Avian influenza H9 vaccine taking DEV as carrier

Abstract

The invention relates to the field of animal health. In particular, the present invention relates to a composition comprising a modified duck enteritis virus (DEV) comprising and capable of expressing a heterologous polynucleotide encoding an antigen of a chicken pathogen. In addition, the present invention relates to a composition comprising the modified DEV as a chicken vector vaccine and uses thereof. More particularly, the present invention relates to a vaccine composition against chicken pathogens comprising a modified DEV that does not exhibit or exhibits reduced pathogenicity in chicken.

Description

Cross-references to related applications

[0001] This application claims priority to PCT / CN2023 / 114926, filed on August 25, 2023, entitled “Compositions comprising modified DEV”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of animal health. In particular, this invention relates to a composition comprising modified duck enteritis virus (DEV) that contains and is capable of expressing heteropolynucleotides encoding antigens of chicken pathogens. Furthermore, this invention relates to a composition comprising modified DEV as a chicken vector vaccine and its use. More particularly, this invention relates to a vaccine composition against chicken pathogens comprising modified DEV that does not show or shows reduced pathogenicity in chickens. Background Technology

[0003] Duck plague, also known as duck viral enteritis, is a disease affecting ducks, geese, and other birds. Anseriformes ( Anseriformes Duck enteritis virus (DEV) is an acute septicemic infection caused by duck enteritis virus, which naturally infects ducks and geese. However, DEV not only causes duck plague in ducks and geese, but it also infects and kills chickens. DEV is also known as duck herpesvirus 1, duck herpesvirus 1, duck viral enteritis virus (DVEV), or duck plague virus (DPV). The complete nucleotide sequence of DEV has been determined and is available online (see, for example, Genbank accession number JQ673560). The viral genome contains approximately 162 kb and encodes nearly 80 different proteins. Several DEV strains have been isolated, such as the Jansen strain, CSC strain, CHv strain, VAC strain, and strain 2085. Complete sequences of several DEV strains are available in Genbank, such as VAC strain: ID EU082088.2; Anaid isolate C-KCE: ID KF263690.1; Anaid strain CHv: ID JQ647509.1; Anaid strain 2085: ID JF999965; Anaid strain CV: ID KJ549663.1 or Anaid strain CSC: ID JQ673560.1.

[0004] As HerpesvirusAs a member of the family, the DEV genome has a stable double-stranded DNA structure and contains multiple non-essential regions for viral replication, which can accommodate the insertion of multiple heterologous genes. DEV strains attenuated through conventional passage in chicken or duck embryos have become promising live vaccine vector systems for developing vaccines against avian diseases. However, attenuation through conventional passage in chicken or duck embryos results in nonspecific and largely unknown genetic modifications, with the risk that such modifications may be lost and the attenuated virus may regain virulence. Currently, the main research focuses on using DEV as a vector to express heterologous genes to develop vaccines against duck diseases. DEV vector vaccines expressing heterologous genes have been reported to be safe in ducks but have shown virulence in chickens (see Wang, J., (2015). Construction of a recombinant duck enteritis virus (DEV) expressing hemagglutinin of H5N1 avian influenza virus based on an infectious clone of DEV vaccine strain and evaluation of its efficacy in ducks and chickens. Virology Journal, 12(1).). Developing a safe and effective live DEV vector that is genetically modified for use in developing vaccines against chicken diseases remains a challenge in the field. Summary of the Invention

[0005] The present invention is based on the surprising discovery that a heterologous gene of interest, for example, a chicken pathogen (e.g., the H9HA gene), can be inserted into the genome of duck enteritis virus (DEV) and can provide protection against the corresponding pathogen (e.g., avian influenza virus H9). The heterologous gene of interest can be inserted into or replace a portion or the entire sequence of a non-essential gene or region in the DEV genome. Specifically, the non-essential gene or region comprises or consists of either the UL2 gene, UL24 gene, UL39 gene, UL40 gene, UL23 gene, US8 gene, US7 gene, and both of the US8 gene, or the UL26-UL27 intergenic region. Such modified DEVs are stable and capable of expressing the heterologous gene of interest encoding the antigen in a manner suitable for inducing protective immunity against the corresponding pathogen, including at very early stages (i.e., on day 0, day 1, day 2, or day 3 post-hatching). Therefore, such modified DEVs can be used as highly effective vaccine candidates for vaccinating chickens and for conferring early protective immunity.

[0006] According to another aspect, another surprising finding upon which this invention is based is that DEVs, particularly those containing and capable of expressing the heterologous gene of interest, can be modified to exhibit reduced or no morbidity or mortality in chickens compared to unmodified DEVs. This attenuation of DEVs in chickens can be achieved by inactivating non-essential genes in the DEV genome, such as i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23, or viii) UL41, which are either alone or in combination with other DEV genes, such as, for example, combinations of: i) US7 and US8; ii) UL24 and UL2; iii) UL40 and UL2; iv) UL23 and UL41; or v) UL41 and US8. While wild-type DEVs are lethal in chicks, these modified DEVs of the present invention are safe and can efficiently deliver and express the gene of interest (e.g., the H9HA gene) in vivo. Specifically, such DEVs (i) are in vivo attenuated for chickens, and (ii) are stable and capable of expressing foreign genes in a manner suitable for inducing protective immunity, including at very early stages (i.e., on day 0, day 1, day 2, or day 3 post-hatching). Furthermore, these modified DEVs retain a rapid growth rate, thereby allowing for high titer production.

[0007] In one aspect, the present invention provides a composition comprising a modified DEV that contains and is capable of expressing a heteropolynucleotide encoding an antigen of a chicken pathogen, wherein the chicken pathogen is an avian influenza virus.

[0008] In one aspect, the present invention provides a composition comprising a modified DEV that contains and is capable of expressing a heteropolynucleotide encoding an antigen of a chicken pathogen, and wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens compared to unmodified wild-type DEV.

[0009] In one aspect, the present invention provides a method for vaccinating chickens by inducing a protective immune response against a chicken pathogen, comprising administering the composition of the present invention at least once.

[0010] In one aspect, the present invention provides a composition of the invention for use in a method of inducing a protective immune response against a pathogen in chickens, wherein such a method comprises or consists of administering the composition of the invention to chickens once or multiple times.

[0011] In one aspect, the present invention provides the use of the compositions of the present invention in the manufacture of a medicament for vaccinating chickens by inducing a protective immune response against chicken pathogens. Attached Figure Description

[0012] Figure 1 : Displays the gene structure diagram of plasmid pB12.

[0013] Figure 2 The diagram shows (A) a bacterial artificial chromosome rDEV4 BAC, (B) a recombinant construct with the deleted gene rDEV4 ΔUL39, and (C) a recombinant construct with the inserted H9HA gene rDEV4 ΔUL39 UL26-H9HA-UL27.

[0014] Figure 3 Results of transfecting DEF (duck embryo fibroblasts) to rescue rDEV4 ΔUL39.

[0015] Figure 4 The results of RFLP analysis of rDEV4 ΔUL39 UL26-H9HA-UL27 by Xho I digestion are shown.

[0016] Figure 5 The results show the mini-F deletion rescued from rDEV4 ΔUL39 UL26-H9HA-UL27 by co-transfection.

[0017] Figure 6 The results show the genetic stability test results of rDEV4 ΔUL39 UL26-H9HA-UL27 by PCR.

[0018] Figure 7 The results show the expression test results of H9HA from rDEV4 ΔUL39 UL26-H9HA-UL27 via IFA.

[0019] Figure 8 : Results showing the HI Ab levels induced by different rDEV4 H9HA vaccine candidate strains in different groups. Detailed Implementation

[0020] Before describing various aspects of the invention, it must be noted that the singular forms “a,” “an,” and “described,” as used herein and in the appended claims, include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to “a gene” includes multiple genes, and a reference to “virus” refers to one or more viruses and their equivalents known to those skilled in the art. The term “and / or” is intended to cover any combination of items connected by the term, which is equivalent to listing all combinations individually. For example, “A, B, and / or C” covers “A,” “B,” “C,” “A and B,” “A and C,” “B and C,” and “A and B and C.” Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the invention, preferred methods, apparatus, and materials are now described. For the purpose of describing and disclosing viral strains, cell lines, vectors, and methods reported in publications, all publications mentioned herein are incorporated herein by reference and may be used in conjunction with the invention. This document should not be construed as an admission that the present invention is not entitled to precede such disclosure by prior invention.

[0021] This invention relates to a modified duck enteritis virus (DEV), wherein the modified virus has one or more specific inactivated genes. The invention demonstrates that by inactivating such genes, viable, stable, and reproducible DEVs can be obtained, and such viruses can be used to create modified DEVs by inserting foreign genetic material. Further results show that such foreign genetic material is highly expressed by such viruses after cell infection, and such expression remains stable over time. Moreover, and remarkably, although natural DEVs and many other deleted DEV constructs produced by the inventors have been found to be pathogenic or lethal in chicks (on day 0, day 1, day 2, or day 3 post-hatching), inactivation of specific genes in the DEV genome generates an attenuated virus that can be safely used to express antigens in chickens. As demonstrated in the examples, compared to the 100% morbidity and 100% mortality caused by unmodified DEV strains in chickens, the morbidity and mortality caused by the modified DEV of the present invention are surprisingly reduced to less than 30%, and in most cases even surprisingly reduced to 0%. Such modified DEVs can be used as safe live virus vectors in the future.

[0022] Furthermore, the inventors have discovered that certain insertion regions of the antigen-coding sequences of chicken pathogens (e.g., the AIV H9 HA gene), combined with the inactivation of specific genes in the DEV genome, result in higher efficacy of protection against the corresponding pathogens (e.g., AIV H9 subtypes). Such modified DEVs can be used to provide safe and effective protection against the corresponding pathogens in chickens. This discovery is entirely surprising and provides significant advantages and practicality to the virus of the present invention. In particular, as shown in the examples, such modified DEVs exhibit a protection rate exceeding 30% against H9N2, thus such modified DEVs can serve as a safe and effective vaccine against H9 virus attack.

[0023] Modified or attenuated DEVs In one aspect, the present invention provides a modified duck enteritis virus (DEV) that contains and is capable of expressing heteropolynucleotides encoding antigens of chicken pathogens.

[0024] The terms “modified” or “recombinant” are used herein to refer to DEVs that have been altered, rearranged, or modified through genetic engineering. However, this term does not refer to changes in polynucleotide, amino acid, or nucleotide sequences caused by naturally occurring events such as spontaneous mutations. For this purpose, DEVs containing one or more inactivated genes and / or heterologous antigens encoding pathogens in their genome, as described below, are also referred to herein as modified or recombinant DEVs. The modified / recombinant DEVs of the present invention can be used as vectors. The terms “modified / recombinant DEV,” “rDEV,” and “modified / recombinant DEV vector” are used interchangeably herein.

[0025] In the context of this invention, a DEV with an "inactivated" gene refers to a DEV that cannot express the functional protein or RNA encoded by said gene. Therefore, an inactivated gene is a mutated, interrupted, substituted, or missing gene that cannot encode the wild-type protein encoded by said gene.

[0026] The term "virus" specifically refers to a viral particle containing nucleic acid molecules (e.g., a genome) encapsulated in a capsid or capsule. The term "virus" also refers to a viral vector or an isolated viral genome.

[0027] As used in this article, the term "DEV" refers to something that belongs to... Herpesviridae ( Herpesviridae In ) Herpesvirus A division ( Alphaherpesvirinae ) within Marek's virus genus ( Mardivirus All viruses of the duck enteritis virus (DEV) species in ).

[0028] The DEV of the present invention can be prepared from any DEV species or strain. In a preferred embodiment, the DEV of the present invention is derived from or prepared from a parent strain selected from the following: Jansen strain, VAC strain (ID EU082088.2), C-KCE strain (ID KF263690.1), CHv strain (ID JQ647509.1), 2085 strain (ID JF999965), CV strain (ID KJ549663.1), or CSC strain (ID JQ673560.1), or with Jansen strain, VAC strain (ID EU082088.2), C-KCE strain (ID KF263690.1), CHv strain (ID JQ647509.1), 2085 strain (ID JF999965), CV strain (ID KJ549663.1), or CSC strain (ID JQ673560.1). JQ673560.1) has at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In a preferred embodiment, the DEV of the present invention is derived from or prepared from the DEV4 strain deposited at the China Center for Type Culture Collection (CCTCC) on August 4, 2023, with CCTCC NO: V202378, or from any DEV strain having at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the DEV4 strain.

[0029] The virus-related term "heteronucleotide" refers to a polynucleotide that is not naturally present in the viral genome, or that is naturally present in the genome but in a different form or location.

[0030] As used in this article, “antigen” refers to, but is not limited to, components that elicit an immune response in the host.

[0031] Chicken pathogens or antigens can originate from viruses, bacteria, fungi, protozoa, etc. In some embodiments, the chicken pathogen or antigen is derived from avian influenza virus. In some embodiments, the chicken pathogen or 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. Preferably, the chicken pathogen or antigen is derived from influenza A virus. More preferably, the chicken pathogen or antigen is derived from H9 avian influenza virus. Most preferably, the chicken pathogen or antigen is derived from avian influenza virus H9N2.

[0032] In some embodiments, the antigen of the chicken pathogen is the HA protein of the H9 subtype avian influenza virus (H9 HA protein). In some embodiments, the antigen of the pathogen is the HA protein of the H9N2 avian influenza virus.

[0033] In some embodiments, the H9 HA protein has an amino acid sequence that is 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% identical to that of SEQ ID NO:1.

[0034] In some embodiments, the complete H9 HA coding sequence has a nucleotide sequence that is 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% identical to SEQ ID NO:2.

[0035] Sequence identity between two polypeptide / nucleotide sequences indicates the percentage of identical amino acids / nucleotides 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 commonly used to determine the identity of amino acid / nucleotide sequences. For instance, identity can be determined using the BLAST procedure in the NCBI database. For determining sequence identity, see, for example, Computational Molecular Biology, Lesk, AM (ed.), Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW (ed.), Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG (ed.), 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. (ed.), M Stockton Press, New York, 1991.

[0036] As used herein, it should be understood in particular that the term “sequence identity with the sequence of SEQ ID NO: X” is equivalent to the terms “sequence identity with the sequence of SEQ ID NO: X over the length of SEQ ID NO: X” or “sequence identity with the sequence of SEQ ID NO: X over the full length of SEQ ID NO: X”. In this context, “X” is any integer, such as 1 or 2, such that “SEQ ID NO: X” represents any SEQ ID NO referred to herein.

[0037] In some implementations, the heteropolynucleotide is typically operatively linked to the promoter. The promoter can be any natural or synthetic promoter derived from a cellular or viral gene. Examples of suitable promoters include, for example, the cytomegalovirus (CMV) immediate early promoter, the mouse CMV promoter, the guinea pig CMV promoter, the SV40 promoter, the human herpesvirus type III glycoprotein B (HHV3gB) promoter, pseudorabies virus promoters such as the glycoprotein X promoter, the herpes simplex virus-1α4 promoter, the Marek's disease virus glycoprotein A (or gC) promoter, the Marek's disease virus glycoprotein B promoter, the Marek's disease virus glycoprotein E promoter, the Marek's disease virus glycoprotein I promoter, the infectious laryngotracheitis virus glycoprotein B promoter, the infectious laryngotracheitis virus glycoprotein E promoter, the infectious laryngotracheitis virus glycoprotein D promoter, the infectious laryngotracheitis virus glycoprotein I promoter, vaccinia H6, and combinations thereof. In some embodiments, a heteropolynucleotide encoding an antigen of the pathogen is typically operably linked to the mCMV promoter. In some embodiments, a heteropolynucleotide encoding H9 HA is typically operably linked to the mCMV promoter.

[0038] In some embodiments, the heteropolynucleotide is operatively linked to a transcription terminator. The transcription terminator may be derived from human herpes simplex virus (HSV), the thymidine kinase (TK) gene, the feline herpesvirus (FHV) glycoprotein B (gB) gene, the human cytomegalovirus (hCMV) immediate early (IE) gene, strain AD 169, or simian virus 40 (SV40), or may be a synthetic terminator. In some embodiments, the heteropolynucleotide is operatively linked to the SV40 polyadenylate signal. In some embodiments, the heteropolynucleotide encoding H9 HA is typically operatively linked to the SV40 polyadenylate signal.

[0039] One embodiment of the present invention provides a modified DEV that comprises (and is capable of expressing) a heteropolynucleotide encoding the H9 HA protein. In some embodiments, the heteropolynucleotide encoding the H9 HA protein is operatively linked to a mouse CMV promoter, and thus the expression of the H9 HA protein is regulated by the mouse CMV promoter. In some embodiments, the heteropolynucleotide encoding the H9 HA protein is operatively linked to an SV40 polyadenylate signaling group, and thus the expression of the H9 HA protein is regulated by the SV40 polyadenylate signaling group. In some embodiments, the heteropolynucleotide encoding the H9 HA protein is operatively linked to both a mouse CMV promoter and an SV40 polyadenylate signaling group, and thus the expression of the H9 HA protein is regulated by both the mouse CMV promoter and the SV40 polyadenylate signaling group.

[0040] In some embodiments, a modified DEV containing (and capable of expressing) a heteropolynucleotide encoding the H9 HA protein contains an expression cassette that, in the 5' to 3' direction, contains, in the following order: a) a promoter, b) a heteropolynucleotide, c) a transcription terminator. In some embodiments, a modified DEV containing (and capable of expressing) a heteropolynucleotide encoding the H9 HA protein contains, in the following order, in the 5' to 3' direction: a) a mouse CMV promoter, b) a heteropolynucleotide encoding the H9 HA protein, c) a transcription terminator. In some embodiments, a modified DEV containing (and capable of expressing) a heteropolynucleotide encoding the H9 HA protein contains, in the following order, in the 5' to 3' direction: a) a mouse CMV promoter, b) a heteropolynucleotide encoding the H9 HA protein, c) an SV40 polyadenylate signal.

[0041] In some implementations, heteropolynucleotides are expressed after the modified DEV has been transfected into a suitable host cell.

[0042] In a particular embodiment, a heteropolynucleotide is inserted into a non-essential gene or region of the modified DEV. In a particular embodiment, the heteropolynucleotide inserts into or replaces a portion or the entire sequence of a non-essential gene or region of the modified DEV. In a particular embodiment, the non-essential gene or region of the modified DEV genome is selected from the group consisting of both the US7 and US8 genes, the UL2 gene, the UL24 gene, the UL39 gene, the UL40 gene, the UL23 gene, the US8 gene, or the UL26-UL27 intergenic region. In a particular embodiment, the heteropolynucleotide is located in a gene or region selected from the group consisting of both the US7 and US8 genes, the UL2 gene, the UL24 gene, the UL39 gene, the UL26-UL27 intergenic region, the UL40 gene, the UL23 gene, and the US8 gene. In a particular implementation, a heteropolynucleotide is inserted or replaced with a portion or the entire sequence of one or more genes selected from the group consisting of both the US7 and US8 genes, the UL2 gene, the UL24 gene, the UL39 gene, the UL40 gene, the UL23 gene, and the US8 gene in the DEV genome, or inserted into the UL26-UL27 intergenic region of the DEV genome.

[0043] US7, US8, UL2, UL24, UL40, UL39, UL23, UL41, UL26, and UL27 are highly conserved among DEV strains. It should be understood that those skilled in the art can readily identify the exact location of the US7, US8, UL2, UL24, UL40, UL39, UL23, UL41, UL26, and UL27 genes in any DEV strain using the information contained in this application and common general knowledge, or through sequence alignment. For example, the exact location of the US7, US8, UL2, UL24, UL40, UL39, UL23, UL41, UL26, and UL27 genes can be identified by referring to the DEV strain with Genbank accession number EU082088.2.

[0044] In some embodiments, the heteropolynucleotide is located in the regions of both the US7 and US8 genes. In a particular embodiment, the DEV of the present invention has an inactivated US8 gene, and the heteropolynucleotide is inserted into the US7 gene sequence of the DEV viral genome in addition to the existing US7 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the US7 gene region of the DEV viral genome after a partial or complete deletion of the US7 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a partial or complete sequence of the US7 gene (thus inactivating the gene by replacing the gene sequence), or is located in a mutated US7 gene sequence. In a particular embodiment, the DEV of the present invention has an inactivated US7 gene, and the heteropolynucleotide is inserted into the US8 gene sequence of the DEV viral genome in addition to the existing US8 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the US8 gene region of the DEV viral genome after a partial or complete deletion of the US8 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a partial or complete sequence of the US8 gene (thus inactivating the gene by replacing the gene sequence), or is located in a mutated US8 gene sequence. In a particular implementation, a heteropolynucleotide is inserted into the US7 and US8 gene regions of the DEV virus genome after a partial or complete deletion of the US7 and US8 gene sequences, or replaces the entire sequence of the US7 and US8 genes.

[0045] In some embodiments, the heteropolynucleotide is located in the UL2 gene. In specific embodiments, the heteropolynucleotide is inserted into the UL2 gene sequence of the DEV virus genome in addition to the existing UL2 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the UL2 gene region of the DEV virus genome after a partial or complete deletion of the UL2 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a part or the entire UL2 gene sequence (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL2 gene sequence.

[0046] In some embodiments, the heteropolynucleotide is located in the UL24 gene. In specific embodiments, the heteropolynucleotide is inserted into the UL24 gene sequence of the DEV virus genome in addition to the existing UL24 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the UL24 gene region of the DEV virus genome after a partial or complete deletion of the UL24 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a part or the entire UL24 gene sequence (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL24 gene sequence.

[0047] In some embodiments, the heteropolynucleotide is located in the UL39 gene. In specific embodiments, the heteropolynucleotide is inserted into the UL39 gene sequence of the DEV virus genome in addition to the existing UL39 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the UL39 gene region of the DEV virus genome after a partial or complete deletion of the UL39 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a part or the entire UL39 gene sequence (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL39 gene sequence.

[0048] In some embodiments, the heteropolynucleotide is located in the UL26-UL27 intergenic region. In a specific embodiment, the heteropolynucleotide is inserted into the UL26-UL27 intergenic region of the DEV viral genome.

[0049] In some embodiments, the heteropolynucleotide is located in the UL40 gene. In specific embodiments, the heteropolynucleotide is inserted into the UL40 gene sequence of the DEV virus genome in addition to the existing UL40 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the UL40 gene region of the DEV virus genome after a partial or complete deletion of the UL40 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a part or the entire UL40 gene sequence (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL40 gene sequence.

[0050] In some embodiments, the heteropolynucleotide is located in the UL23 gene. In specific embodiments, the heteropolynucleotide is inserted into the UL23 gene sequence of the DEV virus genome in addition to the existing UL23 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the UL23 gene region of the DEV virus genome after a partial or complete deletion of the UL23 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a part or the entire UL23 gene sequence (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL23 gene sequence.

[0051] In some embodiments, the heteropolynucleotide is located in the US8 gene. In specific embodiments, the heteropolynucleotide is inserted into the US8 gene sequence of the DEV virus genome in addition to the existing US8 gene sequence (thus inactivating the gene by interrupting the gene sequence), or inserted into the US8 gene region of the DEV virus genome after a partial or complete deletion of the US8 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a part or the entire sequence of the US8 gene (thus inactivating the gene by replacing the gene sequence), or is located in a mutated US8 gene sequence.

[0052] Furthermore, the modified DEV of the present invention may comprise several heteropolynucleotides encoding one or more antigens of one or more pathogens. In this respect, the several heteropolynucleotides may be inserted into the same position in the virus under the control of a single or several different promoters. Alternatively, the heteropolynucleotides may be inserted into different cloning sites of the virus.

[0053] In some embodiments, the present invention relates to a modified duck enteritis virus (DEV) having an inactivated non-essential gene, wherein inactivation of such a non-essential gene results in attenuation of the modified DEV with respect to chickens. The non-essential genes that result in attenuation upon inactivation within the DEV genome are, for example, i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23, or viii) UL41, which may be alone or in combination with other DEV genes, such as combinations of, for example, the following: i) US7 and US8 genes; ii) UL24 and UL2 genes; iii) UL40 and UL2 genes; iv) UL23 and UL41 genes; or v) UL41 and US8 genes. Although natural DEVs and many other DEV constructs with gene deletions produced by the inventors have been found to be pathogenic or even lethal in chicks (on day 0, day 1, day 2, or day 3 post-hatching), inactivation of specific non-essential genes in the DEV genome generates attenuated DEV viruses that can be safely used to express antigens in chickens. As demonstrated in the examples, the morbidity and mortality rates caused by the modified DEVs of the present invention are surprisingly reduced to less than 30%, and in most cases even surprisingly reduced to 0%, compared to 100% morbidity and 100% mortality rates caused by unmodified DEV strains in chickens.

[0054] Therefore, in some implementations, one or more non-essential genes of the modified DEV are inactivated.

[0055] A "gene" refers to a nucleic acid molecule or sequence that contains an open reading frame encoding a product such as a polypeptide (e.g., peptide, protein, etc.) or RNA.

[0056] "Non-essential genes / regions" are genes / regions in the modified DEV genome where inactivation (including mutation, interruption, substitution, or deletion) of the gene / region or insertion of heterologous polynucleotides in the gene / region does not prevent the modified DEV from replicating in the host cell.

[0057] In some embodiments, the gene is inactivated by mutation, interruption, substitution, or deletion of a portion or the entire 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 gene sequence is substituted or deleted.

[0058] In a particular embodiment, the gene is inactivated due to one or more mutations in the coding sequence, particularly point mutations that impair the expression of the full-length protein. Such mutations can cause substitutions of essential amino acid residues in the encoded protein, resulting in an inactivated protein.

[0059] In certain implementations, the gene is inactivated due to one or more interruptions in the coding sequence that prevent the expression of the full-length protein. Such interruptions can introduce stop or nonsense codons into the sequence, thereby producing an inactivated protein.

[0060] In another embodiment, the gene is inactivated by deletion of a portion or the entire coding sequence of the gene, more particularly at least 20%, 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% of the coding sequence of the gene. Such deletion removes the coding sequence and thus inhibits the expression of the wild-type protein.

[0061] In another embodiment, the gene is inactivated by replacing a portion or the entire coding sequence of the gene with a heteropolynucleotide, more particularly at least 20%, 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% of the coding sequence of the gene. Such a substitution removes the coding sequence and thus inhibits the expression of the wild-type protein.

[0062] In some embodiments, the inactivated non-essential gene (for chicken-induced attenuation) in the modified DEV is selected from the group consisting of US7, US8, UL2, UL24, UL40, UL39, UL23, and UL41. In some embodiments, the inactivated non-essential gene in such an attenuated DEV is selected from any of the following i)-viii): i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23, and viii) UL41, wherein the inactivated non-essential gene is alone or in combination with one or more other non-essential genes of the DEV. In some embodiments, one or more other non-essential genes of the DEV are different from the first inactivated gene and are selected from i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23, and viii) UL41. In some implementations, any one of the following i)-viii) is inactivated in the modified DEV genome: i) US7 and US8 genes; ii) UL24 and UL2 genes; iii) UL40 and UL2 genes; iv) UL24 gene; v) UL39 gene; vi) UL40 gene; vii) UL23 and UL41 genes; or viii) UL41 and US8 genes.

[0063] The phrase “inactivation of one or more (non-essential) genes in the DEV genome resulting in attenuation of DEV in chickens” means that the modified DEV of the present invention is attenuated in chickens.

[0064] As used herein, the term "attenuated" refers to a modified virus that is substantially non-toxic in chickens, i.e., that does not cause or causes reduced disease, especially not death, in chickens compared to the unmodified wild-type parent virus. More specifically, attenuated viruses can typically replicate in chickens without causing their death. More specifically, the modified DEVs of the present invention are non-virulent or less virulent in chickens compared to the corresponding unmodified wild-type parent DEVs that do not contain inactivating genes in their genome. More specifically, attenuated virus refers to a virus that, when at 10... 4.0 -10 7.0 TCID 50 / Chicken (such as 10) 6.0 TCID 50When injected at a dose of ( / chicken), the virus is non-toxic in chickens. More specifically, attenuated virus refers to a virus that is non-toxic in chickens in at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, more preferably at least 80%, and even more preferably at least 90%, 95%, 97%, 98%, 99%, or more. 4.0 -10 7.0 TCID 50 / Chicken (such as 10) 6.0 TCID 50 At a dose of 10-chicken, the virus is non-toxic in chickens. In some implementations, attenuated virus more specifically refers to a virus that is non-toxic in chickens at a dose of 10--. 4.0 -10 7.0 TCID 50 / eggs (such as 10) 6.0 TCID 50 The most preferred attenuated virus is a virus that is non-toxic in the embryo when injected at a dose of 10% (eggs). This attenuated virus is defined as a virus present in at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, more preferably at least 80%, and even more preferably at least 90%, 95%, 97%, 98%, 99%, or more of the injected eggs. 4.0 -10 7.0 TCID 50 / eggs (such as 10) 6.0 TCID 50 The virus is non-toxic in the embryo at a dose of (egg). The modified DEV of the present invention is also non-toxic to injections after hatching, including on day 0, day 1, day 2, and day 3 (i.e., 0.1 to 72 hours after hatching).

[0065] In some embodiments, modified DEVs containing one or more inactivated non-essential genes as described above have reduced or no mortality and / or morbidity in chickens. More specifically, the modified DEVs of the present invention have reduced or no mortality and / or morbidity in chickens compared to unmodified DEVs. The phrase "modified DEVs have reduced or no mortality in chickens (compared to unmodified DEVs)" means that the modified DEVs of the present invention are attenuated with respect to chickens. Therefore, the modified DEVs of the present invention are attenuated with respect to chickens. More specifically, the chicken mortality and / or morbidity caused by the modified DEVs 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 specifically, the chicken mortality and / or morbidity caused by the modified DEVs 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 the mortality and / or morbidity caused by unmodified DEVs.

[0066] In another aspect, the present invention relates to a modified duck enteritis virus (DEV) comprising a heteropolynucleotide encoding an antigen of a chicken pathogen, wherein the modified DEV comprises an inactivating gene selected from any of the following i)-viii): i) US7 gene and US8 gene; ii) UL24 gene and UL2 gene; iii) UL40 gene and UL2 gene; iv) UL24 gene; v) UL39 gene; vi) UL40 gene; vii) UL23 gene and UL41 gene; or viii) UL41 gene and US8 gene. The heteropolynucleotide encoding the antigen of a chicken pathogen (e.g., avian influenza H9N2) can be any of those described herein (e.g., avian influenza HA H9), can be inserted into any non-essential gene or region of a modified DEV as described herein (e.g., both US7 and US8 genes, UL2 gene, UL24 gene, UL39 gene, UL40 gene, UL23 gene, US8 gene, or the UL26-UL27 intergenic region), and can be expressed under the control of any genetic element (e.g., promoter, terminator, etc.), or is part of any expression cassette as described herein.

[0067] In specific embodiments, the modified DEV of the present invention has a continuous region spanning a portion or the entire sequence of the US7 gene, the entire US7-US8 intergenic region, and a deletion or substitution of a portion or the entire sequence of the US8 gene. In a more preferred embodiment, the modified DEV of the present invention comprises the entire sequence of the US7 gene, all intergenic regions between the US7 and US8 genes, and a deletion of the entire sequence of the US8 gene. In a more preferred embodiment, the modified DEV of the present invention comprises the entire sequence of the US7 gene, all intergenic regions between the US7 and US8 genes, and a substitution of the entire sequence of the US8 gene. A specific example of such a construct is, for example, rDEV4 H9HA ΔUS7US8 (see Example 3).

[0068] In specific embodiments, the modified DEV of the present invention has a portion or the entire sequence of the UL24 gene, and a portion or the entire sequence of the UL2 gene with deletion or substitution. In a more preferred embodiment, the modified DEV of the present invention comprises a deletion of the entire sequence of the UL24 gene and a substitution of the entire sequence of the UL2 gene. A specific example of such a construct is, for example, rDEV4 ΔUL24 H9HA ΔUL2 (see Example 3).

[0069] In specific embodiments, the modified DEV of the present invention has a portion or the entire sequence of the UL40 gene, and a portion or the entire sequence of the UL2 gene with deletion or substitution. In a more preferred embodiment, the modified DEV of the present invention comprises a deletion of the entire sequence of the UL40 gene and a substitution of the entire sequence of the UL2 gene. A specific example of such a construct is, for example, rDEV4 ΔUL40 H9HA ΔUL2 (see Example 3).

[0070] In specific embodiments, the modified DEV of the present invention has a deletion or substitution of a portion or the entire sequence of the UL24 gene. Specific examples of such constructs are, for example, rDEV4 ΔUL24 (see Example 2), rDEV4 H9HA ΔUL24 (see Example 3), or rDEV4 ΔUL24 UL26-H9HA-UL27 (see Example 3).

[0071] In specific embodiments, the modified DEV of the present invention has a deletion or substitution of a portion or the entire sequence of the UL39 gene. Specific examples of such constructs are, for example, rDEV4 ΔUL39 (see Example 2), rDEV4 H9HA ΔUL39 (see Example 3), or rDEV4 ΔUL39 UL26-H9HA-UL27 (see Example 3).

[0072] In specific embodiments, the modified DEV of the present invention has a deletion or substitution of a portion or the entire sequence of the UL40 gene. Specific examples of such constructs are, for example, rDEV4 ΔUL40 (see Example 2) or rDEV4 H9HA ΔUL40 (see Example 3).

[0073] In specific embodiments, the modified DEV of the present invention has a portion or the entire sequence of the UL23 gene, and a portion or the entire sequence of the UL41 gene with deletions or substitutions. In a more preferred embodiment, the modified DEV of the present invention comprises a substitution of the entire sequence of the UL23 gene, and a deletion of the entire sequence of the UL41 gene. A specific example of such a construct is, for example, rDEV4 H9HA ΔUL23 ΔUL41 (see Example 3).

[0074] In specific embodiments, the modified DEV of the present invention has a portion or the entire sequence of the UL41 gene, and a portion or the entire sequence of the US8 gene, with deletions or substitutions. In a more preferred embodiment, the modified DEV of the present invention comprises a deletion of the entire sequence of the UL41 gene and a substitution of the entire sequence of the US8 gene. A specific example of such a construct is, for example, rDEV4 ΔUL41 H9HA ΔUS8 (see Example 3).

[0075] In specific embodiments, the modified DEV of the present invention has a deletion or substitution of a portion or the entire sequence of the UL41 gene. In specific embodiments, the modified DEV of the present invention has a deletion of the entire sequence of the UL41 gene. A specific example of such a construct is, for example, rDEV4 ΔUL41 (see Example 2).

[0076] In alternative embodiments, the modified DEV of the present invention has an inactivated gene, preferably a deleted gene, and contains (and is capable of expressing) a heteropolynucleotide encoding an antigen of a pathogen located in a different gene or region. In this case, the heteropolynucleotide may be cloned to replace a portion or the entire sequence of the different gene, or it may be inserted into the different gene, or inserted into the different gene after a portion or the entire sequence of the different gene has been deleted (thus inactivating the different gene as well). For example, the modified DEV of the present invention has an inactivated UL24 gene and contains (and is capable of expressing) a heteropolynucleotide encoding an antigen of a pathogen located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, or inserted into the UL2 gene (thus inactivating the UL2 gene as well).

[0077] In some embodiments, the preferred modified DEVs of the present invention containing heteropolynucleotides encoding pathogen antigens can provide 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% protective efficacy against the corresponding pathogen. In some embodiments, the modified DEVs of the present invention containing heteropolynucleotides encoding pathogen antigens can provide 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% protective efficacy against avian influenza viruses, particularly H9 subtype A avian influenza virus, and more particularly H9N2 subtype A avian influenza virus. In some embodiments, the modified DEVs of the present invention containing heteropolynucleotides encoding pathogen antigens can provide 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% protective efficacy against the corresponding pathogen in chickens. In some embodiments, the modified DEVs of the present invention containing heteropolynucleotides encoding pathogen antigens can provide 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% protective efficacy against avian influenza viruses, particularly H9 subtype A avian influenza virus, and more particularly H9N2 subtype A avian influenza virus.

[0078] In a particular embodiment, the modified DEV of the present invention is a live viral vector. A “live viral vector” is a virus (in this case, a DEV) capable of replicating in a host when the host is infected with a live virus or the genomic nucleic acid of such a virus, and wherein such a virus encodes, delivers, and expresses a heterologous polynucleotide sequence in such a host.

[0079] In one aspect, the present invention provides a modified DEV of the present invention for use as a vector vaccine in chickens. The term "vector vaccine" is a vaccine that uses a virus (in this case, a DEV) as a vector to deliver and express a polynucleotide sequence encoding an antigen, wherein such an antigen provides protection against a pathogen. The virus used as a vector does not show, or only shows, limited pathogenicity to the target species in which the virus is used as a vector.

[0080] Therefore, in one aspect, the present invention also provides the modified duck enteritis virus (DEV) of the present invention as a live vector vaccine in chickens.

[0081] Viral construction and cloning can be performed using techniques known in the art itself. Gene cloning and plasmid construction are well known to those skilled in the art and can be performed essentially using standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012). Typically, modified viruses can be prepared by homologous recombination between a viral genome and a construct (e.g., a homologous plasmid) containing the nucleic acid to be inserted, flanked by nucleotides from the insertion site to allow recombination. Cloning can be performed with or without the deletion of endogenous sequences.

[0082] In one aspect, the present invention provides a method for preparing the modified DEV of the present invention, comprising inactivating one or more genes of the DEV genome as indicated above. Inactivation of one or more selected genes results in reduced or no mortality in the modified DEV in chickens compared to unmodified DEV.

[0083] Preferred DEV The preferred modified DEV of the present invention comprises a heteropolynucleotide located in both the US7 and US8 genes, replacing a portion or the entire sequence of both the US7 and US8 genes, thereby inactivating the US7 and US8 genes. A specific example of such a construct is, for example, rDEV4 H9HA ΔUS7US8 (see Example 3).

[0084] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene to inactivate the UL2 gene, and also comprises an inactivated UL24 gene, optionally with the UL24 gene deleted. Specific examples of such constructs are, for example, rDEV4 ΔUL24 H9HA ΔUL2 (see Example 3).

[0085] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene to inactivate the UL2 gene, and also comprises an inactivated UL40 gene, optionally with the UL40 gene deleted. Specific examples of such constructs are, for example, rDEV4 ΔUL40 H9HA ΔUL2 (see Example 3).

[0086] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL24 gene, replacing a portion or the entire sequence of the UL24 gene, thereby inactivating the UL24 gene. A specific example of such a construct is, for example, rDEV4 H9HAΔUL24 (see Example 3).

[0087] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL39 gene, replacing a portion or the entire sequence of the UL39 gene, thereby inactivating the UL39 gene. A specific example of such a construct is, for example, rDEV4 H9HAΔUL39 (see Example 3).

[0088] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL26-UL27 intergenic region and also comprises an inactivated UL24 gene, optionally with the UL24 gene deleted. A specific example of such a construct is, for example, rDEV4 ΔUL24 UL26-H9HA-UL27 (see Example 3).

[0089] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL40 gene, replacing a portion or the entire sequence of the UL40 gene, thereby inactivating the UL40 gene. A specific example of such a construct is, for example, rDEV4 H9HAΔUL40 (see Example 3).

[0090] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL26-UL27 intergenic region and also comprises an inactivated UL39 gene, optionally with the UL39 gene deleted. A specific example of such a construct is, for example, rDEV4 ΔUL39 UL26-H9HA-UL27 (see Example 3).

[0091] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the UL23 gene, replacing a portion or the entire sequence of the UL23 gene to inactivate the UL23 gene, and also comprises an inactivated UL41 gene, optionally with the UL41 gene deleted. A specific example of such a construct is, for example, rDEV4 H9HA ΔUL23 ΔUL41 (see Example 3).

[0092] The preferred modified DEV of the present invention comprises a heteropolynucleotide located in the US8 gene, replacing a portion or the entire sequence of the US8 gene to inactivate the US8 gene, and also comprises an inactivated UL41 gene, optionally with the UL41 gene deleted. Specific examples of such constructs are, for example, rDEV4 ΔUL41 H9HA ΔUS8 (see Example 3).

[0093] In the preferred modified DEV of the present invention, the heteropolynucleotide encodes the H9 HA protein.

[0094] host cells The present invention also relates to a host cell that expresses modified DEV as defined above. The present invention also relates to a host cell that expresses modified DEV and heteropolynucleotides as defined above. In some embodiments, the host cell is a CEF cell (Liang Z. et al., Animal (Basel), 2022, 12(24):3523), an EB66 cell (Alexander Nikolay, Applied Microbiology and Biotechnology (2018) 102:8725-8737), a DEF cell (Chenghuai Yang, Arch virol 2015, 160:267-274), an embryonic egg, or a chicken kidney cell (Andres Rodrı'guez-Avila et al., Avian diseases 2007, 51:905-911).

[0095] The modified DEVs of this invention can be propagated in any competent cell culture. After achieving the desired viral growth, the cells can be detached from the wells using a scraper or trypsin, and the infected cells can be separated from the supernatant by centrifugation.

[0096] Examples of competent cells include CEF, EB66, DEF, embryonic eggs, chicken kidney cells, etc. Cells or viruses can be cultured at approximately 37°C in a culture medium (such as MEM containing 5% FBS) for 1 hour to 6 days.

[0097] Composition The present invention also relates to a composition comprising the modified DEV of the present invention.

[0098] As used herein, the term "composition" refers to a composition containing at least one antigen that elicits an immune response in a host to which the composition is administered. Such an immune response may be a cell- and / or antibody-mediated immune response to the compositions of the present invention. The host is also described as an "object." Preferably, any host or object described or mentioned herein is a chicken.

[0099] An “immune response” to a composition is the development of a cellular and / or antibody-mediated immune response in the host to the composition of interest. Typically, an “immune response” includes, but is not limited to, one or more of the following effects: specific targeting of antigens included in the composition of interest, generation of antibodies, B cells, helper T cells, and / or cytotoxic T cells. Preferably, the host will exhibit a therapeutic or protective immune response that enhances resistance to new infections and / or reduces the clinical severity of the disease.

[0100] A “protective immune response” or “protective response” will be demonstrated by a reduction or absence of the clinical signs that the infected host typically displays, faster recovery time and / or reduced duration of infectivity, or reduced pathogen titers in the infected host’s tissues or body fluids or excretions.

[0101] The compositions of the present invention are described as "vaccines" when the host exhibits a protective immune response that enhances resistance to new infections and / or reduces the clinical severity of disease. In one aspect, the compositions of the present invention are vaccines.

[0102] In some embodiments, the compositions of the present invention are vector vaccines. In some embodiments, the compositions of the present invention are vector vaccines in chickens. In some embodiments, modified DEVs are used as carriers in the vector vaccines.

[0103] The compositions and vaccines of the present invention may also contain pharmaceutically or veterinarily acceptable carriers, excipients, mediators, or adjuvants.

[0104] Pharmaceutically or veterinary acceptable carriers, adjuvants, mediators, or excipients are well known to those skilled in the art. For example, pharmaceutically or veterinary acceptable carriers, adjuvants, mediators, or excipients include, but are not limited to, 0.9% NaCl (e.g., saline) solutions or phosphate buffers, poly(L-glutamate), lactated Ringer's injection diluents (sodium chloride, sodium lactate, potassium chloride, and calcium chloride), or polyvinylpyrrolidone. Pharmaceutically or veterinary acceptable carriers, mediators, adjuvants, or excipients can be any compound or combination of compounds that facilitates the administration of the carrier (or the protein expressed in vitro by the carrier of the present invention), facilitates transfection or infection, and / or improves the preservation of the carrier (or protein).

[0105] In some embodiments, the compositions of the present invention comprise a lyophilization protectant. In a particular embodiment, the compositions of the present invention comprise a preservative.

[0106] The compositions of the present invention can be liquids (solutions, suspensions, emulsions) or solids (powders, gels, pastes, oils). The compositions of the present invention can be formulated for any route of administration. Preferably, the compositions can be formulated for oral, nasal, eye drop, spray, drinking water, intraocular, intramuscular, subcutaneous, intradermal, or transdermal administration.

[0107] The compositions of the present invention may contain a suitable dose sufficient to elicit a protective response in chickens. Dosage and dose volume are discussed in the general description herein and may also be determined by those skilled in the art based on this disclosure and their knowledge in the art, without any excessive experimentation. The viral vector may be titrated based on any viral titration method, including but not limited to FFA (Focus Forming Assay) or FFU (Focus Forming Unit), TCID... 50 (50% tissue culture infectious dose, 50% Tissue Culture Infective Dose), PFU (Plaque Forming Units), and FAID 50 (50% fluorescent antibody infection dose, 50% Fluorescent Antibody Infectious Dose), and the VLP generated in vitro can be titrated by hemagglutination assay, ELISA, and electron microscopy. In some embodiments, the modified DEV in the composition is 1 × 10 2 TCID 50 / ml or TCID 50 / g to 1×10 7 TCID 50 / ml or TCID 50The modified DEV in the composition is present at a dose of 1 × 10⁹ g. In some embodiments, the modified DEV in the composition is present at a dose of 1 × 10⁹ g. 4 TCID 50 / ml or TCID 50 / g to 1×10 6 TCID 50 / ml or TCID 50 The modified DEV in the composition is present at a dose of 1 × 10⁹ g. In some embodiments, the modified DEV in the composition is present at a dose of 1 × 10⁹ g. 6 TCID 50 / ml or TCID 50 A dose of / g is present. In some embodiments, the dose volume may be between about 0.01 and about 10 ml, or between about 0.01 and about 5 ml.

[0108] Depending on the vaccination regimen, the compositions of the present invention can be administered in a single dose or repeated doses. Depending on the vaccination regimen, the vaccines of the present invention can be formulated in a single dose or repeated doses.

[0109] Uses and methods In one aspect, the present invention provides a method for inducing a protective immune response against a pathogen in chickens using the modified DEV of the present invention, or the composition of the present invention, or the vector vaccine of the present invention, or the vector vaccine of the present invention, either once or multiple times.

[0110] In one aspect, the present invention provides the modified DEV of the present invention, the composition of the present invention, or the vector vaccine of the present invention for vaccinating chickens by inducing a protective immune response against the pathogen in chickens.

[0111] In one aspect, the present invention provides a method for vaccinating chickens by inducing a protective immune response against a pathogen, the method comprising or consisting of administering at least once a modified DEV of the present invention, a composition of the present invention, or a vector vaccine of the present invention.

[0112] In one aspect, the present invention provides the use of the compositions of the present invention in the manufacture of a medicament for vaccinating chickens by inducing a protective immune response against a pathogen in chickens.

[0113] The term "vaccination" refers to active immunization by administering an immunogenic composition to chickens to be immunized, thereby evoking a protective immune response against antigens included in such immunogenic compositions.

[0114] In some implementation plans, chickens are vaccinated on days 0, 1, 2, 3, 4, 5, 6, or 7.

[0115] In some implementations, the modified DEV, composition, or vector vaccine is administered on day 0, day 1, day 2, day 3, day 4, day 5, day 6, or day 7 post-incubation.

[0116] As indicated in the experimental section, the modified DEV of the present invention is particularly advantageous for chick vaccination (day 0, day 1, day 2, or day 3 post-hatching). In fact, the present invention surprisingly demonstrates that the modified DEV of the present invention is safe when administered to chickens at such an early stage, whereas natural or wild-type DEV is lethal to chickens. This early administration, combined with the early onset of immunity induced by the modified DEV, is particularly beneficial for inducing early protective immunity before chickens may be significantly exposed to pathogens.

[0117] In some embodiments, the pathogen is a chicken pathogen. In some embodiments, the pathogen is an avian influenza virus. In some embodiments, the pathogen is a type A avian influenza virus. In some embodiments, the pathogen is an H9 subtype type A avian influenza virus. In some embodiments, the pathogen is an H9N2 subtype type A avian influenza virus.

[0118] The application or modification of the DEV, composition, or vector vaccine of the present invention results in a reduction in the incidence of specific pathogen infection in chickens, or a reduction in the severity of clinical signs caused by or associated with specific pathogen infection. Preferably, the application or modification of the DEV, composition, or vector vaccine of the present invention results in a reduction in the incidence of specific avian influenza virus infection in chickens, or a reduction in the severity of clinical signs caused by or associated with specific avian influenza virus infection. It should be understood that the application or modification of the DEV, composition, or vector vaccine of the present invention may not be effective in all chickens administered, but a significant portion (e.g., 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 are effectively immunized.

[0119] In some embodiments, the drug, modified DEV, composition, or vector vaccine is administered via oral / nasal route, eye drops, spray, drinking water, intraovarian, intramuscular, subcutaneous, intradermal, or transdermal administration. In some embodiments, the drug, modified DEV, composition, or vector vaccine can be formulated for oral / nasal, eye drops, spray, drinking water, intraovarian, intramuscular, subcutaneous, intradermal, or transdermal administration. However, depending on the nature of the compound and its mode of action, immunogenic compositions may also be administered via other routes.

[0120] In one aspect of the invention, the drug, modified DEV, composition, or vector vaccine is administered once, and is effective by such a single administration.

[0121] However, while single-dose administration is preferred, the drug, modified DEV, composition, or vector vaccine may also be administered two or more times, with the first dose administered before the second (booster) dose. Preferably, the second dose is administered at least 15 days after the first dose. More preferably, the second dose is administered 15 to 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 17 to 30 days after the first dose. Even more preferably, the second dose is administered at least 19 days after the first dose. Even more preferably, the second dose is administered 19 to 25 days after the first dose. Most preferably, the second dose is administered at least 21 days after the first dose. In a preferred aspect of the two-dose regimen, both the first and second doses of the immunogenic composition are administered in the same amount. In addition to the first and second dose regimens, alternative embodiments include subsequent doses. For example, a third, fourth, or fifth dose may be administered in these aspects. Preferably, the subsequent third, fourth, and fifth dose regimens are administered in the same amount as the first dose, and the time range between each dose is consistent with the time range between the first and second doses mentioned above.

[0122] The modified DEV, composition, or vector vaccine of the present invention can be administered at a suitable dose sufficient to elicit a protective response in chickens. Dosage and dose volume are discussed in the general description herein and can also be determined by those skilled in the art based on this disclosure and their knowledge in the art, without any excessive experimentation. In some embodiments, the modified DEV in the composition or vector vaccine is administered at a dose of 1 × 10⁻⁶. 2 TCID 50 / ml or TCID 50 / g to 1×10 7 TCID 50 / ml or TCID 50 It is present at a dose of / g. In some embodiments, the modified DEV in the composition or vector vaccine is present at 1×10 4 TCID 50 / ml or TCID 50 / g to 1×10 6 TCID 50 / ml or TCID 50 It is present at a dose of / g. In some embodiments, the modified DEV in the composition or vector vaccine is present at 1×10 6 TCID 50 / ml or TCID 50 A dose of / g is present. In some embodiments, the dose volume may be between about 0.01 and about 10 ml, or between about 0.01 and about 5 ml.

[0123] The present invention also relates to a vaccination kit for vaccinating chickens by inducing a protective immune response against chicken pathogens, the kit comprising an effective amount of the modified DEV, composition, or vector vaccine as described above, and a device for administering the modified DEV, composition, or vector vaccine to the chickens. For example, such a kit comprises an injection device filled with the modified DEV, composition, or vector vaccine according to the present invention, and instructions for intradermal, subcutaneous, intramuscular, or intraovarian injection. Alternatively, the kit comprises a spray / aerosol or eye drop device filled with the modified DEV, composition, or vector vaccine according to the present invention, and instructions for oral, nasal, oral, or mucosal administration.

[0124] This document also describes the following terms and some of the disclosures of this invention: Clause 1. A composition comprising modified duck enteritis virus (DEV) (as a live vector vaccine in chickens), said modified DEV comprising (and capable of expressing) a heteropolynucleotide encoding an antigen of a pathogen.

[0125] Clause 2. The composition according to Clause 1, wherein the pathogen is a chicken pathogen, or the antigen is an antigen of a chicken pathogen.

[0126] Clause 3. The composition according to any one of the preceding clauses, wherein the pathogen is or the antigen is derived from an avian influenza virus.

[0127] Clause 4. The composition according to any one of the preceding clauses, wherein the pathogen is or the antigen is derived from the group consisting of avian influenza viruses selected from influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

[0128] Clause 5. The composition according to any one of the preceding clauses, wherein the pathogen is or the antigen is derived from H9 avian influenza virus.

[0129] Clause 6. The composition according to any one of the preceding clauses, wherein the pathogen is or the antigen is derived from avian influenza virus H9N2.

[0130] Clause 7. The composition according to any one of the preceding clauses, wherein the antigen of the pathogen is the hemagglutinin (HA) protein of the H9N2 subtype avian influenza virus.

[0131] Clause 8. The composition according to any one of the preceding clauses, wherein the amino acid sequence of the 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.

[0132] Clause 9. The composition according to any one of the preceding clauses, wherein the sequence of the heteropolynucleotide 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.

[0133] Clause 10. The composition according to any one of the preceding clauses, wherein the heteropolynucleotide is inserted into a non-essential gene or region of the modified DEV.

[0134] Clause 11. The composition according to any one of the preceding clauses, wherein the heteropolynucleotide is inserted into or replaces a portion or the entire sequence of the non-essential gene or region of the modified DEV.

[0135] Clause 12. The composition according to any one of the preceding clauses, wherein the non-essential genes or regions of the modified DEV are selected from the group consisting of both the US7 and US8 genes, the UL2 gene, the UL24 gene, the UL39 gene, the UL26-UL27 intergenic region, the UL40 gene, the UL23 gene, and the US8 gene.

[0136] Clause 13. The composition according to any one of the preceding clauses, wherein the heteropolynucleotide is located in a gene or region selected from the group consisting of both the US7 and US8 genes of the modified DEV, the UL2 gene, the UL24 gene, the UL39 gene, the UL26-UL27 intergenic region, the UL40 gene, the UL23 gene, and the US8 gene.

[0137] Clause 14. The composition according to any one of the preceding clauses, wherein the heteropolynucleotide is inserted into or replaces a portion or the entire sequence of one or more genes selected from the group consisting of both US7 and US8 genes, UL2 gene, UL24 gene, UL39 gene, UL40 gene, UL23 gene, and US8 gene in the modified DEV, or is inserted into the UL26-UL27 intergenic region of the modified DEV.

[0138] Clause 15. The composition according to any one of the preceding clauses, wherein one or more non-essential genes of the modified DEV are inactivated.

[0139] Clause 16. The composition according to Clause 15, wherein the inactivated non-essential gene of the modified DEV is selected from the group consisting of US7, US8, UL2, UL24, UL40, UL39, UL23 and UL41.

[0140] Clause 17. The composition according to Clause 15, wherein the inactivated non-essential gene of the modified DEV is selected from any one of the following i)-viii): i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23 and viii) UL41, wherein the inactivated non-essential gene is combined with an inactivated combination of one or more other non-essential genes of the modified DEV.

[0141] Clause 18. The composition according to Clause 17, wherein the one or more additional non-essential genes of the modified DEV are different from the first inactivating gene and are selected from i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23, or viiii) UL41.

[0142] Clause 19. The composition according to any one of the preceding clauses, wherein the modified DEV comprises an inactivating gene selected from any of the following i)-viii): i) US7 gene and US8 gene; ii) UL24 gene and UL2 gene; iii) UL40 gene and UL2 gene; iv) UL24 gene; v) UL39 gene; vi) UL40 gene; vii) UL23 gene and UL41 gene; or viii) UL41 gene and US8 gene.

[0143] Clause 20. The composition according to any one of Clauses 15-19, wherein the non-essential gene within the modified DEV is inactivated by mutation, interruption, substitution, or deletion of a portion or the entire sequence of the inactivated non-essential gene.

[0144] Clause 21. The composition according to any one of Clauses 15-19, 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 non-essential gene within the modified DEV is replaced or deleted.

[0145] Clause 22. The composition according to any one of the preceding clauses, wherein the modified DEV comprises any one of the following: i) Heteronucleotides located in both the US7 and US8 genes, replacing a portion or the entire sequence of both the US7 and US8 genes; ii) A heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and also containing an inactivated UL24 gene, optionally with the UL24 gene deleted; iii) A heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and also containing an inactivated UL40 gene, optionally with the UL40 gene deleted; iv) Heteronucleotides located in the UL24 gene that replace a portion or the entire sequence of the UL24 gene; v) Heteronucleotides located in the UL39 gene that replace a portion or the entire sequence of the UL39 gene; vi) Heteronucleotides located in the UL26-UL27 intergenic region, and also containing an inactivated UL24 gene, optionally with the UL24 gene deleted; vii) Heteronucleotides located in the UL40 gene that replace a portion or the entire sequence of the UL40 gene; viii) The heteropolynucleotide located in the UL26-UL27 intergenic region and also containing the inactivated UL39 gene, optionally with the UL39 gene deleted; ix) a heteropolynucleotide located in the UL23 gene, replacing a portion or the entire sequence of the UL23 gene, and also containing an inactivated UL41 gene, optionally with the UL41 gene deleted; or x) A heteropolynucleotide located in the US8 gene, replacing a portion or the entire sequence of the US8 gene, and also containing an inactivated UL41 gene, optionally with the UL41 gene deleted.

[0146] Clause 23. The composition according to any one of the preceding clauses, wherein the heteropolynucleotide is operatively linked to a promoter.

[0147] Clause 24. The composition according to Clause 23, wherein the promoter is selected from the group consisting of: cytomegalovirus (CMV) immediate early promoter, mouse CMV promoter, guinea pig CMV promoter, SV40 promoter, human herpesvirus type III glycoprotein B (HHV3gB) promoter, pseudorabies virus promoter such as glycoprotein X promoter, herpes simplex virus-1α4 promoter, Marek's disease virus glycoprotein A (or gC) promoter, Marek's disease virus glycoprotein B promoter, Marek's disease virus glycoprotein E promoter, Marek's disease virus glycoprotein I promoter, infectious laryngotracheitis virus glycoprotein B, infectious laryngotracheitis virus glycoprotein E promoter, infectious laryngotracheitis virus glycoprotein D promoter, infectious laryngotracheitis virus glycoprotein I promoter, vaccinia H6, and combinations thereof.

[0148] Clause 25. The composition according to any one of the preceding clauses, wherein the heteropolynucleotide is operatively linked to the SV40 polyadenylate signal.

[0149] Clause 26. The composition according to any one of the preceding clauses, wherein the heteropolynucleotide is expressed after the modified DEV has been transfected into a suitable host cell.

[0150] Clause 27. The composition according to any one of the preceding clauses, wherein the modified DEV is attenuated (regarding chickens).

[0151] Clause 28. The composition according to any one of Clauses 15-27, wherein the inactivation of the selected gene results in DEV attenuation.

[0152] Clause 29. The composition according to any one of the preceding clauses, wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens compared with the unmodified wild-type DEV.

[0153] Clause 30. The composition according to any one of the preceding clauses, wherein the mortality rate in chickens 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 the mortality rate caused by the unmodified DEV.

[0154] Clause 31. The composition according to any one of the preceding clauses further comprises a pharmaceutically or veterinarily acceptable carrier, excipient, mediator, or adjuvant.

[0155] Clause 32. The composition according to any one of the preceding clauses, wherein the composition is a vaccine.

[0156] Clause 33. The composition according to any one of the preceding clauses, wherein the composition is a vector vaccine in chickens.

[0157] Clause 34. The composition according to Clause 33, wherein the modified DEV is used as a carrier.

[0158] Clause 35. A modified DEV (as a live vector vaccine in chickens) comprising (and capable of expressing) a heteropolynucleotide encoding an antigen of a pathogen.

[0159] Clause 36. The modified DEV as described in Clause 35, wherein the pathogen is a chicken pathogen, or the antigen is an antigen of a chicken pathogen.

[0160] Clause 37. The modified DEV as described in Clause 36, wherein the pathogen is an avian influenza virus.

[0161] Clause 38. The modified DEV according to any one of Clauses 35-37, wherein the pathogen is or the antigen is derived from the group consisting of avian influenza viruses selected from influenza A virus, influenza B virus, influenza C virus and influenza D virus.

[0162] Clause 39. The modified DEV according to any one of Clauses 35-38, wherein the pathogen is or the antigen is derived from H9 avian influenza virus.

[0163] Clause 40. The modified DEV according to any one of Clauses 35-39, wherein the pathogen is or the antigen is derived from avian influenza virus H9N2.

[0164] Clause 41. The modified DEV according to any one of Clauses 35-40, wherein the antigen of the pathogen is the hemagglutinin (HA) protein of the H9N2 subtype avian influenza virus.

[0165] Clause 42. The modified DEV according to any one of Clauses 35-41, wherein the amino acid sequence of the 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.

[0166] Clause 43. The modified DEV according to any one of Clauses 35-42, wherein the sequence of the heteropolynucleotide 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.

[0167] Clause 44. A modified DEV according to any one of Clauses 35-43, wherein the heteropolynucleotide is inserted into a non-essential gene or region of the modified DEV.

[0168] Clause 45. A modified DEV according to any one of Clauses 35-44, wherein the heteropolynucleotide is inserted into or replaces a portion or the entire sequence of the non-essential gene or region of the modified DEV.

[0169] Clause 46. The modified DEV according to any one of Clauses 35-45, wherein the non-essential genes or regions of the modified DEV are selected from the group consisting of both the US7 and US8 genes, the UL2 gene, the UL24 gene, the UL39 gene, the UL26-UL27 intergenic region, the UL40 gene, the UL23 gene, and the US8 gene.

[0170] Clause 47. The modified DEV according to any one of Clauses 35-46, wherein the heteropolynucleotide is located in a gene or region selected from the group consisting of both the US7 and US8 genes, the UL2 gene, the UL24 gene, the UL39 gene, the UL26-UL27 intergenic region, the UL40 gene, the UL23 gene, and the US8 gene of the modified DEV.

[0171] Clause 48. The modified DEV according to any one of Clauses 35-47, wherein the heteropolynucleotide is inserted into or replaces a portion or the entire sequence of one or more genes selected from the group consisting of both US7 and US8 genes, UL2 gene, UL24 gene, UL39 gene, UL40 gene, UL23 gene, and US8 gene in the modified DEV, or is inserted into the UL26-UL27 intergenic region of the modified DEV.

[0172] Clause 49. A modified DEV according to any one of Clauses 35-48, wherein one or more non-essential genes of the modified DEV genome are inactivated.

[0173] Clause 50. The modified DEV as described in Clause 49, wherein the inactivated non-essential gene of the modified DEV is selected from the group consisting of US7, US8, UL2, UL24, UL40, UL39, UL23 and UL41.

[0174] Clause 51. The modified DEV according to Clause 50, wherein the inactivated non-essential gene of the modified DEV is selected from any one of the following i)-viii): i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23 and viii) UL41, wherein the inactivated non-essential gene is alone or in combination with one or more other non-essential genes of the modified DEV.

[0175] Clause 52. The modified DEV according to Clause 51, wherein the one or more additional non-essential genes of the modified DEV are different from the first inactivated gene and are selected from i) US7, ii) US8, iii) UL2, iv) UL24, v) UL40, vi) UL39, vii) UL23 or viiii) UL41.

[0176] Clause 53. A modified DEV according to any one of Clauses 35-52, wherein the modified DEV comprises an inactivated non-essential gene selected from any of the following i)-viii): i) the US7 gene and the US8 gene; ii) the UL24 gene and the UL2 gene; iii) the UL40 gene and the UL2 gene; iv) the UL24 gene; v) the UL39 gene; vi) the UL40 gene; vii) the UL23 gene and the UL41 gene; or viii) the UL41 gene and the US8 gene.

[0177] Clause 54. A modified DEV according to any one of Clauses 49-53, wherein the non-essential gene within the modified DEV is inactivated by mutation, interruption, substitution or deletion of a portion or the entire sequence of the non-essential gene.

[0178] Clause 55. A modified DEV according to any one of Clauses 49-54, 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 non-essential gene in the modified DEV is replaced or deleted.

[0179] Clause 56. A modified DEV according to any one of Clauses 35-55, wherein the modified DEV comprises any one of the following: i) Heteronucleotides located in both the US7 and US8 genes, replacing a portion or the entire sequence of both the US7 and US8 genes; ii) A heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and also containing an inactivated UL24 gene, optionally with the UL24 gene deleted; iii) A heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and also containing an inactivated UL40 gene, optionally with the UL40 gene deleted; iv) Heteronucleotides located in the UL24 gene that replace a portion or the entire sequence of the UL24 gene; v) Heteronucleotides located in the UL39 gene that replace a portion or the entire sequence of the UL39 gene; vi) Heteronucleotides located in the UL26-UL27 intergenic region, and also containing an inactivated UL24 gene, optionally with the UL24 gene deleted; vii) Heteronucleotides located in the UL40 gene that replace a portion or the entire sequence of the UL40 gene; viii) The heteropolynucleotide located in the UL26-UL27 intergenic region and also containing the inactivated UL39 gene, optionally with the UL39 gene deleted; ix) a heteropolynucleotide located in the UL23 gene, replacing a portion or the entire sequence of the UL23 gene, and also containing an inactivated UL41 gene, optionally with the UL41 gene deleted; or x) A heteropolynucleotide located in the US8 gene, replacing a portion or the entire sequence of the US8 gene, and also containing an inactivated UL41 gene, optionally with the UL41 gene deleted.

[0180] Clause 57. The modified DEV according to any one of Clauses 35-56, wherein the heteropolynucleotide is operatively linked to a promoter.

[0181] Clause 58. The modified DEV according to Clause 57, wherein the promoter is selected from the group consisting of: cytomegalovirus (CMV) immediate early promoter, mouse CMV promoter, guinea pig CMV promoter, SV40 promoter, human herpesvirus type III glycoprotein B (HHV3gB) promoter, pseudorabies virus promoter such as glycoprotein X promoter, herpes simplex virus-1α4 promoter, Marek's disease virus glycoprotein A (or gC) promoter, Marek's disease virus glycoprotein B promoter, Marek's disease virus glycoprotein E promoter, Marek's disease virus glycoprotein I promoter, infectious laryngotracheitis virus glycoprotein B, infectious laryngotracheitis virus glycoprotein E promoter, infectious laryngotracheitis virus glycoprotein D promoter, infectious laryngotracheitis virus glycoprotein I promoter, vaccinia H6, and combinations thereof.

[0182] Clause 59. The modified DEV according to any one of Clauses 35-58, wherein the heteropolynucleotide is operatively linked to the SV40 polyadenylate signal.

[0183] Clause 60. The modified DEV according to any one of Clauses 35-59, wherein the heteropolynucleotide is expressed after the modified DEV has been transfected into a suitable host cell.

[0184] Clause 61. The modified DEV according to any one of Clauses 35-60, wherein the modified DEV is attenuated (in relation to chickens).

[0185] Clause 62. The modified DEV according to any one of Clauses 49-61, wherein the inactivation of the selected gene results in DEV attenuation.

[0186] Clause 63. The modified DEV according to any one of Clauses 35-62, wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens compared with the unmodified wild-type DEV.

[0187] Clause 64. The modified DEV according to any one of Clauses 35-63, wherein the mortality rate in chickens 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 the mortality rate caused by the unmodified DEV.

[0188] Clause 65. The modified DEV according to any one of Clauses 35-64, used as a carrier vaccine in chickens.

[0189] Clause 66. A method for inducing a protective immune response against a pathogen in chickens using a modified DEV according to any one of Clauses 35-64, a composition according to any one of Clauses 1-34, or a vector vaccine according to Clause 65, wherein such a method comprises or consists of administering to said chickens once or more of the modified DEV according to any one of Clauses 35-64, a composition according to any one of Clauses 1-34, or a vector vaccine according to Clause 65.

[0190] Clause 67. A modified DEV according to any one of Clauses 35-64, or a composition according to any one of Clauses 1-34, or a vector vaccine according to Clause 65, for use in vaccinating chickens by inducing a protective immune response against the pathogen in chickens.

[0191] Clause 68. A method of vaccinating chickens by inducing a protective immune response against a pathogen, comprising or consisting of administering at least once to said chickens a modified DEV according to any one of Clauses 35-64, or a composition according to any one of Clauses 1-34, or a vector vaccine according to Clause 65.

[0192] Clause 69. Use of the modified DEV according to any one of Clauses 35-64 in the manufacture of a composition for vaccinating chickens by inducing a protective immune response against a pathogen in chickens.

[0193] Clause 70. The method and use according to any one of Clauses 66-69, wherein the pathogen is an avian influenza virus.

[0194] Clause 71. The method and use according to Clause 70, wherein the pathogen is an avian influenza virus, optionally type A avian influenza virus, optionally H9 subtype type A avian influenza virus, optionally H9N2 subtype type A avian influenza virus.

[0195] Clause 72. The method and use according to any one of Clauses 66-71, wherein the chickens are 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days old on the day of vaccination.

[0196] Clause 73. The method and use according to any one of Clauses 66-72, wherein the modified DEV, the composition, or the vector vaccine is administered on day 0, day 1, day 2, day 3, day 4, day 5, day 6, or day 7 after hatching.

[0197] Clause 74. The method and use according to any one of Clauses 66-73, wherein the modified DEV, the composition, or the vector vaccine is administered via oral-nasal, eye-drop, spray, drinking water, intraovarian, intramuscular, subcutaneous, intradermal, or transdermal administration.

[0198] Clause 75. A vaccination kit for vaccinating chickens by inducing a protective immune response against a pathogen, comprising an effective amount of a modified DEV according to any one of Clauses 35-64, or a composition according to any one of Clauses 1-34, or a vector vaccine according to Clause 65, and means for administering the modified DEV, the composition, or the vector vaccine to the chickens.

[0199] Clause 76. A host cell that expresses modified DEV according to any one of Clauses 35-64.

[0200] Clause 77. A host cell that expresses the modified DEV and the heteropolynucleotide as described in any one of Clauses 35-64.

[0201] Clause 78. The host cell as described in Clause 76 or 77, wherein the host cell is a CEF cell, an EB66 cell, or a DEF cell.

[0202] Clause 79. A method for preparing a modified DEV according to any one of Clauses 35-64, comprising inactivating one or more genes of the modified DEV genome as defined in any one of Clauses 49-64, wherein, compared with the unmodified DEV, the inactivation of the one or more selected genes results in a reduced or zero mortality rate of the modified DEV in chickens.

[0203] Example The following embodiments further illustrate the invention by way of example. It should be understood that the invention is not limited to any of the embodiments described below. Those skilled in the art will understand that the performance, results, and findings of these embodiments can be adapted and applied in a broader sense, taking into account the general description of the invention.

[0204] Example 1: Construction of a bacterial artificial chromosome (BAC) for DEV The duck plague virus DEV4 strain used in this study was deposited at the China Center for Type Culture Collection (CCTCC) on August 4, 2023, with the serial number CCTCC NO: V202378.

[0205] To attenuate the DEV4 strain and make it a safe vector, a bacterial artificial chromosome system was constructed using the DEV4 strain genome for subsequent gene deletion and insertion. Specific steps included: 1. Using primers shown in Table 1, amplify the homologous arm sequences to the left and right of the mini-F element insertion site (located between UL44 and UL44.5 in the DEV4 genome) by PCR. Simultaneously, introduce a digestion site. Digest the plasmid pB12 containing the mini-F gene fragment using BamHI (constructed according to B. Karsten Tischer et al., 2007, Journal of Virology, pp. 13200-13208, and the gene structure diagram of pB12 is shown in...). Figure 1 (As shown in the image) to obtain mini-F DNA fragments with BamHI on both sides. Then, the obtained DNA fragments from the left and right homologous arms were ligated with the mini-F DNA fragments to obtain a Mini-F transfer vector, which was then transformed into *E. coli* TOP10 competent cells (purchased from Tiangen). Plasmids containing the mini-F transfer vector were extracted and identified by enzymatic digestion.

[0206] Table 1. PCR primers

[0207] 2. Following the instructions of the commercial transfection kit Lipofectamine™ 3000 (purchased from Invitrogen), the linearized mini-F transfer vector obtained in step 1 above and the extracted DEV4 genomic DNA were co-transfected into DEF to construct the recombinant virus rDEV4 (i.e., recombinant DEV4)-BAC via homologous recombination. After screening and purification, the recombinant virus rDEV4-BAC was obtained (see...). Figure 2 A).

[0208] 3. Following the instructions for use with competent cells, the extracted rDEV4-BAC genomic DNA was electroporated into MegaX competent cells (purchased from Invitrogen, catalog number C6400-03). Then, rod granules rDEV4-BAC were extracted and identified.

[0209] 4. The rod-like particles rDEV4-BAC were electroporated into gs1783 competent cells containing redE / T recombinase (Wang et al., 2015, Virology Journal, 12:126) to obtain the GS1783-DEV4-BAC strain.

[0210] Example 2: Construction of a DEV strain with a missing virulence gene Based on the constructed rDEV4-BAC, different virulence-associated genes of DEV4 (including UL24, UL40, UL39, and UL41 (hereinafter referred to as X)) were deleted to construct DEV strains with deleted virulence genes, including rDEV4 ΔUL24, rDEV4 ΔUL40, rDEV4 ΔUL39, and rDEV4 ΔUL41.

[0211] “Δ” refers to the inactivation of the gene in this article (such as deletion or substitution). For example, ΔUL24 means UL24 deletion or substitution.

[0212] The construction method includes the following steps.

[0213] 1. The plasmid pKan synthesized from GenScript company was digested with Not I to obtain a DNA fragment I_SceI-Kana-X (where X represents the virulence gene to be deleted) containing the I_SceI site, the Kana resistance gene, and 50bp homologous arms upstream and downstream of the virulence gene to be deleted.

[0214] 2. Competent GS1783-DEV4-BAC (obtained in Example 1) cells for electroporation were prepared using conventional methods. The I_SceI-Kana-X fragment was then electroporated into the GS178-DEV4-BAC competent cells. Recombinant clones were then selected on LB agar plates with dual resistance to chloramphenicol and kanamycin. Recombinant rod-particle DNA was extracted and analyzed by PCR and RFLP. Thus, the GS1783-DEV4-BAC-ΔX-Kana strain and recombinant rod-particle rDEV4-BAC-ΔX-Kana were obtained. In step 2 of Red recombination, the expression of the homing endonuclease I-SceI was induced using 2% arabinose, leading to cleavage of the I-SceI restriction site upstream of the kanamycin gene and ultimately excision of the kanamycin cassette. Recombinant rod-particle DNA was extracted and analyzed by PCR and RFLP. Then, the GS1783-DEV4-BAC-ΔX strain and rDEV4-BAC-ΔX were obtained.

[0215] 3. In order to salvage the recombinant rDEV4 ΔX (see...) Figure 3 The mini-F sequence was missing, and recombinant rod-like DNA was extracted and co-transfected with the mini-F homologous arm DNA into DEF cells (prepared according to standard methods from 11- or 12-day-old clean duck embryos (purchased from Harbin Veterinary Research Institute)) using lipofectamine 3000 (Invitrogen). After co-transfection, cells were observed to examine for the formation of both GFP-positive and GFP-negative plaques. Limiting dilutions or plaque purification were performed to isolate the GFP-negative recombinant virus, wherein the mini-F containing the EGFP gene was removed via intramolecular homologous recombination. Thus, rDEV4 ΔX was obtained (see [link to relevant documentation]). Figure 2 B), including rDEV4 ΔUL24, rDEV4 ΔUL40, rDEV4 ΔUL39, and rDEV4 ΔUL41.

[0216] Example 3: Construction of recombinant DEV containing the HA gene of avian influenza virus subtype H9N2 Based on DEV strains with missing virulence genes, the HA gene of the H9N2 avian influenza virus subtype was inserted into different sites. The following recombinant viruses were ultimately constructed: rDEV4 H9HA ΔUS7US8, rDEV4 ΔUL24 H9HA ΔUL2, rDEV4 ΔUL40 H9HA ΔUL2, rDEV4 H9HA ΔUL24, rDEV4 H9HA ΔUL39, rDEV4 ΔUL24UL26-H9HA-UL27, rDEV4 H9HA ΔUL40, rDEV4 ΔUL39 UL26-H9HA-UL27, rDEV4 H9HA ΔUL23 ΔUL41, and rDEV4 ΔUL41 H9HA ΔUS8.

[0217] In this embodiment, the construction method will be described using the construction of rDEV4 ΔUL39 UL26-H9HA-UL27 as an example. Specifically, the construction method includes the following steps.

[0218] 1. Using plasmid puc57-mCMV-H9HA-kana-SV40 (constructed by Nanjing GenScript) as a template and L26L27HA-F and L26L27HA-R as primers (see Table 2), a heterologous gene fragment containing the HA gene and the Kana resistance gene UL26-mCMV-H9HA-kana-UL27 was obtained by PCR amplification, with 50 bp homologous arms flanking it. Primers used for constructing other rDEV4 H9HA viruses are also listed in Table 2.

[0219] Table 2. PCR primers

[0220] 2. Competent cells of GS1783-DEV4-BAC-ΔUL39 (obtained in Example 2) for electroporation were prepared using conventional methods. The heterologous gene fragment L26-mCMV-H9HA-kana-UL27 was then electroporated into the GS1783-DEV4-BAC-ΔUL39 competent cells. Recombinant clones were then selected on LB agar plates with dual chloramphenicol and kanamycin resistance. Recombinant band-granule DNA was extracted and analyzed by PCR and RFLP (see [link to PCR]). Figure 4The recombinant rod-like DNA was analyzed using the following methods. Therefore, the recombinant rod-like DNA rDEV4-BAC-ΔUL39-UL26-H9HA-kana-UL27 was obtained. In step 2 of the Red recombination, the expression of the homing endonuclease I-SceI was induced using 2% arabinose, leading to cleavage of the I-SceI restriction site upstream of the kanamycin gene and ultimately resulting in the excision of the kanamycin cassette. The recombinant rod-like DNA was extracted and analyzed by PCR and RFLP. The rod-like DNA rDEV4-BAC-ΔUL39-UL26-H9HA-UL27 was then obtained.

[0221] 3. To rescue the recombinant rDEV4-ΔUL39-UL26-H9HA-UL27 lacking the mini-F sequence, recombinant rod-particle DNA was extracted and co-transfected with the mini-F homologous arm DNA into DEF cells (prepared according to standard methods from 11- or 12-day-old clean duck embryos (purchased from Harbin Veterinary Research Institute)) using lipofectamine 3000 (Invitrogen). After transfection, cells were observed to examine for the formation of both GFP-positive and GFP-negative plaques (see [link to relevant documentation]). Figure 5 Limiting dilution or plaque purification was performed to isolate GFP-negative recombinant viruses, wherein the mini-F containing the EGFP gene was removed via intramolecular homologous recombination. Thus, recombinant DEVs containing the HA gene of the H9N2 subtype of avian influenza virus rDEV4-ΔUL39-UL26-H9HA-UL27 were obtained (see [link to original text]). Figure 2 C).

[0222] 4. Based on steps 1-3 above, additional recombinant viruses expressing the H9N2 HA gene were constructed, including rDEV4 H9HAΔUS7US8, rDEV4 ΔUL24 H9HA ΔUL2, rDEV4 ΔUL40 H9HA ΔUL2, rDEV4 H9HA ΔUL24, rDEV4 H9HA ΔUL39, rDEV4 ΔUL24 UL26-H9HA-UL27, rDEV4 H9HA ΔUL40, rDEV4 H9HA ΔUL23 ΔUL41 and rDEV4 ΔUL41 H9HAΔUS8.

[0223] Example 4: Preparation and in vitro characterization of seed batches of recombinant virus 4.1. Preparation of seed batches of recombinant DEV containing the HA gene of avian influenza virus subtype H9N2 1.4E7 DEF cells were seeded onto 10 cm cell culture dishes. The culture medium was 10 ml MEM + 5% FBS. After culturing at 37°C in a 5% CO2 incubator for 24 hours, the DEF monolayer covered more than 90% of the culture dish. Then, recombinant DEV cells expressing the HA gene of the H9N2 avian influenza virus subtype were seeded. The cells were incubated at 37°C in a 5% CO2 incubator for approximately 4 days until all cells were infected. The cell supernatant was harvested and centrifuged at 3000 rpm for 10 minutes. The supernatant was separated into 1 ml cryovials and stored at -80°C for later use.

[0224] Next, 1.4E7 CEF cells were seeded onto 10 cm cell culture dishes. The culture medium was 10 ml of MEM + 5% FBS. After culturing at 37°C in a 5% CO2 incubator for 24 hours, the CEF cell monolayer covered more than 90% of the culture dish. Recombinant DEV cells, which had been passaged to P7 in DEF, were then seeded. The cells were incubated at 37°C in a 5% CO2 incubator for approximately 4 days until all cells were infected. The cell supernatant was harvested and centrifuged at 3000 rpm for 10 minutes. The supernatant was separated into 1 ml or 4 ml cryovials and stored at -80°C.

[0225] 4.2. In vitro identification of rDEV containing the HA gene of avian influenza virus subtype H9N2 1. Stability of the HA gene. rDEV containing the HA gene of the avian influenza virus subtype H9N2 was passaged sequentially to passage 15 in CEF. DNA was extracted from the recombinant virus at passages 5, 10, and 15 using the QIAamp DNA Mini Kit (QIAGEN). The extracted DNA was amplified by PCR using primers to identify the insertion of the HA gene (see [link to kit]). Figure 6 The PCR products were identified by sequencing, thus indicating that the HA gene is stably present in the genome.

[0226] Table 3. PCR primers

[0227] 2. Sterility and Mycoplasma Testing. The harvested rDEVs containing the HA gene of the H9N2 avian influenza virus subtype were subjected to sterility and mycoplasma testing. Sterility testing was performed using standard methods, and mycoplasma testing was performed using qPCR. Results showed that these recombinant viruses were free from exogenous microbial contamination.

[0228] 3. Determination of rDEV titer containing the HA gene of avian influenza virus subtype H9N2: The titer (TCID) of rDEV containing the HA gene of avian influenza virus subtype H9N2 was determined on a CEF.50 ).

[0229] Table 4. TCID of rDEV 50

[0230] 4. Expression of the HA gene in the H9N2 subtype of avian influenza virus rDEV containing the HA gene of the H9N2 subtype of avian influenza virus was administered at 10 -2 -10 -4 The diluted solution was inoculated into 48-well CEF plates. Four hours after inoculation, the medium was replaced with MEM containing 0.75% methylcellulose and 5% FBS, and the plates were incubated at 37°C for 4 days in a 5% CO2 incubator. The expression of DEV virus and HA gene was detected using indirect immunofluorescence assay (IFA).

[0231] The indirect immunofluorescence assay consisted of the following steps: Remove the cell culture medium. Gently wash the surface once with PBS, add 96% cold ethanol to each well, and fix for 10 minutes at room temperature. Discard the ethanol. Allow the wells to air dry. Then add appropriately diluted chicken anti-DEV serum and polyclonal rabbit antibody against H9N2 HA (Sino Biological Inc., catalog number 11229-RP02) to the wells, and incubate at 37°C for 1 hour. Discard the antibodies. Wash the wells three times with PBS, and add 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 incubate at 37°C for 1 hour. Discard the antibodies. Wash the wells three times with PBS and observe under an inverted fluorescence microscope. All rDEVs containing the HA gene of the H9N2 avian influenza virus subtype showed specific fluorescence for DEV and H9HA. The results indicate that the HA protein was successfully expressed by recombinant virus in CEF (see [link]). Figure 7 ).

[0232] Example 5: Safety of recombinant DEV in chickens 5.1. Introduction In this embodiment, the safety of recombinant DEVs in chicken, including rDEV4 H9HA ΔUS7US8, rDEV4 ΔUL40H9HA ΔUL2, rDEV4 ΔUL24 H9HA ΔUL2, rDEV4 H9HA ΔUL39, rDEV4 H9HA ΔUL40, rDEV4H9HA ΔUL24, rDEV4 ΔUL41 H9HA ΔUS8, rDEV4 H9HA ΔUL23 ΔUL41, rDEV4 ΔUL24UL26-H9HA-UL27, and rDEV4 ΔUL39 UL26-H9HA-UL27, was verified.

[0233] 5.2. Experimental Design On the day of the experiment, 120 one-day-old SPF experimental chickens were randomly divided into 12 groups of 10 chickens each. Groups 1-10 were the inoculation groups, and the corresponding material to be tested was subcutaneously inoculated into the chickens via the neck. Group 11 was the positive control group, and DEV4 virus was subcutaneously inoculated into the chickens of Group 11 via the neck. Group 12 was the negative control group, and the same volume of MEM + 5% FBS (a dilution of the material to be tested) was injected subcutaneously into the chickens via the neck. The specific experimental design and grouping are shown in Table 5.

[0234] Table 5. Experimental Design and Grouping

[0235] On the day of the experiment, as shown in Table 5, each group of experimental chickens was subcutaneously inoculated with 0.5 ml of the test material or its dilution in the neck. After inoculation, all experimental chickens (including those in the negative control group) were observed once a day for 28 consecutive days; and abnormal symptoms of the experimental chickens were recorded, including: depression, head and neck retraction, ruffled feathers, drooping wings, numbness and weakness in both feet, tearing, eyelid edema, nasal discharge, varying degrees of swelling in the head and neck, and a pulsating sensation upon touch.

[0236] 5.3. Morbidity and mortality rates of experimental chickens after inoculation Following inoculation, all chickens in group 11 (positive control group) died. Chickens in group 12 (negative control group) showed no abnormal clinical symptoms or deaths throughout the experiment. Chickens in groups 2-10 showed no abnormal clinical symptoms or deaths throughout the experiment, with morbidity and mortality rates of 0%. Chickens in group 1 (rDEV4 H9HA ΔUS7US8) showed a morbidity and mortality rate of 30%. However, when compared with the DEV4 challenge group, both morbidity and mortality rates were significantly reduced. The morbidity and mortality results for each group are shown in Table 6.

[0237] Table 6. Morbidity and mortality rates of experimental chickens in each group

[0238] 5.4. DEV serum conversion in experimental chickens after inoculation Chickens in group 12 were negative for DEV antibodies throughout the experiment. On day 28 post-inoculation, the sera of chickens in the other groups became positive for DEV antibodies. Successful inoculation of all experimental groups was confirmed without omission. DEV seroconversion in the chickens also demonstrates that these recombinant viruses replicate to a specific degree in vivo.

[0239] 5.5. Conclusion We investigated the safety of rDEV expressing the H9N2 HA protein in chickens. Four aspects were detected and analyzed: morbidity, mortality, and DEV seroconversion in 1-day-old SPF chickens. rDEV4 H9HA ΔUS7US8 resulted in a morbidity and mortality rate of 30% in 1-day-old SPF chickens. Other recombinant viruses did not cause any morbidity or mortality, but did lead to the production of anti-DEV antibodies. The unmodified original DEV4 virus caused 100% mortality in the experimental chickens. In summary, these rDEVs, by deleting different virulence-related genes and inserting the H9HA gene, are completely or partially attenuated compared to wild-type DEV strains, thus providing the possibility of use as a safe live viral vector for chickens.

[0240] We also verified the effect of successive passages on the virulence of the recombinant virus. The results showed that successive passages did not significantly affect the virulence of the recombinant virus.

[0241] Example 6: Horizontal transmission ability of recombinant DEV among chickens 6.1. Experimental Design This embodiment aims to evaluate the horizontal transmissibility of the constructed recombinant DEV among 1-day-old SPF chickens. 105 SPF chickens were randomly divided into 7 groups of 15 chickens each and transferred to appropriate isolators after hatching (1 day old). On the day of the experiment (i.e., the day of hatching), 70 1-day-old chickens were randomly divided into 7 groups of 10 chickens each. Groups 1-5 were the test groups for the live rDEV vector, and the corresponding material to be tested was subcutaneously inoculated into the chickens via the neck. Group 6 was the positive control group, and DEV4 virus was subcutaneously inoculated into the chickens via the neck. Group 7 was the negative control group, and the same volume of MEM solution containing 5% FBS (a dilution of the test material, hereinafter referred to as MEM+5% FBS) was injected subcutaneously into the chickens via the neck. Additionally, on the day of inoculation, 35 homologous 1-day-old SPF chickens from the same batch were selected and placed in separate isolators, randomly divided into 7 groups of 5 chickens each as contact chickens. Twenty-four hours after immunizing the chickens, the exposed chickens in each group were transferred to their respective groups. The specific experimental design and grouping are shown in Table 7.

[0242] Table 7. Experimental Design and Grouping

[0243] Note: *10 immunized chickens and 5 contact chickens; and the contact chickens were fed in the corresponding group 24 hours after the immunized chickens were vaccinated.

[0244] On the day of the experiment, as shown in Table 7, the experimental chickens in groups 1-5 were fed 10 6.0 TCID 50 The chickens were inoculated subcutaneously in the neck with 0.5 ml of the corresponding recombinant DEV. Chickens in group 6 (as the positive control group) were inoculated with 10... 6.0 TCID 50 Chickens were inoculated with 0.5 ml of DEV4 via subcutaneous injection in the neck. Chickens in group 7 (as a negative control group) were inoculated with 0.5 ml of MEM + 5% FBS via subcutaneous injection in the neck.

[0245] After vaccination, all experimental chickens (immunized and exposed chickens, including those in the negative control group) were observed once a day for 28 consecutive days; and abnormal symptoms of the experimental chickens were recorded, including: depression, head and neck retraction, ruffled feathers, drooping wings, numbness and weakness in both feet, tearing, eyelid edema, nasal discharge, varying degrees of swelling in the head and neck, and a pulsating sensation when touched.

[0246] Blood samples were collected from all immunized chickens on day 28 post-vaccination. Serum was separated to detect DEV seroconversion. Blood samples were collected from contact chickens on days 21 and 28 post-vaccination. Serum was separated to detect DEV seroconversion.

[0247] 6.2. DEV serum conversion in experimental chickens after inoculation Chickens in Group 7 (negative control) remained negative for anti-DEV antibodies throughout the experiment; however, on day 28 post-inoculation, the sera of immunized chickens in other groups became positive for anti-DEV antibodies. On days 20 and 27 post-exposure (i.e., days 21 and 28 post-inoculation), the sera of all exposed chickens in all groups were negative for anti-DEV antibodies. The specific results of DEV seroconversion in each group are shown in Table 8.

[0248] Table 8. DEV serum conversion in experimental chickens in each group

[0249] Note: *10 immunized chickens and 5 contact chickens; and the contact chickens were fed in the corresponding group 24 hours after the immunized chickens were vaccinated.

[0250] 6.3. Conclusion Laboratory tests showed no seroconversion in any of the chickens exposed on days 20 and 27 after contact with the inoculated chickens, demonstrating that the recombinant DEVs with missing or inserted H9N2 HA genes involved in this embodiment did not spread horizontally among chickens. Specifically, these recombinant viruses did not cause disease or death in chickens, nor did they cause horizontal transmission among flocks, and could be used as a safe vector in the future.

[0251] Example 7: Efficacy of vaccine candidate strains containing a live rDEV vector expressing H9N2-HA 7.1. Experimental Design In this embodiment, ten candidate vaccine strains containing a live rDEV vector expressing the HA gene of avian influenza virus (subtype H9) were subcutaneously injected into 1-day-old SPF chickens via the neck. On day 28 post-inoculation, the SPF chickens were challenged with an avian influenza virus (subtype H9) challenge strain (A / chicken / Jiangsu / TX10 / 2010 strain). This was to evaluate the immunogenicity of the candidate vaccine strains after challenge.

[0252] The recombinant viruses tested in this embodiment include rDEV4 H9HAΔUS7US8, rDEV4ΔUL40 H9HAΔUL2, rDEV4ΔUL24 H9HAΔUL2, rDEV4 H9HAΔUL39, rDEV4 H9HAΔUL40, rDEV4 H9HA ΔUL24, rDEV4 ΔUL41 H9HA ΔUS8, rDEV4 H9HA ΔUL23 ΔUL41, rDEV4 ΔUL24 UL26-H9HA-UL27, and rDEV4 ΔUL39 UL26-H9HA-UL27.

[0253] On the day of the experiment (i.e., the hatching day of the SPF chickens), 110 one-day-old SPF chickens were randomly divided into 11 groups of 10 chickens each. Groups 1-10 were the test groups containing the vaccine candidate strain with the live rDEV vector, and group 11 was the control group. As shown in Table 9, all experimental chickens were subcutaneously inoculated with the corresponding test material via the neck. All experimental chickens were clinically observed for 28 consecutive days after inoculation. On day 28, all experimental chickens were... 6.0 EID 50 Chickens were challenged by intranasal inoculation with 0.2 ml of H9 subtype avian influenza virus. The experimental design and grouping are shown in Table 9.

[0254] Table 9. Experimental Design and Grouping

[0255] After inoculation, all experimental chickens were observed once a day for 28 consecutive days. Abnormal symptoms were observed in the experimental chickens, including but not limited to: depression, head and neck retraction, ruffled feathers, drooping wings, numbness and weakness in both feet, tearing, eyelid edema, nasal discharge, varying degrees of swelling in the head and neck, and a pulsating sensation upon touch.

[0256] Blood samples were collected from all chickens on day 28 post-vaccination (before attack); serum was collected by centrifugation for the detection of HI antibodies against H9 subtype avian influenza.

[0257] On day 5 post-challenge, throat and cloacal swabs were collected from all experimental chickens for H9 virus isolation. If the swabs were negative for virus isolation, the candidate vaccine strain was deemed to provide protection for the chickens. The specific procedure was as follows: On day 5 post-challenge, throat and cloacal swabs were collected from all experimental chickens. The collected swabs were placed in centrifuge tubes containing 1.6 ml of swab buffer containing six antibiotics. All swab samples were inoculated into chicken embryos for virus isolation. The procedure was as follows: Each swab sample was inoculated into five 9-11 day old SPF chicken embryos at 0.2 ml / embryo through the allantoic cavity, and the embryos were incubated for 96 hours. The HA titer of all allantoic fluid was then measured. An embryo was considered positive for H9 virus isolation if the HA titer of the allantoic fluid from at least one embryo inoculated with each swab sample was not less than 1:16. Samples negative for virus isolation were retested after one blind passage. If the virus isolation test is still negative after blind transmission, it is determined to be negative for H9 virus isolation; if the virus isolation test is positive after blind transmission, it is determined to be positive for H9 virus isolation.

[0258] 7.2. Levels of anti-H9 antibodies in experimental chickens On day 28 post-vaccination, blood was collected from chickens in each group; and serum was separated to detect anti-H9 antibody levels. Results showed that all chickens in group 11 (control group) were negative for H9 hemagglutination inhibition (HI) antibodies; the mean HI antibody titers in groups 4 and 7 were less than 4 log2; and the mean HI antibody titers in the other groups were greater than or equal to 4 log2. The mean HI antibody titers in the serum of each group are shown in Table 10. Figure 8 middle.

[0259] Table 10. Mean HI antibody titer in serum of each experimental chicken group

[0260] 7.3. Protection rate of recombinant virus against H9N2 On day 5 post-challenge, throat and cloacal swabs were collected from all experimental chickens for H9N2 virus isolation. Results showed that 10 chickens in group 11 (challenge control group) were positive for virus isolation, meaning the positive rate for virus isolation in the challenge control group was 100%; and the challenge control was effective. The results of H9N2 virus isolation from the swab samples and the protection rate against H9N2 in each group are shown in Table 11.

[0261] Table 11. H9N2 virus isolation and protection rate against H9N2

[0262] 7.4. Conclusion Vaccine candidate strains containing live rDEV vectors expressing the HA gene of avian influenza virus (subtype H9) can provide protection against H9N2 to varying degrees, up to 90%, demonstrating the effectiveness of these different insertion sites. These are the first candidate vaccines to use DEV as a vector for expressing the H9N2-HA protein. Overall, this invention provides a novel, safe, and effective chicken vaccine based on a DEV vector.

Claims

1. A composition comprising modified duck enteritis virus (DEV), said modified DEV comprising and capable of expressing a heteropolynucleotide encoding an antigen of a chicken pathogen, wherein, The pathogen in the chicken is an avian influenza virus.

2. The composition according to claim 1, wherein, The pathogen in the chicken is avian influenza A virus.

3. The composition according to claim 1, wherein, The pathogen in the chicken is the H9 avian influenza virus.

4. The composition according to claim 1, wherein, The pathogen in the chicken is the avian influenza virus H9N2.

5. The composition according to claim 1, wherein, The antigen described in the chicken pathogen is the hemagglutinin (HA) protein of the avian influenza virus H9N2.

6. The composition according to claim 1, wherein, The amino acid sequence of the antigen of the chicken pathogen has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

7. The composition according to claim 1, wherein, The sequence of the heteropolynucleotide has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:

2.

8. The composition according to any one of the preceding claims, wherein, The heteropolynucleotide is located in a non-essential gene or region of the modified DEV.

9. The composition according to claim 8, wherein, The heteropolynucleotide insertion or replacement is a portion or the entire sequence of the non-essential gene or region of the modified DEV.

10. The composition according to claim 8 or 9, wherein, The non-essential genes or regions of the modified DEV are selected from the group consisting of both the US7 and US8 genes, the UL2 gene, the UL24 gene, the UL39 gene, the UL40 gene, the UL23 gene, the US8 gene, or the UL26-UL27 gene intergenic region.

11. The composition according to any one of the preceding claims, wherein, The non-essential genes of the modified DEV are inactivated.

12. The composition according to claim 11, wherein, The inactivated non-essential genes of the modified DEV are selected from the group consisting of US7, US8, UL2, UL24, UL40, UL39, UL23 and UL41.

13. The composition according to claim 12, wherein, The inactivated non-essential gene of the modified DEV is selected from any of the following i)-viii): i)US7, ii)US8, iii)UL2, iv)UL24, v)UL40, vi)UL39, vii)UL23, and viii)UL41, and the inactivated non-essential gene is combined with one or more other non-essential genes of the modified DEV.

14. The composition according to claim 13, wherein, The modified DEV has one or more additional non-essential genes that are different from the first inactivated gene and are selected from i)US7, ii)US8, iii)UL2, iv)UL24, v)UL40, vi)UL39, vii)UL23, or viiii)UL41.

15. The composition according to any one of the preceding claims, wherein, The modified DEV contains an inactivated non-essential gene selected from any of i)-viii): i) US7 and US8 genes; ii) UL24 and UL2 genes; iii) UL40 and UL2 genes; iv) UL24 gene; v) UL39 gene; vi) UL40 gene; vii) UL23 and UL41 genes; or viii) UL41 gene and US8 gene.

16. The composition according to any one of claims 11-15, wherein, The non-essential genes within the modified DEV are inactivated by mutation, interruption, substitution, or deletion of a portion or the entire sequence of the non-essential gene.

17. The composition according to any one of claims 11-16, 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 non-essential gene in the modified DEV is replaced or deleted.

18. The composition according to any one of claims 11-16, wherein, The modified DEV is attenuated.

19. The composition according to claim 18, wherein, Compared with the unmodified wild-type DEV, the modified DEV according to any one of claims 11-16 has a reduced mortality rate or no mortality rate in chickens.

20. The composition according to any one of the preceding claims, wherein, The modified DEV includes any of the following: i) Heteronucleotides located in both the US7 and US8 genes, replacing a portion or the entire sequence of both the US7 and US8 genes; ii) A heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and also containing an inactivated UL24 gene, optionally with the UL24 gene deleted; iii) A heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and also containing an inactivated UL40 gene, optionally with the UL40 gene deleted; iv) Heteronucleotides located in the UL24 gene that replace a portion or the entire sequence of the UL24 gene; v) Heteronucleotides located in the UL39 gene that replace a portion or the entire sequence of the UL39 gene; vi) Heteronucleotides located in the UL26-UL27 intergenic region, and also containing an inactivated UL24 gene, optionally with the UL24 gene deleted; vii) Heteronucleotides located in the UL40 gene that replace a portion or the entire sequence of the UL40 gene; viii) The heteropolynucleotide located in the UL26-UL27 intergenic region and also containing the inactivated UL39 gene, optionally with the UL39 gene deleted; ix) a heteropolynucleotide located in the UL23 gene, replacing a portion or the entire sequence of the UL23 gene, and also containing an inactivated UL41 gene, optionally with the UL41 gene deleted; or x) A heteropolynucleotide located in the US8 gene, replacing a portion or the entire sequence of the US8 gene, and also containing an inactivated UL41 gene, optionally with the UL41 gene deleted.

21. The composition according to any one of the preceding claims, wherein, The heteropolynucleotide is operatively linked to the promoter.

22. The composition according to claim 21, wherein, The promoters are selected from the group consisting of: cytomegalovirus (CMV) immediate early promoter, mouse CMV promoter, guinea pig CMV promoter, SV40 promoter, human herpesvirus type III glycoprotein B (HHV3gB) promoter, pseudorabies virus promoter (e.g., glycoprotein X promoter), herpes simplex virus-1α4 promoter, Marek's disease virus glycoprotein A (or gC) promoter, Marek's disease virus glycoprotein B promoter, Marek's disease virus glycoprotein E promoter, Marek's disease virus glycoprotein I promoter, infectious laryngotracheitis virus glycoprotein B, infectious laryngotracheitis virus glycoprotein E promoter, infectious laryngotracheitis virus glycoprotein D promoter, infectious laryngotracheitis virus glycoprotein I promoter, vaccinia H6, and combinations thereof.

23. The composition according to any one of the preceding claims, wherein, The heteropolynucleotide is operatively linked to the SV40 polyadenylate signal.

24. The composition according to any one of the preceding claims further comprises a pharmaceutically or veterinarily acceptable carrier, excipient, mediator, or adjuvant.

25. The composition according to any one of the preceding claims, wherein, The composition is a vaccine.

26. The composition according to any one of the preceding claims, wherein, The composition is a vector vaccine for chickens.

27. The composition according to any one of the preceding claims, wherein, The modified DEV is used as a carrier.

28. A method of vaccinating chickens by inducing a protective immune response against a chicken pathogen, comprising administering at least once the composition according to any one of claims 1-27.

29. The composition according to any one of claims 1-27, in the method of inducing a protective immune response against chicken pathogens, wherein, The method includes administering one or more doses to the chicken. The composition according to any one of claims 1-27, or by applying it to the chicken once or multiple times. The composition according to any one of claims 1-27.

30. The method according to claim 28 or the use according to claim 29, wherein, The chicken pathogen is the H9 avian influenza virus, optionally the H9N2 avian influenza virus.

31. The method according to claim 28 or 30, or the use according to claim 29 or 30, wherein, The chickens were 1, 2, 3, 4, 5, 6, or 7 days old on the day of vaccination.

32. The method according to any one of claims 28 or 30-31, or the use according to any one of claims 29-31, wherein, The application is administered via the mouth, nose, eye drops, spray, drinking water, intraocularly, intramuscularly, subcutaneously, intradermally, or transdermally.

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