WO1995008622A1 - Recombinant infectious laryngotracheitis virus and uses thereof - Google Patents
Recombinant infectious laryngotracheitis virus and uses thereof Download PDFInfo
- Publication number
- WO1995008622A1 WO1995008622A1 PCT/US1994/010628 US9410628W WO9508622A1 WO 1995008622 A1 WO1995008622 A1 WO 1995008622A1 US 9410628 W US9410628 W US 9410628W WO 9508622 A1 WO9508622 A1 WO 9508622A1
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- ser
- infectious laryngotracheitis
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- leu
- ala
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Classifications
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- 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
- C12N2710/16022—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
- C12N2710/16041—Use of virus, viral particle or viral elements as a vector
- C12N2710/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Definitions
- the present invention involves recombinant infectious laryngotracheitis (ILT) viruses useful in vaccines to protect poultry from naturally-occurring infectious laryngotracheitis virus and other poultry diseases.
- ILT infectious laryngotracheitis
- the ability to isolate viral DNA and clone this isolated DNA into bacterial plasmids has greatly expanded the approaches available to make viral vaccines.
- the methods used to make the present invention involve modifying cloned viral DNA sequences by insertions, deletions and single or multiple base changes.
- the modified DNA is then reinserted into the viral genome to render the virus non-pathogenic.
- the resulting live virus may then be used in a vaccine to elicit an immune response in a host animal and to protect the animal against a disease.
- Herpetoviridae is an example of a class of viruses amenable to this approach. These viruses contain 100,000 to 200,000 base pairs of DNA as their genetic material. Importantly, several regions of the genome have been identified that are nonessential for the replication of virus in vitro in cell culture. Modifications in these regions of the DNA may lower the pathogenicity of the virus, i.e., attenuate the virus. For example, inactivation of the thymidine kinase gene renders human herpes simplex virus non-pathogenic (1) , and pseudorabies virus of swine non-pathogenic (2) .
- herpesviruses contain non ⁇ essential regions of DNA in various parts of the genome. Some of these regions are associated with virulence of the virus, and modification of them leads to a less- pathogenic virus, from which a vaccine may be derived.
- Infectious laryngotracheitis virus (ILTV) , an alpha herpesvirus (9) , is an important pathogen of poultry in the USA, Europe, and Australia, responsible for egg production losses and death (10) . It causes an acute disease of chickens which is characterized by respiratory depression, gasping and expectoration of bloody exudate. Viral replication is limited to cells of the respiratory tract wherein infection of the trachea gives rise to tissue erosion and hemorrhage.
- ILTV has been analyzed at the molecular level. Restriction maps of the ILTV genome have been reported 20 (22-26) . The DNA sequence of several genes have been identified, i.e., thymidine kinase (27, 28), glycoprotein gB (27, 29, 30), ribonucleotide reductase (27, 31), capsid p40 (31, 32) .
- the unique short region of the ILT virus genomic DNA contains genes that * are associated with ILTV virulence and that a deletion in those genes leads to an attenuated ILTV.
- the ILT virus results in an attenuated virus, which is useful as a vaccine against subsequent attack by a virulent ILTV strains.
- Applicants also found that a deletion in the glycoprotein gl gene of the unique short region also attenuates the ILTV.
- a deletion in the US2 gene, the UL-47 like gene, and the glycoprotein g60 gene of the unique short region will also attenuate the ILTV.
- ILTV can become latent in healthy animals which makes them potential carriers of the virus. For this reason, it is clearly advantageous to be able to distinguish animals vaccinated with non-virulent virus from animals infected with disease-causing wild-type or naturally- occurring virus.
- the development of differential vaccines and companion diagnostic tests has proven valuable in the management of pseudorabies disease (55) .
- differential diagnostics A similar differential marker vaccine would be of great value in the management of ILTV caused disease.
- the construction of differential diagnostics has focused on the deletion of glycoproteins.
- the glycoprotein chosen to be the diagnostic marker should have the following characteristics: (1) the glycoprotein and its gene should be non-essential for the production of infectious virus in tissue culture; (2) the glycoprotein should elicit a major serological response in the animal; and (3) the glycoprotein should not be one that makes a significant contribution to the protective immunity.
- ILT virus glycoproteins Three major ILT virus glycoproteins, gB (29, 30), gC (27,
- the ILTV gB gene is an 5 essential gene and would not be appropriate as deletion marker genes.
- the gC gene of herpesviruses has been shown to make a significant contribution to protective immunity as a target of neutralizing antibody (56) and as a target of cell-mediated immunity (57) . 10 Therefore, the gC gene is not desirable as a deletion marker gene.
- glycoprotein encoding genes cited above it is not known whether or not they would be suitable 15 candidates for deletion in order to construct a recombinant ILT virus which can be used as a diagnostic vaccine.
- glycoprotein encoding genes located within the unique short region of the ILT viral genome which could be safely deleted in order to construct a recombinant ILT virus that can be used as a diagnostic vaccine. These are the glycoprotein gG gene and the glycoprotein gl
- infectious laryngotracheitis viruses described above may be used as vectors for the delivery of vaccine antigens from microorganisms causing important poultry diseases.
- Other viral antigens which may be included in a multivalent vaccine with an ILTV vector include infectious bronchitis virus (IBV) , Newcastle disease virus (NDV) , infectious bursal disease virus (IBDV) , and Marek 1 s disease virus (MDV) .
- IBV infectious bronchitis virus
- NDV Newcastle disease virus
- IBDV infectious bursal disease virus
- MDV Marek 1 s disease virus
- Such multivalent recombinant viruses would protect against ILT disease as well as other diseases.
- infectious laryngotracheitis viruses may be used as vectors 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 ILTV 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 (39) , ribozymes (40) , suppressor tRNAs (41) , 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
- the present invention provides a recombinant, attenuated 10 infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene. This attenuated virus is useful as a vaccine against infectious laryngotracheitis virus. 15
- the present invention also provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion or other alteration in the glycoprotein gG 20 gene so that upon replication, the recombinant virus produces no glycoprotein gG.
- the present invention also provides a method for distinguishing chickens or other poultry vaccinated with 25 a recombinant infectious laryngotracheitis virus which produces no glycoprotein gG from those infected with a naturally-occurring infectious laryngotracheitis virus.
- the present invention also provides a recombinant, 30 attenuated infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the US2 gene, UL47-like gene, ORF4 gene or glycoprotein g60 gene.
- the present invention also discloses four non-essential regions of the infectious laryngotracheitis viral genome : human cytomegalovirus immediately early (HCMV IE) promoter, pseudorabies virus glycoprotein gX (PRV gX) promoter, and infectious bovine herpesvirus virus 1.1 VP8 (BHV-1.1 VP8) . These regions may be used as insertion sites for a foreign gene in constructing a recombinant infectious laryngotracheitis virus vector.
- HCMV IE human cytomegalovirus immediately early
- PRV gX pseudorabies virus glycoprotein gX
- BHV-1.1 VP8 infectious bovine herpesvirus virus 1.1 VP8
- nucleotide sequences of Figure 1 begin with the internal repeat sequence and end within the terminal repeat sequence.
- the unique short region begins at base pair 274 of this Figure.
- FIG. 1 I restriction enzyme map of the infectious laryngotracheitis virus (ILTV) USDA 83-2 genome.
- the upper diagram identifies the unique long (U L ) , internal
- Fragment “L” is the 2.5 kb Asp718 I fragment
- fragment “H” is the 5164 bp Asp718 I fragment
- fragment “G” is the 8.0 kb .Asp718 I fragment.
- the fragments marked with asterisks contain a hypervariable region of approximately 900 bp that is repeated from one to 12 times. Since no one size predominates, these fragments appear in submolar amounts that are not well resolved on an ethidium bromide stained gel. The position of these repeats is indicated in the figure by the crooked dashed lines.
- the unique short region contains 13 methionine initiated open reading frames
- ORF ORF of greater than or equal to 110 amino acids (excluding smaller nested ORFs) . All 13 ORFs were aligned to the Entrez release 6.0 virus division of the Genbank DNA database utilizing the IBI MacVector Protein to DNA alignment option (default settings) . Eight of the ORFs exhibited significant homology to one or more other virus genes: unique short (US2) , protein kinase (PK) , unique long 47-like (UL47-like) , and glycoproteins gG, g60, gD, gl, and gE.
- US2 unique short
- PK protein kinase
- UL47-like unique long 47-like
- glycoproteins gG, g60, gD, gl, and gE glycoproteins
- FIG. 4 Detailed description of the DNA insertion in Homology Vector 472-73.27. Diagram showing the orientation of DNA fragments assembled in plas id 472-73.27. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 20, 21, 22 and 23). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction.
- infectious laryngotracheitis virus ILTV
- human cytomegalovirus immediate early HCMV IE
- pseudorabies virus PRV
- lacZ lactose operon Z gene
- poly A polyadenylation signal
- BP base pairs
- Homology Vector 501-94 Diagram showing the orientation of DNA fragments assembled in plas id 501-94. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 24, 25, 26, and 27) . The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction.
- infectious laryngotracheitis virus ILTV
- human cytomegalovirus immediate early HCMV
- IE pseudorabies virus
- PRV pseudorabies virus
- lacZ lactose operon Z gene
- E. coli Escherichia coli
- polyadenylation signal poly A
- TK thymidine kinase
- BP base pairs
- FIG. 6 Detailed description of the DNA insertion in Homology Vector 544-55.12. Diagram showing the orientation of DNA fragments assembled in plasmid 544-55.12. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 28, 29, 30, and 31). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction.
- infectious laryngotracheitis virus ILTV
- HSV-1 herpes simplex virus type 1
- PRV pseudorabies virus
- uidA Escherichia coli
- poly A polyadenylation signal
- BP base pairs
- Homology Vector 562-61.IF Diagram showing the orientation of DNA fragments assembled in plasmid 562-61.IF. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 32, 33, 34 35, 36 and 37). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction. The following abbreviations are used, infectious laryngotracheitis virus
- ILTV herpes simplex virus type 1
- HSV-1 herpes simplex virus type 1
- PRV pseudorabies virus
- uidA Escherichia coli
- poly A polyadenylation signal
- BP base pairs
- Figure 8 Detailed description of the DNA insertion in Homology Vector 560-52.Fl. Diagram showing the orientation of DNA fragments assembled in plasmid 560-52.Fl. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 38, 39, 40, 41, and 42). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The location of several gene coding regions and regulatory elements is also given.
- ILTV herpes simplex virus type 1
- HSV-1 herpes simplex virus type 1
- PRV pseudorabies virus
- uidA ,9-glucuronidase gene
- Escherichia coll E. coli
- poly A polyadenylation signal
- Figure 9 Detailed description of the DNA insertion in Homology Vector 579-14.G2. Diagram showing the orientation of DNA fragments assembled in plasmid 579-14.G2. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO:
- Escherichia coli Escherichia coli (E. coli) , polyadenylation signal (poly A) , and base pairs (BP) .
- FIG. 10 Detailed description of the DNA insertion in Plas id Vector 544-39.13. Diagram showing the orientation of DNA fragments assembled in plasmid 544-39.13. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO:
- Plasmid Vector 388-65.2 Diagram showing the orientation of DNA fragments assembled in plasmid 388-65.2. The origin of each fragment is indicated in the table. The sequences located at each of the junctions between fragments are also shown (SEQ ID NO's: 51, 52, 53, and 54). The restriction sites used to generate each fragment as well as the synthetic linker sequences which were used to join the fragments are described for each junction. The synthetic linker sequences are underlined by a heavy bar. The location of several gene coding regions and regulatory elements is also given. Restriction sites in brackets [] indicate the remnants of sites which were destroyed during construction.
- HCMV IE human cytomegalovirus immediate early
- lacZ lactose operon Z gene
- E. coli Escherichia coli
- PRV pseudorabies virus
- poly A polyadenylation signal
- BP base pairs
- the present invention provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein gG gene. Said deletion attenuates the virus, rendering it suitable for use as a vaccine against infectious laryngotracheitis virus.
- a preferred embodiment of this invention is a recombinant infectious laryngotracheitis designated S- ILT-014 (ATCC Accession No. XXXX.
- S-ILT-014 virus has been deposited 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. on September 22, 1993 under ATCC Accession No. ) .
- Another preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus designated S-ILT-002.
- a recombinant infectious laryngotracheitis virus is a live infectious laryngotracheitis 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 HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT ILTV in Materials and Methods, and the virus has not had genetic material essential for the replication of the infectious laryngotracheitis virus deleted.
- the present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the US2 gene.
- a recombinant infectious laryngotracheitis virus designated S-ILT-009.
- the present invention further provides a recombinant laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the ORF4 gene.
- the present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the UL47-like gene.
- the present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene, a deletion in the ORF4 gene, and a deletion in the UL47-like gene.
- a preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus designated S-ILT-015.
- the present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the glycoprotein g60 gene.
- a preferred embodiment of this invention is a recombinant infectious laryngotracheitis virus designated S-ILT-017.
- the present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the glycoprotein gG gene and a deletion in the glycoprotein gl gene.
- the present invention further provides a recombinant infectious laryngotracheitis virus which comprises the infectious laryngotracheitis viral genome containing a deletion in the glycoprotein gG gene and a deletion in the thymidine kinase (TK) gene.
- TK thymidine kinase
- the present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis virus genome which contains a deletion in the unique short region of the viral genome, wherein the deletion in the glycoprotein gG gene, and which also contains an insertion of a foreign gene.
- the foreign gene is inserted into a non-essential site of the infectious laryngotracheitis viral genome in such a way that it is capable of being expressed in a recombinant infectious laryngotracheitis infected host cell.
- a non-essential site of the infectious laryngotracheitis viral genome is a region of the viral genome which is not necessary for viral infection and replication.
- the following non-essential sites of the infectious laryngotracheitis viral genome are preferred sites for inserting a foreign gene into the virus : the thymidine kinase (TK) gene, the US2 gene, the UL47-like gene, the 0RF4 gene, the glycoprotein gG gene, the glycoprotein g60 gene, and the glycoprotein gl gene.
- TK thymidine kinase
- the foreign gene which is inserted into a non-essential site in the infectious laryngotracheitis viral genome, may encode a screenable marker, such as E. coll B- galactosidase or E. coli B-glucuronidas ⁇ .
- the foreign gene which is inserted into a non-essential site in the infectious laryngotracheitis viral genome may encode an antigenic polypeptide which, when introduced into the host cell, induces production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.
- antigenic polypeptide may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus.
- 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.
- the foreign gene may be put under control of an endogenous upstream infectious laryngotracheitis 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, and BHV-1.1 VP8 promoter.
- the present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis 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, so that upon replication, the recombinant virus produces no glycoprotein gG.
- the following recombinant viruses are preferred embodiments of this invention: A recombinant infectious laryngotracheitis virus designated S-ILT-002, S-ILT-014, S-ILT-009, S-ILT-015, and S-ILT-017.
- the present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis 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 infectious laryngotracheitis virus comprising the infectious laryngotracheitis 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.
- the present invention further provides a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the US2 gene, UL47- like gene, or glycoprotein g60 gene. It is contemplated that a deletion in any one of these genes will attenuate the virus, rendering it suitable to be used as a vaccine against infectious laryngotracheitis virus.
- the present invention further provides a recombinant infectious laryngotracheitis virus which comprises a foreign gene inserted within the unique short region of the infectious laryngotracheitis viral genome, provided, however, that the insertion is not in the protein kinase gene, the glycoprotein gD gene, the glycoprotein gE gene and the ORF10 gene.
- Preferred insertion sites are the US2 gene, the UL47-like gene, the ORF4 gene and the glycoprotein g60 gene.
- a foreign gene 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 infectious laryngotracheitis virus of the present invention.
- the foreign gene thus inserted may encode a screenable marker, such as E. coli ⁇ -galactosidase or E. coll B>- glucuronidase.
- the foreign gene thus inserted may encode an antigenic polypeptide which, when introduced into the host cell, induces production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.
- antigenic polypeptide may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus.
- 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.
- the foreign gene thus inserted may be put under control of an endogenous upstream infectious laryngotracheitis virus promoter, or it may be put under control of a heterologous upstream promoter.
- the heterologous upstream promoter may be the HCMV IE promoter, the PRV gX promoter or BHV-1.1 VP8 promoter.
- the present invention further provides a vaccine for infectious laryngotracheitis virus which comprises a suitable carrier and an effective immunizing amount of any of the recombinant infectious laryngotracheitis virus of the present invention.
- This vaccine may contain either inactivated or live recombinant virus.
- Suitable carriers for the recombinant virus are well known in the art and include 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 stablizing, hydrolyzed proteins.
- the inactivated vaccine uses tissue culture fluids directly after inactivation of the virus.
- the present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the glycoprotein gG gene.
- a preferred embodiment of this invention is a vaccine which comprises a suitable carrier and an effective immunizing amount of any one of the following viruses: recombinant infectious laryngotracheitis viruses designated S-ILT-014, S-ILT- 002, S-ILT-009, S-ILT-015 and S-ILT-017.
- the present invention further provides a multivalent vaccine for infectious laryngotracheitis virus and for one or more of other avian diseases which comprises an effective immunizing amount of a recombinant virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region, wherein the deletion is in the glycoprotein gG gene, and an insertion of a foreign gene into a non-essential site of the viral genome.
- the foreign gene encodes an antigenic polypeptide which induces host cell production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.
- the foreign gene may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus, 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., Elmerla spp.,
- Hlstomonas spp. Trichomonas spp. , poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.
- the present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome containing 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, so that upon replication, the recombinant virus produces no glycoprotein gG.
- a preferred embodiment of this invention is a vaccine which comprises a suitable carrier and an effective immunizing amount of any one of the following viruses: recombinant infectious laryngotracheitis viruses designated S-ILT-014, S-ILT- 002, S-ILT-009, S-ILT-015 and S-ILT-017.
- the present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis 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 vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis 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 the glycoprotein gl gene so that upon replication, the recombinant virus produces no glycoprotein gG and glycoprotein gl.
- the present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the US2 gene, UL47-like gene, or glycoprotein g60 gene.
- the present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains a deletion in the unique short region of the viral genome, wherein the deletion is in the US2 gene, ORF4 gene, UL47-like gene, or glycoprotein g60 gene, and insertion of a foreign gene into a non- essential site in the viral genome.
- the foreign gene encodes an antigenic polypeptide which induces host cell production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.
- the foreign gene may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus, avian encephalo yelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent. Salmonella spp. , E. coli, Paste rella spp., Bordetella spp., Eimeria spp.,
- Histomonas spp. Trichomonas spp. , poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.
- the present invention further provides a vaccine which comprises a suitable carrier and an effective immunizing amount of a recombinant infectious laryngotracheitis virus comprising the infectious laryngotracheitis viral genome which contains an insertion of a foreign gene into a non-essential site in the viral genome.
- the foreign gene encodes an antigenic polypeptide which induces host cell production of protective antibodies against an avian disease causing agent from which the antigen is derived or derivable.
- the foreign gene may be derived or derivable from infectious bronchitis virus, Newcastle disease virus, infectious bursal disease virus, and Marek's disease virus, avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian influenza virus, avian adenovirus, fowl pox virus, avian coronavirus, avian rotavirus, chick anemia agent.
- Histomonas spp. Trichomonas spp, Poultry nematodes, cestodes, trematodes, poultry mites/lice, poultry protozoa.
- the present invention further provides a method of immunizing an animal against infectious laryngotracheitis virus which comprises administering to chickens or other poultry an effective immunizing dose of any of the vaccines of the present invention.
- the present invention further provides a method for distinguishing chickens or other poultry which are vaccinated with an effective immunizing amount of a recombinant virus which produces no glycoprotein gG from those which are infected with a naturally-occurring infectious laryngotracheitis virus.
- This method comprises analyzing a sample of body fluid from the chickens or other poultry for the presence of glycoprotein gG of the infectious laryngotracheitis virus and at least one other antigen normally expressed in chickens or other poultry infected by a naturally- occurring infectious laryngotracheitis virus.
- glycoprotein gG The presence of antigen which is normally expressed in chickens or other poultry infected by a naturally- occurring infectious laryngotracheitis virus and the absence of glycoprotein gG in the body fluid is indicative of being vaccinated with the recombinant vaccine and not infected with a naturally-occurring infectious laryngotracheitis virus.
- the presence of glycoprotein gG and the antigen in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glycoprotein gG.
- the present invention further provides a method for distinguishing chickens or other poultry which are vaccinated with an effective immunizing amount of a recombinant infectious laryngotracheitis virus which produces no glycoprotein gl from those which are infected with a naturally-occurring infectious laryngotracheitis virus.
- This method comprises analyzing a sample of body fluid from the chickens or other poultry for the presence of glycoprotein gl of the infectious laryngotracheitis virus and at least one other antigen normally expressed in chickens or other poultry infected by a naturally-occurring infectious laryngotracheitis virus.
- the presence of the antigen which is normally expressed in chickens or other poultry infected by a naturally-occurring infectious laryngotracheitis virus and the absence of glycoprotein gl in the body fluid is indicative of being vaccinated with the recombinant vaccine and not infected with a naturally-occurring infectious laryngotracheitis virus.
- the presence of the antigen and glycoprotein gl in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glycoprotein gl.
- the present invention further provides a method for distinguishing chickens or other poultry which are vaccinated with an effective immunizing amount of a recombinant virus which produces no glycoprotein gG and no glycoprotein gl from those which are infected with a naturally-occurring infectious laryngotracheitis virus.
- This method comprises analyzing a sample of body fluid from the chickens or other poultry for the presence of glycoprotein gG and gl of the infectious laryngotracheitis virus and at least one other antigen normally expressed in an animal infected by a naturally- occurring infectious laryngotracheitis virus.
- the presence of the antigen which is normally expressed in chickens or other poultry by a naturally-occurring infectious laryngotracheitis virus and the absence of glycoprotein gG and gl in the body fluid is indicative of being vaccinated with the vaccine and not infected with a naturally-occurring infectious laryngotracheitis virus.
- the presence of the antigen and glycoprotein gG and gl in the body fluid may be determined by detecting in the body fluid antibodies specific for the antigen and glycoprotein gG and gl.
- the present invention further provides a homology vector for producing a recombinant infectious laryngotracheitis virus by inserting a foreign DNA into the unique short region of the infectious laryngotracheitis genomic DNA, which comprises a double-stranded DNA molecule consisting
- the foreign gene may encode a screenable marker, such as E. coll B-galactosidase or E. coll B- glucuronidase.
- the present invention further provides a homology vector for producing a recombinant infectious laryngotracheitis virus by deleting DNA which encodes a screenable marker, which has been inserted into the infectious laryngotracheitis virus genomic DNA, which comprises a double stranded DNA molecule consisting essentially of a double-stranded DNA to be deleted, which is flanked on each side by a double stranded DNA homologous to the infectious laryngotracheitis virus glycoprotein gG gene, glycoprotein gl gene, US2 gene, or UL-47 like gene.
- Preferred embodiments of this invention are the homology vectors designated Homology Vector 544-55.12, Homology
- Infectious laryngotracheitis virus stock samples were prepared by infecting primary chicken embryo kidney cells (CEK; obtained from Spaf s, Inc.) or primary chicken kidney cells (CK; obtained from chicks hatched from fertile eggs supplied by Hyvac) (50) in 225 cm 2 flasks with 0.5 ml of viral stock containing 10 5 -10 6 pfu in IX Eagle's Basal Medium (modified) with Hank's salts
- BME bromoethylamine
- FBS fetal bovine serum
- P/S pennicillin/streptomycin
- sodium bicarbonate stock 1% sodium bicarbonate stock
- Viral stocks were then harvested 4-5 days later. Infected media and cells were resuspended in complete medium containing 20% sterile whole milk and stored frozen at -70*C.
- the pellets were resuspended in 1 ml/flask of a buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA, and 1.5 mM MgCl 2 and were incubated for 15 minutes at 4°C. Twenty five ⁇ ls of 20% NP40 per flask was added, and the mixture was then homogenized in a dounce homogenizer using an A pestle. The preparation was centrifuged at 1700 x g for 10 minutes at 4°C and the supernatant was retained.
- the cell media supernatants were centrifuged at 23,500 x g for 30 minutes, and drained well. The pellet was resuspended in the above proteinase K-containing mixture as described.
- the DNA pellets were resuspended in 20 ⁇ l TE/flask and could be used at this point for further experiments or treated further to remove RNA with pancreatic RNase A, followed by phenol extraction and ethanol precipitation to obtain the DNA.
- To prepare viral DNA minipreps infected 10 cm. dishes were scraped into conical centrifuge tubes and centrifuged 5 minutes at 1000 x g. Cell media supernatants were kept and treated as above. The cell pellets were each resuspended in 0.5 ml of 10 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.5% NP40, and incubated 10 minutes at room temperature.
- DNA was resuspended in buffer containing 50 mM Tris pH 7.4, 50 mM KC1, 5 mM MgCl , and 400 micromolar each of the four deoxyribonucleotides.
- Ten units of Klenow DNA polymerase (Gibco BRL) were added and the reaction was allowed to proceed for 15 minutes at room temperature.
- the DNA was phenol extracted and ethanol precipitated as above.
- Sequencing was performed using the Sequenase Kit (US Biochemicals) and c 35 S-dATP (New England Nuclear) . Reactions using both the dGTP mixes and the dITP mixes were performed to clarify areas of compression. Alternatively, compressed areas were resolved on forma ide 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. Sequence obtained was assembled and compared using Dnastar software. Manipulation and comparison of sequences obtained was performed with IBI
- 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 (42, 43).
- the polymerase chain reaction (PCR) was used to introduce restriction sites convenient for the manipulation of various DNAs (44) .
- 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 variation. SOUTHERN BLOTTING OF DNA.
- Southern blotting was taken from Maniatis et al . (1982) and Sambrook, et.al. (1989) (42, 43). DNA was blotted to nylon membrane (Biorad Zetaprobe) in 0.4M NaOH and prehybridized for 5 minutes in a solution containing 0.25
- DNA TRANSFECTION FOR GENERATING RECOMBINANT ILT VIRUS The method is based upon the CaCl 2 procedure of Chen and Okayama (1987) (45) with the following modifications. Generation of recombinant ILT virus is dependent upon homologous recombination between ILT viral DNA and the plasmid homology vector containing the desired foreign DNA flanked by the appropriate herpesvirus cloned sequences. Plasmid DNA (10-40 mg) was added to 250 ml of a solution having a final concentration of 0.25 M CaCl 2 .
- the transfection stock was harvested, aliquoted, and frozen at -70 ⁇ C.
- PROCEDURE FOR GENERATING ILTV SUBGENOMIC DNA FRAGMENTS The ability to generate herpesviruses by cotransfection of cloned overlapping subgenomic fragments has been demonstrated for pseudorabies virus (46) . 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. We have used the procedure of overlapping cosmids to map restriction enzyme sites.
- a library of subclones containing overlapping ILTV subgenomic fragments was generated as follows. USDA ILTV
- Strain 83-2 has been designated S-ILT-001.
- ILTV DNA obtained from S-ILT-001
- 10 mM Tris-HCl pH 8.0, 1 mM EDTA (TE) was sheared by passing it twice through a 25 guage needle as previously described (46) .
- the DNA was centrifuged through a 15-40% glycerol gradient in 50 mM Tris-HCl pH 8.0, 1 mM EDTA, and 0.3 M NaCl for 5.5 hours at 274,000 x g. Fractions were analyzed on a 0.3% agarose gel, and those containing
- DNA of 35-50 kb were pooled, diluted twofold with TE, and precipitated with one tenth volume of 3 M sodium acetate and 2.5 volumes of ethanol.
- the tubes were centrifuged for one hour at 109,000 x g at 10 ⁇ C .
- Pellets were resuspended, transferred to microfuge tubes, and precipitated with one tenth volume of 3 M sodium acetate and 2.5 volumes of ethanol.
- the DNA was resuspended in TE. DNA ends were made blunt ended by the POLYMERASE FILL-IN REACTION.
- the DNA was purified by extraction with both buffer saturated phenol and ether, precipitated with sodium acetate and ethanol as above, and resuspended in TE.
- the vector used was pSY1626, which was made as follows. Cosmid pHC79 (Gibco BRL) was cut with Hlndlll and Aval to remove the tetracycline gene, and the ends were filled in with Klenow polymerase (FILL IN REACTION) .
- the polylinker from pWE15 (Stratagene) was ligated into this vector. The polylinker was isolated by digestion with Ec ⁇ Rl, the ends were filled in with Klenow polymerase (FILL IN REACTION) , and the fragment was purified on a LMP-agarose gel. DNA ligation was performed in the presence of melted agarose.
- the resulting cosmid, pSY1005 was modified at the Ec ⁇ Rl site to create pSY1626 by blunt-ended insertion of a 1.5 kb J ⁇ i ⁇ dlll—Ba ⁇ HI fragment from pNEO (P-L Biochemicals) containing the neomycin resistance gene.
- pSY1626 was cut and made blunt at the BamHI site, and ligated with sheared ILTV fragments as described above.
- the ligation mixture was packaged using Gigapack XL (Stratagene) according to the manufacturers instructions.
- the packaging mixture was added to AG1 cells (Stratagene) grown in the presence of maltose, and colonies were selected on LB plates containing kanamycin.
- Cosmid subclones containing ILTV DNA were identified by comparing restriction enzyme maps of individual cosmid clones to each other and to ILVTV genomic DNA to obtain a contiguous sequence of ILTV genomic DNA.
- the substrate X-Glucuro Chx (5-bromo-4- chloro-3-indolyl-,9-D-glucuronic acid Cyclohexylammonium salt, Biosynth AG) was used. Plaques that expressed active marker enzyme turned blue. The blue plaques were then picked onto fresh cells and purified by further blue plaque isolation. In recombinant virus strategies in which the enzymatic marker gene was removed, the assay involves plaque purifying white plaques from a background of parental blue plaques. Viruses were typically purified with five to ten rounds of plaque purification.
- 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 NaCl/ 5mM MgC12), 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 (lOmM
- ILTV gG was purified from the media of cells infected with either wild type ILTV or with FPV or SPV vectors expressing ILTV gG. Cells were allowed to go to complete cytopathic effect (CPE) , the media was poured off, and cell debris was pelleted in a table-top centrifuge. The media was concentrated in an
- the plasmid 501-94 was constructed for the purpose of deleting a portion of the thymidine kinase (TK) gene coding region from the ILT virus (28) . It incorporates the HCMV IE promoter and a screenable marker, the B. coli lacZ gene, flanked by ILT virus DNA. The HCMV IE promoter- ⁇ , coli lacZ gene is inserted in the opposite transcriptional orientation to the ILTV TK gene. Upstream of the marker gene is an approximately 1087 base pair fragment of ILTV DNA which includes the first 77 amino acid codons of the ILTV TK gene.
- TK thymidine kinase
- Downstream of the lacZ gene is an approximately 675 base pair fragment of ILTV DNA which includes 80 amino acid codons at the 3* end of the ILTV TK gene.
- this plasmid When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding for amino acids 78 to 285 of the ILTV TK gene with DNA coding for the lacZ gene.
- the lacZ marker gene is under the control of the human cytomegalovirus (HCMV) immediate early (IE) gene promoter and also contains the pseudorabies virus (PRV) gX gene polyadenylation signal at the 3' end of the gene.
- HCMV human cytomegalovirus
- IE immediate early
- PRV pseudorabies virus
- the plasmid vector is derived from an approximately 3002 base pair HindJ.ll fragment of pSP64/65 (Promega) . Fragment 1 is an approximately 1087 base pair HindiII to
- Fragment 2 is an approximately 5017 base pair Sail to Sail fragment containing the HCMV IE promoter, ⁇ - galactosidase (lacZ) marker gene, and PRV gX polyadenylation signal (see Figure 5) .
- Fragment 3 is an approximately 675 base pair B ⁇ ll to Hlndlll subfragment of the ILTV 2.4 kb HindiII fragment.
- the plasmid 544-55.12 was constructed for the purpose of deleting a portion of the US2 gene coding region from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli uidA gene flanked by ILT virus DNA. The PRV gX promoter-J?. coli uidA gene is inserted in the opposite transcriptional orientation to the ILTV US2 gene. Upstream of the uidA gene is an approximately 2300 base pair fragment of ILTV DNA which includes 41 amino acid codons at the 3' end of the US2 gene (SEQ ID NO 2: aa.
- Downstream of the uidA gene is an approximately 809 base pair fragment of ILTV DNA which includes 22 amino acid codons it the 5* end of the US2 gene (SEQ ID NO 2: aa. 1-22).
- RECOMBINANT HERPESVIRUS it will replace the ILTV US2 DNA coding for amino acids 23 to 187 with DNA coding for the E. coll uidA gene.
- the uidA marker gene is under the control of the pseudorabies virus (PRV) gX promoter and also contains the herpes simplex virus type 1 thymidine kinase (HSV-1 TK) gene polyadenylation signal at the 3' end of the gene.
- PRV pseudorabies virus
- HSV-1 TK herpes simplex virus type 1 thymidine kinase
- the plasmid vector is derived from an approximately 2958 base pair Asp718I restriction fragment of a pSP18/pSP19 fusion such that the multiple cloning site is Ec ⁇ Rl / Sad /Asp 181 /Sad /Ec ⁇ Rl. Fragment
- Fragment 2 is an approximately 3039 base pair XJal fragment containing the PRV gX promoter, the E. coli uidA gene, and the HSV-1 TK polyadenylation site (See Figure 6) .
- Fragment 3 is an approximately 809 base pair Xbal to Asp718I subfragment of the ILTV 1097 bp
- the plasmid 562-61.IF was constructed for the purpose of deleting part of the gl gene from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli uidA gene, flanked by ILT virus DNA.
- the PRV gX promoter- ⁇ , coli uidA gene is transcribed in the opposite direction to the
- ILTV gl gene promoter The 983 base pair deletion begins 12 base pairs upstream of the translation initiation codon and deletes 324 of 363 amino acid codons at the 5 1 end of the ILTV gl gene (SEQ ID NO 11: aa. 325-363) .
- SEQ ID NO 11: aa. 325-363 SEQ ID NO 11: aa. 325-363
- RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS it will replace the DNA coding for the ILTV gl gene with DNA coding for the E. coli uidA gene.
- a detailed description of the plasmid is given in Figure 7. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42, 43). The plasmid vector is derived from an approximately 2647 base pair Asp718I to HindiII fragment of pUC19. Fragment
- Fragment 1 is an approximately 1619 base pair Asp718I to Xbal subfragment of the ILTV 8.0 kb Asp718I fragment (SEQ ID NO 1: Nucl. 7556-9175).
- Fragment 2 is an approximately 691 base pair XJbal to Xhol fragment (SEQ ID NO 1: Nucl. 9175-9861) generated by the polymerase chain reaction
- the template was the ILTV 8.0 kb Asp718I fragment.
- the upstream primer 92.09 (S ⁇ CCTAGCACCCTTGTATCGCG-S•; SEQ ID NO. 55) sits down at a site 821 base pairs upstream of the ILTV gl gene and synthesizes DNA toward the 3' end of the gene.
- the downstream primer 92.11 (5'-
- CGC£ESS ⁇ STCCCAATGAATAGGCATTGG-3• ; SEQ ID NO. 56) sits down at a site 12 base pairs upstream of the translation start site of the ILTV gl gene and synthesizes DNA toward the 5' end of the gD gene.
- the product of the PCR reaction is 818 base pairs.
- This DNA fragment is digested with XJbal at the 5' end (a restriction enzyme site present in the ILTV DNA) and Xhol at the 3' end (a restriction enzyme site created in the PCR primer—see underlined sequence) to create an approximately 691 base pair XJbal to Xhol fragment.
- Fragment 3 is an approximately 3051 base pair Sail fragment containing the PRV gX promoter, the uidA gene, and the HSV-1 TK polyadenylation site (See Figure 6) .
- Fragment 4 is an approximately 624 base pair Xhol to Hindlll fragment generated by PCR (SEQ ID NO 1: Nucl. 10,847-11,461). The template was the ILTV 8.0 kb Asp718I fragment.
- the upstreamprimer 92.10 (5'-CGCCTCGAGGACCCATGGTTGCGTGCG-3• ; SEQ ID NO. 57) sits down at a site 117 base pairs upstream from the translation termination codon within the ILTV gl gene.
- the downstream primer 92.08 (5 1 - CTCGTCCGAACGAGTTACAG-3•; SEQ ID NO. 58) sits down at a site 604 base pairs downstream of the translation termination site of the ILTV gl gene and within the ILTV gE gene.
- the PCR product (729 base pairs) is digested with Xhol which is a unique site generated by the upstream PCR primer (underlined) and with Hindlll at a site within the ILTV gE gene. Restriction endonuclease digestion with Xhol and HindiII creates an approximately
- Fragment 5 is an approximately 2700 base pair Hindi11 subfragment of the ILTV 8.0 kb Asp718I fragment (SEQ ID NO 1: Nucl. 11,461-13,473 plus unsequenced DNA) .
- the plasmid 472-73.27 was constructed for the purpose of deleting a portion of the glycoprotein G (gG) gene coding region from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coli lacZ gene, flanked by ILT virus DNA.
- GAG glycoprotein G
- the HCMV IE promoter-*, coli lacZ gene is transcribed in the same direction to the ILTV gG gene promoter.
- the 874 base pair deletion of the ILTV gG gene extends from 60 nucleotides upstream of the translation initiation site to 814 nucleotides into the amino acid coding sequence, removing the coding capacity of 271 of 292 amino acids of the gG protein (SEQ ID NO 7) .
- GENERATING RECOMBINANT HERPESVIRUS it will replace the DNA coding for amino acids 1 to 271 of the ILTV gG gene with DNA coding for the E. coll lacZ gene.
- a detailed description of the plasmid is given in Figure 4. It was constructed from the indicated DNA sources utilizing standard recombinant DNA techniques (42, 43) .
- the plasmid vector is derived from an approximately 2686 base pair Asp718I restriction fragment of pUC 19 (Gibco, BRL) .
- Fragment 1 is an approximately 2830 base pair Asp7l8I to Nhel subfragment of the ILTV 5164 bp Asp718I fragment
- Fragment 2 is an approximately 5017 base pair Sail to Sail fragment containing the HCMV IE promoter, E. coli ,9-galactosidase (lacZ) marker gene, and PRV gX polyadenylation signal (see Figure 4) .
- Fragment 3 is an approximately 1709 base pair Sail to Asp718I subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1: Nucl. 5419-6878).
- the plasmid 560-52.Fl was constructed for the purpose of deleting part of the UL47- like gene, all of ORF4, and part of the ILTV gG gene from the ILT virus and inserting a foreign DNA. It incorporates a screenable marker, the E. coll uidA gene. flanked by ILT virus DNA.
- the PRV gX promoter-*, coli uidA gene is transcribed in the opposite direction to the ILTV UL47-like, ORF4, and gG gene promoters.
- the 2640 base pair deletion removes 442 of 511 amino acid codons at the 3' end of the UL47-like gene (SEQ ID NO 4), the entire coding sequence of the ORF4 gene (SEQ ID NO 5) and 271 of 293 amino acid codons at the 5' end of the ILTV gG gene (SEQ ID NO 7) .
- this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding for the ILTV UL47-like, ORF4 and gG genes with DNA coding for the PRV gX promoter-*, coli uidA gene.
- the plasmid vector is derived from an approximately 2958 base pair Asp7l8I restriction fragment of pSP18/pSP19 such that the multiple cloning site is EcoRl /Sad/ As p7181 /SacI/EcoRI .
- Fragment 1 is an approximately 1066 base pair Asp718I to BssHII subfragment of the ILTV 5164 bp Asp718I fragment
- Fragment 2 is an approximately 123 base pair Sail to Bell subfragment of the ILTV 5164 bp Asp718I fragment.
- Fragment 3 is an approximately 3027 base pair BamHI fragment containing the PRV gX promoter, the uid ⁇ gene, and the HSV-1 TK polyadenylation site (See Figure 8) .
- Fragment 4 is an approximately 1334 base pair BelI to Asp718I subfragment of the ILTV,5164 bp Asp718I fragment (SEQ ID NO 1: Nucl. 5544-6878 ) .
- the plasmid 579-14.G2 was constructed for the purpose of deleting the entire gG gene and a portion of the g60 gene from the ILT virus and inserting a foreign DNA. It incorporates a PRV gX promoter and a screenable marker, the E. coli uidA gene, flanked by ILT virus DNA.
- the PRV gX promoter-*, coll uidA gene is transcribed in the same direction to the ILTV gG and g60 gene promoters.
- the 3351 base pair deletion includes the entire coding sequence of the ILTV gG gene (SEQ ID NO 7) and 733 of 986 amino acid codons from the 5' end of the g60 gene (SEQ ID NO 8).
- this plasmid When this plasmid is used according to the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT HERPESVIRUS, it will replace the DNA coding for the ILTV gG gene and amino acids 1 to 733 of the ILTV g60 gene with DNA coding for the *. coli uidA gene.
- a detailed description of the plasmid is given in Figure 9. It was constructed from the indicated DNA sources utilizing standard recombinant
- the plasmid vector pUC19 (Gibco, BRL) is derived from an approximately 2677 base pair Asp718I to BamHI fragment. Fragment 1 is an approximately 2830 base pair Asp7l8I to Nhel subfragment of the ILTV 5164 bp Asp718I fragment (SEQ ID NO 1: Nucl.
- Fragment 2 is an approximately 3051 base pair Sail fragment containing the PRV gX promoter, *. coli ?- glucuronidase (uidA) marker gene, and an HSV-1 TK polyadenylation site (See Figure 9) .
- Fragment 3 is an approximately 1709 base pair Sail to BamHI subfragment of the ILTV 4545 base pair BamHI fragment (SEQ ID NO 1: Nucl. 7895-9604).
- Plasmid 544-39.13 contains the ⁇ - glucuronidase expression cassette consisting of the PRV gX promoter, *. coli ,9-glucuronidase (uidA) marker gene, and an HSV-1 TK polyadenylation site.
- UidA *. coli ,9-glucuronidase
- HSV-1 TK polyadenylation site A detailed description of the marker gene is given in FIGURE 10. It was constructed utilizing standard recombinant DNA techniques (42, 43) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGURE 10.
- the plasmid vector pSP71 (Promega) is derived from an approximately 3066 base pair
- Fragment 1 is an approximately 422 base pair Sail to *coRI restriction subfragment of the PRV BamHI restriction fragment /10 (47) . Note that the *coRI site was introduced at the location indicated in FIGURE 12 by PCR cloning. Fragment 2 is an approximately
- Fragment 3 is an approximately 784 base pair Xmal subfragment of the HSV-1 BamHI restriction fragment Q (48) . Note that this fragment is oriented such that the polyadenylation sequence (AATAAA) is located closest to the junction with the *. coli uidA gene.
- PLASMID 388-65.2 Plasmid 388-65.2 contains the ⁇ - galactosidase expression cassette consisting of the HCMV immediate early (IE) promoter, the *.
- FIGURE 11 A detailed description of the /9-galactosidase expression cassette is given in FIGURE 11. It was constructed utilizing standard recombinant DNA techniques (42, 43) by joining restriction fragments from the following sources with the synthetic DNA sequences indicated in FIGURE 11.
- the plasmid vector pSP72 (Promega) is derived from an approximately 3076 base pair PstI to PstI fragment. Fragment 1 is a 1154 base pair PstI to Avail fragment derived from a HCMV 2.1 kb PstI fragment containing the HCMV IE promoter.
- Fragment 2 is a 3010 base pair BamHI to PvuII fragment derived from plasmid pJF751 (49) containing the *. coll lacZ gene.
- Fragment 3 is an approximately 750 base pair iVdel to Sail fragment derived from PRV BamHI ⁇ 7 which contains the carboxy-terminal 19 amino acids and the polyadenylation signal of the PRV gX gene.
- EHV- Equine herpesvirus MDV- Mareks disease virus
- HSV-l- Herpes Simplex virus 1 HSV-l- Herpes Simplex virus 1
- PRV-R Herpes Simplex virus
- HSV-2 Herpes Simplex virus 2; VZV- Varicella-Zoster virus;
- the US2 gene consists of 690 base pairs and codes for a protein 229 amino acids in length and molecular weight approximately 25,272 daltons (SEQ. ID. NO. 12, 13).
- the ILTV US2 is homologous to the Equine herpesvirus(EHV)-1 and EHV-4 US2 proteins.
- the US2 gene is transcribed from nucleotide 970 to 281 on the reverse complement strand of the ILTV unique short region (SEQ. ID. NO. 1) .
- the function of the US2 gene product is unknown.
- the protein kinase gene consists of 1431 base pairs from nucleotide 1059 to 2489 and codes for a protein 476 amino acids in length and molecular weight approximately 54,316 daltons (SEQ. ID. NO. 2) .
- the ILTV protein kinase is homologous to the protein kinases from Mareks disease virus (MDV), Equine herpesvirus(EHV)-1 and -4, Pseudorabies virus (PRV) , Varicella-Zoster virus (VZV) , Simian varicella virus (SW) , and Herpes Simplex virus(HSV)-l and -2.
- the UL47-like gene is unique in its location within the unique short region of ILT virus.
- the UL47-like gene in all other known herpesviruses is located within the unique long sequence.
- the UL47-like gene consists of 1533 base pairs from nucleotide 2575 to 4107 and codes for a protein 510 amino acids in length and molecular weight approximately 57,615 daltons (SEQ. ID. NO. 3).
- ORF4 codes for a protein of unknown function.
- ORF4 consists of 333 base pairs from nucleotide 4113 to 4445 and codes for an open reading frame 110 amino acids in length and molecular weight approximately 12,015 daltons (SEQ. ID. NO. 4) .
- 0RF4 Reverse Complement codes for a protein of unknown function.
- 0RF4 RC consists of 380 base pairs from nucleotide 4519 to 4139 and codes for an open reading frame 126 amino acids in length and molecular weight approximately 13,860 daltons (SEQ. ID. NOS. 14, 15).
- the gG gene consists of 879 base pairs from nucleotide 4609 to 5487 and codes for a glycoprotein 292 amino acids in length and molecular weight approximately 31,699 daltons (SEQ. ID. NO. 5) .
- ILTV gG glycoprotein is homologous to PRV gX, Bovine herpesvirus(BHV)-1.3 gG, EHV-1 gG and EHV-4 gG.
- Recombinant ILTV gG protein produced in a Swinepox virus vector or a Fowlpox virus vector can be purified (see Materials and Methods) and reacts to peptide antisera to ILTV gG.
- the peptide antisera reacts to ILTV gG from wild type virus, but not to viruses deleted for the ILTV gG gene. Deletion of the gG gene results in an attenuated ILT virus that is useful as a vaccine against ILT disease in chickens (see table in Example 6) and also serves as a negative marker to distinguish vaccinated from infected animals.
- the g60 gene has been identified as glycoprotein 60 (33, 53) .
- the g60 gene consists of 2958 base pairs from nucleotide 5697 to 8654 and codes for a glycoprotein 985 amino acids in length and molecular weight approximately
- ORF6 RC consists of 878 base pairs from nucleotide 7826 to 6948 and codes for an open reading frame 292 amino acids in length and molecular weight approximately 32,120 daltons (SEQ. ID. NO. 16, 17) .
- the ILTV ORF6 RC shares limited homology to portions of the HSV-l and HSV-2 ribonucleotide reductase large subunit (UL39) .
- the expression of the gD glycoprotein in vectored fowlpox virus or herpesvirus of turkeys (33) is sufficient to raise a protective immune response in the chicken.
- the gD gene consists of 1305 base pairs from nucleotide 8462 to 9766 and codes for a glycoprotein 434 amino acids in length and molecular weight approximately 48,477 daltons (SEQ. ID. NO. 10, 11).
- the ILTV gD glycoprotein is homologous to the PRV g50, and the gD from HSV-l, MDV, IPV, and BHV-1.1.
- Monoclonal antibodies raised to ILT virus react specifically with gD protein from ILTV and also react to ILTV gD protein expressed in a Herpesvirus of Turkeys (HVT) virus vector.
- HVT Herpesvirus of Turkeys
- the gl gene consists of 1089 base pairs from nucleotide 9874 to 10,962 and codes for a glycoprotein 362 amino acids in length and molecular weight approximately 39,753 daltons (SEQ. ID. NO. 7) .
- the ILTV gl glycoprotein is homologous to the VZV gl.
- Recombinant ILTV gl protein expressed in a swinepox virus vector reacts to convalescent sera from ILTV-infected chickens. Deletion of the gl gene results in an attenuated ILT virus that is useful as a vaccine against ILT disease in chickens.
- Recombinant viruses deleted for gl are safe in animal trials when vaccinated by a natural route directly into the respiratory tract, whereas parental virus causes lesions in 90% of the birds inoculated via the same route. Deletion of the gl gene serves as a negative marker to distinguish vaccinated from infected animals.
- ORF8 Reverse Complement codes for a protein of unknown function.
- ORF8 RC consists of 533 base pairs from nucleotide 11,150 to 10,617 and codes for an open reading frame 177 amino acids in length and molecular weight approximately 19,470 daltons (SEQ. ID. NO. 18, 19).
- the gE gene consists of 1500 base pairs from nucleotide 11,159 to 12,658 and codes for a glycoprotein 499 amino acids in length and molecular weight approximately 55,397 daltons (SEQ. ID. NO. 8) .
- the ILTV gE glycoprotein is homologous to the gE glycoproteins from VZV, Simian herpesvirus (SHV) , EHV-1, HSV-l, and PRV.
- the ILTV gE is a neutralizing antigen useful as a subunit vaccine.
- ORFIO consists of 783 base pairs from nucleotide 12,665 to 13,447 and codes for a protein 261 amino acids in length and molecular weight approximately 27,898 daltons (SEQ. ID. NO. 9).
- S-ILT-004 is an infectious laryngotracheitis virus (ILTV) that has an approximately 620 base pair deletion of the thymidine kinase (TK) gene (28) .
- the gene for *. coll ⁇ - galactosidase (lacZ) was inserted in the place of the TK gene and is under the control of the HCMV immediate early (IE) promoter. Transcription of the HCMV IE promoter-lac
- Z gene is in the opposite orientation to the TK promoter.
- S-ILT-004 was constructed using homology vector 501-94 (see Materials and Methods) and S-ILT-001 (USDA ILTV Strain 83-2) in the HOMOLOGOUS RECOMBINATION PROCEDURE
- the transfection stock was screened by the Bluogal" SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.
- the result of blue plaque purification was recombinant virus S-ILT- 004.
- This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of the ⁇ -galactosidase (lacZ) marker gene and the deletion of approximately 619 base pairs of the TK gene.
- the remaining TK gene sequence codes for protein including amino acids 1 to 77, and amino acids 286 to 363.
- the HCMV IE promoter-lacZ gene is in the opposite orientation to the TK gene transcription.
- S-ILT-004 is attenuated by deletion of the ILTV TK gene, but retains other genes known to be involved in the immune response in chickens to ILT virus. Therefore, S- ILT-004 may be useful as a killed vaccine to protect chickens from ILT disease.
- S-ILT-009 is an infectious laryngotracheitis virus (ILTV) that has an approximately 498 base pair deletion of the ILTV US2 gene and an approximately 874 base pair deletion of the ILTV gG gene.
- the gene for *. coli ,9-glucuronidase (uidA) was inserted in the place of the US2 gene and is under the control of the pseudorabies virus (PRV) gX promoter.
- PRV pseudorabies virus
- S-ILT-009 was constructed using homology vector 544-55.12 (see Materials and Methods) and S-ILT-002 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT VIRUS.
- S-ILT-002 was constructed as described in Example 5 (S-ILT-014) .
- the transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.
- the resulting purification of a blue plaque was recombinant virus S- ILT-009. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure.
- a deletion of the HCMV IE promoter-lacZ gene was detected within the existing ILTV gG deletion.
- the remaining insert into the ILTV gG deletion contains approximately 2000 base pairs of DNA of which all of the lacZ gene and part of the PRV gX polyadenylation site are missing.
- the deletion was characterized by detailed restriction mapping and determined to be slightly different from the S-ILT-014 deletion (See Example 5) .
- S-ILT-009 is attenuated by deletion of the ILTV US2 and gG genes, but retains other genes known to be involved in the immune response in chickens to ILT virus. Therefore,
- S-ILT-009 is useful as an attenuated live vaccine or as a killed vaccine to protect chickens from ILT disease as shown in the table. Since S-ILT-009 does not express the ILTV gG genes, it is utilized as a negative marker to distinguish vaccinated animals from infected animals as described previously.
- S-ILT-Oil is an infectious laryngotracheitis virus (ILTV) that has an approximately 983 base pair deletion of the ILTV gl gene.
- the gene for *. coli ⁇ -glucuronidase (uidA) was inserted in the place of the gl gene and is under the control of the pseudorabies virus (PRV) gX promoter.
- PRV gX promoter-uidA gene is in the opposite orientation to the direction of transcription of the ILTV gl promoter.
- S-ILT-011 was constructed using homology vector 562-61.IF
- RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT VIRUS The transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES. The result of blue plague purification was recombinant virus S-ILT-011. This virus was characterized by restriction mapping and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of the -glucuronidase (uidA) marker gene and the deletion of approximately 983 base pairs of the ILTV gl gene which deletes 325 of 363 amino acid codons from the 5' end of the gl gene.
- uidA -glucuronidase
- S-ILT-Oil is attenuated and is useful as a killed vaccine to protect chickens from ILT disease.
- S-ILT-011 shows a small plaque phenotype in tissue culture which is indicative of slow viral growth and attenuation. Since S-ILT-011 does not express the ILTV gl gene, it may be utilized as a negative marker to distinguish vaccinated animals from infected animals. As indicated in Example 1, ILTV-infected chickens make antibodies against ILTV gl protein.
- Example 5 S - ILT- 013
- S-ILT-013 is an infectious laryngotracheitis virus (ILTV) that has an approximately 983 base pair deletion of the ILTV gl gene and an approximately 874 base pair deletion of the ILTV gG gene (and a deletion of the HCMV IE promoter lacZ marker gene making the lacZ gene nonfunctional) .
- the gene for E. coli / S-glucuronidase (uidA) was inserted in the place of the gl gene and is under the control of the pseudorabies virus (PRV) gX promoter.
- PRV pseudorabies virus
- S-ILT-013 was constructed using homology vector 562-61.IF
- S-ILT-013 is attenuated and is useful as a killed vaccine to protect chickens from ILT disease.
- S-ILT-013 shows a small plaque phenotype in tissue culture which is indicative of slow viral growth and attenuation. Since S-ILT-013 does not express the ILTV gl or gG genes, ILTV gl and gG may be utilized as negative markers to distinguish vaccinated animals from infected animals.
- S-ILT-014 is an infectious laryngotracheitis virus (ILTV) that has an approximately 874 base pair deletion of the ILTV gG gene and a deletion of the inserted HCMV IE promoter lacZ marker gene making the lacZ gene nonfunctional.
- ILTV infectious laryngotracheitis virus
- S-ILT-014 was derived from a purified S-ILT-002 virus stock in which a deletion of the HCMV IE promoter lacZ marker gene occurred.
- S-ILT-002 was constructed using homology vector 472-73.27 (See Materials and Methods) and S-ILT-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT VIRUS.
- the virus S-ILT-002 has a 874 base pair deletion within the ILTV gG gene and an insertion of the *. coli 3-galactosidase (lacZ) gene in place of the ILTV gG gene.
- lacZ *. coli 3-galactosidase
- a white plaque was picked which contained a deletion of the lacZ gene within the ILTV gG deletion.
- This virus S-ILT-014, was characterized by restriction mapping, DNA SEQUENCING and the SOUTHERN BLOTTING OF DNA procedure. This analysis confirmed the presence of an approximately 874 base pair deletion of the ILTV gG gene and approximately 1956 base pair insertion of a partial HCMV IE promoter lacZ marker gene DNA (2958 base pairs deleted) .
- the remaining HCMV IE promoter lacZ marker gene DNA consists of an approximately 686 base pair DNA fragment of the approximately 1154 base pair HCMV IE promoter and an approximately 1270 base pair DNA fragment containing approximately 520 base pairs of the 3010 base pair ⁇ - galactosidase (lacZ) marker gene and all of the approximately 750 base pair PRV gX polyadenylation signal.
- S-ILT-014 is useful as an attenuated live vaccine or as a killed vaccine to protect chickens from ILT disease as indicated in the table below. Since S-ILT-014 does not express the ILTV gG gene and ILTV-infected chickens make antibodies to gG as indicated in Example 1, ILTV gG is utilized as a negative marker to distinguish vaccinated animals from infected animals.
- S-ILT-015 is an infectious laryngotracheitis virus (ILTV) that has an approximately 2640 base pair deletion of the UL47-like gene, the ORF4 gene, and ILTV gG gene.
- the gene for E. coli /S-glucuronidase (uidA) was inserted in the place of the UL47-like, ORF4, and gG genes and is under the control of the pseudorabies virus (PRV) gX promoter.
- PRV pseudorabies virus
- the PRV gX promoter-uidA gene is in the opposite orientation to the direction of transcription of the ILTV UL47-like, ORF4, and gG promoters.
- S-ILT-015 was constructed using homology vector 560-52.Fl
- the transfection stock was screened by the X-Gluc SCREEN FOR
- S-ILT-015 is useful as an attenuated live vaccine or as a killed vaccine to protect chickens from ILT disease as indicated in the table below. Since S-ILT-015 does not express the ILTV gG gene, ILTV gG is utilized as a negative marker to distinguish vaccinated animals from infected animals. EFFICACY OF RECOMBINANT LIVE ILT VIRUS S-ILT-015 AGAINST VIRULENT INFECTIOUS LARYNGOTRACHEITIS VIRUS
- S-ILT-017 is an infectious laryngotracheitis virus (ILTV) that has an approximately 3351 base pair deletion of the ILTV gG gene and the g60 gene.
- the gene for E. coli ⁇ - glucuronidase (uidA) was inserted in the place of the ILTV gG and g60 genes and is under the control of the pseudorabies virus (PRV) gX promoter.
- PRV pseudorabies virus
- S-ILT-017 was constructed using homology vector 579-14.G2 (see Materials and Methods) and S-ILT-001 in the HOMOLOGOUS RECOMBINATION PROCEDURE FOR GENERATING RECOMBINANT VIRUS.
- the transfection stock was screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES.
- the result of blue plaque purification was recombinant virus S-ILT-017.
- S-ILT-017 is attenuated by deletion of the ILTV g60 and gG genes, but retains other genes known to be involved in the immune response in chickens to ILT virus. Therefore, S-ILT- 017 may be used as a killed vaccine to protect chickens from ILT disease. Since S-ILT-017 does not express the ILTV gG or g60 genes, it is used as a negative marker to distinguish vaccinated animals from infected animals.
- IBV infectious bronchitis virus
- a homology vector is used to generate ILT viruses containing the IBV Arkansas spike protein gene.
- the recombinant ILT virus contains a deletion of one or more ILTV genes, including gG, US2, UL47-like, and ORF4, and the insertion of two foreign genes: the E. coli 3-glucuronidase gene (uidA) and the IBV Arkansas spike protein gene.
- the uidA gene is under the control of the PRV gX promoter and the IBV Arkansas spike protein gene is under the control of the HCMV IE promoter.
- a DNA fragment containing the HCMV-IE promoter, the IBV Arkansas spike protein and the HSV-l TK polyadenylation signal is inserted into a restriction enzyme site at the position of the deletion of the ILTV gG gene in the ILTV homology vector.
- a DNA fragment containing the PRV gX promoter and the E. coli ⁇ - glucuronidase (uidA) gene is inserted into a unique restriction enzyme site within the ILTV homology vector.
- a recombinant virus is constructed by combining the final homology vector containing the IBV Arkansas spike gene and the E.
- the transfection stock is screened by the X-Gluc SCREEN FOR RECOMBINANT HERPESVIRUS EXPRESSING ENZYMATIC MARKER GENES to detect the presence of the uidA gene and by the BLACK PLAQUE ASSAY FOR FOREIGN GENE EXPRESSION to detect the presence of the IBV Arkansas spike protein.
- a similar strategy is used to construct recombinant ILT viruses carrying the IBV SI protein from Arkansas, Massachusetts,or Connecticut serotypes, IBV matrix protein from Arkansas, Massachusetts, or Connecticut serotypes, and IBV nucleocapsid from Arkansas, Massachusetts, or Connecticut serotypes.
- the strategy is also used to construct recombinant ILT viruses carrying the Newcastle Disease virus (NDV) HN and F genes and the Infectious Bursal Disease virus (IBDV) polyprotein or portions thereof.
- the strategy is also used to construct recombinant ILT viruses carrying the Mareks Disease virus (MDV) gA, gD, and gB genes.
- Recombinant ILT virus carrying these antigens are valuable as a multivalent vaccine to protect chickens from diseases caused by ILTV and one or more of the viruses IBV, NDV, IBDV, or MDV. Since the ILTV vaccines described here do not express ILTV gG, it is useful as a negative marker to distinguish vaccinated animals from infected animals.
- Vaccines utilizing ILTV to express antigens from various disease causing microorganisms Vaccines utilizing ILTV to express antigens from various disease causing microorganisms.
- Antigens from the following microorganisms are utilized to develop poultry vaccines: Chick anemia agent, Avian encephalomyelitis virus, Avian reovirus, Avian paramyxoviruses, Avian influenza virus ,Avian adenovirus, Fowl pox virus, Avian coronavirus, Avian rotavirus, Salmonella spp., E coli., Pasteurella spp., Haemophilus spp. , Chlamydia spp. , Mycoplasma spp. , Campylobacter spp.
- MOLECULE TYPE DNA (genomic)
- HYPOTHETICAL NO
- GGTCGCCCCC CTCACCGAAC CGAATCACGG GTCTGCACAT CCTGGGAAGG GAAAACAGCT 780 CCCCGGAAAC TTCGTACAGA GATGCCGGGC GCACGATTAC CGATAATGTA CTCGGACGAT 840
- ATC CAG GAA ATC CTT CGG TAC CCG GAT AAT ACG TAC ATG TTA ACG CAG 1745 He Gin Glu He Leu Arg Tyr Pro Asp Asn Thr Tyr Met Leu Thr Gin 215 220 225
- GGC TGT AAA GTG ACC GAG TAC ACG TTC TCC GCC TCG AAC AGA CTA ACC 10230 Gly Cys Lys Val Thr Glu Tyr Thr Phe Ser .Ala Ser Asn Arg Leu Thr 105 110 115 GGA CCT CCA CAC CCG TTT AAG CTC ACT ATA CGA AAT CCT CGT CCG AAC 10278 Gly Pro Pro His Pro Phe Lys Leu Thr He Arg Asn Pro Arg Pro Asn 120 125 130 135
- GGT AAC TAT AAA ATG TAC TTC GTC GTC GCG GGG GTG GCC GCG ACG TGC 12325 Gly Asn Tyr Lys Met Tyr Phe Val Val Ala Gly Val Ala Ala Thr Cys 375 380 385
- MOLECULE TYPE protein
- MOLECULE TYPE protein
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
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- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Biotechnology (AREA)
- Communicable Diseases (AREA)
- Physics & Mathematics (AREA)
- Pulmonology (AREA)
- Oncology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU78386/94A AU7838694A (en) | 1993-09-24 | 1994-09-16 | Recombinant infectious laryngotracheitis virus and uses thereof |
EP94929265A EP0723584A4 (en) | 1993-09-24 | 1994-09-16 | Recombinant infectious laryngotracheitis virus and uses thereof |
JP7509884A JPH09505726A (en) | 1993-09-24 | 1994-09-16 | Recombinant infectious laryngotracheitis virus and their use |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12659793A | 1993-09-24 | 1993-09-24 | |
US08/126,597 | 1993-09-24 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1995008622A1 true WO1995008622A1 (en) | 1995-03-30 |
Family
ID=22425710
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1994/010628 WO1995008622A1 (en) | 1993-09-24 | 1994-09-16 | Recombinant infectious laryngotracheitis virus and uses thereof |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0723584A4 (en) |
JP (1) | JPH09505726A (en) |
AU (1) | AU7838694A (en) |
CA (1) | CA2172387A1 (en) |
WO (1) | WO1995008622A1 (en) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996000791A1 (en) * | 1994-06-30 | 1996-01-11 | The Board Of Trustees Of The University Of Illinois | Recombinant infectious laryngotracheitis virus and vaccine |
EP0822980A1 (en) * | 1995-03-23 | 1998-02-11 | Syntro Corporation | Recombinant infectious laryngotracheitis virus and uses thereof |
US6033670A (en) * | 1996-12-16 | 2000-03-07 | Merial | Recombinant live avian vaccine, using as vector the avian infectious laryngotracheitis virus |
US6180671B1 (en) | 1998-03-10 | 2001-01-30 | Beth Israel Deaconess Medical Center, Inc. | Methods for treating disorders in which docosahexaenoic acid (DHA) levels are affected |
US7205398B2 (en) | 2002-05-24 | 2007-04-17 | Schering-Plough Animal Health Corporation | Eta-1 gene and methods for use |
US20130129780A1 (en) * | 2010-08-02 | 2013-05-23 | University Of Georgia Research Foundation, Inc. | Modified infectious laryngotracheitis virus (iltv) and uses thereof |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1990002802A2 (en) * | 1988-09-13 | 1990-03-22 | Institute For Animal Health Limited | Viral nucleotide sequences |
US4980162A (en) * | 1986-07-17 | 1990-12-25 | Juridical Foundation The Chemosero-Therapeutic Research Institute | Live vaccine for contagious diseases of chickens |
WO1992003554A1 (en) * | 1990-08-24 | 1992-03-05 | Arthur Webster Pty. Ltd. | Infectious laryngotracheitis virus vaccine |
US5182210A (en) * | 1987-10-23 | 1993-01-26 | National Research Development Corporation | Fowlpox virus promoters |
US5231023A (en) * | 1990-07-30 | 1993-07-27 | Akzo N.V. | Recombinant Marek's disease virus |
US5279965A (en) * | 1991-04-05 | 1994-01-18 | Keeler Jr Calvin L | Recombinant infectious laryngotracheitis virus |
US5310671A (en) * | 1988-06-24 | 1994-05-10 | British Technology Group Limited | Fowlpox virus non-essential regions |
-
1994
- 1994-09-16 AU AU78386/94A patent/AU7838694A/en not_active Abandoned
- 1994-09-16 EP EP94929265A patent/EP0723584A4/en not_active Withdrawn
- 1994-09-16 CA CA002172387A patent/CA2172387A1/en not_active Abandoned
- 1994-09-16 JP JP7509884A patent/JPH09505726A/en active Pending
- 1994-09-16 WO PCT/US1994/010628 patent/WO1995008622A1/en not_active Application Discontinuation
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4980162A (en) * | 1986-07-17 | 1990-12-25 | Juridical Foundation The Chemosero-Therapeutic Research Institute | Live vaccine for contagious diseases of chickens |
US5182210A (en) * | 1987-10-23 | 1993-01-26 | National Research Development Corporation | Fowlpox virus promoters |
US5310671A (en) * | 1988-06-24 | 1994-05-10 | British Technology Group Limited | Fowlpox virus non-essential regions |
WO1990002802A2 (en) * | 1988-09-13 | 1990-03-22 | Institute For Animal Health Limited | Viral nucleotide sequences |
US5231023A (en) * | 1990-07-30 | 1993-07-27 | Akzo N.V. | Recombinant Marek's disease virus |
WO1992003554A1 (en) * | 1990-08-24 | 1992-03-05 | Arthur Webster Pty. Ltd. | Infectious laryngotracheitis virus vaccine |
US5279965A (en) * | 1991-04-05 | 1994-01-18 | Keeler Jr Calvin L | Recombinant infectious laryngotracheitis virus |
Non-Patent Citations (6)
Title |
---|
AVIAN DISEASES, Volume 27, No. 4, issued 1983, T. IZUCHI et al., "Studies on Live Virus Vaccine Against Infectious Laryngotracheitis of Chickens. I. Biological Properties of Attenuated Strain C7", pages 918-926. * |
AVIAN DISEASES, Volume 35, issued 1991, C.L. KEELER JR. et al., "Identification of the Thymidine Kinase Gene of Infectious Laryngotracheitis Virus", pages 920-929. * |
See also references of EP0723584A4 * |
TRENDS IN BIOTECHNOLOGY, Volume 7, issued October 1989, A. FINKELSTEIN et al., "Live Recombinant Vaccines for Poultry", pages 273-277. * |
VIROLOGY, Volume 202, issued 1994, P. GUO et al., "Construction of Recombinant Avian Infectious Laryngotracheitis Virus Expressing the Beta-Galactosidase Gene and DNA Sequencing of the Insertion Region", pages 771-781. * |
WORLD'S POULTRY SCIENCE JOURNAL, Volume 47, issued July 1991, H.G. GRIFFIN, "Attenuated Salmonella as Live Vaccines: Prospects for Multivalent Poultry Vaccines", pages 131-140. * |
Cited By (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1996000791A1 (en) * | 1994-06-30 | 1996-01-11 | The Board Of Trustees Of The University Of Illinois | Recombinant infectious laryngotracheitis virus and vaccine |
EP0822980A1 (en) * | 1995-03-23 | 1998-02-11 | Syntro Corporation | Recombinant infectious laryngotracheitis virus and uses thereof |
EP0822980A4 (en) * | 1995-03-23 | 2000-04-26 | Syntro Corp | Recombinant infectious laryngotracheitis virus and uses thereof |
US6033670A (en) * | 1996-12-16 | 2000-03-07 | Merial | Recombinant live avian vaccine, using as vector the avian infectious laryngotracheitis virus |
US6180671B1 (en) | 1998-03-10 | 2001-01-30 | Beth Israel Deaconess Medical Center, Inc. | Methods for treating disorders in which docosahexaenoic acid (DHA) levels are affected |
US6552081B1 (en) | 1998-03-10 | 2003-04-22 | Beth Israel Deaconess Medical Center, Inc. | Methods for treating disorders in which docosahexaenoic acid (DHA) levels are affected |
US7205398B2 (en) | 2002-05-24 | 2007-04-17 | Schering-Plough Animal Health Corporation | Eta-1 gene and methods for use |
US7780965B2 (en) | 2002-05-24 | 2010-08-24 | Schering-Plough Animal Health Corp. | Eta-1 gene and methods for use |
US8188236B2 (en) | 2002-05-24 | 2012-05-29 | Intervet Inc. | Eta-1 gene and methods for use |
US20130129780A1 (en) * | 2010-08-02 | 2013-05-23 | University Of Georgia Research Foundation, Inc. | Modified infectious laryngotracheitis virus (iltv) and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
EP0723584A4 (en) | 2000-12-20 |
EP0723584A1 (en) | 1996-07-31 |
JPH09505726A (en) | 1997-06-10 |
AU7838694A (en) | 1995-04-10 |
CA2172387A1 (en) | 1995-03-30 |
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