragment of PRV BamHI #7
  • Fragment 10 is an approximately 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment.
  • the homology vector 510-36.2F was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli ⁇ -galactosidase gene and a hepatitis B core antigen-FeLV g70 envelope fusion polypeptide flanked by FHV DNA.
  • the FeLV g70 envelope epitope (FEE #1) polypeptide consists of a 14 amino acid antigenic epitope (NH 2 -MGPNLVLPDQKPPS-COOH; Ref. 21; Figure 2, SEQ ID NO: 8) .
  • the homology vector 510- 36.2F contains eight copies of FeLV g70 envelope epitope #1 fused in frame at the carboxy terminus of the hepatitis B core antigen polypeptide.
  • the foreign genes were inserted into Pstl and Sail sites in the FHV homology vector 416-80.21.
  • a detailed description of the plasmid 510-36.2F is given in Figures 8A-8E. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of pSP65.
  • Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 1154 base pair Pstl to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 3 is an approximately 408 base pair fragment of FeLV g70 envelope gene DNA (8 copies of FEE #1; Figure 2) .
  • Fragment 4 is an approximately 590 base pair BamHI to Kpnl fragment of hepatitis B core antigen gene DNA.
  • Fragment 5 is an approximately 543 base pair iVdel to Kpnl subfragment- of PRV BamHI #7 [29] .
  • Fragment 6 is an approximately 423 base pair Sail to BamHI subfragment of PRV BamHI #10 [29] .
  • Fragment 7 is an approximately 3388 base pair BamHI to Ball subfragment of pJF751 [28] .
  • Fragment 8 is an approximately 784 base pair Smal to Smal subfragment of HSV-1 BamHI Q [30] .
  • Fragment 9 is an approximately 221 base pair Kpnl to Sail subfragment of PRV BamHI #7
  • Fragment 10 is an approximately 5200 base pair
  • the homology vector 416-88.2L was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli ⁇ -galactosidase gene flanked by FHV DNA. The foreign gene was inserted into a Pstl site in the FHV homology vector 416-80.21.
  • a detailed description of the plasmid 416-88.2L is given in Figures 5A-5C. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2817 base pair Sacl to PvuII restriction endonuclease fragment of a pSP65.
  • Fragment 1 is an approximately 1417 base pair Sacl to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 423 base pair Sail to BamHI subfragment of PRV BamHI #10 [29] .
  • Fragment 3 is an approximately 3388 base pair BamHI to Ball subfragment of pJF751 [28] .
  • Fragment 4 is an approximately 751 base pair Ndel to Sail subfragment of PRV BamHI #7 [29] .
  • Fragment 5 is an approximately 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment.
  • the homology vector 639-85. IH was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli /3-galactosidase gene and a Dirofilaria immi tis DNA clone encoding a 22 kd immunogenic polypeptide (DiPLA2) flanked by FHV DNA. The foreign genes were inserted into Notl site in the FHV homology vector 644-09.B2. A detailed description of the plasmid 639-85.IH is given in Figures 9A-9E. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2958 base pair Asp718I to Asp718I restriction endonuclease fragment of a pSP18/19.
  • Fragment 1 is an approximately 1415 base pair Asp718I to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 179 base pair Pstl to EcoRV subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 3 is an approximately 423 base pair Sail to BamHI subfragment of PRV BamHI #10 [29] .
  • Fragment 4 is an approximately 3006 base pair BamHI to PvuII subfragment of pJF751 [28] .
  • Fragment 5 is an approximately 751 base pair Wdel to Sail subfragment of PRV BamHI #7 [29] .
  • Fragment 6 is an approximately 977 base pair EcoRV to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 7 is an approximately 618 base pair EcoRI to Xhol fragment of the D. immi tis DiPLA2 gene [34] .
  • Fragment 8 is an approximately 2205 base pair Sail to Asp718I subfragment of the FHV EcoRI E fragment .
  • the homology vector 669-42.04 was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli -galactosidase gene and a feline immunodeficiency virus (FIV) protease
  • PCR PCR
  • the foreign genes were inserted into the iVotl site in the FHV homology vector 644-09.B2.
  • a detailed description of the plasmid 669-42.04 is given in Figures 12A-12E. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques [22, 23] .
  • the plasmid vector is derived from an approximately 2958 base pair Asp718I to Asp718I restriction endonuclease fragment of a pSP18/19.
  • Fragment 1 is an approximately 1415 base pair Asp718I to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 221 base pair Sail to Kpnl subfragment of PRV BamHI #7 [29] .
  • Fragment 3 is an approximately 1154 base pair Pstl to Avail subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 4 is an approximately 1853 base pair Bgrlll to iVcol fragment of the FIV gag gene [32] .
  • the 3' end of the gene is an approximately 1360 base pair Xmal to Ncol subfragment of the FIV PPR genomic cDNA.
  • the 5' end of the gene (an approximately 560 base pair Bglll to Xmal fragment) was synthesized by PCR.
  • the template for the PCR reaction was FIV strain PPR genomic cDNA [32] .
  • the upstream primer 03 (5' -GCGAGATCTGAAAATGGCCATTAAGAGATG-3' ; SEQ ID NO: 3) was synthesized to correspond to DNA from the 5' end of the FIV gag gene starting at nucleotide 653 of FIV strain PPR genomic cDNA (amino acid 10 of FIV gag protein) and to introduce a Bglll site at the 5' end.
  • the downstream primer 04 (5' -GTGCTGCAGTAAAATAGGG-3 ' ; SEQ ID NO: 4) was synthesized to correspond to DNA starting at nucleotide 1323 of FIV PPR genomic cDNA and is positioned downstream of an Xmal site at nucleotide 1207.
  • Fragment 5 is an approximately 784 base pair Smal to Smal subfragment of the HSV-1 BamHI Q fragment [30] .
  • Fragment 6 is an approximately 543 base pair Kpnl to Wdel subfragment of PRV BamHI #7 [29] .
  • Fragment 7 is an approximately 3006 base pair PvuII to BamHI subfragment of pJF751 [28] .
  • Fragment 8 is an approximately 423 base pair BamHI to Sail subfragment of PRV BamHI #10 [29] .
  • Fragment 9 is an approximately 2205 base pair Sail to Asp718I subfragment of the FHV EcoRI E fragment.
  • the homology vector 725-26.A10 was constructed for the purpose of deleting a portion of the gE coding region from the feline herpesvirus and inserting a foreign DNA. It incorporates an E. coli ⁇ -galactosidase gene and a feline immunodeficiency virus (FIV) envelope (env) gene flanked by FHV DNA.
  • the FIV env gene was synthesized by the polymerase chain reaction (PCR) .
  • the foreign genes were inserted into a Wbtl site in the FHV homology vector 644-09.B2 ( Figures 13A-13E) .
  • Figures 13A-13E A detailed description of the plasmid 639-85.IH is given in Figures 9A-9E.
  • the plasmid vector is derived from an approximately 2958 base pair Asp718I to Asp718I restriction endonuclease fragment of a pSP18/l9.
  • Fragment 1 is an approximately 1415 base pair Asp718I to Smal subfragment of the FHV Sail B fragment.
  • Fragment 2 is an approximately 423 base pair Sail to BamHI subfragment of PRV BamHI #10 [29] .
  • Fragment 3 is an approximately 3006 base pair BamHI to PvuII subfragment of pJF751 [28] .
  • Fragment 4 is an approximately 751 base pair Ndel to Sail subfragment of- PRV BamHI #7 [29] .
  • Fragment 5 is an approximately 784 base pair Smal to Smal subfragment of the HSV-1 BamHI Q [30] .
  • Fragment 6 is an approximately 2564 base pair BamHI to BamHI fragment of the FIV env gene [32] synthesized by PCR. The template for the PCR reaction was FIV strain PPR genomic cDNA [32] .
  • the upstream primer 10/93.21BW (5' -GCCCGGATCCTATGGCAGAAGGGTTTGCAGC- 3' ; SEQ ID NO: 5) was synthesized corresponding to the 5' end of the FIV env gene starting at nucleotide 6263 of FIV strain PPR genomic cDNA, and the procedure introduced a BamHI site at the 5' end. The BamHI site was destroyed during the cloning of the PCR fragment .
  • the downstream primer 10/93.20BW (5'- CCGTGGATCCGGCACTCCATCATTCCTCCTC-3' ; SEQ ID NO: 6) was synthesized corresponding to the 3' end of the FIV env gene starting at nucleotide 8827 of FIV PPR genomic cDNA, and the procedure introduced a BamHI site at the 3' end.
  • Fragment 7 is an approximately 1154 base pair Avail to Pstl subfragment of the HCMV 2.1 kb Pstl E fragment [31] .
  • Fragment 8 is an approximately 2205 base pair Sail to Asp718I subfragment of the FHV EcoRI E fragment .
  • Example 1 Recombinant feline herpesvirus (FHV) containing a deletion of the entire gE gene and an insertion of a foreign DNA sequence into that site will replicate in cats and is useful as a vaccine.
  • FHV feline herpesvirus
  • Recombinant FHV (SEQ ID NOs: 1 and 2) is non essential for replication of the recombinant FHV.
  • Recombinant FHV expressing foreign genes for viral, bacterial or parasite antigens protect against disease in dogs and cats.
  • Recombinant FHV which was isolated contains a deletion of the gE gene within the unique short and an insertion of a foreign gene into that gE site or any nonessential site will replicate in cats and is useful as a vaccine.
  • Recombinant FHV containing a deletion of the gE gene within the unique short and an insertion of a foreign gene into that site is useful as a vaccine.
  • Recombinant FHV expressing foreign genes for viral, bacterial, or parasite antigens is useful as a vaccine to protect against disease in cats, dogs, humans, horses, cattle, swine and poultry.
  • Homology vectors 416-80.21, 644-09.Al and 644-09.B2 are deleted for the entire gE gene and are useful in the present invention for insertion and expression of foreign genes in recombinant FHV.
  • Homology vectors 416- 80.21, 644-09.Al and 644-09.B2 carry a 1638 base pair deletion of the gE gene from the Smal site in the FHV Sail B fragment to the Sail site in the FHV EcoRI E fragment (see Figures 4, 10 and 11) .
  • the sequence of the gE gene (SEQ ID NOs: 1 and 2) includes the coding sequence for the 531 amino acid open reading frame of the gE gene and also includes the Smal site in the FHV Sail B fragment and the Sail site in the FHV EcoRI E fragment which define the endpoints of the deletion of the gE gene.
  • homology vectors 416-80.21, 644-09.Al and 644-09.B2 contain an approximately 1415 base pair Asp718I to Smal subfragment of FHV Sail B containing the entire coding sequence of the gl gene (370 amino acids) .
  • homology vector 416-80.21 contains an approximately 5200 base pair Sail to PvuII subfragment of the FHV EcoRI E fragment which contains unique short and terminal repeat sequences.
  • homology vectors 644-09.Al and 644-09.B2 To the opposite side of the FHV gE deletion in homology vectors 644-09.Al and 644-09.B2, an approximately 2205 base pair Sail to Asp718I subfragment of the FHV EcoRI E fragment contains unique short and terminal repeat sequences.
  • Recombinant FHV containing a deletion of the gG gene within the unique short and an insertion of a foreign gene into that site will replicate in cats and is useful as vaccine.
  • Recombinant FHV expressing foreign genes for viral, bacterial, or parasite antigens is useful as a vaccine to protect against disease in cats, dogs, humans, horses, cattle, swine and poultry.
  • the ability to isolate a gG-deleted FHV confirms that the FHV gG gene is non-essential for replication of the recombinant FHV.
  • a homology vector containing a deletion of the gG gene and an insertion of a foreign gene is constructed.
  • a recombinant FHV is isolated utilizing the gG deleted homology vector and virus S- FHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS.
  • Recombinant FHV containing a deletion of the gl gene within the unique short or a deletion in the repeat region, and an insertion of a foreign gene into that site will replicate in cats and is useful as a vaccine.
  • Recombinant FHV expressing foreign genes for viral, bacterial, or parasite antigens is useful as a vaccine to protect against disease in cats, dogs, humans, horses, cattle, swine and poultry.
  • the ability to isolate a gl-deleted FHV or repeat- deleted FHV confirms that the FHV gl gene or FHV repeat sequence is non-essential for replication of the recombinant FHV.
  • a homology vector containing a deletion of the gl gene or repeat sequence, and an insertion of a foreign gene is constructed.
  • a recombinant FHV is isolated utilizing the gl deleted homology vector or the repeat-deleted homology vector, and virus S-FHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS.
  • Recombinant FHV containing a deletion of the gE gene and a deletion of the gG and/or gl genes within the unique short and an insertion of a foreign gene into a nonessential site and is useful as a vaccine to protect against disease in cats, dogs, humans, horses, cattle, swine and poultry.
  • Recombinant viruses S-FHV-003, S-FHV-004 and S-FHV-006 are each useful as a vaccine in cats against feline leukemia and feline rhinotracheitis.
  • S-FHV-003 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of a foreign gene at the gE deletion site.
  • a fusion gene containing four copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2, SEQ ID NOs: 7 and 8) is fused in the correct translational reading frame to the 3' end of the E. coli lacZ gene ( / S-galactosidase) .
  • the FeLV env-lacZ fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-003 was derived from S-FHV-000 (ATCC No. 636) . This was accomplished utilizing the homology vector 411-91.01A (see Materials and Methods) and virus S-FHV- 000 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-003. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FeLV env-lacZ fusion gene and the deletion of the 1638 base pair gE gene.
  • S-FHV-003 was assayed for expression of FeLV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Polyclonal mouse anti FeLV EE#1 peptide antisera was shown to react specifically with S-FHV-003 plaques and not with S-FHV- 000 negative control plaques. Every viral plaque of S- FHV-003 that was analyzed from all stages of propagation reacted with the antiserum indicating that the virus was stably expressing the FeLV foreign gene.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines .
  • S-galactosidase-FeLV env fusion gene product cells were infected with S- FHV-003 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal mouse anti FeLV EE#1 peptide antisera was used to detect expression of ⁇ - galactosidase-FeLV envelope fusion protein. The lysate from S-FHV-003 infected cells exhibited a band at approximately 150 kd which is the expected size of the 3-galactosidase-FeLV env fusion protein.
  • S-FHV-004 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of a foreign gene at the gE deletion site.
  • a fusion gene containing four copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2, SEQ ID NOs: 7 and 8) , one copy of FeLV EE #3 ( Figure 2, SEQ ID NOs: 9 and 10) , one copy of FeLV EE #1, and one copy of FeLV EE #3 are fused in the correct translational reading frame to the 3' end of the E. coli lacZ gene ( ⁇ - galactosidase) .
  • the FeLV env-lacZ fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-004 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 411-91.01B (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-004. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FeLV env-lacZ fusion gene and the deletion of the 1638 base pair gE gene.
  • Table 1 shows the results of vaccinating cats with recombinant FHV S-FHV-003 or S-FHV-004 (4 cats in each group) .
  • Cats were vaccinated intramuscularly with approximately 1 X IO 6 PFU of S-FHV-003 or S-FHV-004 on Day 0, Day 20, and Day 59.
  • Sera from cats bled on days 0, 29, 59 and 69 were assayed for antibodies to FeLV gp70 protein.
  • ELISA ASSAY detected FeLV gp70 -specif ic antibodies in the serum of S-FHV-003 and S-FHV-004 vaccinated cats.
  • S-FHV-006 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of a foreign gene at the gE deletion site.
  • a fusion gene containing four copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2 SEQ ID NOs: 7 and 8) is fused in the correct translational reading frame to the 3' end of the E. coli lacZ gene ( / ⁇ -galactosidase) .
  • the FeLV env-lacZ fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-006 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 411-91.01A (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-006. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FeLV env-lacZ fusion gene and the deletion of the 1638 base pair gE gene.
  • S-FHV-006 was assayed for expression of FeLV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Polyclonal mouse anti FeLV EE#1 peptide antisera was shown to react specifically with S-FHV-006 plaques and not with S-FHV- 000 negative control plaques. Every viral plaque of S- FHV-006 that was analyzed from all stages of propagation reacted with the antiserum indicating that the virus was stably expressing the FeLV foreign gene.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines.
  • FHV-006 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis.
  • Recombinant feline herpesviruses express antigenic epitopes of the feline leukemia virus envelope gene fused to the pseudorabies virus glycoprotein C (PRV gC) gene in animals.
  • Recombinant viruses S-FHV-007, S-FHV- 009 and S-FHV-012 are each useful as a vaccine in cats against feline leukemia and feline rhinotracheitis.
  • S-FHV-007 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of a foreign gene at the gE deletion site.
  • a fusion gene containing six copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2 SEQ ID NOs: 7 and 8) is fused in the correct translational reading frame within the PRV gC gene.
  • the six copies of FeLV EE#1 are in frame between amino acids 1 to 313 and amino acids 421 to 467 of the PRV glycoprotein C.
  • the FeLV env-PRV gC fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-007 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 478-17.IT (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-007. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure.
  • S-FHV-007 was assayed for expression of FeLV-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Polyclonal mouse anti FeLV EE#1 peptide antisera was shown to react specifically with S-FHV-007 plaques and not with S-FHV- 000 negative control plaques. Every viral plaque of S- FHV-007 that was analyzed from all stages of propagation reacted with the antisera indicating that the virus was stably expressing the FeLV envelope peptide.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines.
  • S-FHV-009 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of a foreign gene at the gE deletion site.
  • a fusion gene containing eight copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2, SEQ ID NOs: 7 and 8) is fused in the correct translational reading frame within the PRV gC gene.
  • the eight copies of FeLV EE#1 are in frame between amino acids 1 to 313 and amino acids 421 to 467 of the PRV glycoprotein C.
  • the FeLV env-PRV gC fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-009 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 478-17.IT (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-009. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FeLV env-PRV gC fusion gene and the deletion of the 1638 base pair gE gene.
  • S-FHV-009 was assayed for expression of FeLV-and PRV- specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Polyclonal mouse anti FeLV EE#1 peptide antisera and polyclonal goat 282 antiserum reactive against denatured PRV gC antigen [33] were shown to react specifically with S- FHV-009 plaques and not with S-FHV-000 negative control plaques. Every viral plaque of S-FHV-009 that was analyzed from all stages of propagation reacted with the antisera indicating that the virus was stably expressing the FeLV envelope peptide and the PRV glycoprotein C.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines.
  • S-FHV-012 is a recombinant feline herpesvirus that has a deletion of the gE gene and the insertion of a foreign gene at the gE deletion site.
  • a fusion gene containing four copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2, SEQ ID NOs: 7 and 8) is fused in the correct translational reading frame within the PRV gC gene.
  • the four copies of FeLV EE#1 are in frame between amino acids 1 to 313 and amino acids 421 to 467 of the PRV glycoprotein C.
  • the FeLV env-PRV gC fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-012 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 478-10.11 (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-012. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FeLV env-PRV gC fusion gene and the deletion of the 1638 base pair gE gene.
  • S-FHV-012 was assayed for expression of FeLV-and PRV- specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Polyclonal mouse anti FeLV EE#1 peptide antisera and polyclonal goat 282 antiserum reactive against denatured PRV gC antigen [33] were shown to react specifically with S- FHV-012 plagues artd not with S-FHV-000 negative control plaques. Every viral plaque of S-FHV-012 that was analyzed from all stages of propagation reacted with the antisera indicating that the virus was stably expressing the FeLV envelope peptide and the PRV glycoprotein C.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines.
  • Recombinant feline herpesviruses express antigenic epitopes of the feline leukemia virus envelope gene fused to the hepatitis B virus core antigen gene in animals.
  • Recombinant viruses S-FHV-008 and S-FHV-011 are each useful as a vaccine in cats against feline leukemia and feline rhinotracheitis.
  • S-FHV-008 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of a foreign gene at the gE deletion site.
  • a fusion gene containing four copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2, SEQ ID NOs: 7 and 8) is fused with the correct translational reading frame to the 5' end of the hepatitis B core antigen gene.
  • the FeLV env-hepatitis B core antigen fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-008 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 510-36.1C (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-008. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FeLV env-hepatitis B core antigen fusion gene and the deletion of the 1638 base pair gE gene.
  • S-FHV-008 was assayed for expression of FeLV- and hepatitis B-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Polyclonal mouse anti FeLV EE#1 peptide antisera and polyclonal rabbit anti-hepatitis B core antigen antisera were shown to react specifically with S-FHV- 008 plaques and not with S-FHV-000 negative control plaques. Every viral plaque of S-FHV-008 that was analyzed from all stages of propagation reacted with the antiserum indicating that the virus was stably expressing the FeLV env-hepatitis B core antigen fusion foreign gene.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines.
  • FeLV env-hepatitis B core antigen fusion gene product cells were infected with S-FHV-008 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal mouse anti FeLV EE#1 peptide antisera and polyclonal rabbit anti hepatitis B core antigen antisera were used to detect expression of FeLV env-hepatitis B core antigen fusion protein.
  • the lysate from S-FHV-008 infected cells exhibited a band at approximately 32 kd which is the expected size of the FeLV env (4 copies of FeLV EE#D- hepatitis B core antigen fusion protein.
  • S-FHV-011 is a recombinant feline herpesvirus that has a deletion of the gE gene within the unique short region and the insertion of two foreign genes at the gE deletion site.
  • a fusion gene containing eight copies of the feline leukemia virus envelope epitope #1 (FeLV EE #1; Figure 2, SEQ ID NOs: 7 and 8) is fused with the correct translational reading frame to the 5' end of the hepatitis B core antigen gene.
  • the FeLV env- hepatitis B core antigen fusion gene is under the transcriptional control of the HCMV immediate early promoter.
  • S-FHV-011 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 510-36.2F (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-011. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FeLV env-hepatitis B core antigen fusion gene and the deletion of the 1638 base pair gE gene.
  • S-FHV-011 was assayed for expression of FeLV- and hepatitis B-specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Polyclonal mouse anti FeLV EE#1 peptide antisera and polyclonal rabbit anti-hepatitis B core antigen antisera were shown to react specifically with S-FHV- 011 plaques and not with S-FHV-000 negative control plaques. Every viral plaque of S-FHV-011 that was analyzed from all stages of propagation reacted with the antiserum indicating that the virus was stably expressing the FeLV env-hepatitis B core antigen fusion foreign gene.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines.
  • FeLV env-hepatitis B core antigen fusion gene product cells were infected with S-FHV-011 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal mouse anti FeLV EE#1 peptide antisera and polyclonal rabbit anti hepatitis B core antigen antisera were used to detect expression of FeLV env-hepatitis B core antigen fusion protein. The lysate from S-FHV-011 infected cells exhibited a band at approximately 40 kd which is the expected size of the FeLV env (8 copies of FeLV EE#1) -hepatitis B core antigen fusion protein.
  • Example 7 Recombinant feline herpesvirus express the E. coli lacZ (/3-galactosidase) gene in animals.
  • Recombinant viruses S-FHV-005 and S-FHV-010 are each useful as a vaccine in cats against feline rhinotracheitis.
  • S-FHV-005 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of a foreign gene at the gE deletion site.
  • the foreign gene is an E. coli lacZ ( / S-galactosidase) gene under the transcriptional control of the PRV gX promoter.
  • S-FHV-005 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 416-88.2L (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-005.
  • This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure.- This analysis confirmed the insertion of the E. coli lacZ ( ⁇ - galactosidase) genes and the deletion of the 1638 base pair gE gene.
  • S-FHV-010 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of an unique Sfil restriction endonuclease site at the gE deletion site.
  • S-FHV-010 was derived from S-FHV-005. This was accomplished utilizing the virus S-FHV-005 which was digested with Sfil and religated to remove the PRV gX promoter-E. coli lacZ marker cassette leaving a unique Sfil restriction endonuclease site within the FHV gE deletion site. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final-result of white plaque purification was the recombinant virus designated S-FHV-010. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the Sfil restriction endonuclease site and the deletion of the 1638 base pair gE gene.
  • S-FHV-014 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of two foreign genes at the gE deletion site.
  • the Dirofilaria immi tis 22 kD (DiPLA2) gene is under the transcriptional control of the HCMV immediate early promoter and the E. coli lacZ ( / ⁇ -galactosidase) gene is under the transcriptional control of the PRV gX promoter.
  • S-FHV-014 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 639-85.1H (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-014. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the D. immi tis 20/22 kD (DiPLA2) and the E. coli lacZ (/S-galactosidase) genes and the deletion of the 1638 base pair gE gene.
  • S-FHV-014 was assayed for expression of Dirofilaria immi tis 22kD (DiPLA2) -specific antigens using the BLACK PLAQUE SCREEN FOR FOREIGN GENE EXPRESSION IN RECOMBINANT FHV.
  • Rabbit anti-recombinant 22kd (Dipla2) antisera was shown to react specifically with S-FHV-014 plaques and not with S-FHV-000 negative control plaques. Every viral plaque of S-FHV-014 that was analyzed from all stages of propagation reacted with the antiserum indicating that the virus was stably expressing the D. immi tis 22kD (DiPLA2) foreign gene.
  • the assays described here were carried out in CRFK cells, indicating that CRFK cells would be a suitable substrate for the production of FHV recombinant vaccines.
  • D. immi tis 20/22kD DiPLA2 To confirm the expression of the D. immi tis 20/22kD DiPLA2 gene product, cells were infected with S-FHV-014 and samples of infected cell lysates were subjected to SDS-polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal dog convalescent anti -Dirofilaria immi tis antisera was used to detect expression of D. immi tis 20/22kD DiPLA2 protein. The lysate from S-FHV- 014 infected cells exhibited bands at approximately 26kd, 28kd, 32kd which correspond to glycosylated forms of the D.
  • S-FHV-014 is useful as a vaccine in dogs and cats against heartwor disease caused by the D. immi tis parasite. Cats vaccinated with S-FHV-014 demonstrated significant protection from infection after challenge with the D. immi tis parasite compared to unvaccinated control cats.
  • Example 9 Recombinant feline herpesviruses express feline immunodeficiency virus (FIV) and the E. coli lacZ ( ⁇ - galactosidase) genes in animals.
  • Recombinant viruses S- FHV-016 and S-FHV-017 are useful as a vaccine in cats against feline rhinotracheitis and disease caused by feline immunodeficiency virus.
  • S-FHV-016 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of two foreign genes at the gE deletion site.
  • the FIV gag (p50; protease) gene is under the transcriptional control of the HCMV immediate early promoter and the E. coli lacZ ( / ⁇ -galactosidase) gene is under the transcriptional control of the PRV gX promoter.
  • S-FHV-016 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 669-42.04 (see Materials and Methods) and virus S-FHV- 001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-016. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FIV gag (p50) gene and the E. coli lacZ (3-galactosidase) gene and the deletion of the 1638 base pair gE gene.
  • FIV gag (p50) gene product cells were infected with S-FHV-016 and samples of infected cell lysates were subjected to SDS- polyacrylamide gel electrophoresis. The gel was blotted and analyzed using the WESTERN BLOTTING PROCEDURE. Polyclonal cat anti-FIV (PPR strain) sera was used to detect expression of FIV gag protein. The lysate from S-FHV-016 infected cells exhibited a band at 50 kd which is the expected size of the unprocessed form of the FIV gag protein and a fainter 32 to 35 kd band which may be the result of aberrant cTeavage of the gag protein. In addition, monoclonal antibodies to FIV capsid reacted with a 50 kd protein (the expected size of the FIV gag protein) on a Western blot.
  • S-FHV-017 is a recombinant feline herpesvirus that has a deletion of the gE gene and an insertion of two foreign genes at the gE deletion site.
  • HCMV immediate early promoter and the E. coli lacZ ( ⁇ - galactosidase) gene is under the transcriptional control of the PRV gX promoter.
  • S-FHV-017 was derived from S-FHV-001 (NVSL strain) . This was accomplished utilizing the homology vector 725-26.A10 (see Materials and Methods) and virus S- FHV-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS. The transfection stock was screened by the SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The final result of blue plaque purification was the recombinant virus designated S-FHV-017. This virus was characterized by restriction endonuclease mapping and the SOUTHERN BLOTTING DNA procedure. This analysis confirmed the insertion of the FIV env gene and the E. coli lacZ ( / ⁇ -galactosidase) gene and the deletion of the 1638 base pair gE gene.
  • Recombinant feline herpesvirus expressing antigens from disease causing microorganisms In addition to recombinant feline herpesvirus expressing antigens from feline leukemia virus, feline immunodeficiency virus and Dirofilaria immi tis (heartworm) .
  • Aadditional antigens from disease causing microorganisms in cats include, but art not limited to Dirofilaria immi tis p39 and 22kD antigens, feline infectious peritonitis virus, calicivirus, rabies, virus, feline parvovirus (panleukopenia virus) , feline coronavirus and feline Chla ydia, Toxoplasma gondii.
  • Disease causing microorganisms in dogs include, but are not limited to canine distemper, canine adenovirus type 1 (hepatitis) , adenovirus type 2 (respiratory disease) , parainfluenza, leptospira canicola, icterohemorragia, parvovirus, coronavirus, borrelia burgdorferi, canine herpesvirus, bordetella bronchiseptica and rabies virus.
  • Recombinant feline herpesviruses are useful for expressing antigens from disease causing microorganisms from animals in addition to dogs and cats.
  • Recombinant feline herpesvirus is useful as a vaccine in animals including but not limited to humans, horses, cattle, swine and poultry.
  • Recombinant feline herpesvirus is useful as a vaccine against equine diseases when foreign antigens from the following diseases or disease organisms are expressed in the feline herpesvirus vector: equine influenza, equine herpesvirus-1 and equine herpesvirus-4.
  • Recombinant feline herpesvirus is useful as a vaccine against bovine diseases when foreign antigens from the following diseases, or disease organisms are expressed in the feline herpesvirus vector: bovine viral diarrhea virus, bovine respiratory syncytial virus, bovine parainfluenza virus.
  • Recombinant feline herpesvirus is useful as a vaccine against swine diseases when foreign antigens from the following diseases or disease organisms are expressed in the feline herpesvirus vector: pseudorabies virus, porcine reproductive and respiratory syndrome (PRRS/SIRS) , hog cholera virus, swine influenza virus, swine parvovirus, swine rotavirus.
  • Recombinant feline herpesvirus is useful as a vaccine against poultry diseases when foreign antigens from the following diseases or disease organisms are expressed inthe feline herpesvirus vector: infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, Marek's disease virus, infectious laryngotracheitis virus.
  • Recombinant feline herpesvirus is useful as a vaccine against human diseases.
  • human influenza is a rapidly evolving virus whose neutralizing viral epitopes are rapidly changing.
  • a useful recombinant feline herpesvirus vaccine is one in which the influenza neutralizing eptiopes are quickly changed to protect against new strains of influenza.
  • Human influenza HA and NA genes are cloned into the recombinant feline herpesvirus.
  • Recombinant feline herpesvirus is useful as a vaccine against other human diseases when foreign antigens from the following diseases or disease organisms are expressed in the feline herpesvirus vector: hepatitis B virus surface and core antigens, hepatitis C virus, herpes simplex virus, human herpesviruses, herpes simplex virus-1, herpes simplex virus-2, human herpesvirus-6, human herpesvirus-7, human cytomegalovirus, Epstein-Barr virus, Varicella-Zoster virus, human immunodeficiency virus, human influenza, measles virus, hantaan virus, pneumonia virus, rhinovirus, poliovirus, human respiratory syncytial virus, retrovirus, human T-cell leukemia virus, rabies virus, mumps virus, malaria
  • Recombinant feline herpesviruses coexpressing a species-specific cytokine and an antigen from a disease causing microorganism are useful to stimulate an increased cell-mediated and humoral immune response in the animal and increases the efficacy of the recombinant feline herpesvirus as a vaccine.
  • Cytokines which are expressed in feline herpesvirus include but are not limited to interleukin-2, - interleukin-6, interleukin-12, interferon and granulocyte-macrophage colony stimulating factor.
  • MOLECULE TYPE DNA (genomic)
  • CTACTGTGAC CTACCCCGGG GTGGTAATAA CAATACTATC GAATAGCCAA CA ATG 415
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline immunodeficiency virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline immunodeficiency virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline immunodeficiency virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline immunodeficiency virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline Leukemia Virus
  • ORGANISM Feline Leukemia Virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline herpesvirus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ORGANISM Human cytomegalovirus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM E. coli; Feline leukemia virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline leukemia virus; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline herpesvirus; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM E. coli; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline herpesvirus
  • SEQUENCE DESCRIPTION SEQ ID NO:23: TTCACAGTAT CTACTGTGCA CCTACCCCGG GGATCCTCTA GAGTCGACCT GCAGCCCAAG 60 CTAGCTTGGC CTCGAGGCCA AGCTAGCTTG GGCTGCAGGT CGACGAGTTC TAGCAC 116
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline herpesvirus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Human cytomegalovirus; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHET CAL NO
  • ANTI -SENSE NO
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline leukemia virus; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM E. coli
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Herpes simplex virus type 1; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline herpesvirus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Human cytomegalovirus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Feline leukemia virus; Hepatitis B virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Hepatitis B virus; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM E. coli
  • Herpes simplex virus type 1 SEQUENCE DESCRIPTION: SEQ ID NO:40: GGGCAGCGTT GGGTCCTGGG ACTCTAGAGG ATCCCCGGGA GATGGGGGAG GCTAACTGAA 60
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Herpes simplex virus type 1; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI - SENSE NO
  • ORGANISM Feline herpesvirus,- Human cytomegalovirus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Human cytomegalovirus; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus
  • ORGANISM E. coli; Pseudorabies virus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Pseudorabies virus,- Human cytomegalovirus
  • MOLECULE TYPE DNA (genomic)
  • ORGANISM Human cytomegalovirus; Dirofilaria immitis

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Abstract

La présente invention présente un herpès-virus félin recombinant qui comprend le génome viral de l'herpès-virus félin. Ce génome contient, quant à lui, une délétion dans son unique région courte, cette délétion se trouvant dans le gène de la glycoprotéine E (gE).
PCT/US1995/013975 1994-10-26 1995-10-26 Herpes-virus felin recombinant WO1996013575A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU41971/96A AU4197196A (en) 1994-10-26 1995-10-26 Recombinant feline herpes virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US32988394A 1994-10-26 1994-10-26
US08/329,883 1994-10-26

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WO1996013575A1 true WO1996013575A1 (fr) 1996-05-09

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PCT/US1995/013975 WO1996013575A1 (fr) 1994-10-26 1995-10-26 Herpes-virus felin recombinant

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120052089A1 (en) * 2010-08-31 2012-03-01 Michel Bublot Newcastle disease virus vectored herpesvirus vaccines
CN117085119A (zh) * 2023-10-07 2023-11-21 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 一株表达猫杯状病毒vp1基因重组猫疱疹病毒疫苗及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5252717A (en) * 1990-08-24 1993-10-12 Board Of Trustees Operating Michigan State University Marek's disease herpesvirus DNA segments encoding glycoproteins, gD, gI and gE
US5266489A (en) * 1990-03-12 1993-11-30 Rhone Merieux Recombinant herpesviruses, in particular for the production of vaccines, process for preparing them, plasmids produced during this process and vaccines obtained

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5266489A (en) * 1990-03-12 1993-11-30 Rhone Merieux Recombinant herpesviruses, in particular for the production of vaccines, process for preparing them, plasmids produced during this process and vaccines obtained
US5252717A (en) * 1990-08-24 1993-10-12 Board Of Trustees Operating Michigan State University Marek's disease herpesvirus DNA segments encoding glycoproteins, gD, gI and gE

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120052089A1 (en) * 2010-08-31 2012-03-01 Michel Bublot Newcastle disease virus vectored herpesvirus vaccines
WO2012030720A1 (fr) * 2010-08-31 2012-03-08 Merial Limited Vaccins contre le virus herpétique à base de vecteurs du virus de la maladie de newcastle
CN103269714A (zh) * 2010-08-31 2013-08-28 梅里亚有限公司 以新城疫病毒为载体的疱疹病毒疫苗
US8986706B2 (en) * 2010-08-31 2015-03-24 Merial, Inc. Newcastle disease virus vectored herpesvirus vaccines
US9446118B2 (en) 2010-08-31 2016-09-20 Merial, Inc. Newcastle disease virus vectored herpesvirus vaccines
EP3156070A3 (fr) * 2010-08-31 2017-06-14 Merial Inc. Vaccins du virus de l'herpès vectorisé par le virus de la maladie de newcastle
EP4014989A1 (fr) * 2010-08-31 2022-06-22 Boehringer Ingelheim Animal Health USA Inc. Vaccins du virus de l'herpès vectorisé par le virus de la maladie de newcastle
CN117085119A (zh) * 2023-10-07 2023-11-21 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 一株表达猫杯状病毒vp1基因重组猫疱疹病毒疫苗及其应用
CN117085119B (zh) * 2023-10-07 2024-02-09 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) 一株表达猫杯状病毒vp1基因重组猫疱疹病毒疫苗及其应用

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