WO2025044920A2 - Nouveaux vecteurs du virus de l'entérite du canard pour vaccins aviaires - Google Patents

Nouveaux vecteurs du virus de l'entérite du canard pour vaccins aviaires Download PDF

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WO2025044920A2
WO2025044920A2 PCT/CN2024/114146 CN2024114146W WO2025044920A2 WO 2025044920 A2 WO2025044920 A2 WO 2025044920A2 CN 2024114146 W CN2024114146 W CN 2024114146W WO 2025044920 A2 WO2025044920 A2 WO 2025044920A2
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gene
dev
modified
chicken
day
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WO2025044920A3 (fr
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Chengtai MA
Yifan Huangfu
Ning Chen
Xiaoping CUI
Sidi JU
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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Priority to CN202480054079.8A priority Critical patent/CN121843706A/zh
Publication of WO2025044920A2 publication Critical patent/WO2025044920A2/fr
Publication of WO2025044920A3 publication Critical patent/WO2025044920A3/fr
Priority to MX2026002234A priority patent/MX2026002234A/es
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    • C12N2800/50—Vectors for producing vectors

Definitions

  • the present invention is based on the surprising finding that the DEV comprising an inactive gene in its genome, such as i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene, alone or in combination with other DEV genes such as for example the combinations of i) US7 gene and US8 gene; ii) UL24 gene and UL2 gene; iii) UL40 gene and UL2 gene; iv) UL23 gene and UL41 gene; or v) UL41 gene and US8 gene results in a reduced or no mortality in chicken as compared to the non-modified DEV.
  • the DEV comprising an inactive gene in its genome, such as i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene;
  • the DEVs of the invention are safe and can effectively deliver and express a gene of interest in vivo.
  • such DEVs are (i) attenuated in vivo with respect to chicken, and (ii) are stable and capable of expressing foreign genes in a manner suitable for inducing protective immunity, including at very early stage (i.e., at day 0, day 1, day 2, or day 3 post-hatch) .
  • these modified DEVs retain a fast growth rate, allowing high titer production.
  • Such modified DEVs thus represent very potent vectors for vaccinating non-human animals, particularly poultry, and for conferring early protective immunity.
  • the present invention provides a modified Duck Enteritis Virus (DEV) , wherein one or more genes of the DEV genome selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41, and US8 is inactivated, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • the present invention provides a modified Duck Enteritis Virus (DEV) , comprising an inactivated gene selected from any one of i) -vii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, alone or in combination with any other non-essential gene of DEV and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • the present invention provides a modified Duck Enteritis Virus (DEV) , comprising inactivated gene (s) selected from any one of i) -xii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viii) US7 gene and US8 gene; ix) UL24 gene and UL2 gene; x) UL40 gene and UL2 gene; xi) UL23 gene and UL41 gene; or xii) UL41 gene and US8 gene, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • the present invention provides a modified Duck Enteritis Virus (DEV) , comprising inactivated gene (s) as defined above, wherein the modified DEV further comprises a heterologous polynucleotide coding for a heterologous antigen of a pathogen.
  • DEV Duck Enteritis Virus
  • the present invention provides a modified Duck Enteritis Virus (DEV) as a live vector vaccine in chicken, wherein the modified DEV comprises an inactivated gene selected from any one of i) -vii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene; and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • the present invention provides a modified Duck Enteritis Virus (DEV) as a live vector vaccine in chicken, wherein the modified DEV comprises an inactivated gene selected from any one of i) -vii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, alone or in combination with any other non-essential gene of DEV and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • the present invention provides a composition, comprising the modified DEV of the present invention.
  • the present invention provides the modified DEV of the invention, or the composition of the invention, or the vector vaccine of the invention, for use in a method for inducing a protective immune response in chicken against a pathogen, wherein such method comprises or consists of one or more administration of the modified DEV of the invention, or the composition of the invention, or the vector vaccine of the invention to chicken.
  • the present invention provides a method of vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen, comprising at least one administration of the composition of the present invention.
  • the present invention provides use of the modified DEV of the present invention in the manufacture of a composition for vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen.
  • Figure 1 shows gene structure map of plasmid pB12.
  • Figure 2 illustrates schematic diagrams of (A) bacterial artificial chromosome rDEV4 BAC, (B) recombinant construct with deleted gene rDEV4 ⁇ UL39 and (C) recombinant construct with inserted H9HA gene rDEV4 ⁇ UL39 UL26-H9HA-UL27.
  • Figure 3 shows the result of transfection of DEFs (duck embryo fibroblasts) to rescue rDEV4 ⁇ UL39.
  • Figure 4 shows the result of RFLP analysis of rDEV4 ⁇ UL39 UL26-H9HA-UL27 by Xho I digestion.
  • Figure 5 shows the result of mini-F deletion to rescue rDEV4 ⁇ UL39 UL26-H9HA-UL27 by co-transfection.
  • Figure 6 shows the result of the genetic stability test of rDEV4 ⁇ UL39 UL26-H9HA-UL27 by PCR.
  • Figure 7 shows the result of the expression test of H9HA from rDEV4 ⁇ UL39 UL26-H9HA-UL27 by IFA.
  • Figure 8 shows the result of HI Ab level of different groups induced by different rDEV4 H9HA vaccine candidate strains.
  • A, B and/or C encompasses “A” , “B” , “C” , “A and B” , “A and C” , “B and C” , and “A and B and C” .
  • all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the virus strains, the cell lines, vectors, and methodologies as reported in the publications which might be used in connection with the invention. None herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
  • the invention relates to a modified Duck Enteritis Virus (DEV) , wherein said modified virus has specific inactive gene (s) .
  • DEV Duck Enteritis Virus
  • the invention indeed shows that by inactivating such gene (s) , viable, stable and replicative DEVs can be obtained, and that such viruses may be used to create recombinant DEVs by insertion of foreign genetic material. The results further show that such foreign genetic material is highly expressed from such viruses upon cell infection, and that such expression remains stable over time.
  • the present invention provides a modified Duck Enteritis Virus (DEV) , wherein one or more genes of the DEV genome selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41, and US8 is inactivated, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • recombinant or “modified” refer to a DEV that has been altered, rearranged, or modified by genetic engineering. However, the term does not refer to alterations in polynucleotide, amino acid sequence, or nucleotide sequence that result from naturally occurring events, such as spontaneous mutations.
  • the DEV comprising one or more inactivated genes in its genome and/or a heterologous polynucleotide coding for a heterologous antigen of a pathogen as described below is also called a recombinant or modified DEV herein.
  • the terms “recombinant/modified DEV” , “rDEV” and “recombinant/modified DEV vector” are used interchangeably herein.
  • modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • the modified DEV of the present invention is attenuated with respect to chicken.
  • the modified DEV of the present invention is attenuated with respect to chicken.
  • 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 a viral vector or an isolated viral genome.
  • a “gene” designates a nucleic acid molecule or sequence which comprises an open reading frame encoding a product, such as a polypeptide (e.g., a peptide, protein, etc. ) or an RNA.
  • DEV refers to all viruses belonging to species of Duck enteritis virus (DEV) in the genus Mardivirus within subfamily Alphaherpesvirinae of the family Herpesviridae.
  • the DEVs of the invention may be prepared from any DEV species or strain.
  • the DEV of the invention is derived or prepared from a parental strain selected from the Jansen strain, the VAC strain (ID EU082088.2) , the C-KCE strain (ID KF263690.1) , the CHv strain (ID JQ647509.1) , the 2085 strain (ID JF999965) , the CV strain (ID KJ549663.1) or the CSC strain (ID JQ673560.1) , or any DEV strain having at least 90%sequence identity to the Jansen strain, the VAC strain (ID EU082088.2) , the C-KCE strain (ID KF263690.1) , the CHv strain (ID JQ647509.1) , the 2085 strain (ID JF999965) , the CV strain (ID KJ549663.1) or the CSC strain (ID JQ673560.1) , more preferably at least 95%, at least 96%, at
  • the DEV of the invention is derived or prepared from the DEV4 strain, which is deposited at China Center for Type Culture Collection (CCTCC) on August 4, 2023 under CCTCC NO: V202378, or any DEV strain having at least 90%sequence identity to the DEV4 strain, more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.
  • CTCC China Center for Type Culture Collection
  • the inactivated gene (s) in the DEV genome is non-essential gene (s) .
  • non-essential gene/region is a gene/region in the modified DEV genome in which inactivation (including mutation, interruption, replacement or deletion) on that gene/region or insertion of a heterologous polynucleotide into that gene/region does not prevent the modified DEV from replicating in a host cell.
  • the inactivation of one or more genes of DEV genome as indicated above causes the attenuation of the DEV with respect to chicken.
  • the phrase “The inactivation of one or more genes of DEV genome as indicated above causes the attenuation of the DEV with respect to chicken” means that the modified DEVs of the present invention are attenuated with respect to chicken.
  • the modified DEVs of the present invention are attenuated with respect to chicken.
  • the term "attenuated” as used herein 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 chicken as compared to the non-modified wildtype parent virus. More particularly, an attenuated virus can typically replicate in a chicken without causing death thereof. More particularly, the attenuated DEV of the present invention has no or lower virulence in chicken, than the corresponding non-modified wildtype parent DEV which does not comprise the inactivated gene (s) in its genome.
  • an attenuated virus designates a virus that is not virulent in a chicken when injected at a dose of 10 4.0 -10 7.0 TCID 50 /chicken, such as 10 6.0 TCID 50 /chicken. More particularly, 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 /chicken in at least 10%injected chickens, in at least 20%injected chickens, in at least 30%injected chickens, in at least 40%injected chickens, in at least 50%injected chickens, in at least 60%injected chickens, in at least 70%injected chickens, more preferably in at least 80%injected chickens, 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.
  • an attenuated virus designates a virus that is not virulent in an embryo at a dose of 10 4.0 -10 7.0 TCID 50 /egg, such as 10 6.0 TCID 50 /egg in at least 10%injected eggs, in at least 20%injected eggs, in at least 30%injected eggs, in at least 40%injected eggs, in at least 50%injected eggs, in at least 60%injected eggs, in at least 70%injected eggs, more preferably in at least 80%injected eggs, even more preferably in at least 90%, 95%, 97%, 98%, 99%or more.
  • the modified viruses of the invention are also not virulent for injection post-hatch, including at Day 0, Day 1, Day 2, Day 3 post-hatch (i.e., between 0.1 and 72 hours post-hatch) .
  • the modified DEV has a reduced or no mortality and/or morbidity in chicken. More particularly, the modified DEV of the present invention has a reduced or no mortality and/or morbidity as compared to the non-modified DEV in chicken. More particularly, the mortality and/or morbidity in chicken caused by the modified DEV of the present invention is 0%, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, or less than 70%. More particularly, the mortality and/or morbidity in chicken caused by the modified DEV of the present invention is 0%, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, or less than 70%of that caused by the non-modified DEV.
  • a DEV with an "inactive" gene designates a DEV that cannot express a functional protein or RNA encoded by said gene.
  • An inactive gene thus designates a mutated, an interrupted, a replaced or a deleted gene that cannot encode a wild-type protein encoded by said gene.
  • the inactivated gene in the DEV genome is selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41, and US8.
  • the inactivated gene (s) is/are selected from any one of i) -xii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viii) US7 gene and US8 gene; ix) UL24 gene and UL2 gene; x) UL40 gene and UL2 gene; xi) UL23 gene and UL41 gene; or xii) UL41 gene and US8 gene.
  • the present invention relates to a modified Duck Enteritis Virus (DEV) , wherein any one of i) -xii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viii) US7 gene and US8 gene; ix) UL24 gene and UL2 gene; x) UL40 gene and UL2 gene; xi) UL23 gene and UL41 gene; or xii) UL41 gene and US8 gene is inactivated in the DEV genome, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • UL41, US3, UL24, UL40, UL39, UL23, US7, US8, and UL2 are highly conserved between DEV strains. It is understood that the skilled artisan may easily identify the exact location of the UL41, US3, UL24, UL40, UL39, UL23, US7, US8, and UL2 gene in any DEV strain using the information contained in the present application and general common knowledge, or by sequence alignment. For example, the exact location of the UL41, US3, UL24, UL40, UL39, UL23, US7, US8, and UL2 gene may be identified by reference to a DEV strain with Genbank accession No. EU082088.2.
  • the gene is inactivated by mutation, interruption, replacement or deletion of a portion of or the whole sequence of the gene. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%of the sequence of the gene is replaced or deleted.
  • the gene is inactive as a result of one or more mutations in the coding sequence, particularly point mutations in the coding sequence that prevent the expression of a full length protein.
  • Such mutations may cause substitution of essential amino acid residue (s) in the encoded protein, resulting in an inactive protein.
  • the gene is inactive as a result of one or more interruptions in the coding sequence that prevent the expression of a full length protein.
  • interruptions may introduce a stop or non-sense codon in the sequence, resulting in an inactive protein.
  • the gene is inactive as a result of a deletion of a portion of the (coding) sequence of said gene or the whole (coding) sequence of said gene, more particularly of at least 20%of the (coding) sequence of the gene, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, up to 100%.
  • deletion removes the coding sequence and thus prevents the expression of a wild-type protein.
  • the gene is inactive as a result of a replacement of a portion of the (coding) sequence of said gene or the whole (coding) sequence of said gene with a heterologous polynucleotide, more particularly of at least 20%of the (coding) sequence of the gene, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, up to 100%.
  • a heterologous polynucleotide more particularly of at least 20%of the (coding) sequence of the gene, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or at least 95%, up to 100%.
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL41 gene. In a specific embodiment, the modified DEV of the invention has a deletion of the whole sequence of the UL41 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL41 (see Example 2) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the US3 gene. In a specific embodiment, the modified DEV of the invention has a deletion of the whole sequence of the US3 gene.
  • a specific example of such a construct is e.g. rDEV4 ⁇ US3 (see Example 2) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL24 gene. In a specific embodiment, the modified DEV of the invention has a deletion or replacement of the whole sequence of the UL24 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL24 (see Example 2) , rDEV4 H9HA ⁇ UL24 (see Example 3) or rDEV4 ⁇ UL24 UL26-H9HA-UL27 (see Example 3) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL40 gene. In a specific embodiment, the modified DEV of the invention has a deletion or replacement of the whole sequence of the UL40 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL40 (see Example 2) or rDEV4 H9HA ⁇ UL40 (see Example 3) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL39 gene. In a specific embodiment, the modified DEV of the invention has a deletion or replacement of the whole sequence of the UL39 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL39 (see Example 2) , rDEV4 H9HA ⁇ UL39 (see Example 3) or rDEV4 ⁇ UL39 UL26-H9HA-UL27 (see Example 3) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL23 gene. In a specific embodiment, the modified DEV of the invention has a deletion or replacement of the whole sequence of the UL23 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL23 (see Example 2) , or rDEV4 H9HA ⁇ UL23 (see Example 3) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL24 gene, and a portion of or the whole sequence of the UL2 gene.
  • the modified DEV comprises a deletion of the whole sequence of the UL24 gene, and a replacement of the whole sequence of the UL2 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL24 H9HA ⁇ UL2 (see Example 3) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL40 gene, and a portion of or the whole sequence of the UL2 gene.
  • the modified DEV comprises a deletion of the whole sequence of the UL40 gene, and a replacement of the whole sequence of the UL2 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL40 H9HA ⁇ UL2 (see Example 3) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL23 gene, and a portion of or the whole sequence of the UL41 gene.
  • the modified DEV comprises a replacement of the whole sequence of the UL23 gene, and a deletion of the whole sequence of the UL41 gene.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ UL23 ⁇ UL41 (see Example 3) .
  • the modified DEV of the invention has a deletion or replacement of a portion of or the whole sequence of the UL41 gene, and a portion of or the whole sequence of the US8 gene.
  • the modified DEV comprises a deletion of the whole sequence of the UL41 gene, and a replacement of the whole sequence of the US8 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL41 H9HA ⁇ US8 (see Example 3) .
  • the modified DEV is a live virus vector.
  • a “live virus vector” is virus (in the present case a DEV) 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 polynucleotide sequence in such host.
  • the modified DEV further comprises a heterologous polynucleotide coding for a heterologous antigen of a pathogen.
  • heterologous polynucleotide in relation to a virus designates a polynucleotide which is not found naturally in the genome of the virus, or which is found naturally in said genome but in a different form or at a different position.
  • an “antigen” as used herein refers to, but is not limited to, components which elicit an immune response in a host.
  • the heterologous polynucleotide is inserted into a non-essential gene or region of the modified DEV.
  • the heterologous polynucleotide is inserted into or in replacement of a portion of or the whole sequence of the non-essential gene or region of the modified DEV.
  • the non-essential gene or region of the modified DEV is selected from the group consisting of both US7 gene and US8 gene, UL2 gene, UL24 gene, UL39 gene, UL40 gene, UL23 gene, US8 gene, or the UL26-UL27 intergenic region.
  • the heterologous polynucleotide is located in a gene or a region selected from the group consisting of both US7 gene and US8 gene, UL2 gene, UL24 gene, UL39 gene, UL26-UL27 intergenic region, UL40 gene, UL23 gene, and US8 gene of the modified DEV genome.
  • the heterologous polynucleotide is inserted into or in replacement of a portion of or the whole sequence of one or more genes of the modified DEV genome selected from the group consisting of both US7 gene and US8 gene, UL2 gene, UL24 gene, UL39 gene, UL40 gene, UL23 gene, US8 gene, or inserted into UL26-UL27 intergenic region of the modified DEV genome.
  • the heterologous polynucleotide is located in UL23 gene.
  • the heterologous polynucleotide is inserted into the UL23 gene sequence of the DEV viral genome, in addition to the existing UL23 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the UL23 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the UL23 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the UL23 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated UL23 gene sequence.
  • the heterologous polynucleotide is located in a region of both US7 gene and US8 gene.
  • the DEV of the invention has inactive US8 gene, and the heterologous polynucleotide is inserted into the US7 gene sequence of the DEV viral genome, in addition to the existing US7 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the US7 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the US7 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the US7 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated US7 gene sequence.
  • the DEV of the invention has inactive US7 gene
  • the heterologous polynucleotide is inserted into the US8 gene sequence of the DEV viral genome, in addition to the existing US8 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the US8 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the US8 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the US8 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated US8 gene sequence.
  • the heterologous polynucleotide is inserted into the US7 and US8 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the US7 and US8 gene, or in replacement of the whole sequence of the US7 and US8 gene.
  • the heterologous polynucleotide is located in UL2 gene.
  • the heterologous polynucleotide is inserted into the UL2 gene sequence of the DEV viral genome, in addition to the existing UL2 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the UL2 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the UL2 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the UL2 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated UL2 gene sequence.
  • the heterologous polynucleotide is located in UL24 gene.
  • the heterologous polynucleotide is inserted into the UL24 gene sequence of the DEV viral genome, in addition to the existing UL24 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the UL24 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the UL24 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the UL24 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated UL24 gene sequence.
  • the heterologous polynucleotide is located in UL39 gene.
  • the heterologous polynucleotide is inserted into the UL39 gene sequence of the DEV viral genome, in addition to the existing UL39 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the UL39 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the UL39 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the UL39 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated UL39 gene sequence.
  • the heterologous polynucleotide is located in UL26-UL27 intergenic region. In a particular embodiment, the heterologous polynucleotide is inserted into the UL26-UL27 intergenic region of the DEV viral genome.
  • the heterologous polynucleotide is located in UL40 gene.
  • the heterologous polynucleotide is inserted into the UL40 gene sequence of the DEV viral genome, in addition to the existing UL40 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the UL40 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the UL40 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the UL40 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated UL40 gene sequence.
  • the heterologous polynucleotide is located in US8 gene.
  • the heterologous polynucleotide is inserted into the US8 gene sequence of the DEV viral genome, in addition to the existing US8 gene sequence (thus rendering the gene inactive by interrupting the gene sequence) , or inserted into the US8 gene region of the DEV viral genome after the deletion of a portion of or the whole sequence of the US8 gene (thus rendering the gene inactive by deleting the gene sequence) , or in replacement of a portion of or the whole sequence of the US8 gene (thus rendering the gene inactive by replacing the gene sequence) , or located in a mutated US8 gene sequence.
  • the DEV of the invention has an inactive gene, preferably a deleted gene, and contains a heterologous polynucleotide coding for an antigen of a pathogen located in a different gene or region.
  • the heterologous polynucleotide may be cloned in replacement of a portion of or the whole sequence of said different gene, or it may be inserted within said different gene, or inserted into said different gene after the deletion of a portion of or the whole sequence of said different gene (thus rendering the different gene also inactive) .
  • the DEV of the invention has an inactive UL24 gene, and contains a heterologous polynucleotide coding for an antigen of a pathogen located in UL2 gene, in replacement of a portion of or the whole sequence of the UL2 gene or inserted within UL2 gene (thus rendering UL2 gene also inactive) .
  • the DEVs of the invention may comprise several heterologous polynucleotides coding for one or more antigens of one or more pathogens.
  • the several heterologous polynucleotides may be inserted in the same position in the virus, under the control of a single or several distinct promoters.
  • the heterologous polynucleotides may be inserted into different cloning sites of the virus.
  • the heterologous polynucleotide is expressed after the modified DEV has been transfected into a suitable host cell.
  • the present invention provides the modified DEV of the present invention for use as vector vaccine in chicken.
  • vector vaccine is a vaccine that uses a virus (in the present case a DEV) as vector to deliver and express a polynucleotide sequence coding for an antigen, wherein such antigen provides protection against a pathogen.
  • the virus that is used as a vector shows no or only limited pathogenicity to the target species in which the virus is used as a vector.
  • the present invention also provides a modified Duck Enteritis Virus (DEV) as a live vector vaccine in chicken, wherein the modified DEV comprises an inactivated gene selected from any one of i) -vii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene; and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • the present invention also provides a modified Duck Enteritis Virus (DEV) as a live vector vaccine in chicken, wherein the modified DEV comprises an inactivated gene selected from any one of i) -vii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, alone or in combination with any other non-essential gene of DEV and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • the present invention also provides a modified Duck Enteritis Virus (DEV) as a live vector vaccine in chicken, wherein the modified DEV comprises inactivated gene (s) selected from any one of i) -xii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viii) US7 gene and US8 gene; ix) UL24 gene and UL2 gene; x) UL40 gene and UL2 gene; xi) UL23 gene and UL41 gene; or xii) UL41 gene and US8 gene, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • 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) . Typically, 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.
  • a construct e.g., a homology plasmid
  • the present invention provides a method of making the modified DEV of the invention, comprising the inactivation of one or more genes of the DEV genome as indicated above.
  • the inactivation of the one or more selected genes leads to a reduced or no mortality of the modified DEV in chicken as compared to the non-modified DEV.
  • the DEV of the invention may contain any heterologous polynucleotide coding for an antigen of a pathogen.
  • the pathogen may be or the antigen may be derived from viruses, bacteria, fungi, protozoa, etc.
  • the pathogen may be a chicken pathogen.
  • the antigen may be an antigen of a chicken pathogen.
  • the pathogen is or the antigen is derived from avian influenza virus.
  • the pathogen is or the antigen is derived from avian influenza virus selected from the group consisting of Influenza A virus, Influenza B virus, Influenza C virus, and Influenza D virus.
  • the pathogen is or the antigen is derived from Influenza A virus. More preferentially, the pathogen is or the antigen is derived from avian influenza virus type H9. Most preferentially, the pathogen is or the antigen is derived from avian influenza virus H9N2.
  • the antigen of a pathogen is the HA protein of the subtype H9 avian influenza virus (H9 HA protein) . In some embodiments, the antigen of a pathogen is the HA protein of the subtype H9N2 avian influenza virus.
  • the H9 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: 1.
  • the complete H9 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 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: 2.
  • 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 or 2, so that “SEQ ID NO: X” represents any of the SEQ ID NOs mentioned herein.
  • the heterologous polynucleotide 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 Laryngotracheitis Virus glycoprotein B
  • the heterologous polynucleotide coding for an antigen of a pathogen is generally operably linked to mCMV promoter. In some embodiments, the heterologous polynucleotide coding for H9 HA is generally operably linked to mCMV promoter.
  • the heterologous polynucleotide 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.
  • the heterologous polynucleotide is operably linked to an SV40 polyA signal.
  • the heterologous polynucleotide coding for H9 HA is generally operably linked to an SV40 polyA signal.
  • One embodiment of the invention provides a modified DEV comprising and (capable of expressing) a heterologous polynucleotide coding for the H9 HA protein.
  • the heterologous polynucleotide encoding the H9 HA protein is operably linked to the mouse CMV promoter and therefore the expression of the H9 HA protein is regulated by the mouse CMV promoter.
  • the heterologous polynucleotide encoding the H9 HA protein is operably linked to the SV40 polyA signal and therefore the expression of H9 HA protein is regulated by the SV40 polyA signal.
  • the heterologous polynucleotide encoding the H9 HA protein is operably linked to the mouse CMV promoter and the SV40 polyA signal, and therefore the expression of H9 HA protein is regulated by the mouse CMV promoter and the SV40 polyA signal.
  • the modified DEV comprising and (capable of expressing) a heterologous polynucleotide coding for the H9 HA protein comprises an expression cassette containing in 5' to 3' direction in the following order, a) a promoter, b) a heterologous polynucleotide, c) a transcription terminator.
  • the modified DEV comprising and (capable of expressing) a heterologous polynucleotide coding for the H9 HA protein comprises an expression cassette containing in 5' to 3' direction in the following order, a) the mouse CMV promoter, b) the heterologous polynucleotide encoding the H9 HA protein, c) a transcription terminator.
  • the modified DEV comprising and (capable of expressing) a heterologous polynucleotide coding for the H9 HA protein comprises an expression cassette containing in 5' to 3' direction in the following order, a) the mouse CMV promoter, b) the heterologous polynucleotide encoding the H9 HA protein, c) the SV40 polyA signal.
  • the modified DEV of the present invention containing a heterologous polynucleotide coding for an antigen of a pathogen is capable of providing the efficacy of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%in the protection against the corresponding pathogen.
  • the modified DEV of the present invention containing a heterologous polynucleotide coding for an antigen of a pathogen is capable of providing the efficacy of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%in the protection against avian influenza virus, particularly avian influenza A virus of subtype H9, more particularly avian influenza A virus of subtype H9N2.
  • the modified DEV of the present invention containing a heterologous polynucleotide coding for an antigen of a pathogen is capable of providing the efficacy of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%in the protection against the corresponding pathogen in chicken.
  • the modified DEV of the present invention containing a heterologous polynucleotide coding for an antigen of a pathogen is capable of providing the efficacy of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%in the protection against avian influenza virus, particularly avian influenza A virus of subtype H9, more particularly avian influenza A virus of subtype H9N2 in chicken.
  • a preferred modified DEV of the invention wherein the UL41 gene of the DEV genome is inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL41 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL41 (see Example 2) .
  • a preferred modified DEV of the invention wherein the US3 gene of the DEV genome is inactive as a result of a deletion or replacement of a portion of or the whole sequence of the US3 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ US3 (see Example 2) .
  • a preferred modified DEV of the invention wherein the UL24 gene of the DEV genome is inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL24 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL24 (see Example 2) , rDEV4 H9HA ⁇ UL24 (see Example 3) or rDEV4 ⁇ UL24 UL26-H9HA-UL27 (see Example 3) .
  • a preferred modified DEV of the invention wherein the UL40 gene of the DEV genome is inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL40 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL40 (see Example 2) or rDEV4 H9HA ⁇ UL40 (see Example 3) .
  • a preferred modified DEV of the invention wherein the UL39 gene of the DEV genome is inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL39 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL39 (see Example 2) , rDEV4 H9HA ⁇ UL39 (see Example 3) or rDEV4 ⁇ UL39 UL26-H9HA-UL27 (see Example 3) .
  • a preferred modified DEV of the invention wherein the UL23 gene of the DEV genome is inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL23 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL23 (see Example 2) , or rDEV4 H9HA ⁇ UL23 (see Example 3) .
  • a preferred modified DEV of the invention wherein the US7 gene and US8 gene of the DEV genome are inactive as a result of a deletion or replacement of a contiguous region spanning a portion of or the whole sequence of the US7 gene, the entire US7-US8 intergenic region, and a portion of or the whole sequence of the US8 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ US7US8 (see Example 2) and rDEV4 H9HA ⁇ US7US8 (see Example 3) .
  • a preferred modified DEV of the invention wherein the UL24 gene and UL2 gene of the DEV genome are inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL24 gene, and a portion of or the whole sequence of the UL2 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL24 H9HA ⁇ UL2 (see Example 3) .
  • a preferred modified DEV of the invention wherein the UL40 gene and UL2 gene of the DEV genome are inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL40 gene, and a portion of or the whole sequence of the UL2 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL40 H9HA ⁇ UL2 (see Example 3) .
  • a preferred modified DEV of the invention wherein the UL23 gene and UL41 gene of the DEV genome are inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL23 gene, and a portion of or the whole sequence of the UL41 gene.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ UL23 ⁇ UL41 (see Example 3) .
  • a preferred modified DEV of the invention wherein the UL41 gene and US8 gene of the DEV genome are inactive as a result of a deletion or replacement of a portion of or the whole sequence of the UL41 gene, and a portion of or the whole sequence of the US8 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL41 H9HA ⁇ US8 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL23 gene, in replacement of a portion of or the whole sequence of the UL23 gene, rendering the UL23 gene inactive.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ UL23 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in both of the US7 and US8 genes, in replacement of a portion of or the whole sequence of both of the US7 and US8 genes, rendering the US7 and US8 genes inactive.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ US7US8 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL2 gene, in replacement of a portion of or the whole sequence of the UL2 gene, rendering the UL2 gene inactive, and further comprises an inactive UL24 gene, optionally a deleted UL24 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL24 H9HA ⁇ UL2 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL2 gene, in replacement of a portion of or the whole sequence of the UL2 gene, rendering the UL2 gene inactive, and further comprises an inactive UL40 gene, optionally a deleted UL40 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL40 H9HA ⁇ UL2 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL24 gene, in replacement of a portion of or the whole sequence of the UL24 gene, rendering the UL24 gene inactive.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ UL24 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL39 gene, in replacement of a portion of or the whole sequence of the UL39 gene, rendering the UL39 gene inactive.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ UL39 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL26-UL27 intergenic region, and further comprises an inactive UL24 gene, optionally a deleted UL24 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL24 UL26-H9HA-UL27 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL40 gene, in replacement of a portion of or the whole sequence of the UL40 gene, rendering the UL40 gene inactive.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ UL40 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL26-UL27 intergenic region, and further comprises an inactive UL39 gene, optionally a deleted UL39 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL39 UL26-H9HA-UL27 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the UL23 gene, in replacement of a portion of or the whole sequence of the UL23 gene, rendering the UL23 gene inactive, and further comprises an inactive UL41 gene, optionally a deleted UL41 gene.
  • a specific example of such a construct is e.g., rDEV4 H9HA ⁇ UL23 ⁇ UL41 (see Example 3) .
  • a preferred modified DEV of the invention comprises a heterologous polynucleotide located in the US8 gene, in replacement of a portion of or the whole sequence of the US8 gene, rendering the US8 gene inactive, and further comprises an inactive UL41 gene, optionally a deleted UL41 gene.
  • a specific example of such a construct is e.g., rDEV4 ⁇ UL41 H9HA ⁇ US8 (see Example 3) .
  • the heterologous polynucleotide encodes H9 HA protein.
  • the invention also relates to a host cell, expressing the modified DEV as defined above.
  • the invention also relates to a host cell, expressing the modified DEV and the heterologous polynucleotide as defined above.
  • the host cell is CEF cell (Liang Z., et. al, Animal (Basel) , 2022, 12 (24) : 3523) , EB66 cell (Alexander Nikolay, Applied Microbiology and Biotechnology (2016) 102: 8725-8737) , DEF cell (Chenghuai Yang, Arch virol 2015, 160: 267-274) , embryonated egg, or chicken kidney cell (Andres Rodri′guez-Avila et. al, Avian diseases 2007, 51:905-911) .
  • the modified DEV of the present invention may be propagated in any competent cell cultures. After the required growth of the viruses is achieved, the cells may be detached from the wells using a scraper or with trypsin and the infected cells may be separated from the supernatant by centrifugation.
  • competent cell examples include CEF, EB66, 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, which comprises the modified DEV 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 immune response to the composition of the invention.
  • the host is also described as a “subject” .
  • any of the hosts or subjects described or mentioned herein is a chicken.
  • an “immune response" to a composition is the development in the host of a cellular and/or antibody-mediated 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.
  • a "protective immune response” or “protective response” will be demonstrated by either a reduction or lack of clinical signs normally displayed by an infected host, a quicker recovery time and/or a lowered duration of infectivity or lowered pathogen titer in the tissues or body fluids or excretions of the infected host.
  • composition of the invention is described as a “vaccine” .
  • the composition of the present invention is a vaccine.
  • 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. In some embodiments, the modified DEV is used as a vector in the vector vaccine.
  • 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 pharmaceutically or veterinarily acceptable carrier or vehicle or adjuvant or excipients may be any compound or combination of compounds facilitating the administration of the vector (or protein expressed from an inventive vector in vitro) , or facilitating transfection or infection and/or improving the preservation of the vector (or protein) .
  • 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 modified DEV in the composition 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. In some embodiments, the modified DEV in the composition is present in a dose from 1x10 4 TCID 50 /ml or TCID 50 /g to 1x10 6 TCID 50 /ml or TCID 50 /g. In some embodiments, the modified DEV in the composition is present in a dose of 1x10 6 TCID 50 /ml or TCID 50 /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 repeated doses, depending on the vaccination protocol.
  • the 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 modified DEV of the invention, or the composition of the invention, or the vector vaccine of the invention, for the use in a method for inducing a protective immune response in chicken against a pathogen, wherein such method comprises or consists of one or more administration of the modified DEV of the invention, or the composition of the invention, or the vector vaccine of the invention to chicken.
  • the present invention provides the modified DEV of the invention, the composition of the invention, or the vector vaccine of the invention, for use in vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen.
  • the present invention provides a method of vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen, comprising or consisting of at least one administration of the modified DEV of the invention, the composition of the invention, or the vector vaccine of the invention.
  • the present invention provides use of the modified DEV of the present invention in the manufacture of a composition for vaccinating a chicken by inducing a protective immune response in a chicken 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 chicken 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 modified DEV, 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 viruses of the invention are particularly advantageous for vaccinating young chicken (at Day 0, Day 1, Day 2, or Day 3 post-hatch) .
  • the invention surprisingly shows that the modified DEV of the invention is safe upon such early administration to chicken, while native or wild-type DEV is lethal to chicken.
  • Such early administration combined with the early onset of immunity caused by these modified DEVs, is particularly advantageous to induce early protective immunity, before chicken can be substantially exposed to pathogens.
  • the pathogen is avian species pathogen. In some embodiments, the pathogen is a poultry pathogen. In some embodiments, the pathogen is a chicken pathogen. In some embodiments, the pathogen is avian influenza virus. In some embodiments, the pathogen is avian influenza A virus. In some embodiments, the pathogen is avian influenza A virus of subtype H9. In some embodiments, the pathogen is avian influenza A virus of subtype H9N2.
  • the administration, or the modified DEV, the composition or the vector vaccine of the invention results in lessening of the incidence of the particular pathogen infection in a chicken or in the reduction in the severity of clinical signs caused by or associated with the specific pathogen infection.
  • the administration, or the modified DEV, the composition or the vector vaccine of the invention results in lessening of the incidence of the particular avian influenza virus infection in a chicken or in the reduction in the severity of clinical signs caused by or associated with the specific avian influenza virus infection.
  • the administration, or the modified DEV, the composition or the vector vaccine of the invention may not be effective in all chickens administrated, but there is a significant portion (for example, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%) of chickens effectively immunized.
  • the modified DEV, the composition or the vector vaccine is administered by oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal.
  • the modified DEV, the composition or the vector vaccine may be formulated for oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal administration.
  • the immunogenic composition may be administered by other routes as well.
  • the modified DEV, the composition or the vector vaccine is administered once and is efficacious by such single administration.
  • the modified DEV, 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 modified DEV, 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 modified DEV 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.
  • the modified DEV in the composition or the vector vaccine is present in a dose from 1x10 4 TCID 50 /ml or TCID 50 /g to 1x10 6 TCID 50 /ml or TCID 50 /g. In some embodiments, the modified DEV in the composition or the vector vaccine is present in a dose of 1x10 6 TCID 50 /ml or TCID 50 /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.
  • the present invention further relates to vaccination kits for vaccinating a chicken by inducing a protective immune response in a chicken against a chicken pathogen, which comprises an effective amount of the modified DEV, the composition or the vector vaccine as described above and a means for administering said modified DEV, the composition or the vector vaccine to said chicken.
  • kit comprises an injection device filled with the modified DEV, the composition or the vector vaccine according to the invention and instructions for intradermic, subcutaneous, intramuscular, or in ovo injection.
  • the kit comprises a spray/aerosol or eye drop device filled with the modified DEV, the composition or the vector vaccine according to the invention and instructions for oro-nasal administration, oral or mucosal administration.
  • a modified Duck Enteritis Virus (as a live vector vaccine in chicken) , wherein one or more genes of the DEV genome selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41, and US8 is inactivated, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • a modified Duck Enteritis Virus (as a live vector vaccine in chicken) , comprising an inactivated gene selected from any one of i) -vii) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene, in combination with the inactivation of one or more further non-essential genes of DEV, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • DEV Duck Enteritis Virus
  • the one or more further non-essential gene of DEV is different from the first inactivated gene and is selected from i) US7 gene, ii) UL2 gene, iii) UL41 gene; iv) US3 gene; v) UL24 gene; vi) UL40 gene; vii) UL39 gene; viii) UL23 gene; or ix) US8.
  • a modified Duck Enteritis Virus (as a live vector vaccine in chicken) , comprising inactivated gene (s) selected from any one of i) -xi) , i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US7 gene and US8 gene; viii) UL24 gene and UL2 gene; ix) UL40 gene and UL2 gene; x) UL23 gene and UL41 gene; or xi) UL41 gene and US8 gene, and wherein said modified DEV has a reduced or no mortality in chicken (as compared to the non-modified DEV) .
  • DEV Duck Enteritis Virus
  • Clause 6 The modified DEV of any one of the preceding clauses, wherein at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%of the sequence of the gene is replaced or deleted.
  • Clause 8 The modified DEV of clause 7, wherein the heterologous polynucleotide is inserted into a non-essential gene or region of the modified DEV.
  • Clause 9 The modified DEV of clause 7 or 8, wherein the heterologous polynucleotide is inserted into or in replacement of a portion of or the whole sequence of the non-essential gene or region of the modified DEV.
  • Clause 10 The modified DEV of any one of clauses 7-9, wherein the non-essential gene or region of the modified DEV is selected from the group consisting of UL23 gene, both US7 gene and US8 gene, UL2 gene, UL24 gene, UL39 gene, UL40 gene, US8 gene, and a UL26-UL27 intergenic region.
  • Clause 11 The modified DEV of clause 7, wherein the heterologous polynucleotide is located in a gene or a region selected from the group consisting of UL23 gene, both US7 gene and US8 gene, UL2 gene, UL24 gene, UL39 gene, UL40 gene, US8 gene, and a UL26-UL27 intergenic region of the modified DEV genome.
  • Clause 12 The modified DEV of clause 7, wherein the heterologous polynucleotide is inserted into or in replacement of a portion of or the whole sequence of one or more genes selected from the group consisting of UL23 gene, both US7 gene and US8 gene, UL2 gene, UL24 gene, UL39 gene, UL40 gene, and US8 gene of the modified DEV genome, or inserted into UL26-UL27 intergenic region of the modified DEV genome.
  • heterologous polynucleotide located in the UL23 gene in replacement of a portion of or the whole sequence of the UL23 gene;
  • the heterologous polynucleotide located in the UL2 gene in replacement of a portion of or the whole sequence of the UL2 gene, and further comprises an inactive UL24 gene, optionally a deleted UL24 gene;
  • the heterologous polynucleotide located in the UL2 gene in replacement of a portion of or the whole sequence of the UL2 gene, and further comprises an inactive UL40 gene, optionally a deleted UL40 gene;
  • the heterologous polynucleotide located in the UL26-UL27 intergenic region and further comprises an inactive UL24 gene, optionally a deleted UL24 gene;
  • the heterologous polynucleotide located in the UL26-UL27 intergenic region and further comprises an inactive UL39 gene, optionally a deleted UL39 gene;
  • the heterologous polynucleotide located in the UL23 gene in replacement of a portion of or the whole sequence of the UL23 gene, and further comprises an inactive UL41 gene, optionally a deleted UL41 gene; or
  • the heterologous polynucleotide located in the US8 gene in replacement of a portion of or the whole sequence of the US8 gene, and further comprises an inactive UL41 gene, optionally a deleted UL41 gene.
  • Clause 14 The modified DEV of any one of clauses 7-13, wherein the pathogen is a chicken pathogen or the antigen is an antigen of a chicken pathogen.
  • Clause 15 The modified DEV of any one of clauses 7-13, wherein the antigen is derived from avian influenza virus.
  • Clause 16 The modified DEV of any one of clauses 7-13, wherein the antigen is derived from an avian influenza virus selected from the group consisting of an Influenza A virus, an Influenza B virus, an Influenza C virus, and an Influenza D virus.
  • an avian influenza virus selected from the group consisting of an Influenza A virus, an Influenza B virus, an Influenza C virus, and an Influenza D virus.
  • Clause 17 The modified DEV of any one of clauses 7-13, wherein the antigen is derived from avian influenza virus type H9.
  • Clause 18 The modified DEV of any one of clauses 7-13, wherein the antigen is derived from an avian influenza virus H9N2.
  • Clause 19 The modified DEV of any one of clauses 7-13, wherein the heterologous antigen is the Hemagglutinin (HA) protein of the subtype H9N2 avian influenza virus.
  • HA Hemagglutinin
  • Clause 20 The modified DEV of any one of clauses 7-19, wherein the amino acid sequence of the heterologous antigen has 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: 1.
  • Clause 21 The modified DEV of any one of clauses 7-19, wherein the sequence of the heterologous polynucleotide has 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: 2.
  • Clause 22 The modified DEV of any one of clauses 7-21, wherein the heterologous polynucleotide is operably linked to a promoter.
  • the promoter is selected from the group consisting of 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 Laryngotracheitis Virus glycoprotein E promoter, an Infectious Laryngotracheitis Virus glycoprotein D promoter, an Infectious Laryngotracheitis
  • CMV immediate early cytomegalovirus
  • Clause 24 The modified DEV of any one of clauses 7-23, wherein the heterologous polynucleotide is operably linked to an SV40 polyA signal.
  • Clause 25 The modified DEV of any one of clauses 7-24, wherein the heterologous polynucleotide is expressed after the modified DEV has been transfected into a suitable host cell.
  • Clause 26 The modified DEV of any one of clauses 1-25, wherein the modified DEV is attenuated with respect to chicken.
  • Clause 27 The modified DEV of any one of clauses 1-26, wherein the inactivation of the selected genes causes the attenuation of DEV.
  • Clause 28 The modified DEV of any one of clauses 1-27, wherein the mortality in chicken caused by the modified DEV is 0%, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, or less than 70%of that caused by the non-modified DEV.
  • Clause 29 The modified DEV of any one of clauses 1-28 for use as vector vaccine in chicken.
  • Clause 30 A composition, comprising the modified DEV of any one of clauses 1-28.
  • Clause 31 The composition of clause 30, further comprising a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle or adjuvant.
  • Clause 32 The composition of clause 30 or 31, wherein the composition is a vaccine.
  • Clause 33 The composition of any one of clauses 30-32, wherein the composition is a vector vaccine in chicken.
  • Clause 34 The composition of clause 33, wherein the modified DEV is used as a vector.
  • Clause 35 The modified DEV of any one of clauses 1-28, or the composition of any one of clauses 30-34, or the vector vaccine of clause 29, for the use in a method for inducing a protective immune response in chicken against a pathogen, wherein such method comprises or consists of one or more administration of the modified DEV of any one of clauses 1-28, or the composition of any one of clauses 30-34, or the vector vaccine of clause 29 to chicken.
  • Clause 36 The modified DEV of any one of clauses 1-28, or the composition of any one of clauses 30-34, or the vector vaccine of clause 29, for use in vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen.
  • Clause 37 A method of vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen, comprising or consisting of at least one administration of the modified DEV of any one of clauses 1-28, or the composition of any one of clauses 30-34, or the vector vaccine of clause 29 to the chicken.
  • Clause 38 Use of the modified DEV of any one of clauses 1-28 in the manufacture of a composition for vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen.
  • Clause 39 The method and the use of any one of clauses 35-38, wherein the pathogen is avian influenza virus.
  • Clause 40 The method and the use of clause 39, wherein the pathogen is avian influenza virus, optionally avian influenza A virus, optionally avian influenza A virus of subtype H9, optionally avian influenza A virus of subtype H9N2.
  • Clause 41 The method and the use of any one of clauses 35-40, wherein the chicken 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.
  • Clause 42 The method and the use of any one of clauses 35-41, wherein the modified DEV, 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.
  • Clause 43 The method and the use of any one of clauses 35-42, wherein the modified DEV, the composition or the vector vaccine is administrated by oro-nasal, eye drop, spray, drinking water, in ovo, intramuscular, subcutaneous, intradermal, or transdermal.
  • a vaccination kit for vaccinating a chicken by inducing a protective immune response in a chicken against a pathogen which comprises an effective amount of the modified DEV of any one of clauses 1-28, or the composition of any one of clauses 30-34 or the vector vaccine of clause 29 and a means for administering said modified DEV, said composition or said vector vaccine to said chicken.
  • Clause 45 A host cell, expressing the modified DEV of any one of clauses 1-28.
  • Clause 46 A host cell, expressing the modified DEV and the heterologous polynucleotide of any one of clauses 7-28.
  • Clause 47 The host cell of clause 45 or 46, wherein the host cell line is CEF cell, EB66 cell or DEF cell.
  • Clause 48 A method of making the modified DEV of any one of clauses 1-28, comprising the inactivation of one or more genes of the DEV genome as defined in any one of clauses 1-28, wherein said inactivation of the one or more selected genes let to a reduced or no mortality of the modified DEV in chicken as compared to the non-modified DEV.
  • Example 1 Construction of bacterial artificial chromosome (BAC) of DEV
  • the duck plague virus DEV4 strain used in this study was purchased from China Agricultural University in 2017, and deposited at China Center for Type Culture Collection (CCTCC) , Wuhan University, Wuhan 430072 P. R. China, on August 4, 2023 under CCTCC NO: V202378.
  • CTCC China Center for Type Culture Collection
  • a bacterial artificial chromosome system was constructed with the genome of the DEV4 strain for subsequent gene deletion and insertion. The specific steps include:
  • the sequences of the homologous arms on the left and right side of the insertion site (between UL44 and UL44.5 of the DEV4 genome) for mini-F element were amplified by PCR using the primers as shown in Table 1. The digestion sites were introduced simultaneously.
  • the plasmids pB12 (constructed according to B. Karsten Tischer et al, 2007, Journal of Virology, p. 13200-13208 and gene structure map of pB12 is shown in Figure 1) containing the mini-F gene fragments were digested with BamH I, to obtain the mini-F DNA fragments having BamH I on both sides.
  • the obtained DNA fragments of the left and right homologous arms and the mini-F DNA fragment were ligated to obtain the Mini-F transfer vector, and then transformed into E. coli TOP10 competent cells (purchased from Tiangen) .
  • the plasmid with the mini F transfer vector was extracted and identified by enzyme digestion.
  • the linearized mini-F transfer vector obtained in above step 1 and the extracted DEV4 genomic DNA were co-transfected into DEFs according to the instructions of the commercial transfection kit Lipofectamine TM 3000 (purchased from Invitrogen) , to construct the recombinant virus rDEV4 (i.e. recombinant DEV4) -BAC by homologous recombination.
  • the recombinant virus rDEV4-BAC was obtained (see Figure 2A) .
  • the extracted rDEV4-BAC genomic DNA was electroporated into MegaX competent cell (purchased from Invitrogen, catalog No. C6400-03) according to the instruction of the competent cell. Then, the bacmid rDEV4-BAC was extracted and identified.
  • the bacmid rDEV4-BAC was electroporated into gs1783 competent cells containing redE/T recombinase (Wang et al, 2015, Virology Journal, 12: 126) to obtain GS1783-DEV4-BAC strain.
  • Example 2 Construction of DEV strains having deleted virulence genes
  • virulence-associated genes of DEV4 were deleted, including UL41, US3, UL24, UL40, UL39, UL23 and US7US8 (hereinafter referred to as X in general) , so as to construct DEV strains having deleted virulence genes, including rDEV4 ⁇ UL41, rDEV4 ⁇ US3, rDEV4 ⁇ UL24, rDEV4 ⁇ UL40, rDEV4 ⁇ UL39, rDEV4 ⁇ UL23, and rDEV4 ⁇ US7US8.
  • ⁇ refers to the inactivation (such as deletion or replacement) of the gene herein, for example, ⁇ UL24 refers to deletion or replacement of UL24.
  • the construction method comprises the following steps.
  • the DNA fragment I_SceI-Kana-X (X represents the virulence gene to be deleted) containing I_SceI site, Kana resistance gene and 50bp homologous arms of upstream and downstream of the virulence gene to be deleted was obtained by Not I digestion from the plasmid pKan which was synthesized by GenScript company.
  • GS1783-DEV4-BAC obtained in Example 1 competent cells for electroporation were prepared by conventional method. Then, the I_SceI-Kana-X fragment was electroporated into the GS178-DEV4-BAC competent cells. Recombinant clones were then selected on chloramphenicol and kanamycin double-resistant LB agar plates. The recombinant bacmid DNA was extracted and analyzed by both PCR and RFLP methods. Thus, the GS1783-DEV4-BAC- ⁇ X-Kana strain and the recombinant bacmid rDEV4-BAC- ⁇ X-Kana were obtained.
  • 2%arabinose was used to induce expression of the homing endonuclease I-SceI, resulting in the cleavage of the I-SceI restriction site upstream of the kanamycin gene and, ultimately, the excision of the kanamycin cassette.
  • the recombinant bacmid DNA was extracted and analyzed by both PCR and RFLP methods. Then, the GS1783-DEV4-BAC- ⁇ X strain and the bacmid rDEV4-BAC- ⁇ X were obtained.
  • the recombinant bacmid DNA was extracted and co-transfected with mini-F homologous arm DNA into DEFs (prepared from 11-day-old or 12 day-old clean duck embryos (purchased from Harbin Veterinary Research Institute) according to conventional methods) using lipofectamine 3000 (Invitrogen) . After co-transfection, cells were observed to check the formation of both GFP-positive and -negative plaques. Limiting dilution or plaque purification was performed to separate the GFP-negative recombinant virus, in which mini-F containing EGFP gene was removed via intra-molecular homologous recombination mechanism.
  • the rDEV4 ⁇ X was obtained (see Figure 2B) , including rDEV4 ⁇ UL41, rDEV4 ⁇ US3, rDEV4 ⁇ UL24, rDEV4 ⁇ UL40, rDEV4 ⁇ UL39, rDEV4 ⁇ UL23 and rDEV4 ⁇ US7US8.
  • Example 3 Construction of recombinant DEV containing HA gene of subtype H9N2 of avian influenza virus
  • the construction method will be described using the construction of rDEV4 ⁇ UL39 UL26-H9HA-UL27 as an example. Particularly, the construction method comprises the following steps.
  • the heterologous gene fragment comprising HA gene and Kana resistant gene UL26-mCMV-H9HA-kana-UL27 with 50bp homologous arms on both sides was obtained by PCR amplification using the plasmid puc57-mCMV-H9HA-kana-SV40 (constructed by Nanjing GenScript) as a template and L26L27HA-F and L26L27HA-R as primers (see Table 2) .
  • the primers used for the construction of other rDEV4 H9HA viruses are also listed in Table 2.
  • GS1783-DEV4-BAC- ⁇ UL39 obtained in Example 2 competent cells for electroporation were prepared by conventional method. Then, the heterologous gene fragment UL26-mCMV-H9HA-kana-UL27 was electroporated into the GS1783-DEV4-BAC- ⁇ UL39 competent cells. Recombinant clones were then selected on chloramphenicol and kanamycin double-resistant LB agar plates. The recombinant bacmid DNA was extracted and analyzed by both PCR and RFLP (see Figure 4) methods. Thus, the recombinant blacmid rDEV4-BAC- ⁇ UL39-UL26-H9HA-kana-UL27 was obtained.
  • 2%arabinose was used to induce expression of the homing endonuclease I-SceI, resulting in the cleavage of the I-SceI restriction site upstream of the kanamycin gene and, ultimately, the excision of the kanamycin cassette.
  • the recombinant bacmid DNA was extracted and analyzed by both PCR and RFLP methods. Then, the bacmid rDEV4-BAC- ⁇ UL39-UL26-H9HA-UL27 was obtained.
  • the recombinant bacmid DNA was extracted and co-transfected with mini-F homologous arm DNA into DEFs (prepared from 11-day-old or 12 day-old clean duck embryos (purchased from Harbin Veterinary Research Institute) according to conventional methods) using lipofectamine 3000 (Invitrogen) . After transfection, cells were observed to check the formation of both GFP-positive and-negative plaques (see Figure 5) .
  • Limiting dilution or plaque purification was performed to separate the GFP-negative recombinant virus, in which mini-F containing EGFP gene was removed via intra-molecular homologous recombination mechanism.
  • the recombinant DEV containing HA gene of subtype H9N2 of avian influenza virus rDEV4- ⁇ UL39-UL26-H9HA-UL27 was obtained (see Figure 2C) .
  • additional recombinant viruses expressing the H9N2 HA gene were constructed, including rDEV4 H9HA ⁇ UL23, rDEV4 H9HA ⁇ US7US8, rDEV4 ⁇ UL24 H9HA ⁇ UL2, rDEV4 ⁇ UL40 H9HA ⁇ UL2, rDEV4 H9HA ⁇ UL24, rDEV4 H9HA ⁇ UL39, rDEV4 ⁇ UL24 UL26-H9HA-UL27, rDEV4 H9HA ⁇ UL40, rDEV4 H9HA ⁇ UL23 ⁇ UL41 and rDEV4 ⁇ UL41 H9HA ⁇ US8.
  • Example 4 Preparation and in vitro characterization of the seed batch of the recombinant virus
  • 1.4E7 DEFs were inoculated on a 10cm cell culture dish.
  • the medium was 10ml of MEM containing 5%FBS. After 24 hours of incubation at 37°C in a 5%CO2 incubator, the monolayer of DEFs covered more than 90%of the dish.
  • the cells were inoculated with the DEV strains having deleted virulence genes, respectively. The cells were continued to be incubated at 37°C in a 5%CO2 incubator for about 4 days until all the cells were infected.
  • the cell supernatant was harvested and centrifuged at 3000 rpm for 10 min. The supernatant was separated into 1 ml or 4 ml freezing tubes, and stored at -80°C for later use.
  • 1.4E7 DEFs were inoculated on a 10cm cell culture dish.
  • the medium was 10ml of MEM+5%FBS. After 24 hours of incubation at 37°C in a 5%CO2 incubator, the monolayer of DEFs covered more than 90%of the dish. Then the cells were inoculated with the recombinant DEVs expressing HA gene of subtype H9N2 of avian influenza virus, respectively. The cells were continued to incubate at 37°C in a 5%CO2 incubator for about 4 days until all cells were infected. The cell supernatant was harvested and centrifuged at 3000 rpm for 10 min. The supernatant was separated into 1 ml or 4 ml freezing tubes, and stored at -80°C for later use.
  • 1.4E7 CEF cells were inoculated on a 10cm cell culture dish.
  • the medium was 10ml of MEM+5%FBS. After 24 hours of incubation at 37°C in a 5%CO2 incubator, the monolayer of CEF cells covered more than 90%of the dish.
  • the cells were inoculated with the recombinant DEVs that have been passaged to P7 in DEF, respectively.
  • the cells were continued to be incubated at 37°C in a 5%CO2 incubator for about 4 days until all cells were infected.
  • the cell supernatant was harvested and centrifuged at 3000 rpm for 10 min. The supernatant was separated into 1 ml or 4 ml freezing tubes, and stored at -80°C.
  • the rDEVs with deleted virulence genes were continuously passaged to 15th generation in DEF.
  • the DNA of the recombinant viruses in the 5th, 10th, and 15th generations was extracted by QIAamp DNA Mini Kit (QIAGEN) .
  • the extracted DNA was amplified by PCR using primers to identify the deletion of virulence gene (s) .
  • PCR products were identified by sequencing, indicating that the virulence genes were successfully deleted.
  • the harvested rDEVs having deleted virulence genes were subjected to the detection of sterility and mycoplasma. Sterility detection was performed by a conventional method, and mycoplasma detection was performed by qPCR. Results showed that these recombinant viruses had no exogenous microbial contamination.
  • the rDEVs containing the HA gene of subtype H9N2 of avian influenza virus were continuously passaged to 15th generation in CEF.
  • the DNA of the recombinant viruses in the 5th, 10th, and 15th generations was extracted by QIAamp DNA Mini Kit (QIAGEN) .
  • the extracted DNA was amplified by PCR using primers to identify the insertion of HA gene (see Figure 6) . PCR products were identified by sequencing, indicating that the HA gene was stably present in the genome.
  • the rDEV containing the HA gene of subtype H9N2 of avian influenza virus was inoculated into CEFs in 48-well plate at a dilution of 10 -2 -10 -4 .4 hours after inoculation, the medium was replaced with MEM+5%FBS containing 0.75%methylcellulose, and incubation was continued at 37°C in a 5%CO2 incubator for 4 days.
  • the expression of DEV virus and HA gene was detected using an indirect immunofluorescence assay (IFA) .
  • Indirect immunofluorescence assay comprises the following steps. The cell culture medium was removed. The surface was washed slightly with PBS once, each well was added with 96%cold ethanol and fixed at room temperature for 10 minutes. The ethanol was discarded. The wells were dried in air. The wells were then added with appropriate dilutions of chicken anti-DEV serum and polyclonal rabbit antibody against H9N2 HA (Sino Biological Inc, Catalog#11229-RP02) , respectively, and incubated at 37 °C for 1 hour. Then the antibody was discarded.
  • the wells were washed three times with PBS and added with appropriate amounts of anti-chicken IgG and anti-rabbit IgG (Alexa Fluor 594 goat anti-chicken IgG (H+L) and Alexa Fluor 488 donkey anti-rabbit IgG (H+L) (Invitrogen) ) , and incubated at 37 °C for 1 hour. Then the antibody was discarded.
  • the wells were washed three times with PBS and observed under a fluorescence-inverted microscope. All of the rDEVs containing the HA gene of subtype H9N2 of avian influenza virus showed specific fluorescence for DEV and H9HA. The results indicate the successful expression of HA protein from the recombinant viruses in CEF (see Figure 7) .
  • the safety of the recombinant DEVs including rDEV4 ⁇ UL23, rDEV4 ⁇ US7US8, rDEV4 ⁇ US3, rDEV4 ⁇ UL24, rDEV4 ⁇ UL39, rDEV4 ⁇ UL40, rDEV4 ⁇ UL41, rDEV4 H9HA ⁇ UL23, rDEV4 H9HA ⁇ US7US8, rDEV4 ⁇ UL40 H9HA ⁇ UL2, rDEV4 ⁇ UL24 H9HA ⁇ UL2, rDEV4 H9HA ⁇ UL39, rDEV4 H9HA ⁇ UL40, rDEV4 H9HA ⁇ UL24, rDEV4 ⁇ UL41 H9HA ⁇ US8, rDEV4 H9HA ⁇ UL23 ⁇ UL41, rDEV4 ⁇ UL24 UL26-H9HA-UL27 and rDEV4 ⁇ UL39 UL26-H9HA
  • the experimental chickens in each group were subcutaneously inoculated with 0.5 ml of the materials to be tested or their diluents into the necks. After inoculation, all the experimental chickens including those in the negative control group were observed once a day for 21 consecutive days; and abnormal symptoms of the experimental chickens were recorded, including: mental depression, retracted heads and necks, shuffled feathers, drooping wings, numbness and weakness of both feet, tears, palpebral edemas, outflowing of nasal secretions, swelling of the heads and the necks to varying degrees, following a fluctuation feeling with touch, etc.
  • the experimental chickens in Group 20 were negative for the DEV antibody during the whole experiment. On Day 21 after inoculation, the serum of the experimental chickens in other groups became positive for the DEV antibody. It was verified that the chickens in all the experimental groups were successfully inoculated without missing. The DEV sero-conversion in the experimental chickens also proved that these recombinant viruses had a certain degree of replication in vivo.
  • the wild-type DEV4 has strong pathogenicity to the chickens, and resulted in a morbidity of 100%in the 1-day-old SPF chickens. By deleting different genes, the strain can be attenuated to significant degrees, making it a safe live virus vector for use in chicken.
  • Example 6 Horizontal spreading ability of the recombinant DEVs among chickens
  • This example aims to evaluate the horizontal spreading ability of the constructed recombinant DEVs among the 1-day-old SPF chickens.
  • 165 SPF chickens were randomly divided into 11 groups with 15 chickens in each group and transferred to corresponding isolators for feeding after being hatched (1 day old) .
  • 110 1-day-old chickens were randomly divided into 11 groups, with 10 chickens in each group.
  • Groups 1-9 were test groups for live rDEV vectors, and corresponding materials to be tested were inoculated into the chickens subcutaneously through the neck.
  • Group 10 was a positive control group, and the DEV4 virus was inoculated into the chickens subcutaneously through the neck.
  • Group 11 was a negative control group, and the same volume of MEM solution containing 5%FBS (a diluent of the material to be tested, hereinafter referred to as MEM + 5%FBS) was injected into the chickens subcutaneously through the neck.
  • MEM + 5%FBS a diluent of the material to be tested
  • the experimental chickens in Groups 1-9 were subcutaneously inoculated with 0.5 ml of the corresponding recombinant DEV into the necks at an inoculation dose of 10 6.0 TCID 50 /chicken.
  • the chickens in Group 10 as a positive control group, were subcutaneously inoculated with 0.5 ml of DEV4 into the necks at an inoculation dose of 10 6.0 TCID 50 /chicken.
  • the chickens in Group 11, as a negative control group were subcutaneously injected with 0.5 ml of MEM + 5%FBS into the necks.
  • the experimental chickens in Group 11 were negative for the antibody against DEV during the whole experiment; and on Day 14 after inoculating the serum of the immunized chickens in other groups became positive for the antibody against DEV. On Day 13 and Day 20 after contact (i.e., on Day 14 and Day 21 after inoculating the immunized chickens) , the serum of all the contact chickens in all the groups was negative for the antibody against DEV.
  • Table 10 The results of DEV sero-conversion in the experimental chickens in each group are specifically shown in Table 10.
  • Example 7 Efficacy of vaccine candidate strains containing live rDEV vectors expressing H9N2-HA
  • 10 vaccine candidate strains containing live rDEV vectors expressing the HA gene of avian influenza virus (subtype H9) were inoculated subcutaneously to 1-day-old SPF chickens through the neck.
  • the SPF chickens were challenged with an avian influenza virus (subtype H9) challenge strain (A/chicken/Jiangsu/TX10/2010 strain) . It aims to evaluate the immunogenicity of vaccine candidate strains upon the challenge.
  • the recombinant viruses tested in this example included rDEV4 H9HA ⁇ US7US8, rDEV4 ⁇ UL40 H9HA ⁇ UL2, rDEV4 ⁇ UL24 H9HA ⁇ UL2, rDEV4 H9HA ⁇ UL39, rDEV4 H9HA ⁇ UL40, rDEV4 H9HA ⁇ UL24, rDEV4 ⁇ UL41 H9HA ⁇ US8, rDEV4 H9HA ⁇ UL23 ⁇ UL41, rDEV4 ⁇ UL24 UL26-H9HA-UL27, and rDEV4 ⁇ UL39 UL26-H9HA-UL27.
  • each cotton swab sample was inoculated to five 9-11-day-old SPF chicken embryos through allantoic cavities with 0.2 ml per embryo, then the embryos were incubated for 96 h. Then the HA titers of all allantoic fluids were determined. As long as the HA titer of allantoic fluid of one chicken embryo among the chicken embryos inoculated with each cotton swab sample was no less than 1: 16, it could be determined as positive for H9 virus isolation. The samples that were negative for virus isolation were determined again after blind passage for 1 generation. If it was still negative for virus isolation after the blind passage, then it was determined as negative for H9 virus isolation; if it was positive for virus isolation after the blind passage, then it was determined as positive for H9 virus isolation.
  • the vaccine candidate strains containing live rDEV vectors expressing the HA gene of avian influenza virus can provide protection to H9N2 to varying degrees up to 90%, proving that these different insertion sites are effective.

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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 l'entérite du canard modifié (DEV). Plus particulièrement, la présente invention concerne un virus de l'entérite du canard modifié qui ne présente aucune pathogénicité ou une pathogénicité réduite chez le poulet. En outre, la présente invention concerne une composition comprenant le virus de l'entérite du canard modifié de l'invention en tant que vaccin à vecteur pour le poulet, et son utilisation.
PCT/CN2024/114146 2023-08-25 2024-08-23 Nouveaux vecteurs du virus de l'entérite du canard pour vaccins aviaires Pending WO2025044920A2 (fr)

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