Recombinant HVT and its uses

Abstract

The present invention relates to a recombinant herpesvirus comprising a recombinant nucleotide sequence encoding at least two different antigens. The present invention is particularly suitable for generating vaccines for immunizing avian species against avian pathogen(s).

Description

[Technical Field]

[0001] The present invention relates to recombinant avian herpesviruses encoding different antigens and their uses. The present invention is suitable for generating vaccines for immunizing avian species against avian pathogen(s). [Background technology]

[0002] Poultry meat and eggs are important food sources, and their consumption is constantly increasing due to the growth of the human population and their great quality-price ratio. To ensure the health of poultry and food safety and security, poultry vaccine technology has become a global concern.

[0003] Viral vectors expressing pathogen proteins are commonly used as poultry vaccines against target pathogens. Vaccines containing such viral vectors induce the expression of foreign pathogen proteins in infected hosts, which can confer protective immunity.

[0004] Many different classes of viruses are being investigated as candidate vectors for avian vaccination, including adenoviruses, AAV, fowlpox viruses, and herpes viruses.

[0005] Three herpesviruses, MDV1, MDV2, and MDV3 (also known as herpesvirus of turkey (HVT)), have been characterized. There is a high degree of similarity between these viruses (see Kingham et al., Journal of General Virology (2001) 82:1123-1135), and all of these have been used to prepare recombinant viruses incorporating foreign genes from pathogens for use as vaccines in birds, particularly poultry such as chickens.

[0006] Although such vaccine preparations provide efficient results for vaccinating avian species against many diseases, when two or more recombinant herpesviruses, each encoding a different antigen, are injected into birds, competition between pathogens and immunosuppression can occur.

[0007] To overcome such interference and to facilitate vaccination against multiple diseases, various attempts have been made to generate polyvalent herpesviruses encoding several antigens.

[0008] Initial studies inserted several genes into a single cloning site in the genome of herpesviruses (see, e.g., European Patent No. EP 1026246). However, such constructs either did not provide the necessary level of protective immunity or proved unstable, with all or part of the foreign genes being deleted during repeated passage in cultured cells.

[0009] WO 2013 / 144355 and WO 2020 / 127964 report stable herpesviruses encoding multiple foreign antigens obtained using a combination of cloning sites located in non-coding regions of the viral genome.

[0010] WO 2013 / 057236, WO 2013 / 082327, and WO 2013 / 082317 report another approach in the design of multivalent HVTs by cloning at least one gene within the US2 coding sequence of a herpesvirus.

[0011] Given the number of pathogens and species, there is a need in the art for additional recombinant polyvalent herpesviruses that are capable of stably expressing multiple genes in vivo and that are suitable for vaccination of avian species, particularly poultry. Summary of the Invention

[0012] The present invention provides a recombinant avian herpesvirus comprising at least two recombinant nucleotide sequences in at least two separate locations in the viral genome.

[0013] More specifically, the present invention provides a recombinant Herpes Virus of Turkeys (HVT) comprising: (i) a nucleotide sequence encoding the F protein of Newcastle disease virus, or an immunogenic fragment or variant thereof, inserted into a first insertion site of the viral genome; and (ii) a nucleotide sequence encoding the hemagglutinin (HA) protein of subtype H9 avian influenza virus, or an immunogenic fragment or variant thereof, inserted into a second insertion site of the viral genome, wherein the first insertion site and the second insertion site are located in different non-coding regions of the viral genome selected from the non-coding region between UL45 and UL46 and the non-coding region between SORF3 and US2.

[0014] The present invention also relates to a nucleic acid comprising the genome of a recombinant HVT as defined above, and to a vector (such as a plasmid) containing such a nucleic acid.

[0015] The present invention also relates to novel antigens and nucleic acid molecules encoding same.

[0016] The present invention further relates to a cell containing a recombinant HVT or a nucleic acid or vector as defined above.

[0017] A further object of the present invention is a composition comprising a recombinant HVT as defined above and a suitable excipient or diluent.

[0018] A further object of the present invention is a composition comprising a nucleic acid or a cell as defined above and a suitable excipient or diluent.

[0019] Another object of the present invention is a vaccine comprising an effective immunizing amount of a recombinant HVT, nucleic acid and / or cell as defined above.

[0020] A further object of the present invention resides in a recombinant HVT, nucleic acid, or cell as defined above for use in immunizing birds, such as poultry, against Newcastle Disease Virus (NDV) and Avian Influenza Virus (AIV), and / or related diseases.

[0021] A further object of the present invention resides in a recombinant HVT, nucleic acid or cell as defined above for use in protecting birds, such as poultry, against diseases caused by NDV and AIV.

[0022] A further object of the present invention is a vaccine as defined above for use in vaccinating birds, such as poultry, against NDV and AIV.

[0023] A further object of the present invention is a method for vaccinating birds, comprising administering to said birds a composition or a vaccine or virus as defined above.

[0024] A further object of the present invention is a method for inducing an immune response to an antigen in an avian species, comprising administering to said avian species a composition or a vaccine or a virus as defined above.

[0025] The present invention also provides a vaccination kit for immunizing birds, comprising the following components: a. an effective amount of a composition or vaccine, as defined above, and b. A means for administering the composition or vaccine to the bird.

[0026] The present invention can be used in any avian species for vaccination against NDV and / or AIV, and / or related disorders or conditions. The present invention is particularly suitable for vaccinating poultry such as chickens. [Brief explanation of the drawings]

[0027] [Figure 1] 1 shows schematic diagrams of recombinant bivalent HVT constructs (FW205, FW206, FW208, FW209, FW247, FW248, FW249, FW250, FW251, FW252) with the NDV F gene and AIV HA-H9 gene according to the present invention, and recombinant monovalent HVT constructs (FW202, FW204) with the NDV F gene (FW168) or AIV HA-H9 gene. [Figure 2] FIG. 1 shows the results of immunofluorescence assays demonstrating expression of (A) NDV F protein (green fluorescence), (B) AIV HA-H9 protein (red fluorescence), and (C) a mix (yellow) by the rHVT / ND-H9 construct. [Figure 3] 1 shows the results of a Western blot assay detecting expression of NDV F protein by constructs FW168, FW205, FW206, FW208, and FW209. [Figure 4] 1 shows the results of a Western blot assay detecting the expression of AIV HA-H9 protein by constructs FW205, FW206, FW208, and FW209. [Figure 5] Figure 1 shows the results of a Western blot assay detecting expression of NDV F protein by constructs FW247, FW248, FW249, FW250, FW251, FW252, FW168, and FW206. [Figure 6] Figure 1 shows the results of a Western blot assay detecting expression of AIV HA-H9 protein by constructs FW247, FW248, FW249, FW250, FW251, FW252, FW204, and FW206. [Figure 7]Figure 1 shows NDV ELISA titers in chickens vaccinated with constructs FW205, FW206, and FW208 using a commercially available NDV ELISA kit. [Figure 8A] AIV H9 HI titers in chickens vaccinated with constructs FW205 and FW208 (FIG. 8A) and FW206 (FIG. 8B) are shown. [Figure 8B] AIV H9 HI titers in chickens vaccinated with constructs FW205 and FW208 (FIG. 8A) and FW206 (FIG. 8B) are shown. [Figure 9] Figure 1 shows AIV loads in tracheal swabs of chickens vaccinated with constructs FW205, FW206, and FW208 after challenge with AIV of the H9 subtype. [Figure 10] Air sac lesion scores of chickens vaccinated with constructs FW205, FW206, and FW208 after challenge with AIV of the H9 subtype are shown. [Figure 11] Figure 1 shows NDV ELISA titers in chickens vaccinated with constructs FW247, FW248, FW249, FW250, FW251, and FW252 using a commercially available NDV ELISA kit. [Figure 12] Figure 1 shows AIV H9 HI titers in chickens vaccinated with constructs FW247, FW248, FW249, FW250, FW251, and FW252 using a commercially available NDV ELISA kit. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates generally to recombinant avian herpesviruses containing multiple recombinant nucleotide sequences, their production, compositions containing them, and their uses. The present invention also provides novel antigens suitable for generating a potent immune response against AIV.

[0029] definition This disclosure will be best understood by reference to the following definitions: The term "recombinant," in the context of a herpesvirus, refers to a herpesvirus whose genome has been altered by the insertion of at least one nucleotide sequence (e.g., DNA such as a gene) that is not naturally found in the genome of the herpesvirus, or that is naturally found in the genome but in a different form or location. It will be understood that recombinant herpesviruses can be produced by a variety of methods, including the recombinant DNA techniques described therein, and that once produced, they can be replicated without the further use of recombinant DNA techniques.

[0030] As used herein, the terms "nucleic acid," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably and refer to a nucleic acid molecule having a determined sequence, which may be deoxyribonucleotides and / or ribonucleotides. A nucleotide sequence may be initially prepared, for example, by recombinant, enzymatic, and / or chemical techniques, and then replicated in a host cell or in vitro system. A nucleotide sequence preferentially contains an open reading frame encoding a molecule (e.g., a peptide or protein). A nucleotide sequence may contain additional sequences, such as, for example, a promoter, a transcription terminator, a signal peptide, an IRES, etc.

[0031] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably and refer to any molecule comprising a polymer of at least two consecutive amino acids.

[0032] The term "non-coding region" is well known in the art and refers to any region of a viral genome that does not encode a protein. The non-coding region between UL45 (HVT053) and UL46 (HVT054) typically refers to the region that begins immediately 3' from the stop codon of UL45 and ends immediately 5' from the stop codon of UL46 (because both ORFs are in opposite orientations). The non-coding region between SORF3 (HVT087) and US2 (HVT088) typically refers to the region that begins immediately 3' from the start codon of SORF3 and ends immediately 5' from the stop codon of US2.

[0033] An "immunogenic fragment" of an antigen refers to any fragment capable of eliciting an immune response, preferably any fragment containing an epitope, preferably an antigen-specific epitope. Immunogenic fragments generally contain 5 to 50 consecutive amino acid residues of the antigen, such as 5 to 40, or 10 to 40, or 10 to 30, 10 to 25, or 10 to 20. Examples of fragments of a native F protein include any fragment of 10 to 40 consecutive amino acids of SEQ ID NO: 1.

[0034] As used herein, the term "variant" refers to a modified form of a reference antigen or fragment that retains its immunogenic properties. Generally, variants are similar overall and, in many regions, identical to the reference antigen or fragment. By way of example, a variant may exhibit at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity compared to the reference antigen or fragment. Variants specifically refer to antigens that have one, two, three, four, or five altered amino acid residues compared to the reference sequence. Modifications include amino acid deletion(s), substitution(s), and / or addition(s). Variants are intended to retain the immunogenic properties of the reference sequence, such as the ability to induce an immune response against the reference sequence or pathogen. Examples of variants of native F proteins include any protein comprising or consisting of SEQ ID NO: 1 with one, two, or three amino acid substitutions. Examples of fragment variants include proteins consisting of 10 to 40 consecutive amino acids of SEQ ID NO: 1 with one, two, or three amino acid substitutions.

[0035] The term "avian species" is intended to encompass birds of the avian class, i.e., all types of birds, including feathered, winged, bipedal, endothermic, and egg-laying vertebrates. In the context of the present invention, birds or avian species refer more specifically to birds of economic and / or agricultural interest, such as poultry (such as chickens and turkeys), waterfowl (such as ducks and geese), and ornamental birds (such as swans and parrots).

[0036] As used herein, the term "vaccine" refers to an agent that can be used to induce, stimulate, or amplify an immune response in an organism.

[0037] Recombinant HVT The present invention relates to a recombinant HVT containing multiple foreign genes at specific locations. More specifically, the present invention relates to a recombinant HVT containing multiple foreign genes at specific locations. (i) a nucleotide sequence encoding the F protein of Newcastle disease virus, or an immunogenic fragment or variant thereof, inserted into a first insertion site of the viral genome; (ii) a nucleotide sequence encoding a hemagglutinin (HA) protein of a subtype H9 avian influenza virus, or an immunogenic fragment or variant thereof, inserted into a second insertion site of the viral genome; and Regarding recombinant HVT (rHVT), The first and second insertion sites are located in different non-coding regions of the viral genome selected from the non-coding region between UL45 and UL46 and the non-coding region between SORF3 and US2.

[0038] In certain embodiments, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a nucleotide sequence encoding the F protein of Newcastle disease virus, or an immunogenic fragment or variant thereof, inserted into a non-coding region of the viral genome located between UL45 and UL46; (ii) a nucleotide sequence encoding a hemagglutinin (HA) protein of a subtype H9 avian influenza virus, or an immunogenic fragment or variant thereof, inserted into a non-coding region of the viral genome located between SORF3 and US2; Regarding rHVT, including

[0039] In a further particular embodiment, the present invention provides a method for producing a pharmaceutical composition comprising: (i) a nucleotide sequence encoding the F protein of Newcastle disease virus, or an immunogenic fragment or variant thereof, inserted into a non-coding region of the viral genome located between SORF3 and US2; (ii) a nucleotide sequence encoding a hemagglutinin (HA) protein of a subtype H9 avian influenza virus, or an immunogenic fragment or variant thereof, inserted into a non-coding region of the viral genome located between UL45 and UL46; Regarding rHVT, including

[0040] As shown in the Examples, such constructs are genetically stable in CEF cells for at least 10, preferably at least 15, and more preferably at least 20 passages. Such constructs also provide stable co-expression of antigens in CEF cells for at least 10, preferably at least 15, and more preferably at least 20 passages. They can confer strong and sustained expression of genes in vivo sufficient to confer high protective immunity.

[0041] More specifically, the inventors demonstrated that the claimed rHVT accurately expresses both the NDV F antigen and the AIV HA-H9 antigen (FIGS. 2-6). The inventors also demonstrated that the claimed rHVT efficiently induces antibodies against NDV F and AIV HA-H9 (FIGS. 7, 8, 11, and 12) with high HI titers. The data in this application further demonstrate that SPF chickens vaccinated with the bivalent construct of the present invention are highly efficiently protected (e.g., up to 100%) against Newcastle Disease (ND) and AIV after challenge, and that all vaccinated groups have lower AIV viral loads compared to the control group.

[0042] Thus, the claimed rHVT confers highly efficient clinical protection against challenge with NDV and AIV. Thus, the present invention provides novel, effective constructs that can be used to protect avian species from highly relevant pathogens and related disorders.

[0043] The recombinant HVT of the present invention can be prepared from any HVT, preferably a non-pathogenic HVT. An example of a non-pathogenic strain of HVT (MDV3) suitable for use in the present invention is the FC126 strain. The genome sequence of the FC126 strain is available in the art (Afonso et al., supra; Kingham et al., supra). Another suitable HVT strain is, for example, the PB1 strain. Any other non-pathogenic strain is also suitable.

[0044] The location of the target non-coding region in the viral genome can be easily identified by one skilled in the art using the teachings of this application, general knowledge, and sequence information available in the literature. For example, Kingham et al. (supra) report the nucleotide sequence of the FC126 reference strain and the location of most of the ORFs within its genome.

[0045] With reference to the FC126 complete genome (GenBank: AF291866.1), the non-coding region between UL45 and UL46 corresponds to nucleotides 95323 to 95443 of the HVT genome, and the non-coding region between SORF3 and US2 corresponds to nucleotides 139867 to 140064 of the HVT genome. Cloning at any position within these regions is suitable for the present invention.

[0046] NDV F protein As shown, the claimed rHVT contains a recombinant nucleotide sequence encoding the F protein of NDV, or an immunogenic fragment or variant thereof.

[0047] The NDV F protein is the F protein of Newcastle disease virus (also called avian paramyxovirus type 1 virus) and is a class I viral membrane fusion (F) glycoprotein that mediates cell membrane penetration during viral entry into cells. NDV F is a known antigen of NDV. The amino acid sequence of native NDV F protein is well known and published, for example, under numbers such as AAU89279, ABA39232, and AAA46643, as well as any natural variants thereof (polymorphisms, splicing variants, etc.). Exemplary sequences are provided as SEQ ID NO: 1 (protein) and SEQ ID NO: 14 (nucleic acid).

[0048] The protein encoded by the claimed rHVT can be any native NDV F protein, or any immunogenic fragment or variant thereof, capable of inducing an anti-NDV immune response.

[0049] Examples of fragments of a native F protein include any fragment of 10 to 40 consecutive amino acids of SEQ ID NO: 1. Examples of variants of a native F protein include any protein comprising or consisting of SEQ ID NO: 1 with one, two, or three amino acid substitutions. Examples of variant fragments include proteins consisting of 10 to 40 consecutive amino acids of SEQ ID NO: 1 with one, two, or three amino acid substitutions.

[0050] AIV HA protein As shown, the claimed rHVT contains a recombinant nucleotide sequence encoding the surface protein hemagglutinin (HA) of subtype H9 avian influenza virus, or an immunogenic fragment or variant thereof.

[0051] Influenza A viruses are classified into subtypes based on the serological reactivity of the HA surface protein. HA serological subtyping is performed by hemagglutinin inhibition testing. Sixteen HA subtypes, HA1 to HA16, have been recognized for AIVs (David E. Swayne, David L. Suarez, and Leslie D. Simes. (2013). Influenza. In David E. Swayne (ed.). Diseases of Poultry, 13th ed. (pp. 181-218)). Any AIV can be easily classified into any such subtype according to the above techniques and common sense. Subtype H9 further includes a specific subclass designated H9N2. Examples of subtype H9 AIV strains include A / turkey / Wisconsin / 1 / 1966 (H9N2), A / quail / Hong Kong / G1 / 1997 (H9N2), and A / duck / Hong Kong / Y439 / 1997 (H9N2). Preferably, the HA protein is derived from an H9N2 subtype AIV.

[0052] The HA protein can be any native HA protein of subtype H9 AIV, preferably H9N2 subtype AIV.

[0053] Alternatively, the claimed constructs may encode immunogenic fragments or variants (as defined above) of the surface protein hemagglutinin (HA) of H9 subtype AIV, preferably H9N2 subtype AIV, capable of inducing an anti-AIV immune response. In this regard, as detailed in the experimental section, the inventors have designed and synthesized optimized H9 HA antigens with strong immunogenicity and cross-reactivity. Such antigens are disclosed as H9-CS (SEQ ID NO: 2), H9-CNn1 (SEQ ID NO: 3), H9-CNn2 (SEQ ID NO: 4), H9-CNn3 (SEQ ID NO: 5), H9-CNn4 (SEQ ID NO: 6), and H9-CNn5 (SEQ ID NO: 7). The sequences and antigens represent specific objects of the present invention, as well as any vectors containing them and their uses.

[0054] In this regard, the present invention relates to polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NOs: 2 to 7, and any polypeptide having at least 97% amino acid sequence identity, preferably at least 98%, and even more preferably at least 99% amino acid sequence identity over its entire length to any one of SEQ ID NOs: 2 to 7. Amino acid sequence identity can be determined using any known technique or computer program, such as, for example, BLAST.

[0055] A particular object of the invention is a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2. Another specific object of the invention is a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:3. Another specific object of the invention is a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:4. Another specific object of the invention is a polypeptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:5. Another specific object of the invention is a polypeptide comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO:6. Another specific object of the invention is a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:7.

[0056] The present invention also relates to chimeric molecules comprising the above polypeptides conjugated to a structural moiety, which may be a polypeptide.

[0057] The present invention also relates to nucleic acids encoding the polypeptides defined above, as well as any vectors or cells containing such nucleic acids. Preferred nucleic acid molecules of the present invention comprise, consist essentially of, or consist of a sequence selected from any one of SEQ ID NOs: 8 to 13. The nucleic acid may be conjugated to regulatory sequences (such as a promoter and / or terminator) and / or may be comprised in any cloning or expression vector (e.g., a plasmid, virus, BAC, etc.).

[0058] Other recombination sequences A recombinant HVT according to the present invention may further comprise one or more additional sequences encoding, for example, one or more antigens, cytokines, hormones, costimulatory factors, adjuvants, and the like.

[0059] The recombinant nucleotide sequence inserted into the genome can be in any orientation.

[0060] Promoter The inserted nucleic acid sequence may contain (or be operably linked to) regulatory sequences such as a promoter and / or terminator. The promoter used may be either a synthetic or natural promoter, an endogenous promoter, or a heterologous promoter. In principle, any promoter can be used as long as it can function effectively in the target cell or host. In this regard, the promoter may be a eukaryotic, prokaryotic, viral, or synthetic promoter capable of directing gene transcription in avian cells in the context of a polyvalent vector. Furthermore, each inserted nucleic acid sequence may contain a promoter that may be the same or different from each other. In certain embodiments, each inserted nucleic acid sequence contains a different promoter.

[0061] Preferentially, the promoter used for each inserted nucleic acid sequence is selected from the Pec promoter, the cytomegalovirus (CMV) immediate early 1 (ie1) promoter, in particular the murine cytomegalovirus (Mcmv) ie1 promoter or the human cytomegalovirus (Hcmv) promoter, the chicken beta-actin (Bac) promoter, the Simian virus 40 (SV40) promoter, and the Rous sarcoma virus (RSV) promoter, or any fragment thereof that retains promoter activity.

[0062] Preferentially, the NDV F coding sequence and the AIV HA coding sequence are under the control of different promoters.

[0063] In a preferred embodiment, one coding sequence in the rHVT of the present invention is linked to the Pec promoter.

[0064] In another preferred embodiment, one coding sequence in the rHVT of the present invention is linked to a CMV ie1 promoter, particularly a murine cytomegalovirus (Mcmv) ie1 promoter, or a human cytomegalovirus (Hcmv) promoter.

[0065] The nucleic acid sequence of the Pec promoter is shown in SEQ ID NO: 15, and the sequence of the Mcmv ie1 promoter is shown in SEQ ID NO: 16. It should be noted that variants of such sequences that encode functional promoters are known and / or can be designed / tested by the skilled artisan for use in the present invention.

[0066] In a preferred embodiment, the recombinant nucleotide sequence inserted into the non-coding region located between UL45 and UL46 contains the Pec promoter, and the recombinant nucleotide sequence inserted into the non-coding region located between SORF3 and US2 contains the CMV IE1 promoter, in particular the Mcmv ie1 promoter. The results obtained by the inventors show that such promoters are particularly efficient when placed in the cloning site in the context of the polyvalent vectors of the invention.

[0067] In another preferred embodiment, the foreign gene inserted into the non-coding region located between UL45 and UL46 contains the CMV IE1 promoter, in particular the Mcmv ie1 promoter, and the recombinant nucleotide sequence inserted into the non-coding region located between SORF3 and US2 contains the Pec promoter. Results obtained by the present inventors show that such promoters are also particularly efficient when placed in the cloning site in the context of the polyvalent vectors of the present invention.

[0068] Preferably, the recombinant HVT of the present invention comprises (i) a nucleotide sequence encoding the F protein of Newcastle disease virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between UL45 and UL46 under the control of a Pec promoter, and (ii) a nucleotide sequence encoding the hemagglutinin (HA) protein of subtype H9 avian influenza virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between SORF3 and US2 under the control of a CMV IE1 promoter, preferably an Mcmv ie1 promoter.

[0069] In another preferred embodiment, the recombinant HVT of the present invention comprises (i) a nucleotide sequence encoding the hemagglutinin (HA) protein of a subtype H9 avian influenza virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between UL45 and UL46 under the control of a CMV IE1 promoter, preferably the Mcmv ie1 promoter, and (ii) a nucleotide sequence encoding the F protein of a Newcastle disease virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between SORF3 and US2 under the control of a Pec promoter.

[0070] In another embodiment, the recombinant HVT of the present invention comprises (i) a nucleotide sequence encoding the F protein of Newcastle disease virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between UL45 and UL46 under the control of a Pec promoter, and (ii) a nucleotide sequence encoding the hemagglutinin (HA) protein of subtype H9 avian influenza virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between SORF3 and US2 under the control of a CMV IE1 promoter, preferably an Hcmv promoter.

[0071] How to build it The recombinant HVT of the present invention can be prepared using techniques known per se in the art, such as recombinant techniques, homologous recombination, site-specific insertion, mutagenesis, and the like.

[0072] Gene cloning and plasmid construction are well known to those skilled in the art and can be essentially carried out by standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th ed., Cold Spring Harbor Laboratory Press, Woodbury, NY (2012)).

[0073] Herpesviruses can be propagated in any suitable host cell and culture medium. Examples of suitable host and growth conditions for herpesviruses include chicken-derived cells such as chicken embryo fibroblasts (CEF) and chicken kidney cells. These cells can be cultured in a culture medium such as Eagle's MEM or Leibowitz-L-15 / McCoy 5A (1:1 mixture) at approximately 37°C for 3 to 4 days.

[0074] Genomic DNA can be extracted from virus-infected cells according to any conventional method, in particular, after denaturing and removing proteins in a lysis buffer, the DNA can be extracted with phenol and ethanol.

[0075] Typically, recombinant viruses can be prepared by homologous recombination between the viral genome and a construct (e.g., a plasmid) containing the recombinant nucleotide sequence or nucleic acid to be inserted and flanked by nucleotides from the insertion site that enable recombination. Briefly, a sequence containing the target region is first cloned into a plasmid or other suitable vector. Examples of plasmids include pBR322, pBR325, pBR327, pBR328, pUC18, pUC19, pUC7, pUC8, and pUC9; examples of phages include lambda phage and M13 phage; and an example of a cosmid is pHC79. The cloning region should preferably be long enough so that upon insertion of a foreign gene, the sequences flanking the foreign gene are of appropriate length to allow in vitro homologous recombination with the viral genome. Preferably, each flanking sequence is at least approximately 50 nucleotides in length.

[0076] To insert one or more recombinant nucleotide sequences into a target region, mutations can be performed at specific sites in the region to create restriction enzyme cleavage sites. Conventional methods for mutations can be used, including in vitro mutagenesis and PCR, which are commonly used by those skilled in the art. Thus, in PCR, mutations such as deletion, substitution, or addition of one or two nucleotides are made in PCR primers, and then mutations are created using the primers. Alternatively, naturally occurring restriction sites can be used. The foreign gene (and promoter) is then inserted into the insertion site of the viral genome in the plasmid.

[0077] The resulting plasmid can be introduced into HVT-infected cells or HVT genome-transfected cells using any suitable technique (e.g., electroporation, calcium phosphate, lipofectin-based methods, etc.). When the amount of introduced plasmid is within the range of 0.1 μg to 1000 μg, the efficiency of recombinant virus production by recombination between the homologous region of the HVT genome and the plasmid is high in cells. This leads to a recombination event between the plasmid and the viral genome, resulting in the insertion of the recombinant nucleotide sequence into the virus.

[0078] The resulting recombinant viruses can be selected genotypically or phenotypically using known techniques, for example, by hybridization, detection of an enzymatic activity encoded by a gene cointegrated with the recombinant nucleic acid sequence, or immunological selection by detection of an antigenic peptide expressed by the recombinant herpesvirus. Selected recombinant herpesviruses can be grown on a large scale in cell culture. Once produced, the viruses can be propagated in suitable cells.

[0079] Preferred Embodiments The following recombinant HVTs are preferred specific embodiments of the present invention, which, as shown in the examples, enable a strong immune response in vivo against the antigen encoded by each recombinant nucleotide sequence.

[0080] A particularly preferred recombinant HVT (rHVT) of the present invention comprises (i) a nucleotide sequence encoding the F protein of Newcastle disease virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between UL45 and UL46 under the control of the Pec promoter, and (ii) a nucleotide sequence encoding the hemagglutinin (HA) protein of subtype H9 avian influenza virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between SORF3 and US2 under the control of the Mcmv ie1 promoter. Preferably, such a recombinant rHVT is selected from the following bivalent constructs, as described in experimental data: ·HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CS(FW205), ·HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CN(FW206), ·HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn1(FW247), ·HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn2(FW248), ·HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn3(FW249), ·HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn4(FW250), and ·HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn5(FW251).

[0081] In a preferred embodiment, the rHVT of the present invention encodes an HA antigen that comprises, consists essentially of, or consists of a sequence selected from any one of SEQ ID NOs: 2 to 7.

[0082] In a preferred embodiment, the rHVT of the present invention encodes an F antigen that comprises, consists essentially of, or consists of SEQ ID NO: 1 or a naturally occurring variant thereof.

[0083] In a preferred embodiment, an rHVT of the present invention contains a nucleic acid encoding an F antigen that comprises, consists essentially of, or consists of SEQ ID NO:14.

[0084] In a preferred embodiment, the rHVT of the present invention is HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CS (FW205), which comprises the consensus sequence of the hemagglutinin gene of avian influenza virus H9 subtype (H9-CS) of SEQ ID NO:8.

[0085] In another preferred embodiment, the rHVT of the present invention is HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn3 (FW249), which comprises an artificially designed hemagglutinin gene of the avian influenza virus H9 subtype (H9-CNn3) of SEQ ID NO: 11.

[0086] In another preferred embodiment, the rHVT according to the present invention is selected from the following bivalent constructs: HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn1 (FW247), which contains the artificially designed hemagglutinin gene of the avian influenza virus H9 subtype (H9-Cnn1) of SEQ ID NO: 9; HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn2(FW248), which contains the artificially designed hemagglutinin gene of the avian influenza virus H9 subtype (H9-CNn2) of SEQ ID NO: 10; HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn4(FW250), which contains the artificially designed hemagglutinin gene of the avian influenza virus H9 subtype (H9-CNn4) of SEQ ID NO: 12; HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn5(FW251), which contains an artificially designed hemagglutinin gene of avian influenza virus H9 subtype (H9-CNn5) of SEQ ID NO: 13.

[0087] Another preferred recombinant rHVT of the present invention comprises (i) a nucleotide sequence encoding the hemagglutinin (HA) of subtype H9 avian influenza virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between UL45 and UL46 under the control of the Mcmv ie1 promoter, and (ii) a nucleotide sequence encoding the F protein of Newcastle disease virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between SORF3 and US2 under the control of the Pec promoter. Preferably, such a rHVT is selected from the following bivalent constructs, as described in the experimental data: ·HVT / 45-46 Mcmv ie1 H9-CS / 87-88 PecF(FW208), and ·HVT / 45-46 Mcmv ie1 H9-CN / 87-88 PecF(FW209).

[0088] In another embodiment, the rHVT of the present invention comprises (i) a nucleotide sequence encoding the F protein of Newcastle disease virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between UL45 and UL46 under the control of the Pec promoter, and (ii) a nucleotide sequence encoding the hemagglutinin (HA) protein of subtype H9 avian influenza virus or an immunogenic fragment or variant thereof, inserted into the non-coding region between SORF3 and US2 under the control of the Hcmv promoter. Preferably, such a rHVT is the bivalent construct HVT / 45-46 PecF / 87-88 Hcmv H9-CNn1(FW252), as described in the experimental data.

[0089] Particularly preferred rHVTs of the present invention are prepared using strains FC126 or PB1.

[0090] The recombinant HVT of the present invention can be propagated in cell culture. In a preferred embodiment, CEF, embryonated chicken eggs, chicken kidney cells, or the like are used as host cells for propagating the recombinant HVT. The polyvalent recombinant HVT of the present invention can be cultured in a medium such as Eagle's MEM or Leibowitz-L-15 / McCoy 5A (1:1 mixture) medium at approximately 37°C for 3 to 4 days. The resulting infected cells are then suspended in a medium containing 10% dimethyl sulfoxide (DMSO) and frozen and stored under liquid nitrogen.

[0091] Advantageously, the recombinant HVTs of the present invention exhibit a high level of stability. They are genetically stable, meaning that they maintain the inserted gene in avian cells, preferably CEF cells, after 10 or more passages, preferably 15 or more passages, and more preferably even after 20 or more passages. They also provide stable expression of antigens, meaning that they are co-expressed in avian cells, preferably CEF cells, after 10 or more passages, preferably 15 or more passages, and even more preferably even after 20 or more passages. In the context of the present invention, "passage" or "cell passaging" refers to the cultivation of cells under conditions suitable for allowing the cells to grow and survive until they reach 90% to 100% confluence. The passaging process consists of transferring a small number of cells from a previously confluent culture into new culture medium. An aliquot of a previously confluent culture containing a small number of cells can be diluted with a large amount of fresh medium.

[0092] The viruses can be harvested or purified using conventional techniques. They can be stored in any suitable medium, frozen, and / or lyophilized.

[0093] Nucleic Acids and Cells A further object of the present invention relates to any nucleic acid contained in a virus as defined above. The nucleic acid may be single-stranded or double-stranded, DNA or RNA, or a variant thereof.

[0094] The present invention also relates to vectors (eg, plasmids, cosmids, artificial chromosomes, etc.) comprising the nucleic acids of the present invention.

[0095] The present invention also relates to cells containing a recombinant HVT, nucleic acid, or vector of the invention. The cells are typically eukaryotic cells, such as avian cells, or prokaryotic cells (if the vector is suitable for replication or maintenance in such cell types).

[0096] Vaccine Composition The present invention also relates to compositions, such as vaccines, comprising the multivalent recombinant HVT of the present invention, the nucleic acid of the present invention, or the cell of the present invention.

[0097] The vaccine of the present invention typically contains an immunologically effective amount of the above-mentioned recombinant HVT in a pharmaceutically acceptable vehicle.

[0098] Compositions and vaccines according to the invention typically contain a suitable solvent or diluent or excipient, such as, for example, an aqueous buffer or a phosphate buffer. The compositions may also contain additives, such as proteins or peptides derived from animals (e.g., hormones, cytokines, costimulatory factors), nucleic acids derived from viruses and other sources (e.g., double-stranded RNA, CpG), etc., administered with the vaccine in an amount sufficient to enhance the immune response. In addition, combinations of any number of the foregoing substances may provide an immunostimulatory effect and thus form an immunostimulant of the invention.

[0099] The vaccines of the present invention may be further formulated with one or more additional additives to maintain isotonicity, physiological pH, and stability, such as a buffer such as saline (0.85%), phosphate buffered saline (PBS), citrate buffer, Tris(hydroxymethylaminomethane (TRIS), Tris-buffered saline, or an antibiotic such as neomycin or streptomycin.

[0100] The route of administration can be any route, including oral, ocular (e.g., eye drops), oculonasal administration using aerosol, intranasal, in-feed, in water, or cloacal by spray, intraembryonic, topical, or by injection (e.g., intravenous, subcutaneous, intramuscular, intraorbital, intraocular, intradermal, and / or intraperitoneal). One skilled in the art will readily adapt the formulation of the vaccine composition for each type of administration route.

[0101] Each vaccine dose may contain a suitable amount sufficient to induce a protective immune response in the avian species. Optimization of such doses is well known in the art. The amount of antigen per dose can be determined by known methods using antigen / antibody reactions, such as ELISA.

[0102] The vaccines of the present invention can be administered as a single dose or multiple doses, depending on the vaccination protocol.

[0103] The vaccines of the present invention are further advantageous in that they confer up to 100% protection in avian species against targeted avian pathogens after three weeks of vaccination.

[0104] The present invention further relates to the use of the above vaccines for immunizing avian species, such as poultry, against pathogens.

[0105] The present invention further relates to a method of immunizing avian species by administering an immunologically effective amount of a vaccine according to the invention. The vaccine may conveniently be administered intradermally, subcutaneously, intramuscularly, orally, intraembryonic, mucosally, or by oculonasal administration.

[0106] The present invention further relates to vaccination kits for immunizing avian species, comprising an effective amount of the multivalent vaccine described above and means for administering the components to the species. For example, such kits may comprise an injection device loaded with the multivalent vaccine of the invention and instructions for intradermal, subcutaneous, intramuscular, or intraembryonic injection. Alternatively, the kit may comprise a spray / aerosol or eye drop device loaded with the multivalent vaccine of the invention and instructions for oculonasal, oral, or mucosal administration.

[0107] Further aspects and advantages of the present application will now be disclosed in the following examples which illustrate the invention. [Example]

[0108] In the following examples, recombinant HVTs (rHVTs) were prepared and used, which are designated according to the following nomenclature: - HVT / insertion site, promoter, inserted antigen (monovalent construct); - HVT / first insertion site, first promoter, first insertion antigen / second insertion site, second promoter, second insertion antigen (bivalent construct).

[0109] List of rHVTs prepared and used in the examples: FW168:HVT / 45-46 PecF (monovalent) FW202:HVT / 45-46 Mcmv ie1 H9-CN(monovalent) FW204:HVT / 45-46 Mcmv ie1 H9-CS(monovalent) FW205:HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CS (bivalent) FW206:HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CN (bivalent) FW208:HVT / 45-46 Mcmv ie1 H9-CS / 87-88 PecF (bivalent) FW209:HVT / 45-46 Mcmv ie1 H9-CN / 87-88 PecF(bivalent) FW247:HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn1 (bivalent) FW248:HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn2 (bivalent) FW249:HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn3 (bivalent) FW250:HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn4 (bivalent) FW251:HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CNn5 (bivalent) FW252:HVT / 45-46 PecF / 87-88 Hcmv H9-CNn1 (bivalent)

[0110] Example 1: Design and synthesis of consensus sequences for the hemagglutinin gene of avian influenza virus H9 subtype Design and synthesis of a consensus sequence called H9-CS A consensus sequence for the hemagglutinin (HA) gene of avian influenza virus (AIV) H9 subtype was designed based on phylogenetic analysis and synthesized as follows to maximize the breadth of protection among H9N2 AIV isolates.

[0111] First, over 80 HA gene sequences from recent H9N2 strains were collected from public databases. Then, a phylogenetic tree was constructed and the center of tree (COT) and most recent common ancestor (MRCA) sequences were identified. COT and MRCA analyses are computational methods developed to minimize the negative impact of antigenic diversity on vaccine immunogenicity (Kesturu et al., 2006). We also closely examined sequence alignments, particularly in predicted antigenic regions and N-glycosylation sites, to ensure the selection of representative sequences. These analyses led us to design a single sequence, designated H9-CS, that offers broad cross-reactivity to various H9 strains.

[0112] The H9-CS gene sequence was artificially synthesized. The amino and nucleic acid sequences of H9-CS are provided as SEQ ID NO:2 and SEQ ID NO:8, respectively.

[0113] Design and synthesis of sequences H9-CNn1 to H9-CNn5 To maximize the breadth of protection among H9N2 AIV isolates, five AIV H9 subtype antigens were designed and synthesized as follows: The antigens were further designed based on known HA gene sequences from the AIV H9 subtype, referred to herein as H9-CN (A / chicken / Henan / H24 / 2011; GenBank accession number JN804297).

[0114] We collected several sequences of H9 strains, including seven Chinese isolates from 2016. Based on these H9 sequences, we performed computational protein modeling analysis and selected four antigens designated H9-CNn1-4 (SEQ ID NO: 4), H9-CNn2 (SEQ ID NO: 5), H9-CNn3 (SEQ ID NO: 6), and H9-CNn4 (SEQ ID NO: 7). H9-CNn5 was designed by replacing the transmembrane domain (TM) of H9-CNn1 with the transmembrane domain of the H3 subtype of AIV to further enhance molecular stability and cross-reactivity.

[0115] The amino acid sequences of H9-CNn1-5 are provided as SEQ ID NOs: 3 to 7, respectively. Nucleic acid sequences encoding H9-CNn1-5 are provided as SEQ ID NOs: 9 to 13, respectively.

[0116] Example 2: Construction of recombinant HVT 2.1. Construction of homology vectors Plasmid construction is essentially carried out by standard molecular biology techniques (Molecular Cloning: A Laboratory Manual. 4th Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA, 2012).

[0117] Construction of p45 / 46Mcmv ie1 H9-CS and p45 / 46Mcmv ie1 H9-CN The murine cytomegalovirus (Mcmv) ie1 promoter (SEQ ID NO: 16) was synthesized in a pUC18-based vector, resulting in pGI Mcmv ie1.

[0118] A poly A signal (SPA: SEQ ID NO: 17) was also synthesized and inserted into pGI Mcmv ie1 cut with SalI and SfiI, yielding pGI Mcmv ie1 SPA. The Mcmv ie1 promoter-SPA cassette was excised from pGI Mcmv ie1 SPA by BglI digestion and inserted into the SfiI site of p45 / 46Sfi (WO 03 / 064595), yielding p45 / 46 Mcmv ie1 SPA.

[0119] The consensus sequence H9-CS (SEQ ID NO: 8) synthesized in Example 1 was used. The gene sequence H9-CN (A / chicken / Henan / H24 / 2011; GenBank accession number JN804297) referenced in Example 1 was synthesized and used. These HA sequences were digested with XbaI and SalI and then inserted into XbaI- and SalI-cleaved p45 / 46 Mcmv ie1 SPA, thereby generating p45 / 46 Mcmv ie1 H9-CS SPA and p45 / 46 Mcmv ie1 H9-CN SPA, respectively.

[0120] Construction of pHVT87-88 Mcmv ie1 H9-CS and pHVT87-88 Mcmv ie1 H9-CN The Mcmv ie1 promoter-SPA cassette was excised from pGI Mcmv ie1 SPA by BglI digestion and inserted into the SfiI site of pHVT87-88 (WO 2013 / 144355), yielding pHVT87-88 Mcmv ie1 SPA. The H9-CS or H9-CN gene, digested with XbaI and SalI, was then inserted into XbaI and SalI-cut pHVT87-88 Mcmv ie1 SPA, yielding pHVT87-88 Mcmv ie1 H9-CS SPA and pHVT87-88 Mcmv ie1 H9-CN SPA, respectively.

[0121] Construction of pHVT87-88 PecF The Pec promoter-Newcastle disease virus (NDV) F gene-SV40 polyA cassette was removed from p45 / 46PecF (WO 03 / 064595) by BglI digestion and cloned into SfiI-digested pHVT87-88, resulting in pHVT87-88PecF. The NDV F gene used comprises SEQ ID NO:14.

[0122] Construction of pHVT87-88 Mcmv ie1 H9-CNn1 to CNn5 The H9-CNn1 (SEQ ID NO: 9), H9-CNn2 (SEQ ID NO: 10), H9-CNn3 (SEQ ID NO: 11), H9-CNn4 (SEQ ID NO: 12), and H9-CNn5 (SEQ ID NO: 13) genes synthesized in Example 1 were used. These genes were cloned into XbaI- and SalI-cleaved pHVT87-88 Mcmv i e 1 SPA, thereby obtaining pHVT87-88 Mcmv i e 1 H9-CNn1 SPA, pHVT87-88 Mcmv i e 1 H9-CNn2 SPA, pHVT87-88 Mcmv i e 1 H9-CNn3 SPA, pHVT87-88 Mcmv i e 1 H9-CNn4 SPA, and pHVT87-88 Mcmv i e 1 H9-CNn5 SPA.

[0123] Construction of pHVT87-88cmv H9-CNn1 The human cytomegalovirus (Hcmv) promoter was removed from pGICMVpA (WO 2008 / 121329) by BglI and XbaI digestion and inserted into BglI and XbaI cut pHVT87-88 Mcmv ie1 H9-CNn1 SPA, thereby generating pHVT87-88 Hcmv H9-CNn1 SPA.

[0124] 2.2 Construction of recombinant HVT Recombinant HVT (rHVT) was constructed by homologous recombination in cultured cells. HVT DNA was prepared from chicken embryo fibroblasts (CEF) infected with parent HVT as described by Morgan et al. (Avian Diseases, Vol. 34: 345-351, 1990). Approximately 2 μg of HVT DNA and 1 μg of one of the homologous vectors were electroporated using Nucleofector II (Lonza, Basel, Switzerland) to generate approximately 10 7The transfected cells were then transfected into 100 CEF cells. The transfected cells were added to Leibovitz's L-15 (Life Technologies Corp., catalog no. 41300-39), McCoy's 5A medium (Life Technologies Corp., catalog no. 21500-061) (1:1), and 4% fetal bovine serum (LM(+) medium), plated into a 96-well tissue culture plate, and incubated at 37°C in 4-5% CO2 for 5-7 days until HVT plaques became visible. The cells were then detached from the plate by trypsinization, transferred equally to two 96-well plates containing CEF, and incubated for 3-5 days until plaques were observed. Screening was performed by a black plaque assay, which stains only plaques expressing the antigen protein, NDV F protein, or AIV HA protein. Briefly, one of the two plates was fixed with a methanol:acetone mixture (1:2) and incubated with rabbit anti-NDV F protein serum or chicken anti-HA (H9) serum. The plate was then incubated with biotinylated anti-rabbit IgG antibody (Vector Laboratories, catalog no. BA-1000) or biotinylated anti-chicken IgY antibody (Vector Laboratories, catalog no. BA-9010), and finally with the VECTASTAIN ABC-AP kit (Vector Laboratories, catalog no. AK-5000). Antigen-expressing plaques were stained by adding NBT / BCIP solution (Roche Applied Science, catalog no. 1681451). Wells containing stained recombinant plaques were identified, and the cells in the corresponding wells on the other 96-well plate were trypsinized. The cells were then diluted with fresh secondary CEF cells and transferred to a new 96-well plate to complete the first purification step. The purification procedure was repeated until all plaques stained positive in the black plaque assay. Multiple clones were isolated for each construct.

[0125] A list of the constructed rHVTs, their parent viruses, and the homology vectors used is provided in Table 1 below. A diagram showing the genomic structure of the rHVTs is provided in Figure 1. For the construction of a bivalent rHVT (rHVT / ND-H9) expressing both the NDV F gene and the AIV HA-H9 gene, the construction process described above was repeated using viral DNA extracted from a rHVT containing one antigen gene.

[0126] [Table 1]

[0127] Example 3: Expression of inserted antigen by recombinant HVT Expression of the NDV F protein and / or AIV HA-H9 protein by the rHVT constructs prepared in Example 2 was confirmed by immunofluorescence assay (IFA) and Western blot assay. For IFA, CEF monolayers bearing rHVT plaques were fixed with a methanol:acetone mixture (1:2) and incubated with a mixture of rabbit anti-NDV F protein serum and chicken anti-HA (H9) serum. The plates were then incubated with a mixture of Alexa Fluor 488 anti-rabbit IgG antibody (Invitrogen, catalog no. A-11008) and Alexa Fluor 546 anti-chicken IgY antibody (Invitrogen, catalog no. A-11040) and observed under a fluorescence microscope. Specific green (F protein) or red (HA-H9 protein) fluorescence was observed in each rHVT, demonstrating that these rHVTs expressed the antigenic proteins. It was also demonstrated that each plaque of the bivalent rHVT / ND-H9 construct expressed both the F antigen and the HA-H9 antigen (Fig. 2).

[0128] Western blots were performed using recombinant virus-infected CEF cells and rabbit anti-NDV F protein serum or chicken anti-HA (H9) serum. Briefly, CEF cells in 6-well plates were infected with the recombinant virus or one of the parental HVT strains at a multiplicity of infection of approximately 0.1. Three days after inoculation, cells were harvested with trypsin and centrifuged at 913 × g for 5 min. The pellet was washed with PBS and resuspended in 100 μL of PBS. After adding an equal volume of 2×SDS sample buffer (130 mM Tris-Cl (pH 6.8), 6% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.01% bromophenol blue), the cell suspension was boiled for 5 min. Samples were separated by SDS-PAGE using a 10% polyacrylamide gel and transferred to a PVDF membrane (Immobilon-P, Millipore). The membrane was completely dried and then incubated with rabbit anti-NDV F protein serum or chicken anti-HA (H9) serum. After rinsing the antibody, the membrane was incubated with either biotinylated anti-rabbit IgG antibody (Vector Laboratories, catalog number BA-1000) or biotinylated anti-chicken IgY antibody (Vector Laboratories, catalog number BA-9010), followed by incubation with a VECTASTAIN ABC-AP kit (Vector Laboratories, catalog number AK-5000). Proteins bound to the antibodies were visualized by adding NBT / BCIP solution. As shown in Figures 3 to 6, a 60-kilodalton (kDa) protein band for the NDV F protein or a 70-kDa protein band for the AIV HA-H9 protein was observed only in the lanes containing recombinant virus-infected cells, confirming that the rHVT construct prepared in Example 2 expressed antigen proteins of the expected size.

[0129] Example 4: Verification of the genomic structure of recombinant HVT The genomic structure of the rHVT construct prepared in Example 2 was verified by two PCR reactions amplifying the two insertion regions (UL45 / UL46 and SORF3 / US2). The primer pairs used in the PCR reactions were SEQ ID NO: 18 (5'-GGGGAAGTCTTCCGGTTAAGGGAC-3') and SEQ ID NO: 19 (5'-GGTGCAATTCGTAAGACCGATGGG-3') for UL45 / UL46 and SEQ ID NO: 20 (5'-GCGCGACTCCATACATTGA-3') and SEQ ID NO: 21 (5'-AGTCCACATGCACCCCACCTAAAC-3') for SORF3 / US2. The expected size of the PCR product containing the inserted gene was observed for all rHVTs, confirming that these recombinant HVTs had the expected genomic structure.

[0130] Example 5: Genetic stability of recombinant HVT The rHVT constructs prepared in Example 2 were passaged 20 times through CEF to test for genetic stability. All of the rHVTs after 20 passages were tested for genomic structure by PCR as described in Example 4 and for antigen protein expression by IFA and Western blot as described in Example 3. All rHVT constructs were shown to maintain the inserted gene by PCR and to express antigen protein by IFA and Western blot, demonstrating that these rHVTs are genetically stable and provide stable expression of antigen.

[0131] Example 6: Antibody titers in chickens vaccinated with FW205, FW206, or FW208 Constructs FW205, FW206, and FW208 were investigated for their ability to induce antibodies against NDV F and AIV HA-H9. Approximately 1,000 plaque-forming units (PFU) of the rHVT constructs were administered subcutaneously to 1-day-old specific pathogen-free (SPF) chickens. Serum was collected weekly between 2 and 5 weeks of age and tested for specific antibodies against the antigens. Antibodies against NDV F were tested using the ID Screen Newcastle Disease Indirect ELISA kit (IDVet). All tested constructs induced antibodies against the NDV F protein (Figure 7). FW205 induced much higher titers than the other constructs. Antibodies to AIV HA-H9 were tested using inactivated AIV of the H9 subtype by the hemagglutininhibition (HI) test as described in Chapter 3.3.4 (Avian Influenza) of the OIE Terrestrial Manual 2018. All tested constructs were shown to exhibit HI titers between 2 and 5 weeks of age (Figures 8A and 8B).

[0132] Example 7: Protection against ND in chickens vaccinated with FW205, FW206, or FW208 after challenge at 17 days of age The efficacy of constructs FW205 (HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CS), FW206 (HVT / 45-46 PecF / 87-88 Mcmv ie1 H9-CN), and FW208 (HVT / 45-46 Mcmv ie1 H9-CS / 87-88 PecF) was investigated against challenge with a pathogenic NDV strain. One-day-old SPF chickens were vaccinated subcutaneously with approximately 1,000 PFU of one of the rHVT / ND-H9 constructs. 17-day-old chickens were vaccinated with 1000 PFU of rHVT / ND-H9 via intramuscular injection. 5 ELD 50 Mice were challenged with the virulent NDV Herts 33 / 56 strain and observed for clinical signs of Newcastle disease (ND) for 14 days. All constructs provided greater than 70% protection against challenge at a very early age.

[0133] [Table 2] NICC = non-immunized challenge positive control

[0134] Example 8: Protection against ND in chickens vaccinated with FW205, FW206, or FW208 after challenge at 21 days of age The efficacy of FW205, FW206, and FW208 was investigated against challenge with a pathogenic NDV strain. FW168 (HVT / 45-46 PecF) was also tested. One-day-old SPF chickens were subcutaneously vaccinated with approximately 400 PFU of one of the rHVT constructs. 21-day-old chickens were vaccinated with 1000 PFU of the rHVT constructs via intramuscular injection. 5 ELD 50 The mice were challenged with the virulent NDV Herts 33 / 56 strain and observed for clinical signs of Newcastle disease (ND) for 14 days. Constructs FW205 and FW168 provided excellent protection of 100% and 96%, respectively, and the other constructs also provided good protection of over 70%. The results are shown in Table 3 below.

[0135] [Table 3] NICC = non-immunized challenge positive control

[0136] Example 9: Protection against AI in chickens vaccinated with FW205, FW206, or FW208 after challenge at 25 days of age The efficacy of FW205, FW206, and FW208 against challenge with AIV H9 subtype was investigated in commercial broiler chickens. One-day-old commercial broiler chickens were subcutaneously vaccinated with approximately 1,000 PFU of FW205, FW206, or FW208. Chickens were vaccinated at 25 days of age via the intratracheal and intranasal routes for 10 days. 7 EID 50The chickens were challenged with the AIV A / chicken / Saudi Arabia / D1816 / 1 / 1 / 2011 (H9N2) strain. Five days after challenge, tracheal samples were collected and used for AIV quantification by qPCR analysis. Eleven days after challenge, chickens were necropsied and air sac lesions were evaluated. As shown in Figure 9, all vaccinated groups had lower viral loads compared to the control group. FW205 reduced AIV loads in tracheal swabs by 1 log compared to the challenge control group (Figure 9), and the AIV loads in the other two groups vaccinated with either FW206 or FW208 were 0.5 log lower than those in the challenge control (Figure 9). Air sac lesion scores in all vaccinated groups were also substantially lower than those in the challenge control group (Figure 10). These results demonstrated that all tested constructs provided clinical protection against challenge with the AIV H9 subtype.

[0137] Example 10: Antibody titers in chickens vaccinated with FW247, FW248, FW249, FW250, FW251, or FW252 Constructs FW247, FW248, FW249, FW250, FW251, and FW252 were investigated for their ability to induce antibodies against NDV F and AIV HA-H9. Approximately 3,000 PFU of the rHVT constructs were administered subcutaneously to 1-day-old SPF chickens. Sera were collected at 2 and 3 weeks of age and tested for specific antibodies against the antigens. Antibodies against NDV F were tested using the ID Screen Newcastle Disease Indirect ELISA kit (IDVet). Antibodies against AIV HA-H9 were tested by HI test using inactivated AIV of the H9 subtype. All tested constructs induced antibodies against both the NDV F protein (Figure 11) and AIV HA-H9 (Figure 12). Construct FW249 appeared to induce high F and HA-H9 antibodies.

[0138] Example 11: Protection against ND in FW249-vaccinated chickens after challenge at 21 days of age The efficacy of construct FW249 was investigated against challenge with a pathogenic NDV strain. One-day-old SPF chickens were subcutaneously vaccinated with approximately 2,500 PFU of one of the rHVT / ND-H9 constructs. 21-day-old chickens were vaccinated with 10 rHVT / ND-H9 constructs via intramuscular injection. 5 ELD 50 The mice were challenged with the virulent NDV Herts 33 / 56 strain and observed for clinical signs of Newcastle disease (ND) for 14 days. The results are presented in Table 4.

[0139] [Table 4] NICC = non-immunized challenge positive control

[0140] Sequence Listing SEQ ID NO: 1 Name: F protein Type: Amino acid Length: 553 MGSRSSTRIPVPLMLTVRIMLALSCVCPTSSLDGRPLAAAGIVVTGDKAVNIYTSSQTGSIIIKLLPNMPKDKEACAKAPLEAYNRTLTTLLTPLGDSIRRIQESVTTSGGGKQGRLIGAIIGGVALGVATAAQITAA SALIQANQNAANILRLKESIAATNEAVHEVTDGLSQLAVAVGKMQQFVNDQFNKTAQELDCIKITQQVGVELNLYLTELTTVFGPQITSPALTQLTIQALYNLAGGNMDYLLTKLGVGNNQLSSLIGSGLITGNPILY DSQTQLLGIQVTLPSVGNLNNMRATYLETLSVSTTKGFASALVPKVVTQVGSVIEELDTSYCIETDLDLYCTRIVTFPMSPGIYSCLSGNTSACMYSKTEGALTTPYMTLKGSVIANCKMTTCRCADPPGIISQNYGE AVSLIDRQSCNILSLDGITLRLSGEFDATYQKNISIQDSQVIVTGNLDISTELGNVNNSISNALDKLEESNSKLDKVNVKLTSTSALITYIVLTVISLVCGILSLVLACYLMYKQKAQQKTLLWLGNNTLDQMRATTKM SEQ ID NO: 2 Name: (H9-CS) Type: Amino acid Length: 560 METISLMTILLVVTTSNADKICIGHQSTNSTETVDTLTETNVPVTHAKELLHTEHNGMLCATNLGHPLILDTTCTIEGLVYGNPSCDLLLGGREWSYIVERPSAVNGTCYPGNVENLEELRTLFSSSSSYQRIQIFPDTIW NVTYTGTSKSCSDSFYRNMRWLTQKNGVYPVQDAQYTNNRGKDILFVWGIHHPPTDTAQTNLYTRTDTTTSVTTENLDRTFKPLIGPRPLVNGLIGRINYYWSVLKPGQTLRVRSNGNLIAPWFGHVLSGESHGRILKTD LNSGNCVVQCQTEKGGLNSTLPFHNISKYAFGTCPKYIGVKSLKLAIGLRNVPARSSRGLFGAIAGFIEGGWPGLVAGWYGFQHSNDQGVGMAADRDSTQKAVDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINN KIDDQIQDVWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAIEDGKGCFELYHKCDNQCMETIRNGTYNRRKYKEESRLERQKIEGVKLESEGTYKILTIYSTVASSLVLAMVFAAFLFWAMSNGSCRCNICI SEQ ID NO: 3 Name: H9-CNn1 Type: Amino acid Length: 560 MEVVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTTCTIEGLIYGNPSCDLSLEGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIW NVSYDGTSTACSGSFYRSMRWLTRKNGDYPIQDAQYTNNQGKNILFMWGINHPPTDDTQRNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRIDYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTD LKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINN KIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGTNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI SEQ ID NO:4 Name: H9-CNn2 Type: Amino acid Length: 560 MEVVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTTCTIEGLIYGNPSCDLSLEGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIW NVSYDGTSTACSGSFYRSMRWLTRKNGDYPTQDAQYTNNQGKNILFMWGINHPPTDTAQTNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRIDYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTD LKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINN KIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGTNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI SEQ ID NO:5 Name: H9-CNn3 Type: Amino acid Length: 560 MEVVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTTCTIEGLIYGNPSCDLSLEGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIW NVSYDGTSTACSGSFYRSMRWLTQKNNAYPIQDAQYTNNQGKNILFMWGINHPPTDTTQRNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRIDYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTD LKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINN KIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGTNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI SEQ ID NO:6 Name: H9-CNn4 Type: Amino acid Length: 560 MEVVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTIW NVSYDGTSTACSGSFYRSMRWLTRKNGDYPIQDAQYTNNQGKNILFMWGINHPPTDDTQRNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRIDYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKTD LKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINN KIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGTNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI SEQ ID NO:7 Name:H9-CNn5 Type: Amino acid Length: 559 MEVVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTTCTIEGLIYGNPSCDLSLEGREWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSYQRIQIFPDTI WNVSYDGTSTACSGSFYRSMRWLTRKNGDYPIQDAQYTNNQGKNILFMWGINHPPTDDTQRNLYTRTDTTTSVATEEINRIFKPLIGPRPLVNGLMGRIDYYWSVLKPGQTLRIKSDGNLIAPWYGHILSGESHGRILKT DLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMIN NKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGTNAVEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTISFAISCFLLCVVLLGFIMWACQNGSCRCNICI SEQ ID NO:8 Name: H9-CS Type: Nucleotide Length: 1683 SEQ ID NO:9 Name: H9-CNn1 Type: Nucleotide Length: 1683 SEQ ID NO: 10 Name: H9-CNn2 Type: Nucleotide Length: 1683 SEQ ID NO: 11 Name: H9-CNn3 Type: Nucleotide Length: 1683 SEQ ID NO:12 Name: H9-CNn4 Type: Nucleotide Length: 1683 SEQ ID NO: 13 Name:H9-CNn5 Type: Nucleotide Length: 1680 SEQ ID NO: 14 Name: Newcastle disease virus F gene Type: Nucleotide Length: 1662 SEQ ID NO: 15 Name: Pec Promoter Type: Nucleotide Length: 572

[0141] [Table 5] SEQ ID NO: 16 Name: Murine cytomegalovirus ie1 promoter Type: Nucleotide Length: 572

[0142] [Table 6] SEQ ID NO: 17 Name: Poly A signal Type: Nucleotide Length:87

[0143] [Table 7] SEQ ID NO: 18 Name: Primer Type: Nucleotide Length:24 ggggaagtct tccggttaag ggac SEQ ID NO: 19 Name: Primer Type: Nucleotide Length:24 ggtgcaattc gtaagaccga tggg SEQ ID NO: 20 Name: Primer Type: Nucleotide Length:19 gcgcgactcc atacattga SEQ ID NO: 21 Name: Primer Type: Nucleotide Length:24 agtccacatg caccccacct aaac

Claims

1. 1. A recombinant Herpes Virus of Turkeys (rHVT) comprising: (i) a nucleotide sequence encoding the F protein of Newcastle Disease virus, or an immunogenic fragment or variant thereof, inserted into a first insertion site in a non-coding region of the viral genome located between UL45 and UL46, wherein the sequence encoding the F protein is under the control of a Pec promoter; and (ii) a nucleotide sequence encoding the hemagglutinin (HA) protein of subtype H9 avian influenza virus of SEQ ID NO:2, or an immunogenic fragment or variant thereof that exhibits at least 95% sequence identity compared to SEQ ID NO:2 and maintains the immunogenic properties of SEQ ID NO:2, wherein the nucleotide sequence is inserted into a second insertion site in the viral genome located between SORF3 and US2, wherein the sequence encoding the HA protein is under the control of a mouse or human CMV immediate early promoter.

2. The rHVT described in claim 1, wherein the CMV immediate early promoter is a mouse CMV immediate early (mCMV IE1) promoter.

3. The rHVT of claim 2 , wherein the mCMV IE1 promoter comprises the sequence of SEQ ID NO:

16.

4. An rHVT described in any one of claims 1 to 3, wherein the Pec promoter comprises the sequence of SEQ ID NO:

15.

5. A vaccine comprising the rHVT of any one of claims 1 to 4.

6. 6. The rHVT of any one of claims 1 to 5 for use in vaccinating birds, such as poultry.

7. An rHVT described in any one of claims 1 to 5 for use in vaccinating chickens.

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