WO2015013178A1 - Vaccin contre le virus de la laryngotrachéite infectieuse (iltv) faisant appel à un vecteur de virus recombinant de la maladie de newcastle - Google Patents

Vaccin contre le virus de la laryngotrachéite infectieuse (iltv) faisant appel à un vecteur de virus recombinant de la maladie de newcastle Download PDF

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WO2015013178A1
WO2015013178A1 PCT/US2014/047395 US2014047395W WO2015013178A1 WO 2015013178 A1 WO2015013178 A1 WO 2015013178A1 US 2014047395 W US2014047395 W US 2014047395W WO 2015013178 A1 WO2015013178 A1 WO 2015013178A1
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virus
iltv
ndv
rndv
gene
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WO2015013178A9 (fr
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Siba K. Samal
Mallikarjuna Kanabagatte BASAVARAJAPPA
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University Of Maryland
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    • 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
    • A61K39/12—Viral antigens
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16322—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
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    • 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
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18122—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
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2760/00011—Details
    • C12N2760/18011—Paramyxoviridae
    • C12N2760/18111—Avulavirus, e.g. Newcastle disease virus
    • C12N2760/18141—Use of virus, viral particle or viral elements as a vector
    • C12N2760/18143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present application relates to recombinant Newcastle disease viruses useful as vaccine vectors, which when carrying one or more foreign genes, i.e. genes not found naturally in the Newcastle disease virus, are also useful as bivalent or multivalent vaccines .
  • Newcastle disease is a highly contagious viral disease affecting all species of birds. The disease can vary from an asymptomatic infection to a highly fatal disease, depending on the virus strain and the host species. Newcastle disease has a worldwide distribution and is a major threat to the poultry industries of all countries. Based on the severity of the disease produced in chickens, Newcastle disease virus (NDV) strains are grouped into three main pathotypes: lentogenic (strains that do not usually cause disease in adult chickens), mesogenic (s of intermediate virulence) and velogenic (strains that cause high mortality).
  • lentogenic strains that do not usually cause disease in adult chickens
  • mesogenic strains of intermediate virulence
  • velogenic strains that cause high mortality
  • NDV is a member of the genus Rubulavirus in the family Paramyxoviridae .
  • the genome of NDV is a non- segmented, single-stranded, negative-sense RNA of 15186 nucleotides ( Krishnamurthy & Samal, 1998, J Gen Virol 79, 2419-2424; Phillips et al., 1998, Arch Virol 143, 1993-2002; de Leeuw and Peeters, 1999, J Gen Virol 80, 131-136).
  • the genomic RNA contains six genes that encode the following proteins in the order of: the nucleocapsid protein (NP),
  • P phosphoprotein
  • M matrix protein
  • F fusion protein
  • HN haemagglutinin-neuraminidase
  • L large polymerase protein
  • NP nucleocapsid
  • P and L proteins constitute the nucleocapsid.
  • the genomic RNA is tightly bound by the NP protein and together with the P and L proteins form the functional
  • nucleocapsid within which resides the viral
  • the F and HN proteins form the external envelope spikes, where the HN glycoprotein is responsible for attachment of the virus to host cell receptors and the F
  • glycoprotein mediates fusion of the viral envelope with the host cell plasma membrane thereby enabling penetration of the viral genome into the cytoplasm of the host cell.
  • the HN and F proteins are the main targets for the immune response.
  • the M protein forms the inner layer of the virion.
  • NDV follows the general scheme of transcription and replication of other non-segmented negative- strand RNA viruses .
  • the polymerase enters the genome at a promoter in the 3 ' extragenic leader region and proceeds along the entire length by a sequential stop-start mechanism during which the polymerase remains template bound and is guided by short consensus gene start (GS) and gene end (GE) signals. This generates a free leader RNA and six non- overlapping subgenomic mRNAs . The abundance of the various mRNAs decreases with increasing gene
  • RNA replication occurs when the polymerase somehow switches to a read-through mode in which the
  • Infectious laryngotracheitis is an acute respiratory disease of chickens that causes
  • the causative pathogen, ILTV is a member of the genus Iltovirus in the family Herpesviridae (Bagust et al., 2000, supra; Fuchs et al., 2007, Vet Res 38, 261-279).
  • live attenuated vaccines are used to control ILT infections.
  • live-attenuated vaccines are not satisfactory since they can revert to virulence after bird-to-bird passage (Guy et al., 1991, Avian Dis 35, 348-355) and can induce latent infections (Hughes et al., 1991, Arch Virol 121, 213-218).
  • Several alternative strategies have been used to develop improved ILTV vaccines (Mauricio et al., 2013, Avian Pathol 42, 195-205).
  • One of the strategies has been the
  • glycoprotein B has been shown to be a major protective immunogen (Tong et al., 2001, supra; Sun et al., 2008, supra; York et al., 1991, Avian Pathol 20, 693-704), but the role of other glycoproteins in immunity and protection has not been evaluated.
  • the inventors have evaluated the role of three major surface proteins (gB, gC, and gD) of ILTV in
  • Newcastle disease virus NDV
  • gB, gC, and gD of ILTV were constructed and used to immunize chickens.
  • Reverse-genetic techniques were used in making the recombinant NDVs of the present invention from cloned cDNA. This approach involves co-expression of the cloned cDNA of full length NDV genome and nucleocapsid proteins (the NP, P and L proteins) from transfected plasmids using the vaccinia
  • recombinant NDV can be recovered from cDNA and the genome of NDV can be manipulated at the cDNA level.
  • the production of infectious NDV from cloned cDNA can be used to engineer NDV carrying foreign genes.
  • the genome of NDV one can insert foreign sequences into the NDV genome for co- expression.
  • the gene for a protective antigen of another avian pathogen or the genes for avian cytokines can be inserted into the NDV genome for co-expression.
  • the present invention includes
  • NDV vaccines carrying genes encoding immunogens (e.g. immunogenic proteins) for pathogens of interest, such as for influenza virus, infectious bursal disease virus, rotavirus, infectious bronchitis virus, infectious laryngotracheitis virus, chicken anemia virus, Marek's disease virus, avian Leukosis virus, avian adenovirus and avian pneumovirus.
  • immunogens e.g. immunogenic proteins
  • pathogens of interest such as for influenza virus, infectious bursal disease virus, rotavirus, infectious bronchitis virus, infectious laryngotracheitis virus, chicken anemia virus, Marek's disease virus, avian Leukosis virus, avian adenovirus and avian pneumovirus.
  • the present invention also is directed toward a genetically engineered NDV carrying avian cytokine genes.
  • a NDV carrying at least one gene encoding an avian cytokine e.g. an interleukin such as IL-2 and IL-4, can be used as a vaccine.
  • the recombinant NDV prepared by insertion of foreign genes into the NDV genome can express the foreign genes in cells infected by the recombinant NDV. As a result, the recombinant NDV can be used to express proteins of non-avian pathogens or other avian pathogens.
  • One of the objects of the invention is to provide a recombinant Newcastle disease virus (rNDV) comprising NP gene, P gene, M gene, F gene, HN gene and L gene.
  • the Newcastle disease virus contains a tyrosine to alanine substitution in the fusion or "F " gene at amino acid position 527. This tyrosine has been found to be conserved among different strains of NDV. The tyrosine can be substituted to any
  • hydrophobic amino acid selected from the group:
  • NDV F gene is meant use of either the wild-type or the mutant form unless expressly stated.
  • Another object of the present invention is a recombinant antigenomic RNA or cDNA of Newcastle disease virus, comprising NP gene, P gene, M gene, F gene, HN gene and L gene in this order from a 5 ' to 3' direction, said antigenomic RNA further
  • n foreign nucleotide complexes comprising n foreign nucleotide complexes inserted (a) before the NP gene, (b) between the P and M genes, and/or (c) between the HN and L genes, wherein n is 1, 2, 3 or 4 ;
  • each of the foreign nucleotide complexes comprising a Newcastle disease virus gene start sequence, an open reading frame of a foreign gene and a Newcastle disease virus gene end sequence in this order from the 5' to 3' direction, wherein the foreign gene is a gene not found naturally in the Newcastle disease virus;
  • the foreign nucleotide complexes are the same or different; and wherein when 1, 2, 3 or 4 the foreign nucleotide complexes are inserted together or separately before the NP gene, between the P and M genes, or between the HN and L genes, the foreign nucleotide complexes are sequentially linked directly or indirectly.
  • each foreign nucleotide complex has a NDV gene start signal, i.e. GS sequence motif, upstream of the open reading frame (ORF) of the foreign gene and a NDV gene end signal, i.e. GE sequence motif, downstream of the ORF of the foreign gene, each foreign nucleotide complex forms a transcriptional unit or a gene cassette.
  • NDV gene start signal i.e. GS sequence motif
  • NDV gene end signal i.e. GE sequence motif
  • NP-P intergenic region between the NP gene and P gene comprises NP-P intergenic region between the NP gene and P gene, P-M intergenic region between the P gene and M gene, M-F intergenic region between the M gene and F gene, F-HN intergenic region between the F gene and HN gene, and/or HN-L intergenic region between the HN gene and L gene.
  • the foreign nucleotide complexes When one or more of the foreign nucleotide complexes are inserted between the P and M genes, the foreign nucleotide complexes can be inserted into the P-M intergenic region if present.
  • nucleotide complexes are inserted between the HN and L genes, the foreign nucleotide complexes can be inserted into the HN-L intergenic region.
  • one or more of the NP-P intergenic region, P-M intergenic region, M-F intergenic region, F-HN intergenic region, and HN-L intergenic region are replaced with a single nucleotide, dinucleotide or an oligonucleotide of 3-80
  • nucleotides preferably 4-60 nucleotides
  • the oligonucleotide optionally contains one or more restriction sites.
  • the foreign nucleotide complexes When one or more of the foreign nucleotide complexes are inserted before the NP gene, the foreign nucleotide complexes preferably are inserted into a non-coding region immediately before the ORF of the NP gene, so that the ORF of the foreign gene in each of the foreign nucleotide complexes is flanked by NDV gene start and gene end signals and the ORF of the NP gene is preceded by a NDV gene start signal, with the GS-foreign gene ORF-GE structure preceding the GS signal for the NP ORF.
  • a recombinant antigenomic RNA of NDV having one or more foreign nucleotide complexes inserted between P and M genes.
  • the antigenomic RNA or cDNA can be made by inserting the one or more foreign nucleotide
  • the ORF of the foreign gene is preceded by a NDV gene end and NDV gene start signals, resulting in the ORF of the P gene being preceded by a NDV gene end signal, which is followed by a NDV gene start signal, the ORF of the foreign gene, and a NDV gene end signal in that order (the ORF of the following M gene is preceded by a NDV gene start signal). More foreign gene complexes can be inserted after this foreign gene complex.
  • the recombinant antigenomic RNA or cDNA of NDV having one or more foreign nucleotide complexes inserted between P and M genes can be made by inserting the one or more foreign nucleotide complexes into the noncoding region of M gene before the ORF of the M gene.
  • the present invention is also directed toward a process of preparing the recombinant antigenomic RNA of the invention, comprising the following steps: (i) providing a cDNA comprising NP gene, P gene, M gene, F gene, HN gene and L gene in this order, said cDNA further comprising n foreign nucleotide complexes inserted (a) before the NP gene, (b) between the P and M genes, and/or (c) between the HN and L genes, wherein n is 1, 2, 3 or 4;
  • each of the foreign nucleotide complexes comprising a Newcastle disease virus gene start sequence, an open reading frame of a foreign gene and a Newcastle disease virus gene end sequence in this order from the 5' to 3' direction, wherein the foreign gene is a gene not found naturally in the Newcastle disease virus;
  • the cDNA used in step (i), comprising NP gene, P gene, M gene, F gene, HN gene and L gene having the n foreign nucleotide complexes inserted is prepared by (I) constructing a cDNA comprising the NP gene, P gene, M gene, F gene, HN gene and L gene in this order; and thereafter (II) inserting the n foreign nucleotide complexes (a) before the NP gene, (b) between the P and M genes, and/or (c) between the HN and L genes.
  • the cDNA constructed in step (I) and/or the cDNA constructed in step (II) are in a plasmid, such as pBR322 or pGEM-7Z.
  • the cDNA preferably is transcribed in cells expressing a RNA polymerase, such as T7 RNA
  • the present invention is also directed toward a recombinant NDV (rNDV) comprising a recombinant antigenomic RNA carrying one or more foreign genes of the present invention.
  • rNDV recombinant NDV
  • the recombinant NDV can be produced by a process comprising the following steps :
  • step (iii) isolating Newcastle disease virus from a supernatant of the medium of step (ii) to obtain the recombinant Newcastle disease virus.
  • the cells capable of synthesizing T7 RNA polymerase provided in step (i) can be animal cells of an avian or mammalian species, plant cells, or cells from a cell line expressing T7 RNA polymerase.
  • a cDNA encoding a recombinant NDV antigenomic RNA having one or more foreign genes inserted according to the invention a cell containing the cDNA, a plasmid comprising the cDNA, a cell containing the plasmid, a cell containing the recombinant
  • a recombinant NDV containing the recombinant antigenomic RNA of the invention e.g. a recombinant NDV carrying one or more foreign genes recovered from transcription of the cDNA or the plasmid in a competent cell.
  • the recombinant NDV containing the recombinant antigenomic RNA of the invention is preferably substantially purified. Also preferred is a substantially purified recombinant antigenomic RNA of NDV carrying one or more foreign genes prepared according to the invention.
  • the present invention provides a cDNA encoding a recombinant antigenomic NDV RNA having one or more genes from ILTV inserted according to the invention, a cell containing the cDNA, a plasmid comprising the cDNA, a cell
  • a cell containing the recombinant antigenomic RNA, and a recombinant NDV containing the recombinant antigenomic RNA of the invention e.g. a recombinant NDV carrying one or more ILTV genes recovered from transcription of the cDNA or the plasmid in a competent cells.
  • the recombinant NDV, or rNDV, containing the one or more inserted foreign genes can be used as a monovalent vaccine to provide immunity and
  • the present invention includes a bivalent vaccine to provide immunity and protection against NDV and ILTV challenge, the vaccine comprising rNDV having one or more genes from ILTV, gB, gC, gD, preferably gD.
  • the present invention also includes a method of vaccinating an avian animal against Newcastle disease, wherein the avian animal is in need of the vaccination, comprising administering an effective amount of the recombinant NDV optionally carrying one or more foreign genes according to the invention to the avian animal .
  • One of the objects of the invention is a method of treating an avian animal with an avian cytokine, wherein the avian animal is in need of the
  • said method comprising administering an effective amount of the recombinant NDV of the invention carrying one or more foreign genes
  • Another object of the invention is a method of immunizing an avian animal against an avian pathogen selected from the group consisting of influenza virus, infectious bursal disease virus, rotavirus, infectious bronchitis virus, infectious
  • laryngotracheitis virus chicken anemia virus, Marek's disease virus, avian Leukosis virus, avian adenovirus and avian pneumovirus, wherein the avian animal is in need of the immunization, said method comprising administering an effective amount of the recombinant NDV of the invention to the avian animal, wherein one or more the recombinant NDV carries one or more foreign genes encoding one or more immunogenic proteins of the avian pathogen against which the avian animal is immunized.
  • the present invention provides a method of immunizing an avian animal against ILTV, said method comprising administering an effective amount of the recombinant NDV of the present invention, wherein the NDV carries one or more ILTV genes encoding one or more immunogenic ILTV proteins.
  • the ILTV genes are gB, gC, and gD, in any combination.
  • Also within the scope of the invention is a method of immunizing a mammal against a non-avian pathogen, wherein the mammal is in need of the immunization, said method comprising administering an effective amount of the one or more recombinant NDV of the invention to the mammal, wherein the recombinant NDV carries one or more foreign genes encoding one or more immunogenic proteins of the non-avian pathogen, e.g.
  • influenza virus SARS- causing virus, human respiratory syncytial virus, human immunodeficiency virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, poliovirus, rabies virus, Hendra virus, Nipah virus, human parainfluenza 3 virus, measles virus, mumps virus, Ebola virus, Marburg virus, West Nile virus,
  • ILTV gD is a major protective immunogen capable of inducing a protective immune response against ILTV in chickens.
  • Immunization with rNDV expressing ILTV gD induced a higher level of neutralizing antibodies and offered complete protection to chickens against lethal ILTV challenge.
  • the complete protection offered by gD can be attributed to its superior envelope incorporation and cell surface expression leading to induction of a protective immune
  • the ILTV gD protein can be exploited as an effective vaccine antigen for the development of safe vectored vaccines against ILT using viral and nonviral vectors.
  • viral vectors include adenovirus, adeno-associated virus,
  • herpesvirus pox virus, influenza virus, retrovirus, and other recombinant viral vectors known to a person in the art.
  • the present invention provides an ILTV vaccine comprising gD. Also provided is a method for eliciting in a subject an immune response against ILTV , the method
  • nucleic acid comprising a gD encoding nucleic acid comprising administering to a subject a nucleic acid comprising a gD encoding nucleic acid.
  • the nucleic acid comprising a gD encoding nucleic acid can be part of a vector such as a viral vector, capable of producing gD in an immunized avian or non-avian animal.
  • a composition comprising gD can be administered to a subject in need thereof.
  • FIG. 1 Construction of recombinant NDVs expressing ILTV gB, gC, and gD.
  • gC or gD ectodomain of gC or gD respectively, fused to the transmembrane and cytoplasmic tail of the NDV F protein.
  • the inserted foreign ORF was placed under the control of a set of NDV transcriptional gene end (GE) and gene start (GS) signals such that each was expressed as a separate mRNA.
  • GE NDV transcriptional gene end
  • GS gene start
  • FIG 2-1 and 2-2 Western blot and flow cytometry analysis of the rNDVs expressing ILTV proteins.
  • Fig 2-1 Expression of ILTV gB, gC, and gD in DF1 cells and their incorporation into rNDV virions.
  • Fig 2-2 Flow cytometry analysis of the surface expression of ILTV proteins.
  • DF1 cells were infected with rNDV gB (panel A), rNDV gC (panel B) or rNDV gD (panel C) viruses at a MOI of 5, in parallel with cells that were mock-infected or infected with the rNDV LaSota empty vector.
  • the cells were probed with rabbit anti-ILTV sera, followed by incubation with Alexa Fluor 488 conjugated goat anti-rabbit IgG antibody and analyzed by Flowjo program of FACSRIA II flow cytometer. Values represent averages of the results obtained from two independent experiments .
  • FIG. 1 Immunoelectron microscopy of purified virions of rNDV LaSota, rNDV gB, rNDV gC, and rNDV gD, analyzed using rabbit anti ILTV serum against gB (upper panel), gC (middle panel) or gD (lower panel ) .
  • Figure 4-1 Multicycle growth kinetics of rNDVs in nine-day-old SPF embryonated chicken eggs. Nine-day- old embryonated chicken eggs were inoculated with 100 PFU of each virus, and allantoic fluids from three eggs were harvested at different time points (12 h, 24 h, 36 h, 48 h, 60 h, and 72 h) after inoculation. The virus titer in allantoic fluid was determined by TCID50 assay in DF-1 cells.
  • FIG. 5A and 5B ILTV-neutralizing antibody response post-vaccination and clinical signs score evaluation post-ILTV challenge.
  • 5A Chickens were immunized by the oculonasal route with rNDVs either individually or in combination. Sera were taken on days 12 (12 days following primary immunization) and 21 (7 days following booster immunization) post- vaccination and analyzed for the ability to
  • the recombinant antigenomic RNA is from a paramyxovirus, Newcastle disease virus strain LaSota.
  • Other NDV strains for example, Hitchner-Bl (Bl), Clone-30, Strain-F, Strain V4 , Strain V4-HR, Strain-I2 and Ulster (U) can also be used.
  • n is 1, 2, 3 or 4 (preferably 2 or 3 , and more preferably 2) and the foreign nucleotide complexes are different.
  • n is 1, 2, 3 or 4 (preferably 2 or 3 , and more preferably 2) and the foreign nucleotide complexes are the same.
  • n is 1 or 2.
  • the ORF of each of the foreign genes in the inserted foreign nucleotide complexes is no more than about 3000 nucleotides, no more than about 2000 nucleotides, no more than about 1500
  • nucleotides no more than about 1000 nucleotides, no more than about 800 nucleotides, no more than about 500 nucleotides, or no more than about 300
  • nucleotides in length in length.
  • the foreign nucleotide complexes are sequentially linked
  • the foreign nucleotide complexes have a combined length of no more than about 5000 nucleotides, no more than about 4000 nucleotides, no more than about 3000 nucleotides, no more than about 2000 nucleotides, no more than about 1000 nucleotides, or no more than about 800 .
  • the foreign gene inserted in the recombinant antigenomic RNA of the invention preferably encode a substance selected from the group consisting of chloramphenical acetyltransferase , GFP, an avian cytokine, and an immunogenic protein of influenza virus, infectious bursal disease virus, rotavirus, infectious bronchitis virus, infectious
  • Marek's disease virus avian leukosis virus, avian adenovirus, or avian pneumovirus.
  • the foreign gene may encode an immunogenic protein of a non-avian pathogen, e.g. influenza virus, SARS-causing virus, human respiratory syncytial virus, human
  • immunodeficiency virus hepatitis A virus, hepatitis B virus, hepatitis C virus, poliovirus, rabies virus, Hendra virus, Nipah virus, human
  • parainfluenza 3 virus measles virus, mumps virus, Ebola virus, Marburg virus, West Nile disease virus, Japanese encephalitis virus, Dengue virus, Hantavirus, Rift Valley fever virus, Lassa fever virus, herpes simplex virus and yellow fever virus.
  • the foreign genes may encode the same or different avian cytokines, such as avian interleukins , e.g. IL-2 and IL-4.
  • Examples of the foreign gene encoding an immunogenic protein of an avian pathogen are HA or NA gene of influenza virus, VP2 or polyprotein gene of infectious bursal disease virus, S or SI gene of infectious bronchitis virus, glycoprotein gene of infectious laryngotracheitis virus, e.g. gB, gC, gD, the complete genome of chicken anemia virus,
  • glycoprotein gene of Marek's disease virus envelope gene of avian leukosis virus, avian adenovirus, and G or F gene of avian pneumovirus .
  • Examples of the foreign gene encoding an immunogenic protein of a non-avian pathogen are HA or NA gene of influenza virus, S or SI gene of SARS- causing virus, G or F gene of human respiratory syncytial virus, gp60, gpl20 or gp41 gene of human immunodeficiency virus, surface antigen gene of hepatitis A virus, surface antigen gene of hepatitis B virus, surface antigen of hepatitis C virus, capsid proteins gene of poliovirus, G protein gene of rabies virus, G or F protein gene of Hendra virus, G or F protein gene of Nipah virus, HN or F protein gene of human parainfluenza 3 virus, H or F protein gene of measles virus, HN or F protein gene of mumps virus, G protein gene of Ebola virus, G protein gene of Marburg virus, envelope protein gene of West Nile disease virus, envelope protein gene of Japanese encephalitis virus, envelope protein gene of Dengue virus, glycoprotein gene of Hant
  • the present invention is also directed toward an antigenomic RNA of NDV carrying one or more foreign genes inserted before the NP gene, between the P and M genes, and/or between the HN and L genes, wherein at least one of the foreign genes encodes a tumor antigen, such as pglOO, MAGE1, MAGE3 and CDK4.
  • the foreign nucleotide complexes In the recombinant antigenomic RNA of the invention, the foreign nucleotide complexes
  • At least one of the foreign nucleotide complexes is inserted before the NP gene, and/or between the P and M genes. More preferably, at least one of the foreign nucleotide complexes is inserted before the NP gene. In some embodiments of the recombinant antigenomic RNA, at least one of the foreign nucleotide complexes is inserted before the NP gene and at least one of the foreign nucleotide complexes is inserted between the P and M genes. In some embodiments, at least one of the foreign nucleotide complexes is inserted before the NP gene and at least one of the foreign nucleotide complexes is inserted between the HN and L genes.
  • At least one of the foreign nucleotide complexes is inserted before the NP gene, at least one of the foreign nucleotide complexes is inserted between the P and M genes, and at least one of the foreign nucleotide complexes is inserted between the HN and L genes. In yet some embodiments, at least one of the foreign nucleotide complexes is inserted between the P and M genes. Most preferably, the foreign nucleotide complexes are inserted only before the NP gene.
  • NDV grows to very high titers ( ⁇ 10 9 PFU/ml) in many cell lines and eggs and elicits strong humoral and cellular immune responses in vivo. NDV naturally infects via respiratory and alimentary tract mucosal surfaces. NDV replicates in the cytoplasm of
  • recombination making vaccine vectors based on the recombinant NDV carrying foreign genes stable and safe. Due to these characteristics of NDV described herein, recombinant NDVs that can express foreign genes carried in the recombinant NDVs are good vaccines, wherein the foreign genes encode
  • the recombinant NDV of the invention carrying one or more inserted foreign genes show robust expression of the foreign genes. Moreover, the recombinant NDV expressing one or more of the foreign gene can replicate in cell culture and in vivo. NDV recombinants expressing heterologous proteins could be used as multivalent vaccines.
  • recombinant antigenomic RNA carrying one or more foreign genes inserted according to the invention can also be used as an inactivated vaccine.
  • recombinant NDV generated from the recombinant antigenomic RNA carrying one or more foreign genes inserted according to the invention can be
  • the dose of the vaccine or vaccine vector to be used can be readily
  • ILTV gD is a major protective immunogen capable of inducing protective immune responses against ILTV infection in chickens.
  • gD also includes analogs and truncated forms that are immunologically cross-reactive with natural gD.
  • gD is intended gD from other strains of ILTV, or any other newly identified strain or field isolate of ILTV.
  • gD can be used as a homo-oligomer , containing more than one gD monomer, e.g. gD dimers, trimers or tetramers, or any higher-order homo-oligomers of gD.
  • the oligomers may contain one, two, or several different monomers of gD obtained from different strains of ILTV including for example USDA strain, ILTV strain 63140/C/08/BR, Strain A489, Australian CSW-1 ILTV strain, SA-2 ILTV, A-2G ILTV, Serva-ILTV, Strain Vl-99, Strain Ql-96, Strain N3-Q4, Strain S2- 04, Trachivax ILTV vaccine strain, and other strains and field isolates.
  • Such mixed oligomers are still homo-oligomers within the scope of this invention, and may allow more universal diagnosis, prophylaxis or treatment of ILTV,
  • ILTV gD can be recombinantly expressed, isolated and purified using methods well known in the art.
  • the term 'purified' as applied to proteins herein refers to a composition wherein the desired protein comprises at least 35% of the total protein component in the composition.
  • the desired protein preferably comprises at least 40%, more preferably at least about 50%, more preferably at least about 60%, still more preferably at least about 70%, even more preferably at least about 80%, even more preferably at least about 90%, and most preferably at least about 95% of the total protein component.
  • the composition may contain other
  • An 'isolated' protein intends a composition that is at least 35% pure.
  • the term 'essentially purified proteins' refers to proteins purified such that they can be used for in vitro diagnostic methods and as a prophylactic compound. These proteins are substantially free from cellular proteins, vector-derived proteins or other ILTV components.
  • the proteins of the present disclosure are substantially free from cellular proteins, vector-derived proteins or other ILTV components.
  • recombinantly expressed used within the context of the present invention refers to the fact that the proteins of the present invention are produced by recombinant expression methods be it in prokaryotes, or lower or higher eukaryotes.
  • the present invention relates to a DNA or cDNA segment which encodes ILTV gD as described above. Genome sequences fro different strains of ILTV have been published and are publicly available. DNA or nucleic acid sequences to which the invention also relates include fragments of the gD gene containing protective epitopes or antigenic determinants.
  • the sequence of nucleic acids encoding antigens may be generated in any manner, including for example, chemical synthesis or DNA replication or reverse transcription or transcription, which are based on the information provided by the sequence bases in the region(s) from which the polynucleotide is derived. In addition, combinations of regions corresponding to that of the designated sequence may be modified in ways known in the art to be
  • the DNA encoding the desired antigen can be introduced into the cell in any suitable form including the fragment alone, in a vector such as a linearized plasmid, a circular plasmid, a plasmid capable of replication, an episome, RNA, a viral vector, an expression vector, etc.
  • a vector such as a linearized plasmid, a circular plasmid, a plasmid capable of replication, an episome, RNA, a viral vector, an expression vector, etc.
  • Individual expression vectors capable of expressing the genetic material can be produced using standard recombinant techniques. Please see e.g., Maniatis et al., 1985 Molecular Clonings A Laboratory Manual or DNA
  • the DNA alone or in a vector, can be delivered by injection into the tissue of the recipient, oral or pulmonary delivery. Any of these methods can be used to deliver DNA as long as the DNA is expressed and the desired antigen is made in the cell.
  • the present invention more particularly relates to a composition comprising at least one of the above-specified peptides or a recombinant gD protein composition as defined above, for use as a vaccine for immunizing avian subject against ILT, comprising administering a sufficient amount of the composition possibly accompanied by pharmaceutically acceptable adjuvant(s), to produce an immune response.
  • the vaccine composition of the present invention is expected to provide cross-protection against
  • Immunogenic compositions can be prepared according to methods known in the art.
  • the present compositions comprise an immunogenic amount of a recombinant protein or peptides as defined above, usually combined with a pharmaceutically acceptable carrier, preferably further comprising an adjuvant.
  • Pharmaceutically acceptable carriers include any carrier that does not itself induce the
  • Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; and inactive virus particles. Such carriers are well known to those of ordinary skill in the art ,
  • the immunogenic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, preservatives, and the like, may be included in such vehicles .
  • the immunogenic compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to administration may also be prepared.
  • Immunogenic compositions used as vaccines comprise a 'sufficient amount' or 'an immunologically effective amount' of the protein gD or a vector which will produce a sufficient amount of the gD protein in the subject, 'Immunologically effective amount', means that the administration of that amount to an individual , either in a single dose or as part of a series, is effective for treatment, as defined above. This amount varies depending upon the health and physical condition of the subject to be treated, the formulation of the vaccine, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Usually, the amount will vary from 0.01 to 1000 ug/dose, more particularly from about 1.0 to 100 ug/dose most preferably from about 10 to 50 ug/dose.
  • Administration of the compounds or vaccines, disclosed herein may be carried out by any suitable means, including parenteral injection (such as intraperitoneal, subcutaneous, or intramuscular injection), in ovo injection of birds, orally, oculonasal, or by topical application to an airway surface carried out by inhalation administration, such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) as an aerosol suspension, and then causing the subject to inhale the respirable particles.
  • parenteral injection such as intraperitoneal, subcutaneous, or intramuscular injection
  • inhalation administration such as by creating respirable particles of a pharmaceutical formulation (including both solid particles and liquid particles) as an aerosol suspension, and then causing the subject to inhale the respirable particles.
  • respirable particles of a pharmaceutical formulation including both solid particles and liquid particles
  • aerosol suspension a pharmaceutical formulation
  • Oral administration may be in the form of an ingestable liquid or solid formulation .
  • the treatment may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of treatment may be with 1—10 separate doses , followed by other doses given at subsequent time intervals required to maintain and or reinforce the response, for example, at 1-4 months for a second dose, and if needed, a
  • suitable treatment schedules include; (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, ⁇ iii ⁇ 0 and 1 month, (iv) 0 and 6 months, (v) 0 and 14 days, or other schedules sufficient to elicit the desired responses expected to reduce disease symptoms, or reduce severity of disease.
  • the present invention also provides kits which are useful for carrying out the present invention.
  • the present kits comprise a first container means containing the above-described antibodies.
  • the kit also comprises other container means containing solutions necessary or convenient for carrying out the invention.
  • the container means can be made of glass, plastic or foil and can be a vial, bottle, pouch, tube, bag, etc.
  • the kit may also contain written information, such as procedures for carrying out the present invention or analytical information, such as the amount of reagent contained in the first container means .
  • the container means may be in another container means, e.g. a box or a bag, along with the written information. All publications, including, but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as if each individual publication were specifically and individually indicated to be incorporated by reference herein as though fully set forth.
  • Human epidermoid carcinoma, chicken embryo fibroblast, and Vero cells were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and maintained in DMEM with 5% FBS .
  • DMEM Dulbecco's modified Eagle medium
  • FBS fetal bovine serum
  • Chicken embryo liver cells (CELi) and chicken embryo kidney cells (CEK) were harvested from 11 -12 day-old and 18-19 day-old specific pathogen free embryonated chicken eggs, respectively, by
  • the chicken-embryo-origin ILTV vaccine Trachivax was obtained from the Schering- Plough Animal Health Corp, Millsboro, DE .
  • the chicken-embryo-origin ILTV vaccine Trachivax was obtained from the Schering- Plough Animal Health Corp, Millsboro, DE .
  • Vectormune HVT-LT vaccine was obtained from the Ceva Animal Health, Lenexa, KS.
  • the USDA challenge strain of ILTV was obtained from the National Veterinary Services Laboratory, Ames, IA, USA.
  • the USDA ILTV challenge strain was propagated on monolayers of chicken embryo liver cells.
  • Recombinant NDV strains were grown in 9-day-old specific-pathogen-free (SPF) embryonated chicken eggs.
  • the modified vaccinia virus Ankara strain expressing T7 RNA polymerase was grown in primary chicken embryo fibroblast cells.
  • the Freund's complete and the Freund's incomplete adjuvants were obtained from the Sigma-Aldrich, St- Louis, MO.
  • the anti-ILTV antiserum was raised in rabbits against the synthetic peptides of gB, gC, and gD of ILTV.
  • Synthetic peptides of ILTV gB, gC, and gD were obtained from GenScript USA Inc.,
  • anti-ILTV gB, gC, and gD antisera was determined by Western blot analysis.
  • a Anti ILTV titers were determined by ELISA (ProFLOCK® LT ELISA Kit, Synbiotics Corp., San Diego, CA) following the
  • the ILTV gB, gC, and gD (GenBank accession number NC_06623) open reading frames were PCR amplified from the purified ILTV DNA and were subsequently cloned into the pCR 4 TOPO vector
  • the nucleic acid sequence of gB is shown in SEQ ID NO:l, and the amino acid sequence is shown in SEQ ID NO: 2.
  • the nucleic acid sequence of gC in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4.
  • the nucleic acid sequence of gD is shown in SEQ ID NO: 5, and the amino acid sequence is shown in SEQ ID NO: 6.
  • the integrity of the gB, gC, and gD genes was confirmed by sequence analysis. To construct an insert encoding the modified gB glycoprotein, the complete ORF
  • the gB open reading frame (ORF) was amplified by PCR using forward primer ( gBF )
  • glycoprotein gC and gD inserts were constructed by fusing the ectodomain of glycoproteins to the transmembrane domain and cytoplasmic tail (amino acids 497-553) of the NDV F protein by overlapping PCR. Briefly, the gC gene of ILTV was amplified by PCR using a forward primer (gCF) 5'
  • GATCTTAATTAATTAGAAAAAATACGGGTAGAAGGCCACCatqcaqcatcaq agtactgcg 3' (SEQ ID NO: 9) (The primer and its constituents are notated similarly as described for the gBF primer) and a reverse primer (gCl) 5'- GACTGCGGGGAATCCTTGCCGCATTG-3 ' (sequence represents the sequence specific to ILTV gC gene ORF at
  • transmembrane domain and cytoplasmic tail sequences of the NDV F gene was PCR amplified using forward primer (gC2) 5'- CAATGCGGCAAGGATTCCCCGCAGTCagcacatctgctctcattac-3 ' (SEQ ID NO: 10 )( sequence specific to ILTV gC gene overlap is in uppercase and NDV F gene
  • transmembrane-specific sequence is in lower case
  • a reverse primer gCR 5'- gateTTAATTAATCACAT TTTTGTAGTGGCTCTCATCTGATC-3 ' (SEQ ID NO:ll)(the Pad site is italicized and NDV F gene cytoplasmic tail- specific sequence is in uppercase).
  • the ILTV gD gene was amplified by PCR using a forward primer (gDF) 5'- GATCTTAATTAATTAGAAAAAATACGGGTAGAAGGCCGCCACCatqqaccQC catttatttttgag-3 ' (SEQ ID NO: 12) (The primer and its constituents are notated similarly as described for gBF primer) and a reverse primer (gDl) 5'- GGGCATGGA GACGGCATTAGAACT-3 ' (SEQ ID NO : 13 )( sequence
  • NDV F gene represents the sequence specific to ILTV gD gene ORF at position (1030-1053).
  • the transmembrane domain and cytoplasmic tail sequences of the NDV F gene was PCR amplified using forward primer (gD2) 5'- AGTTCTAATGCCGTCTCCATG CCCagcacatctgctctcattacct-3 ' (SEQ ID NO: 14 )( sequence specific to ILTV gD gene overlap is in uppercase and NDV F gene
  • transmembrane-specific sequence is in lower case
  • a reverse primer gDR 5'- gatcTTAATTAATCACATTTTTGTAGTGGCTCTCATCTGATC-3 ' ( SEQ ID NO: 15) (the Pad site is italicized and NDV F gene cytoplasmic tail-specific sequence is in uppercase). Both the fragments were ligated by overlapping PCR by using forward primer gDF and reverse primer gDR. After amplification, 1227-bp PCR product was cloned into pCR-4 Topo vector (Invitrogen) and sequenced to confirm the correct gD gene structure and the absence of any mutations.
  • Immunofluorescence assay was performed to evaluate the cell surface and intracellular expression of ILTV glycoproteins. Briefly, confluent monolayers of vero cells on 4 well Lab-Tek chamber slides were infected with the recombinant viruses at a multiplicity of infection (MOI) of 0.1. At 24 h post-infection, the infected cells were either fixed with 4% paraformaldehyde for 20 min at room
  • glycoproteins to the transmembrane domain and cytoplasmic tail (amino acids 497-553) of the NDV F protein.
  • the inserts bearing the gB, gC, and gD gene of ILTV were cloned at the unique Pad site between P and M genes of full-length NDV plasmid.
  • the resulting plasmids were designated as pNDV gB (SEQ ID NO: 16), pNDV gC (SEQ ID NO: 17), and pNDV gD (SEQ ID NO: 18), respectively, (figure 1) which were used to recover recombinant viruses designated rNDV gB (SEQ ID NO: 19), rNDV gC (SEQ ID NO: 20), and rNDV gD (SEQ ID NO: 21), respectively, following the
  • ILTV gB, gC, and gD The expression of ILTV gB, gC, and gD was examined by Western blot, immunofluorescence, and flow cytometry while their incorporation by rNDVs was evaluated by Western blot and immunoelectron microscopy assays as described (Nayak et al., 2009, supra; Khattar et al., 2010, Vaccine 28, 3159-70; Khattar et al., 2011, J Virol 85, 10529-41; Nayak et al., 2010, J Virol 84, 2408-20), using anti-peptide antisera raised in rabbits against ILTV gB, gC, and gD.
  • DF1 cells were infected with the individual rNDV constructs and 48 h later the cells were collected and processed to prepare cell lysates.
  • ILT virions infected Chicken embryo liver cell lysates were cleared by centrifugation at 4500 x g for 15 min followed by sedimentation of ILTV by centrifugation through a cushion of 40% sucrose in phosphate buffered saline (PBS), and purified in a continuous 20—50% sucrose gradient at 25,000 rpm and 4 °C for 1 and half hour. The virions were resuspended in PBS.
  • Total CEK cell lysates were prepared 24 h after infection with ILTV at a multiplicity (MOI) of 5 PFU per cell. These samples were analyzed by Western blot analysis using rabbit anti-ILTV gB, gC, and gD antisera (see text for details ) .
  • DF1 cells were infected with the rNDV gB (panel A), rNDV gC (panel B) or rNDV gD (panel C) viruses at a MOI of 5, in parallel with cells that were mock-infected or infected with the rNDV LaSota empty vector.
  • the cells were probed with rabbit anti- ILTV sera, followed by incubation with Alexa Fluor 488 conjugated goat anti-rabbit IgG antibody and analyzed by Flowjo program of FACSRIA II flow cytometer. Values represent averages of the results obtained from two independent experiments .
  • expressing ILTV gB, gC, and gD were determined in SPF embryonated chicken eggs (Nayak et al., 2010 supra) .
  • the pathogenicity of recombinant viruses was determined by the mean death time (MDT) test in 9- day-old SPF embryonated chicken eggs (Nayak et al., 2010 , supra) .
  • the immunogenicity and protective efficacy of the recombinant viruses against virulent ILTV and virulent NDV challenges were evaluated in specific pathogen free (SPF) chickens obtained from Charles River Laboratories, Wilmington, MA, USA.
  • SPF pathogen free
  • a total of 140 two-week-old SPF white leghorn chickens were assigned to 10 groups of 14 chickens each and received a prime-boost immunization on days 0 and 14 with the indicated virus by the indicated routes as described below (the day 0 and day 14 doses are identical). Briefly, the control group remained unvaccinated and served later as challenge controls.
  • Group CEO and HVT-LT were vaccinated with the ILTV- CEO vaccine Trachivax and the recombinant herpes virus of turkey expressing laryngotracheitis
  • the groups gB, gC, and gD received a virus rNDV gB, rNDV gC, and rNDV gD, respectively, by oculonasal route with a dose of 10 6 TCID 50 /mL, whereas the groups gB+gC, gB+gD, gC+gD, and gB+gC+gD were immunized through the same route with a multivalent vaccine consisting of a mixture of 10 6 TCID 50 /mL each of rNDV gB and rNDV gC, a mixture of 10 6 TCID 50 /mL each of rNDV gB and rNDV gD, a mixture of 10 6 TCID 50 /mL each of rNDV gC and rNDV gD, and a mixture of 10 6 TCID 50 /mL each of rNDV gC and rNDV gD, and
  • Each oculonasal immunization involved administration of allantoic fluid containing the indicated rNDVs in a total volume of 200 ⁇ 1_ ⁇ (50 ⁇ 1_ ⁇ in each eye and nostril). Blood was collected on days 12 and 21 and sera were
  • Tissue sample (trachea, lungs, and brain) were collected, homogenized in cell culture medium (lgm/lOml) and clarified by centrifugation .
  • the challenge virus titers in tissue samples were
  • ILT challenge virus in a total volume of 200 ⁇ 1_ ⁇ (100 ⁇ 1_ ⁇ intratracheally and 50 ⁇ 1_ ⁇ in each nostril). All birds were observed daily for 14 days post challenge for clinical signs of dyspnea, conjunctivitis,
  • the homogenate was used to determine the challenge virus titers by limiting dilutions in chicken embryo liver cells. The remaining five chickens in each group were observed daily for 14 days for disease signs and mortality following challenge. Virulent NDV Texas-GB challenge
  • VNT neutralization test
  • HI hemagglutination inhibition
  • Inflammatory, necrotic, and ulcerative lesions were scored as 0 (no lesions), + (minimal lesions), ++ (mild lesions), +++ (moderate lesions), and ++++ (severe lesions). Inclusion bodies were scored as either + (present) or —
  • HEp-2 cells (6-well plates) were infected at 1 p.f.u. per cell with modified vaccinia virus (MVA/T7) expressing T7 RNA polymerase.
  • VVA/T7 modified vaccinia virus
  • Lipofectamine Plus (Life Technologies). Four h after transfection, cells were washed and the medium was replaced with 2 ml fresh medium (DMEM with 0% fetal calf serum and 1 ⁇ g/ml acetyl trypsin). Three days post-transfection, the supernatant was harvested for virus, clarified and used to infect fresh HEp-2 cells. Three days later, 100 ⁇ supernatant was taken to inoculate into the allantoic cavity of 10- day-old embryonated SPF eggs. After 96 h, allantoic fluid was harvested and tested for haemagglutinating (HA) activity.
  • HA haemagglutinating
  • a recombinant vaccinia virus-based transfection system was used to recover infectious recombinant NDV from cDNA.
  • HEp-2 cells were infected with recombinant vaccinia virus (MVA/T7) capable of synthesizing T7 RNA polymerase.
  • VVA/T7 recombinant vaccinia virus
  • the cells were transfected with the recombinant NDV encoding the desired foreign antigen, along with plasmids encoding proteins of RNP complex, namely NP (pNP), P (pP), and L (pL) .
  • NP pNP
  • P P
  • L L
  • HA hemagglutination
  • the recovered virus was designated, for example rNDVgB, when the foreign antigen was gB, to distinguish it from the parental wild-type NDV, or in this case, pNDV gB.
  • the Newcastle disease virus Fusion protein (F) is a major contributor to the protective immunity of the NDV vaccine and also the primary determinant of NDV virulence and pathogenicity in chickens.
  • the cytoplasmic tail of the NDV fusion protein contains a tyrosine amino acid at position 527 (of the "F " protein, SEQ ID NO: 25) which is found to be
  • tyrosine was substituted to alanine, cloned into PBR322 to produce pNDVY527A (SEQ ID NO:26) and the resulting Newcastle disease virus, rNDVY527A (SEQ ID NO: 27), with phenotype designated "Y527A” was compared with the wild type Lasota (WT) virus for its ability to multiply in cell culture, fusogenicity , levels of surface expression of a foreign protein, pathogenicity to chicken eggs and chicken embryos, and immunogenicity and protective efficacy in chickens against virulent NDV challenge.
  • WT Lasota
  • Y527A Growth characteristics and fusion activity of Y527A: The multistep growth kinetics and magnitudes of replication of the Y527A and the WT viruses were determined in DF1 cells (data not shown) . Both the viruses replicated exponentially until -40 hpi, after which replication was at a plateau. The magnitudes of replication were similar for WT and the Y527A, however, the titer of the Y527A virus was approximately 1.75 loglO higher than that of WT at 24 hpi. These results suggest that the mutagenesis in Y527A virus did not compromise its ability to multiply in cell culture but the same has been improved slightly over the WT virus.
  • MDT mean embryo death time
  • ICPI ICPI test.
  • MDT values were determined in 9-day-old embryonated chicken eggs (data not shown) .
  • NDV strains are categorized into three pathotypes on the basis of their MDT values: velogenic (less than 60 h), mesogenic (60 to 90 h), and lentogenic (greater than 90 h) .
  • the MDT value of the Y527A mutant (90.60 h) was reduced by 10 h compared to that for WT
  • the groups Y527A and WT received a virus Y527A and WT, respectively, by oculonasal route with a dose of 10 6 TCID50/mL.
  • Each oculonasal immunization involved administration of allantoic fluid containing the indicated recombinant viruses in a total volume of 200 ⁇ (50 ⁇ in each eye and nostril). Blood was collected on day 21 and sera were separated from the blood samples for analyzing antibody response. After 21 days, birds were
  • Tissue sample (trachea, lungs, and brain) were collected, homogenized in cell culture medium (lgm/lOml) and clarified by centrifugation .
  • the challenge virus titers in tissue samples were determined by limiting dilution in DF-1 cells.
  • NDV-specific antibody responses in the sera collected on 21 st day post immunization was assayed using HI test. High levels of NDV-specific serum antibodies were detected for both Y527A and WT groups (data not shown). However, the Y527A group possessed
  • the hyperfusogenic virus developed in this study may be useful in developing NDV as a better vaccine vector and as an oncolytic agent.
  • transmembrane domain and cytoplasmic tail of the foreign envelope protein with those of a NDV
  • envelope protein increased incorporation of the foreign glycoprotein into the NDV virion (Nayak et al . , 2009 , supra) .
  • ILTV gC and gD were achieved when their ectodomain was fused to the cytoplasmic tail and transmembrane domain of NDV F protein creating rNDV gC and rNDV gD (described above, figure 1 ) , respectively.
  • the genetic stability of the ILTV genes was confirmed by passaging the recombinant viruses in embryonated chicken eggs. Our results showed that the integrity of the added genes and the expression of the foreign proteins were preserved even after 10 egg passages.
  • glycoproteins by recombinant viruses
  • ILTV glycoproteins The expression and incorporation of ILTV glycoproteins by recombinant viruses were analyzed by western blot using rabbit anti-ILTV peptide sera. All the three proteins of ILTV that were expressed and incorporated by rNDVs reacted in western blot with the anti-ILTV gB, gC and gD antisera (figure 2- 1).
  • LaSota virus The pathogenicities of the rNDVs were evaluated by MDT test in 9-day old embryonated SPF chicken eggs.
  • the MDTs for the recombinant viruses were 110 h (rNDV LaSota), 125 h (rNDV gB), 124 h (rNDV gC), and 122 h (rNDV gD) which indicated that the rNDVs expressing ILTV proteins were lentogenic viruses (an NDV strain is considered lentogenic or avirulent, if the MDT value is>90 h (Alexander, DJ, 1989, Newcastle disease, p. 114-120. In, HG Purchase et al., Eds. A Laboratory Manual for the Isolation and Identification of Avian Pathogens, 3 rd Ed.
  • NDV-specific antibody responses in the sera collected on 21 st day post immunization was assayed using HI test. High levels of NDV-specific serum antibodies with no statistically significant
  • VNT neutralization test
  • ILT is a highly contagious and economically important disease of poultry world-wide.
  • Currently available vaccine strategies against ILT are not ideal and the knowledge about the protective
  • antigens of ILTV is limited. Therefore, we have used recombinant NDV to evaluate the role of three major ILTV envelope glycoproteins gB, gC, and gD in immunity and protection. These three envelope proteins of ILTV were chosen because they were found to be the major protective antigens in other
  • herpesviruses Fischer et al., 1003, Vaccine 21, 1732-1741; Hong et al., 2002, Vaccine 20, 1205-1214; Lukacs et al., 1985, J Virol 53, 166-173; Hampl et al., 1984, J Virol 52, 583-590; Zuckermann et al., 1990, J Virol 64, 802-812; Ober et al., 1998, J
  • Glycoprotein B (gB) has previously been shown to be an important target for cellular and humoral immune responses capable of conferring protective immunity against ILTV infection (Tong et al, 2001, supra; Sun et al., 2008, supra; York and Fahey,
  • gC in other herpesviruses has been shown to be a target for cellular and humoral immune responses capable of inducing neutralizing antibodies and T-cell immune responses (Fischer et al., 2003, Vaccine 21, 1732- 1741; Hong et al., 2002, Vaccine 20, 1205-1214;
  • rNDV gD elicited immune response specific to NDV and ILTV and provided complete protection against highly virulent NDV and ILTV challenges.
  • ILTV gD is a major protective antigen capable of inducing neutralizing antibodies.
  • the immune response induced by rNDV gC or rNDV gB or multivalent rNDV combinations was not adequate enough to confer complete protection against virulent ILTV challenge.
  • the NDV-vectored vaccine expressing gD alone was superior to a combination vaccine consisting of rNDVs expressing gB, gC, and gD. Therefore, the rNDV-based ILTV gD vaccine generated in this study for the protection of both NDV and ILTV will be highly beneficial to the poultry industry worldwide and could be the promising vaccine candidate to replace the existing ILTV vaccines.

Abstract

Dans cette étude, pour la première fois, l'efficacité protectrice de gD contre la souche fixe de l'ILTV a été évaluée. L'immunisation à l'aide du virus recombinant de la maladie de Newcastle exprimant la gD de l'ILTV a induit un taux plus élevé d'anticorps neutralisants et a permis d'obtenir une protection complète pour les poulets contre la souche fixe de l'ILTV létal. Des utilisations de NDV recombinant en tant que vecteur de vaccin sont également décrites.
PCT/US2014/047395 1999-05-05 2014-07-21 Vaccin contre le virus de la laryngotrachéite infectieuse (iltv) faisant appel à un vecteur de virus recombinant de la maladie de newcastle WO2015013178A1 (fr)

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