WO2025201373A2 - Virus de la maladie de marek recombinant - Google Patents

Virus de la maladie de marek recombinant

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Publication number
WO2025201373A2
WO2025201373A2 PCT/CN2025/084908 CN2025084908W WO2025201373A2 WO 2025201373 A2 WO2025201373 A2 WO 2025201373A2 CN 2025084908 W CN2025084908 W CN 2025084908W WO 2025201373 A2 WO2025201373 A2 WO 2025201373A2
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Prior art keywords
virus
nucleotide sequence
recombinant
recombinant virus
protein
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WO2025201373A3 (fr
Inventor
Sidi JU
Ning Chen
Yifan Huangfu
Chengtai MA
Qingshui ZHANG
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Boehringer Ingelheim Vetmedica China Co Ltd
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Boehringer Ingelheim Vetmedica China Co Ltd
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Publication of WO2025201373A2 publication Critical patent/WO2025201373A2/fr
Publication of WO2025201373A3 publication Critical patent/WO2025201373A3/fr
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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • 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
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/5254—Virus avirulent or attenuated
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
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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
    • C12N2710/16321—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • 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
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16311—Mardivirus, e.g. Gallid herpesvirus 2, Marek-like viruses, turkey HV
    • C12N2710/16341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/16343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
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    • 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

Definitions

  • the present invention relates to the field of animal health.
  • the present invention relates to a recombinant Marek's Disease Virus (MDV) comprising at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10 or a position between UL2 and UL3.
  • MDV Marek's Disease Virus
  • the present invention provides an immunogenic composition comprising the recombinant MDV of the present invention and the use of the immunogenic composition for preventing and/or treating diseases in an animal.
  • Marek's Disease Virus is classified in the genus Mardivirus, belonging to the subfamily Alphaherpesvirinae of Herpesviridae.
  • the viral genome is a double-stranded linear DNA with a full length of approximately 180 kb, encoding 103 proteins.
  • Marek's disease virus can be divided into 3 serotypes. Among them, serotype 1 virus is pathogenic and oncogenic to chicken hosts. According to the pathogenicity and virulence, the virus can be further divided into mild MDV (mMDV) , virulent MDV (vMDV) , and very virulent MDV (vvMDV) , and very virulent plus MDV (vv+MDV) .
  • mMDV mild MDV
  • vMDV virulent MDV
  • vvMDV very virulent MDV
  • v+MDV very virulent plus MDV
  • Serotype 2 Marek's disease virus is non-oncogenic, and serotype 3 Marek's disease virus is not pathogenic to chickens.
  • the vaccines currently used for MD prevention are mainly serotype 1 attenuated vaccines, including the Dutch CVI988 strain (Rispens) and the Chinese 814 strain.
  • Recombinant herpes virus live vector vaccine is a genetically engineered vaccine that has been studied in depth and has broad application prospects. The principle is to insert the protective antigen gene of a certain pathogen through genetic engineering technology into a region not essential for replication of the viral vector, such that the antigen is continuously expressed as the vector replicates, inducing the body to produce corresponding antibodies to exert immune protection.
  • the viral vector of the recombinant live virus vaccine can replicate itself, so usually a lower dose can produce sufficient exogenous proteins in the body to produce prolonged and good immune protection.
  • Recombinant viral vector vaccines can not only induce humoral immunity, but also induce cellular immunity and mucosal immunity. Compared with traditional inactivated vaccines or live attenuated vaccines, different recombinant viruses have been proven to significantly reduce the level of shedding after infection and reduce the viral load in the environment.
  • MDV Serotype 3 Herpesvirus of Turkeys (HVT) and serotype 1 MDV are considered ideal vectors for constructing live vector vaccines. These viruses have been used as vectors to construct different recombinant viruses. Li et al. used the MDV 814 vaccine strain as a vector to successfully construct a recombinant MDV expressing the VP2 gene of infectious bursal disease virus. Studies have shown that the recombinant virus is safe and stable, and can protect chickens well against both Marek's disease and infectious bursal disease.
  • the NDV genome encodes six viral proteins: L protein (large protein) , NP (nucleoprotein) , P protein (phosphorprotein) , M protein (matrix protein) , HN (haemagglutinin-neuraminidase) , F protein (fusion protein) .
  • L protein large protein
  • NP nucleoprotein
  • P protein phosphorprotein
  • M protein matrix protein
  • HN haemagglutinin-neuraminidase
  • F protein fusion protein
  • H9N2 avian influenza is an acute, highly contagious infectious disease caused by Influenza A virus of the Orthomyxoviridae family.
  • AIV (Avian Influenza virus) belongs to the family Orthomyxoviridae and the genus Orthomyxovirus.
  • the virus particles are mostly spherical, with a diameter of 80 to 120 nanometers, and the surface is covered with dense spikes or fibers 10 to 12 nanometers long, including hemagglutinin (HA) and neuraminidase (NA) .
  • the virus particle has a spiral nucleocapsid within the viral envelope.
  • AIV is a single-stranded negative-sense RNA virus whose genome is composed of 8 RNA segments, encoding different proteins.
  • the genome of the virus is very susceptible to mutation, and the mutation rate of the HA gene is high, which is the main reason for the antigenic variation of the virus.
  • Potential glycosylation sites on HA are one of the possible factors affecting the virulence of avian influenza viruses.
  • the amino acids in the HA receptor binding site can affect the receptor binding properties, thereby changing the affinity of viruses to cells and the host range. This may be the real reason why the virulence variation and infection spectrum of H9N2 subtype avian influenza virus continue to expand.
  • the mutation of the NA gene is mainly closely related to the maturation and release of the virus and may affect the replication and spread of the virus.
  • H9N2 subtype avian influenza is a low-pathogenic avian influenza, but it is very harmful to the poultry industry. It not only causes a serious decrease in egg production of laying hens, but also increases the incidence of complex respiratory diseases and mortality in broilers and young chickens. It can also cause immunosuppression in the body, resulting in slow growth of the chickens, causing huge economic losses to the poultry industry.
  • the present invention provides a recombinant Marek's Disease Virus comprising at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10, or a position between UL2 and UL3.
  • the present invention provides an immunogenic composition, comprising the recombinant virus of the present invention, and optionally a pharmaceutical-or veterinary-acceptable carrier or excipient.
  • the present invention provides use of the recombinant virus of the present invention in preparation of an immunogenic composition for inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.
  • the present invention provides the recombinant virus of the present invention or the immunogenic composition of the present invention for use in a method of inducing a protective immune response in a host animal against a pathogen, preferably said animal is an avian, more preferably, a poultry such as a chicken.
  • the present invention provides a method of inducing a protective immune response in a host animal against a pathogen, said method comprising the step of administering to the animal the recombinant virus of the present invention or the immunogenic composition of the present invention, preferably said animal is an avian, more preferably, a poultry such as a chicken.
  • A, B and/or C encompasses “A” , “B” , “C” , “A and B” , “A and C” , “B and C” , and “A and B and C” .
  • “A or B” means either “A” or “B” , without including “A and B” .
  • the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10.
  • virus designates in particular a viral particle comprising a nucleic acid molecule (e.g., a genome) encapsulated in a capsid or capsule.
  • virus also designates an isolated viral genome.
  • the recombinant virus is derived from an attenuated strain of serotype 1 MDV.
  • attenuated strains of serotype 1 MDV include but are not limited to Dutch CVI988 strain (Rispens) (GenBank: DQ530348.1; Comparative full-length sequence analysis of oncogenic and vaccine (Rispens) strains of Marek's disease virus, Nair, 2007) , Chinese 814 strain (GenBank: JF742597.1; Comparative full-length sequence analysis of Marek's disease virus vaccine strain 814, Cheng, 2012) , or SC9-2 strain (Chinese Patent Publication No: CN102628053A) .
  • an attenuated virus refers to a modified virus that is essentially not virulent in chicken, i.e. does not cause or causes reduced illness, especially does not cause death in a host animal, such as chicken, as compared to the non-modified wildtype parent virus. More particularly, an attenuated virus can typically replicate in a in a host animal, such as chicken, without causing death thereof. More particularly, an attenuated virus designates a virus that is not virulent in in a host animal, such as chicken, when injected at a dose of 10 4.0 -10 7.0 TCID 50 /animal, such as 10 6.0 TCID 50 /animal.
  • an attenuated virus designates a virus that is not virulent in a chicken at a dose of 10 4.0 -10 7.0 TCID 50 /chicken, such as 10 6.0 TCID 50 /animal in at least 10%injected chickens, in at least 20%injected animals, in at least 30%injected animals, in at least 40%injected chickens, in at least 50%injected animals, in at least 60%injected animals, in at least 70%injected animals, more preferably in at least 80%injected animals, even more preferably in at least 90%, 95%, 97%, 98%, 99%or more.
  • an attenuated virus more particularly designates a virus that is not virulent in an embryo when injected at a dose of 10 4.0 -10 7.0 TCID 50 /egg, such as 10 6.0 TCID 50 /egg.
  • the recombinant virus is derived from the SC9-2 strain.
  • the SC9-2 strain is deposited according to Budapest Treaty on December 15, 2023 at CHINA CENTER FOR TYPE CULTURE COLLECTION (Wuhan University, Wuhan 430072, P. R. China) , under the accession number: CCTCC No: V2023114.
  • UL55, Lorf10, UL2 and UL3 genes are highly conserved between different MDV strains such as Dutch CVI988 strain (Rispens) , Chinese 814 strain and SC9-2 strain. It is understood that the skilled artisan may easily identify the exact location of the UL55, Lorf10, UL2 and UL3 genes in any MDV strain using the information contained in the present application and general common knowledge, or by sequence alignment.
  • Exemplary amino acid sequence of UL55 of SC9-2 strain is shown in SEQ ID NO: 1.
  • Exemplary nucleotide sequence of UL55 of SC9-2 strain is shown in SEQ ID NO: 2.
  • Exemplary amino acid sequence of Lorf10 of SC9-2 strain is shown in SEQ ID NO: 3.
  • Exemplary nucleotide sequence of Lorf10 of SC9-2 strain is shown in SEQ ID NO: 4.
  • Exemplary nucleotide sequence between UL55 and Lorf10 is shown in SEQ ID NO: 5.
  • Exemplary amino acid sequence of UL2 of SC9-2 strain is shown in SEQ ID NO: 6.
  • Exemplary nucleotide sequence of UL2 of SC9-2 strain is shown in SEQ ID NO: 7.
  • Exemplary amino acid sequence of UL3 of SC9-2 strain is shown in SEQ ID NO: 8.
  • Exemplary sequence of UL3 of SC9-2 strain is shown in SEQ ID NO: 9.
  • Exemplary nucleotide sequence between UL2 and UL3 is shown in SEQ ID NO: 10.
  • the heterologous nucleotide sequence is inserted into the intergenic region between UL55 and Lorf10, or the intergenic region between UL2 and UL3.
  • the heterologous nucleotide sequence is inserted into the intergenic region between UL55 and Lorf10.
  • the heterologous nucleotide sequence is inserted between the sequence encoding the amino acid sequence as shown in SEQ ID NO: 1 (UL55) or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto and the sequence encoding the amino acid sequence as shown in SEQ ID NO: 3 (Lorf10) or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • the heterologous nucleotide sequence is inserted between the sequence encoding the amino acid sequence as shown in SEQ ID NO: 6 (UL2) or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto and the sequence encoding the amino acid sequence as shown in SEQ ID NO: 8 (UL3) or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • the heterologous nucleotide sequence is inserted within the sequence as shown in SEQ ID NO: 5 (between UL55 and Lorf10) or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • the heterologous nucleotide sequence is inserted within the sequence as shown in SEQ ID NO: 10 (between UL2 and UL3) or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • the heterologous nucleotide sequence is inserted into the intergenic region between UL55 and Lorf10, and
  • At least one upstream Lorf10 flanking region selected from the group consisting of: SEQ ID NO: 28, 29 and 30, or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • the heterologous nucleotide sequence is inserted into the intergenic region between UL2 and UL3, and
  • At least one upstream UL3 flanking region selected from the group consisting of: SEQ ID NO: 34, 35 and 36, or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • intergenic region is well known by the person skilled in the art. The term encompasses a region between two genes. By using an intergenic region for the insertion of a heterologous polynucleotide, no essential gene (gene essential for example for viability, infectivity or replication) of the virus is inactivated. Accordingly, an intergenic region can be used for the insertion of a heterologous polynucleotide such as an antigen encoding sequence.
  • one or more nucleotides at or near the insertion site may be deleted.
  • the at least one heterologous nucleotide sequence is an expression cassette of a polypeptide of interest.
  • the expression cassette comprises the coding nucleotide sequence of the polypeptide of interest operably linked to an expression regulatory element, such as a promoter.
  • the heterologous nucleotide sequence is linked to a promoter sequence
  • At least one downstream UL55 flanking region selected from the group consisting of: SEQ ID NO: 25, 26 and 27, or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto, and
  • At least one upstream Lorf10 flanking region selected from the group consisting of: SEQ ID NO: 28, 29 and 30, or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • the heterologous nucleotide sequence is linked to a promoter sequence
  • At least one downstream UL2 flanking region selected from the group consisting of: SEQ ID NO: 31, 32 and 33, or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto, and
  • At least one upstream UL3 flanking region selected from the group consisting of: SEQ ID NO: 34, 35 and 36, or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • an “antigenic polypeptide” or “antigen” as used herein refers to, but is not limited to, components which elicit an immune response in a host.
  • the antigenic polypeptide is an antigenic polypeptide from an avian pathogen.
  • the avian pathogen may be or the antigenic polypeptide may be derived from viruses, bacteria, fungi, protozoa, etc.
  • Specific examples of an avian pathogen include, without limitation, Newcastle disease virus (NDV) , Infectious bursal disease virus (IBDV) , Infectious laryngotracheitis virus (ILTV) , Avian influenza virus and the like.
  • the antigenic polypeptide is selected from the F protein of NDV, the VP2 protein of IBDV, the gB protein, gD protein, gI protein, or gE protein of ILTV, or the hemagglutinin (HA) of Avian influenza virus.
  • the antigenic polypeptide is the F protein of NDV.
  • the F protein of NDV may comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 11.
  • the complete coding sequence of F protein of NDV may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 12.
  • the H9 HA protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 13.
  • the complete H9 HA coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 14.
  • the coding nucleotide sequence of the polypeptide of interest encodes for gD, gI, and partial gE of ILTV.
  • the gD protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 15.
  • the gI protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 17.
  • the complete gI coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 18.
  • the partial gE protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 19.
  • the complete gE protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 20.
  • the complete gE coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 21.
  • Sequence identity between two polypeptide/nucleotide sequences indicates the percentage of amino acids/nucleotides that are identical between the sequences. Methods for evaluating the level of sequence identity between amino acid or nucleotide sequences are known in the art. For example, sequence analysis software is often used to determine the identity of amino acid/nucleotide sequences. For example, identity can be determined by using the BLAST program in the NCBI database.
  • sequence identity For determination of sequence identity, see, e.g., Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.
  • sequence identity with the sequence of SEQ ID NO: X is equivalent to the term “sequence identity with the sequence of SEQ ID NO: X over the length of SEQ ID NO: X” or to the term “sequence identity with the sequence of SEQ ID NO: X over the whole length of SEQ ID NO: X” , respectively.
  • X is any integer, such as 1, 2 or 3, so that “SEQ ID NO: X” represents any of the SEQ ID NOs mentioned herein.
  • the coding nucleotide sequence of the polypeptide of interest is generally operably linked to a promoter.
  • the promoter may be any natural or synthetic promoter, derived from cellular or viral genes. Examples of suitable promoters include, for instance, an immediate early cytomegalovirus (CMV) promoter, mouse CMV promoter, guinea pig CMV promoter, an SV40 promoter, Human Herpesvirus Type III glycoprotein B (HHV3gB) promoter, Pseudorabies Virus promoters such as that of glycoprotein X promoter, Herpes Simplex Virus-1 alpha 4 promoter, a Marek's Disease Virus glycoprotein A (or gC) promoter, a Marek's Disease Virus glycoprotein B promoter, a Marek's Disease Virus glycoprotein E promoter, a Marek's Disease Virus glycoprotein I promoter, an Infectious Laryngotracheitis Virus glycoprotein B, an Infectious
  • CMV immediate early
  • the promoter is selected from the chicken beta-actin (Bac) promoter, the Pec promoter, the Murine Cytomegalovirus (mCMV) immediate-early 1 promoter, the Human Cytomegalovirus (hCMV) promoter, the Simian virus (SV) 40 promoter, the Raus Sarcoma virus (RSV) promoter, and the gD native promotor of ILTV, the gI native promotor of ILTV.
  • Bac chicken beta-actin
  • Pec the Murine Cytomegalovirus
  • mCMV Murine Cytomegalovirus
  • hCMV Human Cytomegalovirus
  • SV Simian virus
  • RSV Raus Sarcoma virus
  • gD native promotor of ILTV the gI native promotor of ILTV.
  • the expression cassette comprises the coding nucleotide sequence of the F protein of NDV operably linked to the SV40 promoter.
  • the expression cassette comprises the coding nucleotide sequence of the gD, gI, and partial gE of ILTV operably linked to the gD native promotor of ILTV.
  • the coding nucleotide sequence is operably linked to a transcription terminator.
  • the transcription terminator may be derived from human Herpes Simplex Virus (HSV) , thymidine kinase (TK) gene, from the glycoprotein B (gB) gene of Feline Herpesvirus (FHV) , from the immediate early (IE) gene of human cytomegalovirus (hCMV) , strain AD 169 or from simian virus 40 (SV40) , or may be a synthetic terminator, such as a synthetic poly A signal (see Levitt N, Briggs D, Gil A, Proudfoot NJ. Definition of an efficient synthetic poly (A) site. Genes Dev.
  • the coding nucleotide sequence is operably linked to a synthetic polyA signal. In some embodiments, the coding nucleotide sequence is operably linked to an SV40 polyA signal.
  • the invention provides a recombinant Marek's Disease Virus (MDV) comprising at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10,
  • MDV Marek's Disease Virus
  • the at least one heterologous nucleotide sequence is an expression cassette of F protein of NDV, in which the coding nucleotide sequence of the F protein is operably linked to the SV40 promoter.
  • the invention provides a recombinant Marek's Disease Virus (MDV) comprising at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10,
  • MDV Marek's Disease Virus
  • the at least one heterologous nucleotide sequence is an expression cassette of HA protein of H9 subtype (preferably H9N2) AIV, in which the coding nucleotide sequence of the HA protein is operably linked to the SV40 promoter.
  • the invention provides a recombinant Marek's Disease Virus (MDV) comprising at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL2 and UL3,
  • MDV Marek's Disease Virus
  • the at least one heterologous nucleotide sequence is an expression cassette of HA protein of H9 subtype (preferably H9N2) AIV, in which the coding nucleotide sequence of the HA protein is operably linked to the SV40 promoter.
  • the at least one heterologous nucleotide sequence is an expression cassette of the gD, gI, and partial gE of ILTV, in which the coding nucleotide sequence of the gD, gI, and partial gE is operably linked to the gD native promotor of ILTV.
  • the invention provides a recombinant Marek's Disease Virus (MDV) comprising at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL2 and UL3,
  • MDV Marek's Disease Virus
  • the at least one heterologous nucleotide sequence is an expression cassette of the gD, gI, and partial gE of ILTV, in which the coding nucleotide sequence of the gD, gI, and partial gE is operably linked to the gD native promotor of ILTV.
  • the heterologous nucleotide sequence is an expression cassette of the HA protein of H9 subtype Avian influenza virus.
  • the expression cassette of the H9 HA protein of Avian influenza virus may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 23.
  • the rMDV of the present invention is a live virus vector.
  • a “live virus vector” is virus (in the present case a MDV) that is competent to replicate in a host when such host is infected with the live virus or the genomic nucleic acid of such virus and wherein such virus encodes, delivers and express a heterologous nucleotide sequence in such host.
  • the present invention provides the rMDV of the present invention for use as vector vaccine in a host animal, such as chicken.
  • vector vaccine is a vaccine that uses virus (in the present case a MDV) as vector to deliver and express a nucleotide sequence coding for an antigenic polypeptide, wherein such antigenic polypeptide provides protection against a pathogen.
  • virus in the present case a MDV
  • the virus that is used as vector shows no or only limited pathogenicity to the target species in which the virus is used as a vector.
  • Virus construction and cloning may be accomplished by techniques known per se in the art. Gene cloning and plasmid construction are well known to one person of ordinary skill in the art and may be essentially performed by standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012) .
  • the recombinant viruses may be prepared by homologous recombination between the viral genome and a construct (e.g., a homology plasmid) comprising the nucleic acid to be inserted, flanked by nucleotides from the insertion site to allow recombination. Cloning can be made with or without the deletion of endogenous sequences.
  • the recombinant viruses may be prepared by BAC technology.
  • the invention also relates to a host cell, expressing the rMDV as defined above.
  • the invention also relates to a host cell, expressing the rMDV and the heterologous polynucleotide as defined above.
  • the host cell is CEF cell (Liang Z., et. al, Animal (Basel) , 2022, 12 (24) : 3523) , DEF cell (Chenghuai Yang, Arch virol 2015, 160: 267-274) , embryonated egg, or chicken kidney cell (Andres Rodr1′guez-Avila et. al, Avian diseases 2007, 51: 905-911) .
  • competent cell examples include CEF, DEF, embryonated egg, chicken kidney cells, and the like.
  • the cells or viruses may be cultured in a culture medium such as MEM containing 5%FBS at about 37°C for 1h to 6 days.
  • the invention also relates to a composition, e.g., an immunogenic composition, which comprises the rMDV of the present invention.
  • composition refers to a composition that comprises at least one antigen, which elicits an immune response in the host to which the composition is administered.
  • immune response may be a cellular and/or antibody-mediated (humoral) immune response to the composition of the invention.
  • the host is also described as a “subject” , “host animal” or “animal” .
  • the host may be an avian, more preferably, a poultry such as a chicken.
  • an “immune response" to a composition is the development in the host of a cellular and/or antibody-mediated (humoral) immune response to a composition of interest.
  • an “immune response” includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and/or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition of interest.
  • the host will display either a therapeutic or protective immune response such that resistance to new infection will be enhanced and/or the clinical severity of the disease reduced.
  • the composition of the present invention is a vector vaccine. In some embodiments, the composition of the present invention is a vector vaccine in chicken.
  • compositions and vaccines of the invention may further comprise a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle, or adjuvant.
  • a pharmaceutically or veterinarily acceptable carrier or adjuvant or vehicles or excipient includes, but is not limited to, 0.9%NaCl (e.g., saline) solution or a phosphate buffer, poly- (L-glutamate) , the Lactated Ringer's Injection diluent (sodium chloride, sodium lactate, potassium chloride, and calcium chloride) , or polyvinylpyrrolidone.
  • the composition of the invention comprises a lyoprotectant. In a particular embodiment, the composition of the invention comprises a preservative.
  • composition of the invention may be liquid (solutions, suspensions, emulsions) or solid (powder, gel, paste, oil) .
  • the composition of the invention may be formulated for any administration route.
  • the composition may be formulated for oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal administration.
  • composition of the invention may contain a suitable dose sufficient to elicit a protective response in a chicken.
  • dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure in conjunction with the knowledge in the art, without any undue experimentation.
  • the viral vector may be titrated based on any virus titration methods including, but not limited to, FFA (Focus Forming Assay) or FFU (Focus Forming Unit) , TCID 50 (50%Tissue Culture Infective Dose) , PFU (Plaque Forming Units) , and FAID 50 (50%Fluorescent Antibody Infectious Dose) , and the VLPs produced in vitro can be titrated by hemagglutination assay, ELISA, and electron microscopy.
  • FFA Fluorescent Antibody Infectious Dose
  • the rMDV in the composition is present in a dose from 1 ⁇ 10 2 TCID50/ml or TCID50/g to 1x10 7 TCID50/ml or TCID50/g. In some embodiments, the rMDV in the composition is present in a dose from 1x10 4 TCID50/ml or TCID50/g to 1x10 6 TCID50/ml or TCID50/g. In some embodiments, the dose volumes can be between about 0.01 and about 10 ml, between about 0.01 and about 5 ml.
  • composition of the invention can be administered in a single dose or in repeated doses, depending on the vaccination protocol.
  • medicament or vector vaccine of the invention can be formulated as single doses or in repeated doses, depending on the vaccination protocol.
  • the present invention provides the rMDV of the invention, the composition of the invention, or the vector vaccine of the invention, for use in vaccinating a host animal by inducing a protective immune response in a host animal against a pathogen.
  • the present invention provides a method of vaccinating a host animal by inducing a protective immune response in a host animal against a pathogen, comprising or consisting of at least one administration of the rMDV of the invention, the composition of the invention, or the vector vaccine of the invention.
  • the present invention provides use of the composition of the present invention in the manufacture of a medicament for vaccinating a host animal by inducing a protective immune response in a host animal against a pathogen.
  • the term "vaccinating” relates to an active immunization by the administration of an immunogenic composition to a chicken to be immunized, thereby causing a protective immune response against the antigen included in such immunogenic composition.
  • the host animal is 0 day-old, 1 day-old, 2 day-old, 3 day-old, 4 day-old, 5 day-old, 6 day-old, or 7 day-old at the day of vaccination.
  • the rMDV, the composition or the vector vaccine is administrated at Day 0 post-hatch, Day 1 post-hatch, Day 2 post-hatch, Day 3 post-hatch, Day 4 post-hatch, Day 5 post-hatch, Day 6 post-hatch, or Day 7 post-hatch.
  • the rMDVs of the invention are particularly advantageous for vaccinating young host animals (at Day 0, Day 1, Day 2, or Day 3 post-hatch) .
  • Such early administration combined with the early onset of immunity caused by the rMDV, is particularly advantageous to induce early protective immunity, before the host animal can be substantially exposed to pathogens.
  • the rMDV is administered in ovo.
  • the administration is performed when embryos are between 15 to 20 days old, preferably at day 17, 18 or 19, most preferably at day 18 of age.
  • the pathogen is an avian pathogen.
  • the pathogen is selected from the group consisting of MDV, Newcastle disease virus (NDV) , Infectious bursal disease virus (IBDV) , Infectious laryngotracheitis virus (ILTV) or Avian influenza virus (AIV) , or any combinations thereof.
  • the pathogen is MDV.
  • the pathogen is ILTV.
  • the pathogen is NDV.
  • the rMDV, the composition or the vector vaccine can also be administered twice or several times, with a first dose being administered prior to the administration of a second (booster) dose.
  • the second dose is administered at least 15 days after the first dose. More preferably, the second dose is administered between 15 and 40 days after the first dose. Even more preferably, the second dose is administered at least 17 days after the first dose. Still more preferably, the second dose is administered between 17 and 30 days after the first dose. Even more preferably, the second dose is administered at least 19 days after the first dose. Still more preferably, the second dose is administered between 19 and 25 days after the first dose. Most preferably the second dose is administered at least 21 days after the first dose.
  • both the first and second doses of the immunogenic composition are administered in the same amount.
  • an alternate embodiment comprises further subsequent doses.
  • a third, fourth, or fifth dose could be administered in these aspects.
  • subsequent third, fourth, and fifth dose regimens are administered in the same amount as the first dose, with the time frame between the doses being consistent with the timing between the first and second doses mentioned above.
  • the rMDV, the composition or the vector vaccine of the invention may be administrated in a suitable dose sufficient to elicit a protective response in a chicken. Doses and dose volumes are herein discussed in the general description and can also be determined by the skilled artisan from this disclosure in conjunction with the knowledge in the art, without any undue experimentation.
  • the rMDV in the composition or the vector vaccine is present in a dose from 1 ⁇ 10 2 TCID 50 /ml or TCID 50 /g to 1x10 7 TCID 50 /ml or TCID 50 /g.
  • At least one upstream Lorf10 flanking region selected from the group consisting of: SEQ ID NO: 28, 29 and 30, or a sequence having at least 80 %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.95%, 99.98%or 99.99%sequence identity thereto.
  • polypeptide of interest is an antigenic polypeptide; preferably, wherein the antigenic polypeptide is an antigenic polypeptide from an avian pathogen.
  • avian pathogen is selected from Newcastle disease virus (NDV) , Infectious bursal disease virus (IBDV) , Infectious laryngotracheitis virus (ILTV) or Avian influenza virus.
  • antigenic polypeptide is selected from the F protein of NDV, the VP2 protein of IBDV, the gB protein gD protein, gI protein, and/or gE protein/partial gE protein of ILTV, or the hemagglutinin (HA) of Avian influenza virus.
  • the F protein of NDV comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 11; or the complete coding sequence of F protein of NDV has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%,
  • HA protein has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 13; or the HA coding sequence has a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 94%, at least 95%, at least 96%
  • the recombinant virus of clause 18, wherein the gI protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 17; or the gI coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 9
  • the recombinant virus of clause 18, wherein the complete gE protein may have an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 20; or the complete gE coding sequence may have a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%,
  • the promoter is selected from the chicken beta-actin (Bac) promoter, the Pec promoter, the Murine Cytomegalovirus (mCMV) immediate-early 1 promoter, the Human Cytomegalovirus (hCMV) promoter, the Simian virus (SV) 40 promoter, the Raus Sarcoma virus (RSV) promoter, and the gD native promotor of ILTV, the gI native promotor of ILTV.
  • the promoter is selected from the chicken beta-actin (Bac) promoter, the Pec promoter, the Murine Cytomegalovirus (mCMV) immediate-early 1 promoter, the Human Cytomegalovirus (hCMV) promoter, the Simian virus (SV) 40 promoter, the Raus Sarcoma virus (RSV) promoter, and the gD native promotor of ILTV, the gI native promotor of ILTV.
  • the expression cassette comprises the coding nucleotide sequence of the F protein of NDV operably linked to the SV40 promoter;
  • the at least one heterologous nucleotide sequence is an expression cassette of F protein of NDV, in which the coding nucleotide sequence of the F protein is operably linked to the SV40 promoter;
  • the at least one heterologous nucleotide sequence is an expression cassette of HA protein of AIV of type H9H2, in which the coding nucleotide sequence of the HA protein is operably linked to the SV40 promoter;
  • the at least one heterologous nucleotide sequence is an expression cassette of HA protein of AIV of type H9H2, in which the coding nucleotide sequence of the HA protein is operably linked to the SV40 promoter;
  • the at least one heterologous nucleotide sequence is an expression cassette of the gD, gI, and partial gE of ILTV, in which the coding nucleotide sequence of the gD, gI, and partial gE is operably linked to the gD native promotor of ILTV; or
  • the recombinant virus comprises at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL2 and UL3,
  • the at least one heterologous nucleotide sequence is an expression cassette of the gD, gI, and partial gE of ILTV, in which the coding nucleotide sequence of the gD, gI, and partial gE is operably linked to the gD native promotor of ILTV.
  • the recombinant virus comprises at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10,
  • the at least one heterologous nucleotide sequence is an expression cassette comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 22;
  • the recombinant virus comprises at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10,
  • the at least one heterologous nucleotide sequence is an expression cassette comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 23;
  • the recombinant virus comprises at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL2 and UL3,
  • the at least one heterologous nucleotide sequence is an expression cassette comprising a nucleotide sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%or 100%sequence identity with SEQ ID NO: 23;
  • the recombinant virus comprises at least one heterologous nucleotide sequence in the genome thereof, wherein the at least one heterologous nucleotide sequence is located at a position between UL55 and Lorf10,
  • the pathogen is selected from MDV, Newcastle disease virus (NDV) , Infectious laryngotracheitis virus (ILTV) or Avian influenza virus.
  • a method of inducing a protective immune response in a host animal against a pathogen comprising the step of administering to the animal the recombinant virus of any one of clauses 1-28 or the immunogenic composition of any one of clauses 31-35, preferably said animal is an avian, more preferably, a poultry such as a chicken.
  • Example 1 Construction of recombinant serotype I MDV expressing the F protein of Newcastle disease virus
  • the original serotype I Marek's disease virus SC9-2 strain was purchased from Shandong Agricultural University (Chinese Patent Publication No: CN102628053A) .
  • the SC9-2 strain used in this study was obtained by continuously passaging and amplifying the original SC9-2 strain on chicken embryo fibroblasts (CEF) . Then safety and efficacy of this strain were tested, and the results showed that the SC9-2 strain can achieve ⁇ 90%protection against Marek's disease virus very virulent strain Md5.
  • Safety experiments on SPF chickens showed that this strain did not cause clinical signs, death or tumors in 1-day-old SPF chickens, proving that this strain is safe for chickens.
  • SC9-2 virus strain used in this study was deposited according to Budapest Treaty on December 15, 2023 at CHINA CENTER FOR TYPE CULTURE COLLECTION (Wuhan University, Wuhan 430072, P.R. China) , under the accession number CCTCC No: V2023114.
  • the linearized GFP transfer vector obtained in step 1 and the SC9-2 genomic DNA obtained in step 2 were co-transfected into monolayer CEF cells according to the instructions of the commercial transfection kit Lipofectamine TM 3000 (purchased from Invitrogen) .
  • Lipofectamine TM 3000 purchased from Invitrogen
  • the supernatant was removed and replaced with cell culture medium containing 5%serum.
  • the recombination results were observed under a fluorescence microscope.
  • the green plaques corresponded to the recombinant virus rSC9-2-GFP.
  • the purified rSC9-2-GFP recombinant virus was amplified on CEF cells for 2 passages and a batch of recombinant virus rSC9-2-GFP P2 was harvested.
  • the rSC9-2-GFP P2 virus was used to infect the pre-seeded CEF cells.
  • the cell culture supernatant was discarded, the cells were digested with trypsin, and the cells were pipet down with cell culture medium MEM and collected into a centrifuge tube. The cells were centrifuged at 500 g for 5 minutes and the supernatant was discarded, leaving only the cells, and then rSC9-2-GFP genomic DNA was extracted with phenol-chloroform.
  • the pUC57-SV40-NDF-PolyASV40 plasmid was double digested with restriction enzymes EcoR I and Hind III, and the ordinary agarose gel DNA recovery kit Gel Extraction Kit was used to recover the digested fragments to obtain foreign gene fragment UL55-SV40-NDF-Lorf10 with 1500bp homology arm at both ends.
  • the inventors constructed 2 recombinant serotype I Marek's disease viruses containing the HA gene of H9N2 subtype avian influenza, namely rSC9-2-UL2-SV40-H9HA-UL3 and rSC9-2-UL55-SV40-H9HA-Lorf10.
  • the recombinant virus rSC9-2-UL2-SV40-H9HA-UL3 was obtained by constructing a bacterial artificial chromosome (BAC) ; while the recombinant virus rSC9-2-UL55-SV40-H9HA-Lorf10 was obtained by homologous recombination method.
  • BAC bacterial artificial chromosome
  • the genome of the SC9-2 strain was firstly constructed as a bacterial artificial chromosome (BAC) system, and then based on the constructed rSC9-2-BAC, the HA gene of the H9N2 subtype avian influenza virus was inserted between the UL2 and UL3 of the SC9-2 genome, and a recombinant Marek's disease virus expressing the HA gene of the H9N2 subtype avian influenza virus was finally constructed: rSC9-2 UL2-SV40-H9HA-UL3.
  • BAC bacterial artificial chromosome
  • the insertion site of mini-F (the main element of bacterial artificial chromosome) selected in this study is between US10 and Sorf3 in the SC9-2 genome.
  • PCR was used to amplify the homology arm sequences on the left and right sides of the insertion site and introduce enzyme cutting sites.
  • electrophoresis and gel cutting were performed to recover the homology arm DNA fragments.
  • the left homology arm DNA fragment was 1400 bp and BamH I and Sac I restriction endonuclease sites were introduced.
  • the right homology arm DNA fragment was 1300 bp and BamHI and Sal I restriction endonuclease sites were introduced.
  • the plasmid pB12 containing the mini-F gene fragment (preserved by the Innovation Center Laboratory of Boehringer Ingelheim Animal Health (China) Co., Ltd. ) was digested with BamHI and electrophoresed and gel recovered to obtain mini-F DNA fragment with BamHI on both sides.
  • the obtained left and right homology arm DNA fragments and the mini-F DNA fragment were ligated and transformed into E. coli TOP10 competent cells.
  • the bacteria were picked and the plasmid was extracted, and the mini-F transfer vector plasmid identified correctly by enzyme digestion was stored in a -20°Crefrigerator for later use.
  • SC9-2 genomic DNA was obtained according to a method mentioned above.
  • step (7) 10%glycerol was added to the bacterial cells obtained in step (7) , the bacterial solution was diluted to 500 ⁇ L, and aliquoted into pre-cooled EP tubes (50 ⁇ L each tube) to obtain gS1783-rSC9-2-BAC electroporation competent cells.
  • step (4) after culturing the bacterial liquid obtained in step (4) at 32°C for 1 hour, 100 ⁇ L of bacterial liquid was added to 900 ul of LB liquid culture medium, mixed well, and 100 ul solution was applied on LB solid medium plate containing chloramphenicol and 1%L-arabinose, cultured at 32°C for 24h to 48h;
  • step (5) the single colonies obtained in step (5) were selected for parallel screening on LB solid medium plates containing chloramphenicol and kanamycin and on LB solid medium plates with chloramphenicol only; the colonies that did not grow on LB solid medium with chloramphenicol and kanamycin but grown on LB solid medium with chloramphenicol only were expanded. Plasmid was extracted using the QIAGEN Plasmid Midi Kit, then identified by PCR and RFLP methods. And the positive clone gS1783-rSC9-2-BAC-UL2-SV40-H9HA-PolyA SV40 -UL3 was obtained.
  • the serotype I Marek's disease virus SC9-2 parent strain was purchased from Shandong Agricultural University (Chinese Patent Publication No: CN102628053A) .
  • the SC9-2 strain used in this study was obtained by continuously passaging and amplifying the original SC9-2 strain on chicken embryo fibroblasts (CEF) . Then safety and efficacy of this strain were tested, and the results showed that the SC9-2 strain can provide ⁇ 90%protection against Marek's disease virus very virulent strain Md5.
  • Safety experiments on one-day-old SPF chickens showed that this strain did not cause clinical sign, death or tumors in SPF chickens, proving that this strain is safe for chickens.
  • pUC57-ILT-gD-gI-/gE/plasmid was synthesized by GenScript Biotechnology Co., Ltd. and preserved at Global Innovation China Center Laboratory of Boehringer Ingelheim Animal Health (China) Co., Ltd.
  • SC9-2 virus was taken out from the liquid nitrogen tank and quickly placed into a 37°C water bath for rapid thawing;
  • step (4) the cell suspension in step (4) was added to two new 96-well cell culture plates seeded with secondary CEF cells (100 ⁇ L/well) , allowing the corresponding wells of the old plate and the new plates in one-to-one correspondence, respectively; the two new plates were labeled as plate "A” and plate "B” and placed in a 37°C, 5%CO 2 incubator for 3 days;
  • the dual indirect immunofluorescence assay was used to stain the fixed cells; diluted anti-ILTV gD protein monoclonal antibody and anti-ILTV gI protein monoclonal antibody were added to the wells (gD protein monoclonal antibody diluted at 1: 500, gI protein monoclonal antibody diluted at 1: 200) , incubated at 37°C for 1 hour; the primary antibodies were discarded and the wells were washed 3 times with PBS; anti-human IgG secondary antibody and anti-pig IgG secondary antibody (Alexa Fluor 488 goat anti-Human IgG (H+L) , purchased from Invitrogen Company, and Dylight 594 goat anti-Pig IgG (H+L) , purchased from Abcam Company) , were added to each well at the same time, incubated at 37°C for 1 hour; the two antibodies were discarded and the wells were washed 3 times with PBS, observed under a fluorescence microscope; wells that shown both green and red flu
  • the wells in the 96-well cell culture plate "B" corresponding to the positive wells in the plate "A” labeled in step (7) were selected; the cell supernatant was discarded, the wells were washed once with sterile PBS, and trypsin (50 ⁇ L/well) was added for digestion at 37°C for 5 minutes; growth solution (150 ⁇ L/well) was added to resuspend the digested cells.
  • step (3) the cell suspension in step (3) was added to two new 96-well cell culture plates seeded with secondary CEF cells (100 ⁇ L/well) , allowing the corresponding holes of the old plate and the new plates in one-to-one correspondence; the two new plates were labeled as plate "A” and plate "B” and placed in a 37°C, 5%CO 2 incubator for 3 days;
  • step (6) the wells in the 96-well cell culture plate "B" corresponding to the positive wells in the plate "A” labeled in step (6) were selected; the cell supernatant was discarded, the wells were washed once with sterile PBS, and trypsin (50 ⁇ L/well) was added for digestion at 37°C for 5 minutes; growth solution (150 ⁇ L/well) was added to resuspend the digested cells;
  • the recombinant serotype I Marek's disease virus rSC9-2 UL55-NDF-Lorf10 containing the F gene of Newcastle disease virus constructed above was continuously passaged and amplified on chicken embryo fibroblasts (CEF) . These continuously passaged viruses were harvested and stored in liquid nitrogen tanks for subsequent efficacy research.
  • CEF chicken embryo fibroblasts
  • the recombinant Marek's disease virus containing the chicken Newcastle disease virus F gene was continuously passaged to Passage 15 on CEFs.
  • rSC9-2 recombinant virus DNA of the 5th, 10th, and 15th passages were extracted using the QIAamp DNA Mini Kit (purchased from QIAGEN Company) and amplified by PCR using primers for identifying the insertion of the F gene. Sequencing of the PCR products confirmed that the F gene inserted in the SC9-2 genome was stable.
  • the recombinant Marek's disease virus containing the Newcastle disease virus F gene was infected into a CEF pre-seeded 24-well plate with a dilution of 10 -2 to10 -4 , then cultured in a 37°C, 5%CO 2 incubator for 5 days, and dual immunofluorescence assay was used to detect the expression of NDV-F protein.
  • Dual immunofluorescence assay steps when observed obvious plaques, the cell culture medium was discarded, the cells were washed lightly with PBS once, then pre-cold 96%ethanol was added to each well for fixing at room temperature for 10 minutes, the 96%ethanol was discarded, and dried naturally; then 200-fold diluted anti-NDV chicken serum and MDV mouse monoclonal antibody (purchased from Shandong Agricultural University) were added, incubated at 37°C for 1 hour; the antibodies were discarded, and cells were washed 3 times with PBS; anti-chicken and anti-mouse secondary antibodies (Alexa Fluor 488 goat anti-chicken IgG (H+L) and Alexa Fluor 596 donkey anti-mouse IgG (H+L) , purchased from Invitrogen) were added, incubated at 37°C for 1 hour; secondary antibodies were discarded and cells were washed three times with PBS and observed under a fluorescence microscope.
  • the recombinant serotype I Marek's disease virus rSC9-2 UL55-SV40-H9HA-Lorf10 and rSC9-2 UL2-SV40-H9HA-UL3 constructed above were passaged and expanded on CEF cells. These continuously passaged viruses were harvested and stored in liquid nitrogen tanks for subsequent efficacy research.
  • the recombinant Marek's disease virus containing the H9N2 avian influenza subtype HA gene was continuously passaged to passage 15 on CEF, and rSC9-2 recombinant viruses DNA of the 5th, 10th, and 15th passages were extracted with QIAamp DNA Mini Kit (purchased from QIAGEN Company) .
  • HA gene insertion was identified by PCR amplification with primers. Sequencing of the PCR products confirmed that the HA gene inserted in SC9-2 genome was stable.
  • the recombinant Marek's disease virus containing the H9HA gene was inoculated into a CEF pre-seeded 24-well plate with a dilution of 10 -2 to10 -4 , cultured in a 37°C, 5%CO 2 incubator for 5 days, and dual immunofluorescence assay was used to detect the expression of H9HA protein.
  • the recombinant serotype 1 Marek's disease virus containing infectious laryngotracheitis virus gD, gI and partial gE was continuously passaged on CEF cells for 15 passages, and the 10th and 15th passages were used to extract DNA using DNA /RNA Virus Mini Kit (purchased from INVITEK, Germany) .
  • the inserted gene was identified by PCR amplification with specific primers. The PCR amplification products were sequenced, and the results showed that the inserted gD, gI and partial gE gene in SC9-2 genome are stable..
  • the serotype 1 Marek's disease virus containing infectious laryngotracheitis virus (ILTV) gD and gI genes was used to infect CEF cells at a dose of 0.001 MOI into a 60mm cell culture dish. After inoculation, the dish was placed at 37°C and 5%CO 2 , cultured for about 3 days, and plaque formation was observed under a white light microscope.
  • ILTV infectious laryngotracheitis virus
  • the cell supernatant in the plate was removed, pre-cold 96%ethanol was added to the plate to fix the cells, placed at room temperature for 10 minutes; the ethanol was removed, and cells were dried naturally at room temperature, and dual immunofluorescence assay was used to detect the protein expression of ILTV gD and gI in the recombinant viruses.
  • Dual immunofluorescence assay steps diluted anti-ILTV gD monoclonal antibody and anti-ILTV gI monoclonal antibody (gD monoclonal antibody diluted at 1: 500, gI monoclonal antibody diluted at 1: 200) were added to the dish respectively, and incubated at 37°C for 1 hours; the primary antibodies were discarded and the cells were washed 3 times with PBS; anti-human IgG secondary antibody (1: 200 dilution, Alexa Fluor 488 goat anti-Human IgG (H+L) , purchased from Invitrogen) and anti-pig IgG secondary antibody (1:200 dilution, Dylight 594 goat anti-Pig IgG (H+L) , purchased from Abcam company) were added to each well., incubated at 37°C for 1 hour; the antibodies were discarded and the cells were washed 3 times with PBS; observed under a fluorescence microscope.
  • the recombinant viruses used in this example include rSC9-2 UL55-SV40-H9HA-Lorf10 and rSC9-2 UL2-SV40-H9HA-UL3 obtained above.
  • oropharyngeal swabs and cloacal swabs of all experimental chickens were collected for H9 virus isolation. If the virus isolation result is negative, the vaccine candidate strain is determined to provide protection to the test chickens.
  • throat and cloaca cotton swabs were collected from all test chickens at 5 days after challenge.
  • the collected cotton swabs were placed into a centrifuge tube containing 1.6 ml of six-antibody buffer.
  • All cotton swab samples were used for virus isolation by inoculation to chicken embryos: each cotton swab sample was inoculated to five 9 to 11-day-old SPF chicken embryos through the allantoic cavity, with 0.2 ml per embryo, incubated for 96 hours, then the allantoic fluid were harvested for HA titers testing. As long as the HA titer of one chicken embryo is more than 1: 16, it can be determined as positive for H9 virus isolation.
  • Samples with negative virus isolation were blindly passaged for one generation, and then determined again. If it is still negative after blind passage, the sample is determined to be negative for H9 virus isolation; if it is positive after blind passage, the sample is determined to be positive for H9 virus isolation.
  • the blood of test chickens in each group was collected before challenge, and the serum was separated for H9 HI antibody test.
  • the results showed that the H9 HI antibodies in the serum of all the chickens in Group 3 (challenge control group) were negative, and the average HI antibody titer of the test chickens in Groups 1 and 2 was lower than 5 log2.
  • the average HI antibody titer of chicken serum used in each group of tests is shown in Table 2.
  • H9N2 virus isolation results were shown in Table 3.
  • the recombinant Marek's disease virus live vector vaccine candidate strains expressing the HA gene of avian influenza virus (H9 subtype) studied in this example can provide different degrees of protection against H9N2 challenge.
  • the recombinant virus used in this example is rSC9-2 UL55-NDF-Lorf10 obtained in the above example.
  • test chickens After vaccination, all test chickens were observed continuously for 28 days. General clinical observations were conducted daily, and abnormalities in spirit, feed intake, breathing, and defecation of the test chickens were recorded.
  • ELISA enzyme-linked immunosorbent assay
  • clinical symptoms include but not limited to: depression, lethargy, ruffled feathers, dyspnea, nervous symptoms (head or muscular tremors, torticollis, paralysis) , oral and nasal salivation, conjunctivitis (need to exclude ammonia cases caused by excessive concentration) , facial swelling, etc.
  • the recombinant Marek's disease virus live vector vaccine candidate strain expressing Newcastle disease virus F gene used in this example can provide 100%protection against ND challenge and is an ideal vaccine candidate strain for ND.
  • Example 7 Efficacy test of recombinant SC9-2 live vector vaccine candidate expressing infectious laryngotracheitis virus gD, gI and partial gE
  • test chickens were collected before challenge, and the serum was separated and used for ILTV antibody detection.
  • the dose of virus challenge is 10 3.0 TCID 50 /bird.
  • all test chickens were clinically observed twice a day (once in the morning and once in the afternoon) for 10 consecutive days.
  • all surviving test chickens in the test groups were euthanized (after clinical observation in the afternoon) .
  • the gross pathology of the trachea was observed in the test chickens that died after the challenge and in the test chickens that were euthanized at the end of the experiment.
  • the morbidity of chickens in different groups was calculated. The results showed that the morbidity of test chickens in the challenge control group was 92%, and the challenge control was effective. The morbidity of chickens in the other three groups and the protection rates of the corresponding vaccine candidate strains are shown in Table 8.

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Abstract

La présente invention se rapporte au domaine de la santé animale. En particulier, la présente invention concerne un virus de la maladie de Marek (MDV) recombinant comprenant au moins une séquence nucléotidique exogène dans son génome, ladite au moins une séquence nucléotidique exogène étant située à une position entre UL55 et Lorf10, ou à une position entre UL2 et UL3. En outre, la présente invention prévoit une composition immunogène comprenant le MDV recombinant de la présente invention et l'utilisation de la composition immunogène pour prévenir et/ou traiter les maladies chez un animal.
PCT/CN2025/084908 2024-03-27 2025-03-26 Virus de la maladie de marek recombinant Pending WO2025201373A2 (fr)

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