New dev vectors for avian vaccines

Abstract

The invention relates to the field of animal health. In particular, the present invention relates to a modified duck enteritis virus (DEV). More specifically, the present invention relates to a modified DEV that does not exhibit or exhibits reduced pathogenicity in chicken. Furthermore, the present invention relates to a composition comprising the modified DEV of the invention as a chicken vector vaccine and the use thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480054079.8 (22) Application Date 2024.08.23 (66) Domestic Priority Data PCT / CN2023 / 114921 2023.08.25 CN (85) PCT International Application Entering National Phase Date 2026.02.14 (86) PCT International Application Application Data PCT / CN2024 / 114146 2024.08.23 (87) PCT International Application Publication Data WO2025 / 044920 EN 2025.03.06 (83) Biological Preservation Information CCTCC NO:V202378 2023.08.04 (71) Applicant Boehringer Ingelheim Animal Health (China) Co., Ltd. Address: No. 299, Xiangtai Road, Taizhou City, Jiangsu Province, 225300 (72) Inventors Ma Chengtai Huangfu Yifan Chen Ning Cui Xiaoping Ju Sidi Zhang Qingshui (74) Patent Agency Yongxin Patent & Trademark Agency Co., Ltd. 72002 Patent Attorney Qu Bin (51) Int.Cl. A61K 39 / 00 (2006.01) A61K 39 / 12 (2006.01) A61K 39 / 255 (2006.01) C07K 14 / 005 (2006.01) C12N 7 / 00 (2006.01) C12N 15 / 86 (2006.01) (54) Invention Title Novel DEV Carrier for Avian Vaccines (57) Abstract This invention relates to the field of animal health. Specifically, the present invention relates to a modified duck enteritis virus (DEV). More specifically, the present invention relates to a modified DEV that does not show or shows reduced pathogenicity in chickens. Furthermore, the present invention relates to a composition comprising the modified DEV of the present invention as a chicken vector vaccine and its use. Claims 2 pages Description 35 pages Sequence Listing (electronic publication) Drawings 6 pages CN 121843706 A 2026.04.10 CN 1 21 84 37 06 A 1. A modified duck enteritis virus (DEV) wherein one or more genes selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41 and US8 in the DEV genome are inactivated, and wherein, compared with unmodified DEV, said modified DEV has reduced mortality or no mortality in chickens.2. A modified duck enteritis virus (DEV) comprising an inactivated gene selected from any one of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene, said inactivated gene in combination with inactivated combinations of one or more additional non-essential genes of the DEV, and wherein, compared with said unmodified DEV, said modified DEV has reduced mortality or no mortality in chickens. 3. The modified DEV according to claim 2, wherein said one or more additional non-essential genes of the DEV are different from the first inactivated gene and are selected from: i) US7 gene; ii) UL2 gene; iii) UL41 gene; iv) US3 gene; v) UL24 gene; vi) UL40 gene; vii) UL39 gene; viiii) UL23 gene; or ix) US8. 4. A modified duck enteritis virus (DEV) comprising an inactivating gene selected from any one of i)-xi), i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US7 and US8 genes; viii) UL24 and UL2 genes; ix) UL40 and UL2 genes; x) UL23 and UL41 genes; or xi) UL41 and US8 genes, and wherein, compared with the unmodified DEV, the modified DEV has a reduced mortality rate or no mortality rate in chickens. 5. The modified DEV according to any one of the preceding claims, wherein the gene is inactivated by mutation, interruption, substitution, or deletion of a portion or the entire sequence of the gene. 6. The modified DEV according to any one of the preceding claims, wherein at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence of the gene is replaced or deleted. 7. The modified DEV according to any one of the preceding claims, wherein the modified DEV further comprises a heteropolynucleotide encoding a chicken heteroantigen. 8. The modified DEV according to any one of the preceding claims, wherein the heteropolynucleotide is inserted into a non-essential gene of the modified DEV. 9. The modified DEV according to claim 7 or 8, wherein the heteropolynucleotide is expressed after the modified DEV has been transfected into a suitable chicken host cell. 10. The modified DEV according to any one of claims 7-9, used as a vector vaccine in chickens.11. A composition comprising the modified DEV according to any one of claims 7-10. 12. The composition according to claim 11, wherein the composition is a vaccine. 13. A method for inducing a protective immune response against a chicken pathogen in chickens using the modified DEV according to any one of claims 7-9, the composition according to claim 11 or 12, or the vector vaccine according to claim 10, wherein the method comprises administering the modified DEV according to any one of claims 7-9, the composition according to claim 11 or 12, or the vector vaccine according to claim 10 to the chickens once or multiple times, or consisting of administering the modified DEV according to any one of claims 7-9, the composition according to claim 11 or 12, or the vector vaccine according to claim 10 to the chickens once or multiple times. 14. A method of vaccinating chickens by inducing a protective immune response against a chicken pathogen, comprising administering the chickens at least once with the modified DEV according to any one of claims 7-9, the composition according to claim 11 or 12, or the vector vaccine according to claim 10. 15. The use according to claim 13, or the method according to claim 14, wherein the chickens are 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days old on the day of vaccination. 16. The use according to claim 13 or 15, or the method according to claim 14 or 15, wherein the administration is performed via oral-nasal route, eye drops, spray, drinking water, intraocular, intramuscular, subcutaneous, intradermal, or transdermal administration. 17. A host cell expressing the modified DEV according to any one of claims 1-9. 18. The host cell according to claim 17, wherein the host cell is a CEF cell, an EB66 cell, or a DEF cell. 19. A method for preparing a modified DEV according to any one of claims 1-9, comprising inactivating one or more genes selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41 and US8 in the DEV genome, 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.Claims 2 / 2 Page 3 CN 121843706 A Cross-Reference to Related Applications for Novel DEV Vectors for Avian Vaccines

[0001] This application claims priority to PCT / CN2023 / 114921, filed August 25, 2023, entitled “Modified DEV Safe for Chickens,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of animal health. Specifically, this invention relates to a modified duck enteritis virus (DEV). More specifically, this invention relates to a modified DEV that does not show or shows reduced pathogenicity in chickens. Furthermore, this invention relates to a composition comprising the modified DEV of the present invention as a chicken vector vaccine and its use therein. Background Art

[0003] Duck plague, also known as duck viral enteritis, is an acute septicemic infection in anseriformes such as ducks and geese. It is caused by duck enteritis virus (DEV), which naturally infects ducks and geese. However, DEV causes duck plague not only in ducks and geese, but 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 a member of the Herpesvirus 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, research mainly focuses on using DEV as a vector to express heterologous genes in order to develop vaccines against duck diseases. It has been reported that DEV vector vaccines expressing heterologous genes are safe in ducks, but show 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, genetically modified for use in developing vaccines against chicken diseases, remains a challenge in this field.

[0005] The present invention is based on the surprising discovery that, compared with unmodified DEVs, the following DEVs result in reduced or no mortality in chickens: containing inactivated genes in their genome, such as i) the UL41 gene; ii) the US3 gene; iii) the UL24 gene; iv) the UL40 gene; v) the UL39 gene; vi) the UL23 gene; vii) the US8 gene, which are inactivated genes alone or in combination with other DEV genes, such as, for example, combinations of the following: 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 UL23 gene and the UL41 gene; or v) the UL41 gene and the US8 gene. Although wild-type DEVs are lethal in chicks, the DEVs of the present invention are safe and can be efficiently delivered and expressed in vivo for the genes of interest. Specifically, such DEVs (i) are in vivo attenuated in 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, allowing for high titer production. Therefore, such modified DEVs represent a highly effective vector for vaccinating non-human animals (particularly poultry) and for conferring early protective immunity.

[0006] In one aspect, the present invention provides a modified duck enteritis virus (DEV) wherein one or more genes selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41 and US8 in the DEV genome are inactivated, and wherein the modified DEV has reduced or no mortality in chickens (compared to unmodified DEV).

[0007] In one aspect, the present invention provides a modified duck enteritis virus (DEV) comprising an inactivated gene selected from any of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, wherein the inactivated gene is alone or in combination with any other non-essential gene of the DEV, and wherein the modified DEV has reduced or no mortality in chickens (compared to unmodified DEV).

[0008] In one aspect, the present invention provides a modified duck enteritis virus (DEV) comprising one or more inactivated genes selected from any one of the following i)-xii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viiii) US7 and US8 genes; ix) UL24 and UL2 genes; x) UL40 and UL2 genes; xi) UL23 and UL41 genes; or xii) UL41 and US8 genes, and wherein the modified DEV has reduced or no mortality in chickens (compared to unmodified DEV).

[0009] In one aspect, the present invention provides a modified duck enteritis virus (DEV) comprising inactivated genes as defined above, wherein the modified DEV further comprises heteropolynucleotides encoding heteroantigens of pathogens.

[0010] In one aspect, the present invention provides a modified duck enteritis virus (DEV) as a live vector vaccine in chickens, wherein the modified DEV contains an inactivating gene selected from any of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, and wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens (compared to unmodified DEV).

[0011] In one aspect, the present invention provides a modified duck enteritis virus (DEV) as a live vector vaccine in chickens, wherein the modified DEV comprises an inactivated gene selected from any of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, the inactivated gene being alone or in combination with any other non-essential gene of the DEV, and wherein the modified DEV has reduced or no mortality in chickens (compared to unmodified DEV).

[0012] In one aspect, the present invention provides a composition comprising the modified DEV of the present invention.

[0013] In one aspect, the present invention provides the modified DEV of the present invention, or the composition of the present invention, or the vector vaccine of the present invention, for use in a method of inducing a protective immune response against a pathogen in chickens, wherein the method comprises or consists of administering to chickens once or multiple times the modified DEV of the present invention, or the composition of the present invention, or the vector vaccine of the present invention.

[0014] In one aspect, the present invention provides a method of vaccinating chickens by inducing a protective immune response against a pathogen in chickens, comprising administering the composition of the present invention at least once.

[0015] In one aspect, the present invention provides the use of the modified DEV of the present invention in the manufacture of a composition for vaccinating chickens by inducing a protective immune response against a pathogen in chickens. Brief Description of the Drawings

[0016] Figure 1: Gene structure diagram showing plasmid pB12.

[0017] Figure 2: Schematic diagram showing (A) bacterial artificial chromosome rDEV4 BAC, (B) recombinant construct having the deleted gene rDEV4 Δ UL39, and (C) recombinant construct having the inserted H9HA gene rDEV4 Δ UL39 UL26-H9HA-UL27.

[0018] Figure 3: Results showing transfection of DEF (duck embryo fibroblasts) to rescue rDEV4 Δ UL39.

[0019] Figure 4: Showing the results of RFLP analysis of rDEV4 Δ UL39 UL26-H9HA-UL27 by Xho I digestion.

[0020] Figure 5: Showing the results of mini-F deletion rescue of rDEV4 Δ UL39 UL26-H9HA-UL27 by co-transfection.

[0021] Figure 6: Showing the results of genetic stability test of rDEV4 Δ UL39 UL26-H9HA-UL27 by PCR.

[0022] Figure 7: Showing the results of the expression test of H9HA from rDEV4 Δ UL39 UL26-H9HA-UL27 by IFA.

[0023] Figure 8: Showing the results of HI Ab levels induced by different rDEV4 H9HA vaccine candidate strains in different groups. Detailed Description

[0024] Before describing various aspects of the invention, it must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “described” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a gene” includes multiple genes, and reference to “virus” is a reference 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 one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this 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 this invention. This document should not be construed as an admission that this invention is not entitled to precede such disclosures by prior invention.

[0025] This invention relates to a modified duck enteritis virus (DEV) wherein the modified virus has a specific inactivating gene. The invention demonstrates that by inactivating such a gene, viable, stable, and reproducible DEVs can be obtained, and such viruses can be used to create recombinant 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. Furthermore, and remarkably, although natural DEV and many other missing 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 for chickens that can be safely used to express antigens in chickens. As demonstrated in the examples, the morbidity and mortality rates caused by the modified DEV of the present invention are surprisingly reduced to less than 60%, and in most cases even surprisingly reduced to 0%, compared to 100% morbidity and 100% mortality rates caused by unmodified DEV strains in chickens. Such modified DEVs can be used as safe live viral vectors in the future.Specification 3 / 35 pages 6 CN 121843706 A

[0026] Modified or Attenuated DEV In one aspect, the present invention provides a modified duck enteritis virus (DEV) wherein one or more genes selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41 and US8 in the DEV genome are inactivated, and wherein the modified DEV has reduced or no mortality in chickens (compared to unmodified DEV).

[0027] The terms “recombinant” or “modified” refer to DEVs that have been altered, rearranged, or modified by 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. In this regard, 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 recombinant or modified DEVs. The terms “recombinant / modified DEV,” “rDEV,” and “recombinant / modified DEV vector” are used interchangeably herein.

[0028] The phrase “that the modified DEV has reduced or no mortality in chickens (compared to unmodified DEV)” means that the modified DEV of the present invention is attenuated with respect to chickens. Therefore, the modified DEV of the present invention is attenuated with respect to chickens.

[0029] The term “virus” specifically refers to a viral particle containing a nucleic acid molecule (e.g., a genome) encapsulated in a capsid or capsule. The term “virus” also refers to a viral vector or an isolated viral genome.

[0030] “Gene” refers to a nucleic acid molecule or sequence containing an open reading frame encoding a product such as a polypeptide (e.g., a peptide, a protein, etc.) or RNA.

[0031] As used herein, the term “DEV” refers to all viruses belonging to the genus Mardivirus of the subfamily Alphaherpesvirinae of the family Herpesviridae.

[0032] The DEVs 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 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.

[0033] The inactivated genes in the DEV genome are non-essential genes.

[0034] As used herein, a “non-essential gene / region” is a gene / region in the modified DEV genome, wherein inactivation (including mutation, interruption, substitution, or deletion) of the gene / region or insertion of a heterologous polynucleotide in the gene / region does not prevent the modified DEV from replicating in the host cell.

[0035] Inactivation of one or more genes in the DEV genome as indicated above results in attenuation of the DEV in chickens. The phrase “inactivation of one or more genes in the DEV genome as indicated above results in attenuation of the DEV in chickens” means that the modified DEV of the present invention is attenuated in chickens. Therefore, the modified DEV of the present invention is attenuated in chickens.

[0036] As used herein, the term “attenuated” means a modified virus that is substantially non-toxic in chickens, i.e., that does not cause or causes a reduced disease, especially not death, in chickens compared to the unmodified wild-type parent virus. More specifically, on page 4 / 35 of CN 121843706 A, the attenuated virus can typically replicate in chickens without causing their death.More specifically, the attenuated DEV of the present invention is non-toxic or less toxic in chickens compared to the corresponding unmodified wild-type parental DEV that does not contain the inactivating gene in its genome. More specifically, the attenuated virus refers to a virus that is non-toxic in chickens when injected at a dose of 104.0–107.0 TCID50 / chicken (such as 106.0 TCID50 / chicken). More specifically, the attenuated virus refers to a virus that is non-toxic in chickens at a dose of 104.0–107.0 TCID50 / chicken (such as 106.0 TCID50 / chicken) 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 of the injected chickens. In some embodiments, the attenuated virus more specifically refers to a virus that is non-toxic in the embryo when injected at a dose of 104.0–107.0 TCID50 / egg (such as 106.0 TCID50 / egg). The most preferred attenuated virus is a virus that is non-toxic in the embryo at a dose of 104.0–107.0 TCID50 / egg (such as 106.0 TCID50 / egg) 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 of the injected eggs. The modified virus of the present invention is also non-toxic to injections after hatching, including on days 0, 1, 2, and 3 after hatching (i.e., 0.1 to 72 hours after hatching).

[0037] The modified DEV has reduced or no mortality and / or morbidity in chickens. More specifically, the modified DEV of the present invention has reduced or no mortality and / or morbidity in chickens compared to unmodified DEV. More specifically, the mortality and / or morbidity in chickens caused by the modified DEV of the present invention is 0%, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, or less than 70%. More specifically, the mortality and / or morbidity in chickens caused by the modified DEV of the present invention is 0%, less than 5%, less than 10%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, or less than 70% of the mortality and / or morbidity caused by unmodified DEV.

[0038] The attenuation of DEV in chickens is caused by the inactivation of one or more genes in the DEV genome.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.

[0039] In some embodiments, the inactivated gene in the DEV genome is selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41, and US8. In some embodiments, the inactivated gene is selected from any of the following i)-xii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viii) US7 and US8 genes; ix) UL24 and UL2 genes; x) UL40 and UL2 genes; xi) UL23 and UL41 genes; or xii) UL41 and US8 genes.

[0040] Therefore, in another aspect, the present invention relates to a modified duck enteritis virus (DEV) wherein any of the following i)-xii) are inactivated in the DEV genome: i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viiii) US7 and US8 genes; ix) UL24 and UL2 genes; x) UL40 and UL2 genes; xi) UL23 and UL41 genes; or xii) UL41 and US8 genes, and wherein the modified DEV has reduced or no mortality in chickens (compared to unmodified DEV).

[0041] UL41, US3, UL24, UL40, UL39, UL23, US7, US8 and UL2 are highly conserved among DEV strains. It should be understood that those skilled in the art can readily identify the exact locations of the UL41, US3, UL24, UL40, UL39, UL23, US7, US8, and UL2 genes in any DEV strain using the information contained in this application and common general knowledge, or by sequence alignment. For example, the exact locations of the UL41, US3, UL24, UL40, UL39, UL23, US7, US8, and UL2 genes can be identified by referring to the DEV strain described on page 5 / 35 of the specification, CN 121843706 A, or Genbank accession number EU082088.2.

[0042] In some embodiments, the genes are 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 replaced or deleted.

[0043] In a particular embodiment, the gene is inactivated due to one or more mutations in the coding sequence, particularly point mutations in the coding sequence that prevent the expression of the full-length protein. Such mutations can cause substitutions of essential amino acid residues in the encoded protein, thereby producing an inactivated protein.

[0044] In a particular embodiment, 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.

[0045] 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.

[0046] In another embodiment, the gene is inactivated by substitution of 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 substitution removes the coding sequence and thus inhibits the expression of the wild-type protein.

[0047] In a specific embodiment, the modified DEV of the present invention has a partial or complete deletion or substitution of the UL41 gene sequence. In a specific embodiment, the modified DEV of the present invention has a complete deletion of the UL41 gene sequence. A specific example of such a construct is, for example, rDEV4 Δ UL41 (see Example 2).

[0048] In a specific embodiment, the modified DEV of the present invention has a partial or complete deletion or substitution of the US3 gene sequence. In a specific embodiment, the modified DEV of the present invention has a complete deletion of the US3 gene sequence. A specific example of such a construct is, for example, rDEV4 Δ US3 (see Example 2).

[0049] In a specific embodiment, the modified DEV of the present invention has a partial or complete deletion or substitution of the UL24 gene sequence. In a specific embodiment, the modified DEV of the present invention has a complete deletion or substitution of the UL24 gene sequence.Specific examples of such constructs are, for example, rDEV4 Δ UL24 (see Example 2), rDEV4 H9HA Δ UL24 (see Example 3), or rDEV4 Δ UL24 UL26-H9HA-UL27 (see Example 3).

[0050] In a specific embodiment, the modified DEV of the present invention has a deletion or substitution of a portion or the entire sequence of the UL40 gene. In a specific embodiment, the modified DEV of the present invention has a deletion or substitution of the entire sequence of the UL40 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL40 (see Example 2) or rDEV4 H9HA Δ UL40 (see Example 3).

[0051] In a specific embodiment, the modified DEV of the present invention has a deletion or substitution of a portion or the entire sequence of the UL39 gene. In a specific embodiment, the modified DEV of the present invention has a deletion or substitution of the entire sequence of the UL39 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL39 (see Example 2), rDEV4 H9HA Δ UL39 (see Example 3), or rDEV4 Δ UL39 UL26-H9HA-UL27 (see Example 3).

[0052] In a specific embodiment, the modified DEV of the present invention has a deletion or substitution of a portion or the entire sequence of the UL23 gene. In a specific embodiment, the modified DEV of the present invention has a deletion or substitution of the entire sequence of the UL23 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL23 (see Example 2), or rDEV4 H9HA Δ UL23 (see Example 3). Specification 6 / 35 pages 9 CN 121843706 A

[0053] In a specific embodiment, 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 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 an even more preferred embodiment, the modified DEV 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. Specific examples of such constructs are, for example, rDEV4 Δ US7US8 (see Example 2) and rDEV4 H9HA Δ US7US8 (see Example 3).

[0054] In a specific embodiment, the modified DEV of the present invention has a partial or complete sequence of the UL24 gene and a partial or complete sequence of the UL2 gene with deletions or substitutions. In a more preferred embodiment, the modified DEV comprises a complete deletion of the UL24 gene and a complete substitution of the UL2 gene. A specific example of such a construct is, for example, rDEV4 Δ UL24 H9HA Δ UL2 (see Example 3).

[0055] In a specific embodiment, the modified DEV of the present invention has a partial or complete sequence of the UL40 gene and a partial or complete sequence of the UL2 gene with deletions or substitutions. In a more preferred embodiment, the modified DEV comprises a complete deletion of the UL40 gene and a complete substitution of the UL2 gene. A specific example of such a construct is, for example, rDEV4 Δ UL40 H9HA Δ UL2 (see Example 3).

[0056] In a specific embodiment, the modified DEV of the present invention has a partial or complete sequence of the UL23 gene and a partial or complete sequence of the UL41 gene with deletions or substitutions. In a more preferred embodiment, the modified DEV 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 Δ UL23 Δ UL41 (see Example 3).

[0057] In a specific embodiment, the modified DEV of the present invention has a partial or complete sequence of the UL41 gene and a partial or complete sequence of the US8 gene with deletion or substitution. In a more preferred embodiment, the modified DEV 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 Δ UL41 H9HA Δ US8 (see Example 3).

[0058] In a particular embodiment, the modified DEV 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 heteropolynucleotide sequence in such a host.

[0059] In a particular embodiment, the modified DEV further comprises a heteropolynucleotide encoding a heteroantigen of the pathogen.

[0060] The virus-related term "heteropolynucleotide" 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.

[0061] As used herein, "antigen" refers to, but is not limited to, a component that elicits an immune response in a host.

[0062] In a particular embodiment, the heteropolynucleotide is inserted into a non-essential gene or region of the modified DEV.In one specific embodiment, a heteropolynucleotide is inserted 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 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.

[0063] 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 of the modified DEV genome. In certain embodiments, a 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, or is inserted into the UL26-UL27 intergenic region of the modified DEV genome.

[0064] In some embodiments, the heteropolynucleotide is located in the UL23 gene. In certain embodiments, the heteropolynucleotide is inserted into the UL23 gene sequence of the DEV viral genome in addition to the existing UL23 gene sequence (thus inactivating the gene by interrupting the gene sequence), or is inserted into the UL23 gene region of the DEV viral genome after a portion or the entire UL23 gene sequence is deleted (thus inactivating the gene by deleting the gene sequence), or replaces a portion or the entire UL23 gene sequence (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL23 gene sequence.

[0065] 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 a 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 one specific embodiment, the heteropolynucleotide is inserted into the US7 and US8 gene regions of the DEV viral 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.

[0066] In some embodiments, the heteropolynucleotide is located in the UL2 gene. In certain 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 partial or complete sequence of the UL2 gene (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL2 gene sequence.

[0067] In some embodiments, the heteropolynucleotide is located in the UL24 gene. In certain 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 partial or complete sequence of the UL24 gene (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL24 gene sequence.

[0068] 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 sequence of the UL39 gene (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL39 gene sequence.

[0069] In some embodiments, the heteropolynucleotide is located in the UL26-UL27 intergenic region.In a particular embodiment, the heteropolynucleotide is inserted into the UL26-UL27 intergenic region of the DEV virus genome.

[0070] In some embodiments, the heteropolynucleotide is located in the UL40 gene. In a particular embodiment, 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 DEV virus genome specification page 8 / 35 11 CN 121843706 A after a partial or complete deletion of the UL40 gene sequence (thus inactivating the gene by deleting the gene sequence), or replaces a partial or complete UL40 gene sequence (thus inactivating the gene by replacing the gene sequence), or is located in a mutated UL40 gene sequence.

[0071] In some embodiments, the heteropolynucleotide is located in the US8 gene. In a particular embodiment, 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 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.

[0072] In an alternative embodiment, the DEV of the present invention has an inactivating gene, preferably a deleted gene, and contains a heteropolynucleotide encoding an antigen of the pathogen located in a different gene or region. In this case, the heteropolynucleotide can be cloned to replace a partial or complete sequence of the different gene, or it can be inserted into the different gene, or inserted into the different gene after a partial or complete deletion of the different gene sequence (thus inactivating the different gene as well). For example, the DEV of the present invention has an inactivated UL24 gene and contains a heteropolynucleotide encoding a pathogen antigen 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).

[0073] Furthermore, the DEV of the present invention may contain several heteropolynucleotides encoding one or more antigens of one or more pathogens. In this respect, 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.

[0074] In some embodiments, the heteropolynucleotides are expressed after the modified DEV has been transfected into suitable host cells.

[0075] 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" refers to a vaccine that uses a virus (in this case, 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.

[0076] Accordingly, in one aspect, the invention also provides a modified duck enteritis virus (DEV) as a live vector vaccine in chickens, wherein the modified DEV contains an inactivating gene selected from any of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, and wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens (compared to unmodified DEV).

[0077] In one aspect, the present invention also provides a modified duck enteritis virus (DEV) as a live vector vaccine in chickens, wherein the modified DEV contains an inactivating gene selected from any of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; or vii) US8 gene, the inactivating gene being alone or in combination with any other non-essential gene of the DEV, and wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens (compared to unmodified DEV).

[0078] In one aspect, the present invention also provides a modified duck enteritis virus (DEV) as a live vector vaccine in chickens, wherein the modified DEV contains an inactivating gene selected from any of the following i)-xii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene; viiii) US7 gene and US8 gene; ix) UL24 gene and UL2 gene; x) UL40 gene and UL2 gene; xi) UL23 gene and UL41 gene; or xii) UL41 gene and US8 gene, and wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens (compared to unmodified DEV). Specification 9 / 35 pages 12 CN 121843706 A

[0079] Virus construction and cloning can be achieved by techniques known in the art itself.Gene cloning and plasmid construction are well known to those skilled in the art and can be performed substantially using standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012). Typically, recombinant viruses can be prepared by homologous recombination between a viral genome and a construct (e.g., a homologous plasmid) containing a nucleic acid to be inserted, with nucleotides from the insertion site side-joined to allow recombination. Cloning can be performed with or without deletion of endogenous sequences.

[0080] 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 a reduced or zero mortality rate in the modified DEV in chickens compared to an unmodified DEV.

[0081] Heteropolynucleotides The DEV of the present invention may contain any heteropolynucleotide encoding an antigen of a pathogen. The pathogen may be, or the antigen may be derived from, viruses, bacteria, fungi, protozoa, etc.

[0082] In some embodiments, the pathogen may be a chicken pathogen. In some embodiments, the antigen may be an antigen of a chicken pathogen. In some embodiments, the pathogen or antigen is derived from an avian influenza virus. In some embodiments, the pathogen or antigen is derived from an avian influenza virus selected from the group consisting of influenza A virus, influenza B virus, influenza C virus, and influenza D virus. Preferably, the pathogen or antigen is derived from an influenza A virus. More preferably, the pathogen or antigen is derived from an H9 avian influenza virus. Most preferably, the pathogen or antigen is derived from an H9N2 avian influenza virus.

[0083] In some embodiments, the antigen of the pathogen is the HA protein (H9 HA protein) of an H9 subtype avian influenza virus. In some embodiments, the antigen of the pathogen is the HA protein of an H9N2 subtype avian influenza virus.

[0084] 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 the sequence of SEQ ID NO:1.

[0085] In some embodiments, the complete H9 HA coding sequence has a nucleotide sequence with 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.

[0086] The “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 typically used to determine the identity of amino acid / nucleotide sequences. For example, identity can be determined by using the BLAST program in the NCBI database. 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, D.W. (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.

[0087] As used herein, it should be particularly understood that the term “sequence identity with the sequence of SEQ ID NO: X” is equivalent, respectively, to the terms “sequence identity with the sequence of SEQ ID NO: X in length” or “sequence identity with the sequence of SEQ ID NO: X in full length” in the specification 10 / 35, CN 121843706 A. In this context, “X” is any integer, such as 1 or 2, such that “SEQ ID NO: X” represents any SEQ ID NO mentioned herein.

[0088] In some embodiments, the heteropolynucleotide is typically operatively linked to a 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 immediate early promoter of cytomegalovirus (CMV), 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.

[0089] 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 glycoprotein B (gB) gene of feline herpesvirus (FHV), the immediate early (IE) gene of human cytomegalovirus (hCMV), 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 generally operatively linked to the SV40 polyadenylate signal.

[0090] One embodiment of the invention provides a modified DEV that contains (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 signal, and thus the expression of the H9 HA protein is regulated by the SV40 polyadenylate signal. In some embodiments, the heteropolynucleotide encoding the H9 HA protein is operatively linked to both a mouse CMV promoter and an SV40 polyadenylate signal, and thus the expression of the H9 HA protein is regulated by both a mouse CMV promoter and an SV40 polyadenylate signal.

[0091] In some embodiments, the modified DEV containing (and capable of expressing) the heteropolynucleotide encoding the H9 HA protein comprises an expression cassette containing, in the 5' to 3' direction, in the following order: a) a promoter, b) a heteropolynucleotide, c) a transcription terminator. In some embodiments, the 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 mouse CMV promoter, b) a heteropolynucleotide encoding the H9 HA protein, and c) a transcription terminator. In some embodiments, the 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 mouse CMV promoter, b) a heteropolynucleotide encoding the H9 HA protein, and c) an SV40 polyadenylate signal.

[0092] In some embodiments, the modified DEV of the present invention containing a heteropolynucleotide encoding an antigen of a pathogen is capable of providing 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 DEV of the present invention containing a heteropolynucleotide encoding an antigen of the pathogen is capable of providing 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% protection 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 DEV of the present invention containing a heteropolynucleotide encoding an antigen of a pathogen 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 DEV of the present invention containing a heteropolynucleotide encoding an antigen of a pathogen 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.

[0093] Preferred DEVs The preferred modified DEVs of the present invention, wherein the UL41 gene of the DEV genome is inactivated due to deletion or substitution of a portion or the entire sequence of the UL41 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL41 (see Example 2).

[0094] A preferred modified DEV of the present invention, wherein the US3 gene of the DEV genome is inactivated due to the deletion or substitution of a portion or the entire sequence of the US3 gene. A specific example of such a construct is, for example, rDEV4 Δ US3 (see Example 2).

[0095] A preferred modified DEV of the present invention, wherein the UL24 gene of the DEV genome is inactivated due to the deletion or substitution of a portion or the entire sequence of the UL24 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL24 (see Example 2), rDEV4 H9HA Δ UL24 (see Example 3), or rDEV4 Δ UL24 UL26-H9HA-UL27 (see Example 3).

[0096] A preferred modified DEV of the present invention, wherein the UL40 gene of the DEV genome is inactivated due to the deletion or substitution of a portion or the entire sequence of the UL40 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL40 (see Example 2) or rDEV4 H9HA Δ UL40 (see Example 3).

[0097] The preferred modified DEV of the present invention is wherein the UL39 gene of the DEV genome is inactivated due to deletion or substitution of a portion or the entire sequence of the UL39 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL39 (see Example 2), rDEV4 H9HA Δ UL39 (see Example 3), or rDEV4 Δ UL39 UL26-H9HA-UL27 (see Example 3).

[0098] A preferred modified DEV of the present invention, wherein the UL23 gene of the DEV genome is inactivated due to deletion or substitution of a portion or the entire sequence of the UL23 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL23 (see Example 2) or rDEV4 H9HA Δ UL23 (see Example 3).

[0099] A preferred modified DEV of the present invention, wherein the US7 and US8 genes of the DEV genome are inactivated due to deletion or substitution of a continuous region spanning a portion or the entire sequence of the US7 gene, the entire US7-US8 intergenic region, and a portion or the entire sequence of the US8 gene. Specific examples of such constructs are, for example, rDEV4 Δ US7US8 (see Example 2) and rDEV4 H9HA Δ US7US8 (see Example 3).

[0100] A preferred modified DEV of the present invention, wherein the UL24 and UL2 genes of the DEV genome are inactivated due to deletion or substitution of a portion or the entire sequence of the UL24 gene and a portion or the entire sequence of the UL2 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL24 H9HA Δ UL2 (see Example 3).

[0101] A preferred modified DEV of the present invention, wherein the UL40 and UL2 genes of the DEV genome are inactivated due to the deletion or substitution of a portion or the entire sequence of the UL40 gene and a portion or the entire sequence of the UL2 gene. Specific examples of such constructs are, for example, rDEV4 Δ UL40 H9HA Δ UL2 (see Example 3).

[0102] A preferred modified DEV of the present invention, wherein the UL23 and UL41 genes of the DEV genome are inactivated due to the deletion or substitution of a portion or the entire sequence of the UL23 gene and a portion or the entire sequence of the UL41 gene. Specific examples of such constructs are, for example, rDEV4 H9HA Δ UL23 Δ UL41 (see Example 3).

[0103] A preferred modified DEV of the present invention, wherein the UL41 gene and the US8 gene of the DEV genome are inactivated by deletion or substitution of a portion or the entire sequence of the UL41 gene and a portion or the entire sequence of the US8 gene. A specific example of such a construct is, for example, rDEV4 Δ UL41 H9HA Δ US8 (see Example 3).

[0104] A 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, thereby inactivating the UL23 gene. A specific example of such a construct is, for example, rDEV4 H9HA Δ UL23 (see Example 3).

[0105] A 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 Δ US7US8 (see Example 3).

[0106] A 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, thereby inactivating the UL2 gene, and also comprises an inactivated UL24 gene, optionally with the UL24 gene deleted. A specific example of such a construct is, for example, rDEV4 Δ UL24 H9HA Δ UL2 (see Example 3).

[0107] A 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, thereby inactivating 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 Δ UL40 H9HA Δ UL2 (see Example 3).

[0108] A preferred modified DEV of the present invention contains 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 H9HA Δ UL24 (see Example 3).

[0109] A preferred modified DEV of the present invention contains 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 H9HA Δ UL39 (see Example 3).

[0110] A preferred modified DEV of the present invention contains a heteropolynucleotide located in the UL26-UL27 intergenic region, and also contains an inactivated UL24 gene, optionally with the UL24 gene deleted. Specific examples of such constructs are, for example, rDEV4 Δ UL24 UL26-H9HA-UL27 (see Example 3).

[0111] A preferred modified DEV of the present invention contains 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 H9HA Δ UL40 (see Example 3).

[0112] A preferred modified DEV of the present invention contains a heteropolynucleotide located in the UL26-UL27 intergenic region, and also contains an inactivated UL39 gene, optionally with the UL39 gene deleted. A specific example of such a construct is, for example, rDEV4 Δ UL39 UL26-H9HA-UL27 (see Example 3).

[0113] A 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, thereby inactivating 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 Δ UL23 Δ UL41 (see Example 3).

[0114] A 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, thereby inactivating the US8 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 Δ UL41 H9HA Δ US8 (see Example 3).

[0115] In the preferred modified DEV of the present invention, the heteropolynucleotide encodes the H9 HA protein. Instructions for Use, Pages 13 / 35, 16 CN 121843706 A

[0116] Host Cell 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 heteropolynucleotide 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 Rodríguez-Avila et al., Avian diseases 2007, 51:905-911).

[0117] The modified DEV of the present 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.

[0118] Examples of competent cells include CEF, EB66, DEF, embryonic eggs, chicken kidney cells, etc. Cells or viruses can be cultured at about 37°C in a culture medium (such as MEM containing 5% FBS) for 1 hour to 6 days.

[0119] Composition The present invention also relates to a composition comprising the modified DEV of the present invention.

[0120] 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 composition of the present invention. The host is also described as an "object." Preferably, any host or object described or mentioned herein is a chicken.

[0121] An "immune response" to a composition is the development of a cell- and / or antibody-mediated immune response in a host to the composition of interest. Generally, an "immune response" includes, but is not limited to, one or more of the following effects: specificity against an antigen 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 disease.

[0122] A “protective immune response” or “protective reaction” will be demonstrated by a reduction or absence of clinical signs typically exhibited by the infected host, a faster recovery time and / or a reduced duration of infectivity, or a reduced pathogen titer in the tissues or body fluids or excretions of the infected host.

[0123] The compositions of the present invention are described as “vaccines” when the host exhibits a protective immune response thereby enhancing resistance to new infections and / or reducing the clinical severity of disease. In one aspect, the compositions of the present invention are vaccines.

[0124] 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 vectors in vector vaccines.

[0125] The compositions and vaccines of the present invention may also comprise pharmaceutically or veterinarily acceptable carriers, excipients, mediators, or adjuvants.

[0126] Pharmaceutically or veterinarily acceptable carriers or adjuvants or mediators or excipients are well known to those skilled in the art. For example, pharmaceutically or veterinarily acceptable carriers, adjuvants, mediators, or excipients include, but are not limited to, 0.9% NaCl (e.g., saline) solution or phosphate buffer, poly(L-glutamate), lactated Ringer's injection diluent (sodium chloride, sodium lactate, potassium chloride, and calcium chloride), or polyvinylpyrrolidone. Pharmaceutically or veterinarily 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). Specification 14 / 35 pages 17 CN 121843706 A

[0127] In some embodiments, the compositions of the present invention comprise a lyophilization protectant. In certain embodiments, the compositions of the present invention comprise a preservative.

[0128] The compositions of the present invention may be liquids (solutions, suspensions, emulsions) or solids (powders, gels, pastes, oils). The compositions of the present invention may be formulated for any route of administration. Preferably, the compositions may be formulated for oral, nasal, eye drop, spray, drinking water, intraocular, intramuscular, subcutaneous, intradermal, or transdermal administration.

[0129] 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 can be titrated using any viral titration method, including but not limited to FFA (Focus Forming Assay) or FFU (Focus Forming Unit), TCID50 (50% Tissue Culture Infective Dose), PFU (Plaque Forming Units), and FAID50 (50% Fluorescent Antibody Infectious Dose). In vitro generated VLPs can be titrated using hemagglutination assays, ELISA, and electron microscopy. In some embodiments, the modified DEV in the composition is present at a dose from 1 × 10² TCID50 / ml or TCID50 / g to 1 × 10⁷ TCID50 / ml or TCID50 / g. In some embodiments, the modified DEV in the composition is present at a dose from 1 × 10⁴ TCID50 / ml or TCID50 / g to 1 × 10⁶ TCID50 / ml or TCID50 / g. In some embodiments, the modified DEV in the composition is present at a dose of 1×10⁶ TCID₅₀ / ml or TCID₅₀ / g. In some embodiments, the dose volume may be between about 0.01 and about 10 ml, or between 0.01 and about 5 ml.

[0130] Depending on the vaccination regimen, the compositions of the present invention may be administered in a single dose or repeated doses. Depending on the vaccination regimen, the vector vaccines of the present invention may be formulated in a single dose or repeated doses.

[0131] Uses and Methods In one aspect, the present invention provides the modified DEV of the present invention, or the compositions of the present invention, or the vector vaccines of the present 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 to chickens once or multiple times the modified DEV of the present invention, or the compositions of the present invention, or the vector vaccines of the present invention.

[0132] In one aspect, the present invention provides a modified DEV of the present invention, a composition of the present invention, or a vector vaccine of the present invention for vaccinating chickens by inducing a protective immune response against a pathogen in chickens.

[0133] In one aspect, the present invention provides a method of vaccinating chickens by inducing a protective immune response against a pathogen in chickens, 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.

[0134] In one aspect, the present invention provides the use of the modified DEV of the present invention in the manufacture of a composition for vaccinating chickens by inducing a protective immune response against a pathogen in chickens.

[0135] The term "vaccination" refers to active immunization by administering an immunogenic composition to chickens to be immunized, thereby eliciting a protective immune response against an antigen included in such immunogenic composition.

[0136] In some embodiments, the chickens are vaccinated on days 0, 1, 2, 3, 4, 5, 6, or 7 days of age.

[0137] In some embodiments, 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-hatching.

[0138] As indicated in the experimental section, the virus of the present invention is particularly advantageous for vaccinating chicks (day 0, day 1, day 2, or day 3 post-hatching). Indeed, the present invention surprisingly shows that the modified DEV of the present invention is safe when administered to chickens at such an early stage, whereas natural or wild-type DEVs are lethal to chickens. This early administration, combined with the early onset of immunity induced by these modified DEVs, is particularly advantageous for inducing early protective immunity before chickens may be significantly exposed to the pathogen.

[0139] In some embodiments, the pathogen is a avian species pathogen. In some embodiments, the pathogen is a poultry pathogen. In some embodiments, the pathogen is a chicken pathogen. In some embodiments, the pathogen is 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.

[0140] The administration of the present invention or modified DEV, composition, or vector vaccine results in a reduction in the incidence of infection with a specific pathogen in chickens, or a reduction in the severity of clinical signs caused by or associated with infection with a specific pathogen.Preferably, the application of the present invention, or the modified DEV, composition, or vector vaccine, results in a reduction in the incidence of a specific avian influenza virus infection in chickens, or a reduction in the severity of clinical signs caused by or associated with a specific avian influenza virus infection. It should be understood that the application of the present invention, or the modified DEV, composition, or vector vaccine, 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.

[0141] In some embodiments, the modified DEV, composition, or vector vaccine is administered via oral-nasal, eye drops, spray, drinking water, intraocular, intramuscular, subcutaneous, intradermal, or transdermal administration. In some embodiments, the modified DEV, composition, or vector vaccine may be formulated for oral-nasal, eye drops, spray, drinking water, intraocular, intramuscular, subcutaneous, intradermal, or transdermal administration. However, depending on the nature and mode of action of the compound, the immunogenic composition may also be administered via other routes.

[0142] In one aspect of the invention, the modified DEV, composition, or vector vaccine is administered once, and such a single administration is effective.

[0143] However, while single-dose administration is preferred, the modified DEV, composition, or vector vaccine may also be administered two or more times, wherein the first dose is 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.

[0144] 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 present at a dose of 1×10² TCID50 / ml or TCID50 / g to 1×10⁷ TCID50 / ml or TCID50 / g. In some embodiments, the modified DEV in the composition or vector vaccine is present at a dose of 1×10⁴ TCID50 / ml or TCID50 / g to 1×10⁶ TCID50 / ml or TCID50 / g. In some embodiments, the modified DEV in the composition or vector vaccine is present at a dose of 1×10⁶ TCID50 / ml or TCID50 / g. 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.

[0145] 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.

[0146] The following terms are also described herein, as well as part of the disclosure of the present invention: Term 1. A modified duck enteritis virus (DEV) (as a live vector vaccine in chickens), wherein the DEV genome is selected from the group consisting of one or more genes selected from US3, UL24, UL40, UL39, UL23, UL41 and US8 that are inactivated, and wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens compared with unmodified DEV.

[0147] Clause 2. A modified duck enteritis virus (DEV) (as a live vector vaccine in chickens) comprising an inactivated gene selected from any of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene, said inactivated gene in combination with one or more other non-essential genes of DEV, and wherein said modified DEV has reduced mortality or no mortality in chickens (compared to said unmodified DEV).

[0148] Clause 3. The modified duck enteritis virus (DEV) according to Clause 2, wherein the one or more additional non-essential genes of the DEV are different from the first inactivating gene and are selected from: i) the US7 gene; ii) the UL2 gene; iii) the UL41 gene; iv) the US3 gene; v) the UL24 gene; vi) the UL40 gene; vii) the UL39 gene; viiii) the UL23 gene; or ix) the US8 gene.

[0149] Clause 4. A modified duck enteritis virus (DEV) (as a live vector vaccine in chickens) comprising an inactivated gene selected from any of the following i)-xi): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US7 and US8 genes; viiii) UL24 and UL2 genes; ix) UL40 and UL2 genes; x) UL23 and UL41 genes; or xi) UL41 and US8 genes, and wherein the modified DEV has a reduced mortality rate or no mortality rate in chickens (compared to the unmodified DEV).

[0150] Clause 5. The modified DEV according to any one of the preceding clauses, wherein the gene is inactivated by mutation, interruption, substitution, or deletion of a portion or the entire sequence of the gene.

[0151] Clause 6. A modified DEV according to any one of the preceding clauses, wherein at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence of the gene is replaced or deleted.

[0152] Clause 7. A modified DEV according to any one of the preceding clauses, wherein the modified DEV further comprises (and is capable of expressing) a heteropolynucleotide encoding a heteroantigen.

[0153] Clause 8. A modified DEV according to Clause 7, wherein the heteropolynucleotide is inserted into a non-essential gene or region of the modified DEV.

[0154] Clause 9. A modified DEV according to Clause 7 or 8, 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.

[0155] Clause 10. The modified DEV according to any one of Clauses 7-9, wherein the non-essential genes or regions of the modified DEV are selected from the group consisting of both the UL23 gene, the US7 gene and the US8 gene, the UL2 gene, the UL24 gene, the UL39 gene, the UL40 gene, the US8 gene, and the UL26-UL27 intergenic region.Specification 17 / 35 pages 20 CN 121843706 A

[0156] Clause 11. The modified DEV according to Clause 7, wherein the heteropolynucleotide is located in a gene or region selected from the group consisting of the UL23 gene, both the US7 gene and the US8 gene, the UL2 gene, the UL24 gene, the UL39 gene, the UL40 gene, the US8 gene, and the UL26-UL27 intergenic region of the modified DEV genome.

[0157] Clause 12. The modified DEV according to Clause 7, 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 the UL23 gene, both the US7 gene and the US8 gene, the UL2 gene, the UL24 gene, the UL39 gene, the UL40 gene, and the US8 gene of the modified DEV genome, or is inserted into the UL26-UL27 intergenic region of the modified DEV genome.

[0158] Clause 13. A modified DEV according to any one of the preceding clauses, wherein the modified DEV comprises any one of the following: i) a heteropolynucleotide located in the UL23 gene, replacing a portion or the entire sequence of the UL23 gene; ii) a heteropolynucleotide located in both the US7 and US8 genes, replacing a portion or the entire sequence of both the US7 and US8 genes; iii) a heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and further comprising an inactivated UL24 gene, optionally with the UL24 gene deleted; iv) a heteropolynucleotide located in the UL2 gene, replacing a portion or the entire sequence of the UL2 gene, and further comprising an inactivated UL40 gene, optionally with the UL40 gene deleted; v) a heteropolynucleotide located in the UL24 gene, replacing a portion or the entire sequence of the UL24 gene; vi) a heteropolynucleotide located in the UL39 gene, replacing a portion or the entire sequence of the UL39 gene; vii) a heteropolynucleotide located in the UL26-UL27 intergenic region and further comprising an inactivated UL24 gene, optionally with the UL24 gene deleted; viii) a heteropolynucleotide located in the UL40 gene, replacing a portion or the entire sequence of the UL40 gene; ix) a heteropolynucleotide located in the UL26-UL27 intergenic region and further comprising an inactivated UL39 gene, optionally with the UL39 gene deleted; x) a heteropolynucleotide located in the UL23 gene, replacing a portion or the entire sequence of the UL23 gene, and further comprising an inactivated UL41 gene, optionally with the UL41 gene deleted; or xi) a heteropolynucleotide located in the US8 gene, replacing a portion or the entire sequence of the US8 gene, and further comprising an inactivated UL41 gene, optionally with the UL41 gene deleted.

[0159] Clause 14. A modified DEV according to any one of Clauses 7-13, wherein the pathogen is a chicken pathogen, or the antigen is an antigen of a chicken pathogen.

[0160] Clause 15. The modified DEV according to any one of Clauses 7-13, wherein the antigen is derived from an avian influenza virus.

[0161] Clause 16. The modified DEV according to any one of Clauses 7-13, wherein the antigen is derived from an avian influenza virus selected from the group consisting of influenza A virus, influenza B virus, influenza C virus, and influenza D virus.

[0162] Clause 17. The modified DEV according to any one of Clauses 7-13, wherein the antigen is derived from the H9 type avian influenza virus, page 18 / 35 of the specification, 21 CN 121843706 A.

[0163] Clause 18. The modified DEV according to any one of Clauses 7-13, wherein the antigen is derived from avian influenza virus H9N2.

[0164] Clause 19. The modified DEV according to any one of Clauses 7-13, wherein the heterologous antigen is a hemagglutinin (HA) protein of H9N2 subtype avian influenza virus.

[0165] Clause 20. The modified DEV according to any one of Clauses 7-19, wherein the amino acid sequence of the heterologous antigen has at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity with SEQ ID NO:1.

[0166] Clause 21. The modified DEV according to any one of Clauses 7-19, 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 22. The modified DEV according to any one of Clauses 7-21, wherein the heteropolynucleotide is operatively linked to a promoter.

[0168] Clause 23. The modified DEV according to Clause 22, 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.

[0169] Clause 24. The modified DEV according to any one of Clauses 7-23, wherein the heteropolynucleotide is operatively linked to the SV40 polyadenylate signal.

[0170] Clause 25. The modified DEV according to any one of Clauses 7-24, wherein the heteropolynucleotide is expressed after the modified DEV has been transfected into a suitable host cell.

[0171] Clause 26. The modified DEV according to any one of Clauses 1-25, wherein the modified DEV is attenuated for chickens.

[0172] Clause 27. The modified DEV according to any one of Clauses 1-26, wherein the inactivation of the selected gene results in attenuation of the DEV.

[0173] Clause 28. A modified DEV according to any one of Clauses 1-27, 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.

[0174] Clause 29. A modified DEV according to any one of Clauses 1-28, used as a carrier vaccine in chickens.

[0175] Clause 30. A composition comprising a modified DEV according to any one of Clauses 1-28.

[0176] Clause 31. A composition according to Clause 30, further comprising a pharmaceutically or veterinarily acceptable carrier, excipient, mediator, or adjuvant. Specification 19 / 35 pages 22 CN 121843706 A

[0177] Clause 32. A composition according to Clause 30 or 31, wherein the composition is a vaccine.

[0178] Clause 33. The composition according to any one of Clauses 30-32, wherein the composition is a vector vaccine for chickens.

[0179] Clause 34. The composition according to Clause 33, wherein the modified DEV is used as a vector.

[0180] Clause 35. A modified DEV according to any one of Clauses 1-28, or a composition according to any one of Clauses 30-34, or a vector vaccine according to Clause 29, used in a method for inducing a protective immune response against a pathogen in chickens, wherein such a method comprises or consists of administering to chickens once or more of the modified DEV according to any one of Clauses 1-28, or the composition according to any one of Clauses 30-34, or the vector vaccine according to Clause 29.

[0181] Clause 36. A modified DEV according to any one of Clauses 1-28, or a composition according to any one of Clauses 30-34, or a vector vaccine according to Clause 29, for use in vaccinating chickens by inducing a protective immune response against a pathogen in chickens.

[0182] Clause 37. A method of vaccinating chickens by inducing a protective immune response against a pathogen in chickens, comprising or consisting of administering at least once to said chickens a modified DEV according to any one of Clauses 1-28, or a composition according to any one of Clauses 30-34, or a vector vaccine according to Clause 29.

[0183] Clause 38. Use of a modified DEV according to any one of Clauses 1-28 in the manufacture of a composition for vaccinating chickens by inducing a protective immune response against a pathogen in chickens.

[0184] Clause 39. A method and use according to any one of Clauses 35-38, wherein said pathogen is an avian influenza virus.

[0185] Clause 40. The method and use according to Clause 39, wherein the pathogen is an avian influenza virus, optionally type A avian influenza virus, optionally H9 subtype A avian influenza virus, optionally H9N2 subtype A avian influenza virus.

[0186] Clause 41. The method and use according to any one of Clauses 35-40, wherein the chickens are vaccinated on days 0, 1, 2, 3, 4, 5, 6, or 7.

[0187] Clause 42. The method and use according to any one of Clauses 35-41, 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.

[0188] Clause 43. The method and use according to any one of Clauses 35-42, wherein the modified DEV, the composition, or the vector vaccine is administered via oral-nasal, ocular, spray, drinking water, intraocular, intramuscular, subcutaneous, intradermal, or transdermal administration.

[0189] Clause 44. A vaccination kit for vaccinating chickens by inducing a protective immune response against a pathogen in chickens, comprising an effective amount of the modified DEV according to any one of Clauses 1-28, or the composition according to any one of Clauses 30-34, or the vector vaccine according to Clause 29, and means for administering the modified DEV, the composition, or the vector vaccine to the chickens.

[0190] Clause 45. A host cell expressing the modified DEV according to any one of Clauses 1-28.

[0191] Clause 46. A host cell expressing the modified DEV and any heteropolynucleotide according to any one of Clauses 7-28.

[0192] Clause 47. A host cell according to Clause 45 or 46, wherein the host cell line is a CEF cell, an EB66 cell, or a DEF cell.

[0193] Clause 48. A method for preparing a modified DEV according to any one of Clauses 1-28, comprising inactivating one or more genes of the DEV genome as defined in any one of Clauses 1-28, 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.

[0194] Examples The following examples further illustrate the invention by way of example. It should be understood that the invention is not limited to any of the examples described below. Those skilled in the art will understand that the performance, results, and findings of these examples can be adapted and applied in a broader sense in light of the general description of the invention.

[0195] Example 1: Construction of the bacterial artificial chromosome (BAC) of DEV The duck plague virus DEV4 strain used in this study was purchased from China Academy of Advanced Technology and Research in 2017 and deposited at the China Center for Type Culture Collection (CCTCC) on August 4, 2023, with CCTCC NO: V202378.

[0196] To attenuate the DEV4 strain and make it a safe vector, a bacterial artificial chromosome system was constructed using the genome of the DEV4 strain for subsequent gene deletion and insertion. The specific steps included: 1. Using primers shown in Table 1, the homologous arm sequences on the left and right sides of the mini-F element insertion site (located between UL44 and UL44.5 in the DEV4 genome) were amplified by PCR. A digestion site was also introduced. The plasmid pB12 containing the mini-F gene fragment (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) was digested with BamHI to obtain a mini-F DNA fragment flanked by BamHI. The obtained DNA fragments from the left and right homologous arms were then ligated with the mini-F DNA fragment to obtain a Mini-F transfer vector, which was then transformed into E. coli TOP10 competent cells (purchased from Tiangen). The plasmid containing the mini-F transfer vector was extracted and identified by enzymatic digestion.

[0197] Table 1. PCR Primers

[0198] 2. According to 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 cells to construct recombinant virus rDEV4 (i.e., recombinant DEV4)-BAC through homologous recombination. After screening and purification, recombinant virus rDEV4-BAC was obtained (see Figure 2A).

[0199] 3. According to the instructions of the competent cells, the extracted rDEV4-BAC genomic DNA was electroporated into MegaX competent cells (purchased from Invitrogen, catalog number C6400-03). Then, rod-particle rDEV4-BAC was extracted and identified.

[0200] 4. The rod-like rDEV4-BAC was electroporated into gs1783 competent cells containing redE / T recombinase (Wang et al., 2015, Virology Journal, 12:126) to obtain the GS1783-DEV4-BAC strain.

[0201] Example 2: Construction of DEV strains with missing virulence genes Based on the constructed rDEV4-BAC, different virulence-associated genes of DEV4 (including UL41, US3, UL24, UL40, UL39, UL23 and US7US8 (hereinafter commonly referred to as X)) are deleted to construct DEV strains with missing virulence genes, including rDEV4 Δ UL41, rDEV4 Δ US3, rDEV4 Δ UL24, rDEV4 Δ UL40, rDEV4 Δ UL39, rDEV4 Δ UL23 and rDEV4 Δ US7US8.

[0202] “Δ” refers to the inactivation of the gene described herein (such as deletion or substitution), for example, Δ UL24 refers to the deletion or substitution of UL24.

[0203] The construction method includes the following steps.

[0204] 1. Obtain the 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, from the plasmid pKan synthesized from GenScript company by Not I digestion.

[0205] 2. Prepare GS1783-DEV4-BAC (obtained in Example 1) competent cells for electroporation using conventional methods. Then, electroporate the I_SceI-Kana-X fragment into GS178-DEV4-BAC competent cells. Recombinant clones are then selected on LB agar plates with dual resistance to chloramphenicol and kanamycin.Recombinant band-particle DNA was extracted and analyzed by PCR and RFLP. Thus, the GS1783-DEV4-BAC-ΔX-Kana strain and recombinant band-particle rDEV4-BAC-ΔX-Kana were obtained. In step 2 of Red recombination, the expression of 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 band-particle DNA was extracted and analyzed by PCR and RFLP. Then, the GS1783-DEV4-BAC-ΔX strain and band-particle rDEV4-BAC-ΔX were obtained.

[0206] 3. To rescue recombinant rDEV4 ΔX (see Figure 3) and remove the mini-F sequence, 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 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. Therefore, rDEV4 Δ X (see Figure 2B) was obtained, including rDEV4 Δ UL41, rDEV4 Δ US3, rDEV4 Δ UL24, rDEV4 Δ UL40, rDEV4 Δ UL39, rDEV4 Δ UL23 and rDEV4 Δ US7US8.

[0207] 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 avian influenza virus subtype H9N2 was inserted into different sites. The following recombinant viruses were finally constructed: rDEV4 H9HA Δ UL23, rDEV4 H9HA Δ US7US8, rDEV4 Δ UL24 H9HA Δ UL2, rDEV4 Δ UL40 H9HA Δ UL2, rDEV4 H9HA Δ UL24, rDEV4 H9HA Δ UL39, rDEV4 Δ UL24 UL26‑H9HA‑UL27, rDEV4 H9HA Δ UL40, rDEV4 Δ UL39 UL26‑H9HA‑ UL27, rDEV4 H9HA Δ UL23 Δ UL41, and rDEV4 Δ UL41 H9HA Δ US8.

[0208] In this embodiment, the construction method using rDEV4 Δ UL39 UL26-H9HA-UL27 will be described as an example. Specifically, the construction method includes the following steps.

[0209] 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 is obtained by PCR amplification, with 50bp homologous arms on both sides. Primers used for constructing other rDEV4 H9HA viruses are also listed in Table 2.

[0210] Table 2. PCR Primer Specification 22 / 35 pages 25 CN 121843706 A

[0211] 2. GS1783-DEV4-BAC-Δ UL39 (obtained in Example 2) competent cells for electroporation were prepared using conventional methods. Then, the heterologous gene fragment L26-mCMV-H9HA-kana-UL27 was electroporated into GS1783-DEV4-BAC-Δ UL39 competent cells. Recombinant clones were then selected on LB agar plates with dual resistance to chloramphenicol and kanamycin. Recombinant rod-granule DNA was extracted and analyzed by PCR and RFLP (see Figure 4). Thus, recombinant rod-granule rDEV4-BAC-Δ UL39-UL26-H9HA-kana-UL27 was obtained. In step 2 of the Red recombination, the expression of nested endonuclease I-SceI was induced using 2% arabinose, which led to the cleavage of the I-SceI restriction site upstream of the kanamycin gene and ultimately the excision of the kanamycin cassette. Recombinant rod-granule DNA was extracted and analyzed by PCR and RFLP. Then, rod-granule rDEV4-BAC-ΔUL39-UL26-H9HA-UL27 was obtained.

[0212] 3. In order to rescue the recombinant rDEV4-ΔUL39-UL26-H9HA-UL27 and remove the mini-F sequence, recombinant rod-granule DNA was extracted and co-transfected with the mini-F homologous arm DNA into DEF (prepared according to conventional 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 the formation of both GFP-positive and GFP-negative plaques (see 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 Figure 2C).

[0213] 4. Based on steps 1-3 above, additional recombinant viruses expressing the H9N2 HA gene were constructed, including rDEV4 H9HA Δ UL23, rDEV4 H9HA Δ US7US8, rDEV4 Δ UL24 H9HA Δ UL2, rDEV4 Δ UL40 H9HA Δ UL2, rDEV4 H9HA Δ UL24, rDEV4 H9HA Δ UL39, rDEV4 Δ UL24 UL26-H9HA-UL27, rDEV4 H9HA Δ UL40, rDEV4 H9HA Δ UL23 Δ UL41 and rDEV4 Δ UL41 H9HA Δ US8.

[0214] Example 4: Preparation and In Vitro Characterization of Recombinant Virus Seed Batch 4.1. Preparation of Recombinant DEV Seed Batch with Deleted Virulence Gene rDEV4 Δ X (X represents the deleted gene) 1.4E7 DEF was seeded onto 10cm cell culture dishes. The culture medium was 10ml of MEM containing 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. Cells were seeded with DEV strains containing the deleted virulence gene. 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 for later use.

[0215] 4.2. Preparation of seed batches of recombinant DEV containing the HA gene of avian influenza virus subtype H9N2: 1.4E7 DEF cells were seeded onto 10cm cell culture dishes. The culture medium was 10ml 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, cells were seeded with recombinant DEV expressing the HA gene of avian influenza virus subtype H9N2. 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 1ml or 4ml cryovials and stored at -80°C for later use.

[0216] Next, 1.4E7 CEF cells were seeded onto 10cm 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 that had been passaged to P7 in DEF were seeded. The cells were incubated in a 5% CO2 incubator at 37°C for about 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.

[0217] 4.3. In vitro identification of rDEV vector 1. rDEV with the deleted virulence gene was passaged in DEF for 15 generations. DNA of the recombinant virus in generations 5, 10 and 15 was extracted using the QIAamp DNA Mini Kit (QIAGEN). The extracted DNA was amplified by PCR using primers to identify the deletion of the virulence gene. The PCR product was identified by sequencing to indicate successful deletion of the virulence gene.

[0218] Table 3. PCR primer instructions 24 / 35 pages 27 CN 121843706 A

[0219] 2. The harvested rDEVs with the deleted virulence gene were subjected to sterility and mycoplasma testing. Sterility testing was performed by conventional methods, and mycoplasma testing was performed by qPCR. The results showed that these recombinant viruses were free of exogenous microbial contamination.

[0220] 3. Titer determination of rDEVs with the deleted virulence gene: The titer (TCID50) of rDEVs with the deleted virulence gene was determined on DEF.

[0221] Table 4. TCID50 of rDEVs

[0222] 4.4. In vitro identification of rDEVs containing the HA gene of the avian influenza virus subtype H9N2 1. Stability of the HA gene. rDEVs containing the HA gene of the avian influenza virus subtype H9N2 were passaged continuously in CEF up to the 15th generation. DNA was extracted from the 5th, 10th, and 15th generation recombinant viruses 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 Figure 6). The PCR products were identified by sequencing, indicating the stable presence of the HA gene in the genome.

[0223] Table 5. PCR Primer Instructions 25 / 35 pages 28 CN 121843706 A Instructions 26 / 35 pages 29 CN 121843706 A

[0224] 2. Sterility and Mycoplasma Detection. The harvested rDEVs containing the HA gene of the avian influenza virus subtype H9N2 were subjected to sterility and mycoplasma testing. Sterility testing was performed using standard methods, and mycoplasma testing was performed by qPCR. The results showed that these recombinant viruses were free from exogenous microbial contamination.

[0225] 3. Titer determination of rDEV containing the HA gene of avian influenza virus subtype H9N2: The titer (TCID50) of rDEV containing the HA gene of avian influenza virus subtype H9N2 was determined on CEF.

[0226] Table 6. TCID50 of rDEV

[0227] 4. Expression of rDEV containing the HA gene of avian influenza virus subtype H9N2: rDEV containing the HA gene of avian influenza virus subtype H9N2 was inoculated into CEF of 48-well plates at a dilution of 10⁻²-10⁻⁴. Four hours after inoculation, the medium was replaced with MEM containing 0.75% methylcellulose + 5% FBS, and incubated for 4 days at 37°C in a 5% CO₂ incubator. The expression of DEV virus and HA gene was detected using indirect immunofluorescence assay (IFA).

[0228] The indirect immunofluorescence assay included the following steps. Cell culture medium was removed. The surface was gently washed once with PBS, and 96% cold ethanol was added to each well and fixed for 10 minutes at room temperature. The ethanol was discarded. The wells were allowed to air dry. Then, chicken anti-DEV serum and polyclonal rabbit antibody against H9N2 HA (Sino Biological Inc, catalog number 11229-RP02) at appropriate dilutions were added to the wells, and incubated at 37°C for 1 hour. The antibodies were then discarded. The wells were washed three times with PBS, and 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)) were added and incubated at 37°C for 1 hour. The antibodies were then discarded. The wells were washed three times with PBS and observed 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 the recombinant virus in CEF (see Figure 7).

[0229] Example 5: Safety of Recombinant DEVs in Chickens 5.1. Introduction In this example, recombinant DEVs in chickens were verified, including rDEV4 Δ UL23, rDEV4 Δ US7 US8, rDEV4 Δ US3, rDEV4 Δ UL24, rDEV4 Δ UL39, rDEV4 Δ UL40, rDEV4 Δ UL41, rDEV4 H9HA Δ UL23, rDEV4 H9HA Δ US7 US8, rDEV4 Δ UL40 H9HA Δ UL2, rDEV4 Δ UL24 H9HA Δ UL2, rDEV4 H9HA Δ UL39, rDEV4 H9HA Δ UL40, rDEV4 H9HA Δ UL24, rDEV4 Δ UL41 H9HA Δ US8, rDEV4 H9HA Δ Safety of UL23 Δ UL41, rDEV4 Δ UL24 UL26-H9HA-UL27 and rDEV4 Δ UL39 UL26-H9HA-UL27.

[0230] 5.2. Experimental Design On the day of the experiment, 200 one-day-old SPF experimental chickens were randomly divided into 20 groups, with 10 chickens in each group. Groups 1-18 were the inoculation groups, and the corresponding materials to be tested were subcutaneously inoculated into the chickens through the neck. Group 19 was the positive control group, and DEV4 virus was subcutaneously inoculated into the chickens of Group 19 through the neck. Group 20 was the negative control group, and the same volume of MEM+5% FBS (the dilution of the materials to be tested) was subcutaneously injected into the chickens through the neck. The specific experimental design and grouping are shown in Table 7.

[0231] Table 7. Experimental Design and Grouping Instructions 28 / 35 pages 31 CN 121843706 A

[0232] On the day of the experiment, as shown in Table 7, 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 21 consecutive days; and abnormal symptoms of the experimental chickens were recorded, including: depression, head and neck retraction, ruffled feathers, drooping wings, numbness and weakness of both feet, tearing, eyelid edema, nasal discharge, swelling of the head and neck to varying degrees, and a pulsating sensation when touched.

[0233] 5.3. Morbidity and Mortality of Experimental Chickens After inoculation, all experimental chickens in Group 19 (positive control group) died after inoculation. The experimental chickens in Group 20 (negative control group) did not show any abnormal clinical symptoms or die during the entire experiment.Chickens in groups 4, 5, and 6 (rDEV4 Δ UL24, rDEV4 Δ UL39, rDEV4 Δ UL40) and groups 10–18 expressing H9N2 HA showed no abnormal clinical symptoms or mortality throughout the experiment, with morbidity and mortality rates of 0%. Chickens in other experimental groups (rDEV4 Δ UL23, rDEV4 Δ US7US8, rDEV4 Δ UL41, rDEV4 H9HA Δ US7US8) showed some levels of morbidity and mortality. However, these were significantly lower than those in the DEV4 challenge group. The morbidity and mortality results for each group are shown in Table 8.

[0234] Table 8. Morbidity and mortality rates of experimental chickens in each group. (Instructions for use, pages 29 / 35, CN 121843706 A)

[0235] 5.4. DEV seroconversion in experimental chickens after inoculation. The experimental chickens in group 20 were negative for DEV antibodies throughout the experiment. On day 21 after inoculation, the sera of the experimental chickens in other groups became positive for DEV antibodies. It was confirmed that all chickens in the experimental groups were successfully inoculated without omission. DEV seroconversion in the experimental chickens also demonstrated that these recombinant viruses replicate to a certain extent in vivo.

[0236] 5.5. Conclusion. Wild-type DEV4 is highly pathogenic to chickens and causes 100% morbidity in 1-day-old SPF chickens. By deleting different genes, the strain can be significantly attenuated, making it a safe live viral vector for chickens.

[0237] 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.

[0238] Example 6: Horizontal Transmission Ability of Recombinant DEV among Chickens 6.1. Experimental Design This example aims to evaluate the horizontal transmission ability of the constructed recombinant DEV among 1-day-old SPF chickens. 165 SPF chickens were randomly divided into 11 groups of 15 chickens each, and were transferred to corresponding isolators for feeding after hatching (1 day old). On the day of the experiment (i.e., the day the chickens hatched), 110 1-day-old chickens were randomly divided into 11 groups of 10 chickens each. Groups 1-9 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 10 was the positive control group, and DEV4 virus was subcutaneously inoculated into the chickens via the neck. Group 11 was the negative control group, and the same volume of MEM solution containing 5% FBS (the dilution of the test material, hereinafter referred to as MEM+5% FBS) was subcutaneously injected into the chickens via the neck. In addition, on the day of vaccination, 55 one-day-old SPF chickens from the same batch were selected and placed in separate isolators, randomly divided into 11 groups of 5 chickens each, as contact chickens. 24 hours after vaccination, the contact chickens in each group were transferred to their respective groups.The specific experimental design and grouping are shown in Table 9, on pages 30 / 35 of CN 121843706 A.

[0239] Table 9. Experimental Design and Grouping

[0240] Note: *10 immunized chickens and 5 contact chickens; and the contact chickens were fed in the corresponding groups 24 hours after immunization.

[0241] On the day of the experiment, as shown in Table 9, the experimental chickens in groups 1-9 were subcutaneously inoculated with 0.5 ml of the corresponding recombinant DEV in the neck at an inoculation dose of 106.0 TCID50 / chicken. The chickens in group 10 (as the positive control group) were subcutaneously inoculated with 0.5 ml of DEV4 in the neck at an inoculation dose of 106.0 TCID50 / chicken. The chickens in group 11 (as the negative control group) were subcutaneously injected with 0.5 ml of MEM+5% FBS in the neck.

[0242] After inoculation, all experimental chickens (immunized chickens and contact chickens, including those in the negative control group) were observed once a day for 21 consecutive days; and abnormal symptoms of the experimental chickens were recorded, including: depression, head and neck retraction, ruffled feathers, drooping wings, numbness and weakness in both feet, tearing, eyelid edema, nasal discharge, swelling of varying degrees of head and neck, and a pulsating sensation upon touch.

[0243] On day 14 after inoculation, blood samples were collected from all immunized chickens. Serum was separated to detect DEV seroconversion. On days 14 and 21 after inoculation, blood samples were collected from contact chickens. Serum was separated to detect DEV seroconversion.

[0244] 6.2. DEV seroconversion in experimental chickens after inoculation The experimental chickens in group 11 (negative control) were negative for anti-DEV antibodies throughout the experiment; and on day 14 after inoculation, the serum of immunized chickens in other groups became positive for anti-DEV antibodies. On days 13 and 20 after contact (i.e., days 14 and 21 after immunization), the sera of all contacted chickens in all groups were negative for anti-DEV antibodies. The results of DEV seroconversion in the experimental chickens in each group are shown in Table 10.

[0245] Table 10. DEV seroconversion in experimental chickens in each group Instruction manual 31 / 35 pages 34 CN 121843706 A

[0246] Note: *10 immunized chickens and 5 contact chickens; and the contact chickens were fed in the corresponding groups 24 hours after immunization.

[0247] 6.3. Conclusion According to laboratory tests, no seroconversion was detected in all contact chickens on days 13 and 20 after contact with the immunized chickens, which proves that the recombinant DEVs with missing or inserted H9N2 HA genes involved in this embodiment did not spread at the chicken level. In particular, these recombinant viruses do not cause disease or death in chickens, nor do they cause horizontal transmission among flocks, and can be used as safe vectors in the future.

[0248] Example 7: Efficacy of vaccine candidate strains containing live rDEV vectors expressing H9N2-HA 7.1. Experimental Design In this example, 10 vaccine candidate strains containing live rDEV vectors expressing the HA gene of avian influenza virus (subtype H9) were subcutaneously inoculated into 1-day-old SPF chickens via the neck. On day 28 post-inoculation, SPF chickens were challenged with an avian influenza virus (subtype, instructions page 32 / 35, CN 121843706 A H9) challenge strain (A / chicken / Jiangsu / TX10 / 2010 strain). The aim was to evaluate the immunogenicity of the vaccine candidate strains after challenge.

[0249] The recombinant viruses tested in this embodiment include rDEV4 H9HAΔ US7US8, rDEV4Δ UL40 H9HAΔ UL2, rDEV4 Δ UL24 H9HA Δ UL2, rDEV4 H9HA Δ UL39, rDEV4 H9HA Δ UL40, rDEV4 H9HA Δ UL24, rDEV4 Δ UL41 H9HA Δ US8, rDEV4 H9HA Δ UL23 Δ UL41, rDEV4 Δ UL24 UL26-H9HA-UL27, and rDEV4 Δ UL39 UL26-H9HA-UL27.

[0250] On the day of the experiment (i.e., the day the SPF chickens hatched), 110 one-day-old SPF chickens were randomly divided into 11 groups, with 10 chickens in each group. Groups 1-10 were test groups for vaccine candidate strains containing live rDEV vectors, and group 11 was the control group. As shown in Table 11, all experimental chickens were subcutaneously inoculated with the corresponding test material via the neck. After inoculation, all experimental chickens were clinically observed for 28 consecutive days. On day 28, all experimental chickens were inoculated with 0.2 ml of H9 subtype avian influenza virus via nasal drops at a challenge dose of 106.0 EID50 / chicken. The experimental design and grouping are shown in Table 11.

[0251] Table 11. Experimental Design and Grouping

[0252] After inoculation, all experimental chickens were observed once a day for 28 consecutive days. Abnormal symptoms of the experimental chickens were observed, 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, swelling of the head and neck to varying degrees, and a pulsating sensation upon touch.

[0253] On day 28 post-vaccination (before challenge), blood samples were collected from all chickens; serum was collected by centrifugation for the detection of HI antibodies against H9 subtype avian influenza.

[0254] On day 5 post-challenge, throat and cloacal swabs were collected from all experimental chickens for H9 virus isolation. If the results were negative for virus isolation, the vaccine candidate strain was determined to provide protection for the experimental chickens. The specific operating steps are as follows: On day 5 post-challenge, throat and cloacal swabs were collected from all experimental chickens.The collected cotton swabs were placed in centrifuge tubes containing 1.6 ml of swab buffer containing six antibiotics. All cotton swab samples were inoculated into chicken embryos for virus isolation. The procedure was as follows: Each cotton swab sample was inoculated into five 9-11 day old SPF chicken embryos at a rate of 0.2 ml / embryo through the allantoic cavity, and the embryos were then incubated for 96 hours. The HA titer of all allantoic fluid was then measured. A chicken embryo in which at least one of the inoculated cotton swab samples had an HA titer of at least 1:16 in its allantoic fluid was considered positive for H9 virus isolation. Samples that were negative for virus isolation were tested again after one blind passage. If the virus isolation was still negative after blind passage, it was considered negative for H9 virus isolation; if the virus isolation was positive after blind passage, it was considered positive for H9 virus isolation.

[0255] 7.2. Anti-H9 antibody levels in experimental chickens On day 28 after inoculation, blood was collected from each group of experimental chickens; and serum was separated to detect the level of anti-H9 antibodies. The results showed that the serum of all experimental chickens in group 11 (control group) was negative for H9 hemagglutination inhibition (HI) antibodies; the average HI antibody titer of experimental chickens in groups 4 and 7 was less than 4 log2; and the average HI antibody titer of experimental chickens in other groups was greater than or equal to 4 log2. The average HI antibody titer in the serum of each group of experimental chickens is shown in Table 12 and Figure 8.

[0256] Table 12. Average HI antibody titer in the serum of each group of experimental chickens

[0257] 7.3. Protection rate of recombinant virus against H9N2 On day 5 after challenge, throat and cloacal swabs were collected from all experimental chickens for H9N2 virus isolation. The results showed that 10 experimental chickens in group 11 (attack control group) were positive for virus isolation, that is, the positive rate of virus isolation in the experimental chickens in the attack control group was 100%; and the attack control was effective. The results of H9N2 virus isolation in cotton swab samples and the protection rate against H9N2 in each group are shown in Table 13.

[0258] Table 13. H9N2 virus isolation and protection rate against H9N2 Specification 34 / 35 pages 37 CN 121843706 A

[0259] 7.4. Conclusion The vaccine candidate strains containing the live rDEV vector expressing the HA gene of avian influenza virus (subtype H9) can provide protection against H9N2 to varying degrees, up to 90%, proving that these different insertion sites are effective.Instruction manual, page 35 / 35; 38 CN 121843706 A; Figure 1; Instruction manual, Figure 1 / 6; 39 CN 121843706 A; Figure 2; Instruction manual, Figure 2 / 6; 40 CN 121843706 A; Figure 3; Instruction manual, Figure 3 / 6; 41 CN 121843706 A; Figure 4; Instruction manual, Figure 4 / 6; 42 CN 121843706 A; Figure 5; Figure 6; Instruction manual, Figure 5 / 6; 43 CN 121843706 A; Figure 7; Figure 8; Instruction manual, Figure 6 / 6; 44 CN 121843706 A.

Claims

1. A modified duck enteritis virus (DEV), wherein, One or more genes selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41 and US8 in the DEV genome are inactivated, and wherein the modified DEV has reduced or no mortality in chickens compared with unmodified DEV.

2. A modified duck enteritis virus (DEV) comprising an inactivating gene selected from any of the following i)-vii): i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US8 gene, wherein the inactivating gene is combined with an inactivating combination of one or more other non-essential genes of the DEV, and wherein, Compared to the unmodified DEV, the modified DEV has a reduced mortality rate or no mortality rate in chickens.

3. The modified DEV according to claim 2, wherein, The one or more additional non-essential genes of DEV are different from the first inactivated gene and are selected from: i) the US7 gene; ii) the UL2 gene; iii) the UL41 gene; iv) the US3 gene; v) the UL24 gene; vi) the UL40 gene; vii) the UL39 gene; viiii) the UL23 gene; or ix) the US8 gene.

4. A modified duck enteritis virus (DEV) comprising an inactivating gene selected from any one of i)-xi). i) UL41 gene; ii) US3 gene; iii) UL24 gene; iv) UL40 gene; v) UL39 gene; vi) UL23 gene; vii) US7 and US8 genes; viii) UL24 and UL2 genes; ix) UL40 gene and UL2 gene; x) UL23 and UL41 genes; or xi)UL41 gene and US8 gene, And among them, Compared to the unmodified DEV, the modified DEV has a reduced mortality rate or no mortality rate in chickens.

5. The modified DEV according to any one of the preceding claims, wherein, The gene is inactivated by mutation, interruption, substitution, or deletion of a portion or the entire sequence of the gene.

6. The modified DEV according to any one of the preceding claims, wherein, At least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence of the gene is replaced or deleted.

7. The modified DEV according to any one of the preceding claims, wherein, The modified DEV also contains a heteropolynucleotide encoding a chicken heteroantigen.

8. The modified DEV according to any one of the preceding claims, wherein, The heteropolynucleotide is inserted into the non-essential gene of the modified DEV.

9. The modified DEV according to claim 7 or 8, wherein, The heteropolynucleotide was expressed after the modified DEV had been transfected into suitable chicken host cells.

10. The modified DEV according to any one of claims 7-9, used as a carrier vaccine in chickens.

11. A composition comprising the modified DEV according to any one of claims 7-10.

12. The composition according to claim 11, wherein, The composition is a vaccine.

13. A method for inducing a protective immune response against a chicken pathogen in chickens using the modified DEV according to any one of claims 7-9, the composition according to claim 11 or 12, or the vector vaccine according to claim 10, wherein, The method includes administering one or more doses to the chicken. The modified DEV according to any one of claims 7-9, the composition according to claim 11 or 12, or the vector vaccine according to claim 10, or administered to the chickens once or multiple times. The modified DEV according to any one of claims 7-9, the composition according to claim 11 or 12, or the vector vaccine according to claim 10.

14. A method of vaccinating chickens by inducing a protective immune response against a chicken pathogen, comprising administering the chickens at least once with a modified DEV according to any one of claims 7-9, a composition according to claim 11 or 12, or a vector vaccine according to claim 10.

15. The use according to claim 13, or the method according to claim 14, wherein, The chickens were 1, 2, 3, 4, 5, 6, or 7 days old on the day of vaccination.

16. The use according to claim 13 or 15, or the method according to claim 14 or 15, wherein, The application is administered via the mouth, nose, eye drops, spray, drinking water, intraocularly, intramuscularly, subcutaneously, intradermally, or transdermally.

17. A host cell that expresses the modified DEV according to any one of claims 1-9.

18. The host cell according to claim 17, wherein, The host cells are CEF cells, EB66 cells, or DEF cells.

19. A method for preparing a modified DEV according to any one of claims 1-9, comprising inactivating one or more genes selected from the group consisting of US3, UL24, UL40, UL39, UL23, UL41 and US8 in the DEV genome, wherein, Compared to the unmodified DEV, the inactivation of one or more selected genes results in a reduced or zero mortality rate in the modified DEV in chickens.

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