Recombinant hvt vectors expressing antigens of avian pathogens and uses thereof

Abstract

The present invention relates to recombinant HVT vectors expressing antigens of avian pathogens and uses thereof. In particular, the present invention provides recombinant herpesvirus comprising and expressing a turkey (HVT) vector of an antigen of an avian pathogen, compositions comprising the recombinant HVT vectors, multivalent vaccines comprising the recombinant HVT vectors and one or more wild-type viruses or recombinant vectors. The present invention also provides methods of vaccinating against multiple avian pathogens and methods of making the recombinant HVT vectors.

Description

[0001] This application is a divisional application of the invention patent application with application number 201280066323.X, application date November 29, 2012, entitled "Recombinant HVT vector expressing antigens of avian pathogens and its use therein".

[0002] Cross-reference related applications

[0003] This application claims priority to U.S. Provisional Application No. 61 / 564,877, filed November 30, 2011, and U.S. Provisional Application No. 61 / 694,957, filed August 30, 2012. Technical Field

[0004] This invention relates to recombinant viral vectors for inserting and expressing foreign genes to serve as safe immune vectors for protection against infection by a variety of pathogens. It also relates to multivalent compositions or vaccines comprising one or more recombinant viral vectors for protection against infection by a variety of pathogens. This invention relates to methods for preparing and using recombinant viral vectors. Background Technology

[0005] Poultry vaccination is widely used to protect poultry populations from devastating diseases, including Newcastle disease (ND), infectious bursal disease (IBD), Marek's disease (MD), infectious bronchitis (IB), infectious laryngotracheitis (ILT), and avian influenza (AI). ND is caused by avian paramyxovirus type 1 (APMV-1) (also known as ND virus (NDV)) belonging to the Paramyxoviridae family. MD is caused by chicken herpesvirus type 2 (Herpesviridae family), also known as MD virus serotype 1 (MDV1). IB is caused by IB virus (IBV) belonging to the Coronaviridae family, ILT is caused by chicken herpesvirus 1 (Herpesviridae family), also known as ILT virus (ILTV), and AI is caused by AI virus (AIV) belonging to the Orthomyxoviridae family.

[0006] Many recombinant avian viral vectors have been proposed for use in inoculating chickens against these avian pathogens. The viral vectors used include fowlpox virus, particularly fowlpox virus (EP-A-0,517,292), Marek's disease virus such as serotypes 2 and 3 (HVT) (WO-A-87 / 04463), or alternatively ITLV, NDV, and avian adenovirus. When some of these recombinant avian viral vectors are used for inoculation, they show visible levels of protection.

[0007] Several recombinant turkey herpesvirus (HVT, also known as turkey herpesvirus 1 or MDV serotype 3) vectors expressing antigens from various pathogens (US Patent Nos. 5,980,906, 5,853,733, 6,183,753, 5,187,087) have been developed and licensed. Of particular interest is the HVT vector expressing the IBDV VP2 protective gene, which has shown clear advantages over classic IBD vaccines (Bublot et al., J. Comp. Path. 2007, Vol. 137, pp. 81-84; US 5,980,906). Other target HVT vectors are those expressing protective genes for NDV (Morgan et al. 1992, Avian Dis. 36, 858-70; US 6,866,852; US 5,650,153) or ILTV (Johnson et al. 2010, Avian Dis. 54, 1251-1259; US 6,299,882; US 5,853,733). One of the practical problems with using several HVT-based recombinant vaccines together is their interference. When two HVT recombinants expressing different antigens are mixed, they induce lower protective effects against at least one disease (Rudolf Heine 2011; Issues of the Poultry Recombinant Viral Vector Vaccines which May Cause an Effect on the Economic Benefits of those Vaccines; Paper presented at the XVII World Veterinary Association (WVPA) meeting in Cancún, Mexico, August 14-18, 2011; Slacum G, Hein R. and Lynch P., 2009, The compatibility of HVT recombinants with other Marek's disease vaccines, 58th Western Poultry Disease Conference, Sacramento, CA, USA, March 23-25, p84).

[0008] The combination of HVT and SB-1, and chicken herpesvirus 3 (MDV serotype 2 or MDV-2) vaccine strains has shown a synergistic effect in protecting against MD (Witter and Lee, 1984, Avian Pathology 13, 75-92). To illustrate the problem of interference, it is beneficial to evaluate HVT virus as a vaccine vector expressing one or more protective antigens against a variety of avian pathogens.

[0009] The SB-1 genome was cloned and characterized in a bacterial artificial chromosome (BAC) (Petherbridge et al., J. Virol. Methods 158, 11-17, 2009; Singh et al., Research in Veterinary Science 89, 140-145, 2010). The MDV2 SB-1 sequence was recently obtained and analyzed (Spatz and Schat, Virus Gene 42, 331-338, 2011). The deletion of glycoprotein E in the SB-1 virus was described by Petherbridge et al. (J. Virol. Methods 158, 11-17, 2009). However, no studies have reported using SB-1 as a viral vector to express exogenous protective genes.

[0010] Given the potential impacts of animal pathogens such as NDV and IBDV on veterinary public health and the economy, there is a need for efficient methods to prevent infection and protect animals. There is a need for solutions to effective vector vaccines and appropriate methods for preparing combinations that can mitigate the interference problems observed between two HVT-based vector vaccines. Summary of the Invention

[0011] This invention demonstrates surprising results in which multivalent compositions or vaccines containing single or dual HVT vectors effectively protect animals from a variety of avian pathogens without interference. Surprising results have also been observed when different combinations of promoters, codon-optimized genes, polyA tails, and insertion sites confer varying levels of potency and stability on the in vivo expression of one or more heterologous genes.

[0012] This invention relates to a recombinant HVT vector comprising one or more heterologous polynucleotides encoding and expressing at least one antigen of an avian pathogen.

[0013] The present invention provides compositions or vaccines comprising one or more recombinant HVT vectors, said vectors comprising one or more heteropolynucleotides encoding and expressing at least one antigen of an avian pathogen.

[0014] The present invention provides a multivalent composition or vaccine comprising one or more recombinant HVT vectors and one or more recombinant SB1 vectors, wherein the recombinant HVT vectors comprise a heteropolynucleotide encoding and expressing at least one antigen of an avian pathogen, and the recombinant SB1 vectors comprise a heteropolynucleotide encoding and expressing at least one antigen of an avian pathogen.

[0015] This invention relates to methods for inoculating animals or inducing immunogenic or protective responses in animals, comprising at least one administration of the composition or carrier of the invention. Attached Figure Description

[0016] The following detailed description, given by way of example and not intended to limit the invention to the specific embodiments described, can be understood in conjunction with the accompanying drawings (incorporated herein by reference), wherein:

[0017] Figure 1 It is a table showing the SEQ ID NO assigned to each DNA and protein sequence.

[0018] Figure 2 Describe the genomic structure of HVT and its insertion site.

[0019] Figure 3 A plasmid map depicting pHM103.

[0020] Figure 4 Describe the PCR analysis results of vHVT114.

[0021] Figure 5 Describe the results of the dual immunofluorescence assay.

[0022] Figure 6 Describe the Southern imprint results for vHVT114.

[0023] Figure 7 Describe the results of immunoprecipitation and Western blot analysis of vHVT114.

[0024] Figure 8 Western blot analysis of immunoprecipitation samples from vHVT306-infected cells.

[0025] Figure 9 Western blot analysis of immunoprecipitation samples from vSB1-009 infected cells.

[0026] Figure 10 The results of the study on the resistance of vHVT304 and vHVT114 to attacks by NDV ZJ1 and CA02 are described.

[0027] Figure 11 illustrates the virus shedding results after NDV CA02 and ZJ1 attacks.

[0028] Figure 12 illustrates the virus shedding results after the NDV Chimalhuacan attack.

[0029] Figure 13 Displays sequence alignment and percentage identity.

[0030] Figure 14Displays DNA and protein sequences. Invention Details

[0031] It should be noted that in this disclosure, and particularly in the claims, terms such as “comprising,” “including,” “containing,” etc., may have the meanings in U.S. patent law; for example, they may mean “comprising,” “containing,” “containing,” etc.; terms such as “consistently composed of” and “consistently composed of” have their meanings in U.S. patent law, for example, they allow the inclusion of elements not expressly cited, but do not include elements found in the prior art or that affect the essential or novel features of the invention.

[0032] Unless otherwise specified, technical terms are used according to common usage. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes V., Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0033] Unless the context explicitly indicates otherwise, the singular terms “a,” “an,” and “the” include their plural forms. Similarly, unless explicitly indicated otherwise, the word “or” is intended to include “and.” The word “or” refers to any one member of a particular list, and also includes any combination of members of that list.

[0034] The term "animal" is used herein to include all mammals, birds, and fish. Animals used herein may be selected from equines (e.g., horses), canids (e.g., dogs, wolves, foxes, steppe wolves, jackals), felines (e.g., lions, tigers, domestic cats, wildcats, other large cats, and other felines including cheetahs and lynxes), bovines (e.g., cattle), swine (e.g., pigs), sheep (e.g., sheep, goats, alpacas, bison), birds (e.g., chickens, ducks, geese, turkeys, quails, pheasants, parrots, sparrows, eagles, crows, ostriches, emus, and cassowaries), primates (e.g., lemurs, tarsiers, monkeys, gibbons, apes), humans, and fish. The term "animal" also includes individuals at all developmental stages, including the embryonic and fetal stages.

[0035] The terms “polypeptide” and “protein” are used interchangeably in this document to refer to polymers of consecutive amino acid residues.

[0036] The terms “nucleic acid,” “nucleotide,” and “polynucleotide” are used interchangeably and refer to RNA, DNA, cDNA, or cRNA and their derivatives, such as those nucleic acids containing a modified backbone. It should be understood that this invention provides polynucleotides containing sequences complementary to those described herein. The term “polynucleotide” as used in this invention includes a forward strand (5' to 3') and a reverse complementary strand (3' to 5'). Polynucleotides according to the invention can be prepared in various ways (e.g., by chemical synthesis, by gene cloning, etc.) and can take various forms (e.g., linear or branched, single-stranded or double-stranded, or hybrids thereof, primers, probes, etc.).

[0037] The terms “genomic DNA” or “genome” are used interchangeably to refer to the heritable genetic information of a host organism. Genomic DNA includes DNA in the cell nucleus (also known as chromosomal DNA), as well as DNA in plastids (e.g., chloroplasts) and other organelles (e.g., mitochondria). In this invention, genomic DNA or genome also refers to viral RNA. RNA can be positive-sense or negative-sense RNA. The term “genomic DNA” in this invention includes genomic DNA containing a sequence complementary to the sequence described herein. The term “genomic DNA” also refers to messenger RNA (mRNA), complementary DNA (cDNA), and complementary RNA (cRNA).

[0038] The term "gene" is widely used to refer to any segment of a polynucleotide that is associated with a biological function. Therefore, a gene or polynucleotide includes introns and exons (as in a genomic sequence) or coding-only sequences (as in cDNA), such as open reading frames (ORFs) that begin with a start codon (methionine codon) and end with a stop signal (stop codon). Genes and polynucleotides may also include regions that regulate their expression, such as transcription initiation, translation, and transcription termination. This includes promoter and ribosome-binding regions (generally, these regulatory elements are located approximately 60 to 250 nucleotides upstream of the start codon in a coding sequence or gene; Doree SM et al.; Panther K et al.; Chung JY et al.) and transcription terminators (generally, terminators are located approximately 50 nucleotides downstream of the stop codon in a coding sequence or gene; Ward CK et al.). A gene or polynucleotide also refers to a nucleic acid segment that expresses mRNA or functional RNA or encodes a specific protein, including regulatory sequences.

[0039] As used herein, the term "heterologous DNA" refers to DNA derived from a different organism, such as a different cell type or a species different from the recipient. The term also refers to DNA or fragments thereof on the same genome as the host DNA, wherein the heterologous DNA is inserted into a region of the genome that differs from its original location.

[0040] As used herein, the term "antigen" or "immunogen" refers to a substance that induces a specific immune response in a host animal. Antigens may include whole organisms (killed, attenuated, or live); subunits or parts of an organism; recombinant vectors containing inserts with immunogenic properties; DNA fragments or segments capable of inducing an immune response upon presentation to a host animal; polypeptides, epitopes, haptens, or any combination thereof. Alternatively, an immunogen or antigen may include a toxin or antitoxin.

[0041] As used herein, the term "immunogenic protein or peptide" includes polypeptides that are immunologically active in the sense that, once administered to a host, they can elicit a humoral and / or cellular immune response against the protein. Preferably, a protein fragment is a fragment that has substantially the same immunological activity as the whole protein. Thus, a protein fragment according to the invention comprises or is substantially composed of at least one epitope or antigenic determinant. As used herein, "immunogenic" protein or polypeptide includes the full-length sequence of a protein, its analogues, or immunogenic fragments thereof. "Immunogenic fragment" means a protein fragment that includes one or more epitopes, thereby eliciting the aforementioned immune response. Such fragments can be identified using many epitope mapping techniques known in the art. For example, linear epitopes can be determined, for example, by simultaneously synthesizing a large number of peptides (which correspond to portions of the protein molecule) on a solid support and reacting the peptides with an antibody (while the peptides remain attached to the support). Similarly, conformational epitopes can be readily identified by determining the spatial conformation of amino acids (e.g., by X-ray crystallography and two-dimensional nuclear magnetic resonance).

[0042] The term “immunogenic protein or peptide” also refers to the deletion, addition, and substitution of sequences, provided that the polypeptide can produce an immune response as defined herein. The term “conserved alteration” means the substitution of an amino acid residue or a nucleotide in a nucleic acid sequence with another biologically similar residue such that the encoded amino acid residue remains unchanged or is replaced by another biologically similar residue. In this respect, particularly preferred substitutions are generally conserved in nature, i.e., those that occur within a family of amino acids. For example, amino acids are generally classified into four families: (1) acidic—aspartic acid and glutamic acid; (2) basic—lysine, arginine, histidine; (3) nonpolar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conserved alterations include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue; or the substitution of one polar residue for another polar residue, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine; or similar conserved substitutions of structurally related amino acids that do not have a major effect on biological activity. Therefore, proteins having a substantially identical amino acid sequence to the reference sequence but with a few amino acid substitutions that do not substantially affect the immunogenicity of the protein are within the definition of a reference polypeptide. All polypeptides resulting from these modifications are included herein. The term "conserved alteration" also includes the substitution of an unsubstituted parental amino acid with a substituted amino acid, provided that antibodies generated against the substituted polypeptide also elicit an immune response against the unsubstituted polypeptide.

[0043] The term "epitope" refers to a site on an antigen or hapten to which a specific B cell and / or T cell responds. This term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." Antibodies that recognize the same epitope can be identified in a simple immunoassay that demonstrates the ability of one antibody to block the binding of another antibody to a target antigen.

[0044] An "immune response" to a composition or vaccine is the development of a cellular and / or antibody-mediated immune response in the host against the target composition or vaccine. Typically, an "immune response" includes, but is not limited to, one or more of the following effects: the formation of antibodies specifically against antigens contained in the target composition or vaccine; the production of B cells, helper T cells, and / or cytotoxic T cells. Preferably, the host will exhibit a therapeutic or protective immune response, thereby enhancing resistance to new infections and / or reducing the clinical severity of the disease. Such protection can be demonstrated by the reduction or absence of symptoms typically exhibited by the infected host, faster recovery time in the infected host, and / or lower viral titers.

[0045] The terms “recombinant” and “genetically modified” are used interchangeably to refer to any modification, alteration, or engineering of a polynucleotide or protein in its natural form or structure, or any modification, alteration, or engineering of a polynucleotide or protein in its natural environment or surroundings. Modification, alteration, or engineering of a polynucleotide or protein may include, but is not limited to, the deletion of one or more nucleotides or amino acids, the deletion of an entire gene, codon optimization of a gene, conserved substitution of amino acids, or the insertion of one or more heterologous polynucleotides.

[0046] The term "dual HVT construct" or "dual HVT vector" refers to an HVT viral vector containing two heterologous polynucleotides.

[0047] The terms "multivalent vaccine or composition," "combo vaccine or composition," and "multivalent vaccine or composition" are used interchangeably to refer to a composition or vaccine comprising more than one composition or vaccine. A multivalent vaccine or composition may comprise two, three, four, or more compositions or vaccines. A multivalent vaccine or composition may comprise a recombinant viral vector, an active or attenuated or killed wild-type virus, or a mixture of a recombinant viral vector and a wild-type virus present in an active or attenuated or killed form.

[0048] One embodiment of the present invention provides a recombinant HVT virus vector comprising one or more heteropolynucleotides encoding and expressing at least one antigen or polypeptide of an avian pathogen. The HVT strain used for the recombinant virus vector can be any HVT strain, including but not limited to HVT strain FC126 (Igarashi T. et al., J. Gen. Virol. 70, 1789-1804, 1989).

[0049] Another embodiment of the invention provides a recombinant SB-1 viral vector comprising one or more heteropolynucleotides encoding and expressing at least one antigen or polypeptide of an avian pathogen. The SB-1 strain can be any SB-1 strain, including but not limited to commercially available Marek's disease vaccine (SB-1 vaccine) (Merial Select Inc., Gainesville, GA 30503, USA) and SB-1 strains having a genomic sequence as defined by GenBank accession number HQ840738.1.

[0050] Genes encoding antigens or polypeptides may be those encoding the following proteins: Newcastle disease virus fusion protein (NDV-F), Newcastle disease virus hemagglutinin neuraminidase (NDV-HN), Marek's disease virus glycoprotein C (gC), Marek's disease virus glycoprotein B (gB), Marek's disease virus glycoprotein E (gE), Marek's disease virus glycoprotein I (gI), Marek's disease virus glycoprotein H (gH) or Marek's disease virus glycoprotein L (gL), infectious bursal disease virus (IBDV) VP2, IBDV VPX, IBDV VP3, IBDV VP4, ILTV glycoprotein B, ILTV glycoprotein I, ILTV UL32, ILTV glycoprotein D, ILTV glycoprotein E, ILTV glycoprotein C, influenza hemagglutinin (HA), influenza neuraminidase (NA), or mycoplasma gallisepticum (MG) or mycoplasma synoviae (MG). The protective gene or combination thereof of synoviae (MS). The antigen or polypeptide can be any antigen from a poultry pathogen selected from avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian metapneumovirus, avian influenza virus, avian adenovirus, fowlpox virus, avian coronavirus, avian rotavirus, chicken anemia virus, avian astrovirus, avian parvovirus, coccidiosis virus (a species of Eimeria sp.), a species of Campylobacter sp., a species of Salmonella sp., a species of Pasteurella sp., a species of Avibacterium sp., Mycoplasma synoviae, a species of Clostridium sp., and Escherichia coli.

[0051] Furthermore, the aforementioned homologs of antigens or polynucleotides are intended to be within the scope of this invention. As used herein, the term "homolog" includes orthologs, analogs, and paralogs. The term "analog" refers to two polynucleotides or polypeptides that have the same or similar functions but have evolved separately in unrelated organisms. The term "ortholog" refers to two polynucleotides or polypeptides from different species but that have evolved from a common ancestral gene through speciation. Typically, orthologs encode polypeptides with the same or similar functions. The term "paralog" refers to two polynucleotides or polypeptides that are related through replication within the genome. Paralogs typically have different functions, but these functions can be related. Analogs, orthologs, and paralogs of wild-type polypeptides may differ from wild-type polypeptides in post-translational modifications, amino acid sequence differences, or both. Specifically, the homologs of the present invention generally exhibit at least 80-85%, 85-90%, 90-95%, or 95%, 96%, 97%, 98%, or 99% sequence identity with all or part of the polynucleotide or polypeptide sequence of the above-mentioned antigens, and may exhibit similar functions.

[0052] In one embodiment, the present invention provides a recombinant HVT or SB-1 viral vector comprising one or more heteropolynucleotides encoding and expressing an NDV-F antigen or polypeptide. In one aspect of this embodiment, the NDV-F antigen or polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 2, 4, 6, 33, 35, or 37, or a conserved variant, allelic variant, homolog of one of these polypeptides, or an immunogenic fragment comprising at least 8 or at least 10 consecutive amino acids of said polypeptide, or a combination of these polypeptides. In another aspect of this embodiment, the heteropolynucleotide encodes an NDV-F antigen or polypeptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 2, 4, 6, 33, 35, or 37. In another aspect of this embodiment, the heteropolynucleotide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the polynucleotide having the sequence shown in SEQ ID NO: 1, 3, 5, 32, 34, or 36.

[0053] Variants include allelic variants. The term "allelic variant" refers to a polynucleotide or polypeptide that contains a change in the amino acid sequence of a protein and is present within a natural population (e.g., a species or variant of a virus). Such natural allelic variations typically result in 1-5% variation within the polynucleotide or polypeptide. Allelic variants can be identified by sequencing the target nucleic acid sequences of many different species, which can be readily performed by using hybridization probes to identify the loci of the same gene in those species. Any and all such nucleic acid variations and resulting amino acid polymorphisms or variations that result from natural allelic variations and do not alter the functional activity of the target gene are intended to be included within the scope of this invention.

[0054] The term "identity" in relation to sequences can refer, for example, the number of identical nucleotide or amino acid positions divided by the number of nucleotides or amino acids in the shorter of the two sequences, where the alignment of the two sequences can be determined according to the Wilbur and Lipman algorithm. Sequence identity or sequence similarity between two amino acid sequences, or sequence identity between two nucleotide sequences, can be determined using the Vector NTI software package (Invitrogen, 1600 Faraday Ave., Carlsbad, CA). When an RNA sequence is considered similar to or has a certain degree of sequence identity or homology with a DNA sequence, thymine (T) in the DNA sequence is considered equivalent to uracil (U) in the RNA sequence. Therefore, RNA sequences are included within the scope of this invention and can be derived from DNA sequences, wherein thymine (T) in the DNA sequence is considered equivalent to uracil (U) in the RNA sequence.

[0055] The polynucleotides disclosed herein include degenerate sequences that are a result of the genetic code (e.g., optimal codon selection for a particular host). As used herein, “optimized” refers to a polynucleotide genetically engineered to increase its expression in a given species. To provide an optimal polynucleotide encoding the NDV-F polypeptide, the DNA sequence of the NDV-F protein gene may be modified to 1) contain codons preferred by genes highly expressed in a particular species; 2) contain A+T or G+C content in a nucleotide base composition similar to that found in the species in a substantially similar manner; 3) form the initial sequence of the species; or 4) eliminate sequences that cause RNA destabilization, inappropriate polyadenylation, degradation, and termination, or the formation of secondary structure hairpins or RNA splicing sites. Increased expression of the NDV-F protein in the species may be achieved by using the frequency distribution of codon usage in eukaryotes and prokaryotes or in a particular species. The term “frequency of preferred codon usage” refers to the preference for a specific amino acid shown by a particular host cell in the use of nucleotide codons. There are 20 naturally occurring amino acids, most of which are designated by more than one codon. Therefore, all degenerate nucleotide sequences are included in this disclosure, provided that the amino acid sequence of the NDV-F polypeptide encoded by the nucleotide sequence is not functionally altered.

[0056] Successful expression of heteropolynucleotides (HMNs) by recombinant / modified infectious viruses requires two conditions. First, the HMN must be inserted into or introduced into a region of the viral genome to maintain the viability of the modified virus. The second condition for expression of the inserted HMN is the presence of regulatory sequences that allow gene expression in a viral context (e.g., promoters, enhancers, donor and acceptor splicing sites and introns, Kozak translation initiation concordance sequences, polyadenylation signals, and non-translational sequence elements).

[0057] The insertion site can be any non-essential region of the HVT genome, including but not limited to the region between the ATG and UL of ORF UL55 and the adjacent repeat region (US5,980,906), IG1 locus, IG2 locus, IG3 locus, UL43 locus, US10 locus, SORF3 / US2 locus (see [link to documentation]). Figure 2 ).

[0058] Generally, it is advantageous to use strong promoters that are functional in eukaryotic cells. Promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoters, guinea pig CMV promoters, SV40 promoters, pseudorabies virus promoters such as glycoprotein X promoters, herpes simplex virus-1 promoters such as α4 promoters, Marek's disease virus (including MDV-1, MDV-2, and HVT) promoters such as promoters driving the expression of glycoproteins gC, gB, gE, or gI, infectious laryngotracheitis virus promoters such as promoters of glycoprotein gB, gE, gI, gD genes, or other herpesvirus promoters.

[0059] One embodiment of the present invention provides a recombinant HVT vector comprising a heteropolynucleotide encoding and expressing an NDV-F antigen or polypeptide. In one aspect of this embodiment, the polynucleotide encoding the NDV-F polypeptide is efficiently linked to an SV40 promoter having the sequence shown in SEQ ID NO:9, thereby regulating the expression of the NDV-F antigen or polypeptide by the SV40 promoter. In another aspect of this embodiment, the expression of the NDV-F antigen or polypeptide is regulated by an SV40polyA signal having the sequence shown in SEQ ID NO:11. In yet another aspect of this embodiment, the polynucleotide encoding the NDV-F polypeptide is efficiently linked to an MDV gB promoter having the sequence shown in SEQ ID NO:38, thereby regulating the expression of the NDV-F antigen or polypeptide by the MDV gB promoter.

[0060] Another embodiment of the invention provides a recombinant dual HVT vector comprising a first heteropolynucleotide encoding and expressing an NDV-F antigen or polypeptide and a second polynucleotide encoding and expressing an IBDV VP2 antigen or polypeptide. In one aspect of this embodiment, the NDV-F antigen or polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 2, 4, 6, 33, 35, or 37. In another aspect of this embodiment, the IBDV VP2 antigen or polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with a polypeptide having the sequence shown in SEQ ID NO: 8 or 42. In another aspect, the polynucleotide encoding the NDV-F polypeptide is effectively linked to the SV40 promoter having the sequence shown in SEQ ID NO:9, thereby regulating the expression of the NDV-F antigen or polypeptide by the SV40 promoter. In another aspect, the expression of the NDV-F antigen or polypeptide is regulated by the SV40 polyA signal having the sequence shown in SEQ ID NO:11 or the synthetic polyA signal having the sequence shown in SEQ ID NO:12. In another aspect, the expression of the IBDV VP2 antigen or polypeptide is regulated by the CMV-IE promoter having the sequence shown in SEQ ID NO:10 and the SV40 polyA signal having the sequence shown in SEQ ID NO:11.

[0061] Another embodiment of the invention provides a recombinant dual HVT vector comprising two polynucleotides encoding and expressing an IBDV VP2 antigen or polypeptide. In one aspect of the embodiment, the IBDV VP2 antigen or polypeptide has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO: 8 or 42. In one aspect, the polynucleotide encoding the first IBDV VP2 antigen or polypeptide is effectively linked to a CMV-IE promoter having the sequence shown in SEQ ID NO: 10, and the polynucleotide encoding the second IBDV VP2 antigen or polypeptide is effectively linked to a guinea pig CMV promoter having the sequence shown in SEQ ID NO: 43. In another aspect, the expression of the first IBDV VP2 antigen or polypeptide is regulated by a CMV-IE promoter having the sequence shown in SEQ ID NO:10 and an SV40 polyA signal having the sequence shown in SEQ ID NO:11, and the expression of the second IBDV VP2 antigen or polypeptide is regulated by a guinea pig CMV promoter having the sequence shown in SEQ ID NO:43 and a synthetic polyA signal having the sequence shown in SEQ ID NO:12. In another aspect of this embodiment, a polynucleotide encoding the IBDV VP2 antigen or polypeptide may be inserted into one or more locus regions selected from IG1, IG2, US10, SORF3-US2, and gD of the HVT genome. In one embodiment, the present invention relates to a pharmaceutical composition or vaccine comprising one or more of the recombinant HVT or SB-1 viral vectors of the present invention and pharmaceutically or veterinarily acceptable vectors, excipients, mediators, or adjuvants.

[0062] In another embodiment, the present invention provides a composition or vaccine comprising an HVT viral vector containing a polynucleotide encoding the NDV-F antigen, an SV40 promoter, and optionally a pharmaceutically or veterinary acceptable vector, excipient, medium, or adjuvant. In another embodiment, the present invention provides a pharmaceutical composition or vaccine comprising a first HVT vector containing a polynucleotide encoding the NDV-F antigen, a second HVT vector containing a polynucleotide encoding the IBDV VP2 antigen, and optionally a pharmaceutically or veterinary acceptable vector, excipient, medium, or adjuvant. In another embodiment, the present invention provides a pharmaceutical composition or vaccine comprising an HVT vector containing a polynucleotide encoding the NDV-F antigen, an SB-1 vector containing a polynucleotide encoding the NDV-F antigen, and optionally a pharmaceutically or veterinary acceptable vector, excipient, medium, or adjuvant. The pharmaceutical compositions or vaccines of the present invention may comprise a first HVT carrier containing a polynucleotide encoding the NDV-F antigen, a second HVT carrier containing a polynucleotide encoding the IBDV VP2 antigen, an SB-1 carrier containing a polynucleotide encoding the NDV-F antigen, optionally a pharmaceutically or veterinarily acceptable carrier, excipient, mediator, or adjuvant.

[0063] In another embodiment, the present invention provides a composition or vaccine comprising a dual HVT viral vector, comprising: i) a first heteropolynucleotide encoding and expressing an NDV-F antigen or polypeptide; ii) a second polynucleotide encoding and expressing an IBDV VP2 antigen or polypeptide; and iii) optionally a pharmaceutically or veterinary-acceptable vector, excipient, medium, or adjuvant. In another embodiment, the present invention provides a composition or vaccine comprising a dual HVT viral vector containing two polynucleotides encoding and expressing an IBDV VP2 antigen or polypeptide, and optionally a pharmaceutically or veterinary-acceptable vector, excipient, medium, or adjuvant. In another embodiment, the composition comprising a dual HVT viral vector further comprises an HVT vector containing a polynucleotide encoding an IBDV VP2 antigen or an SB-1 vector containing a polynucleotide encoding an NDV-F antigen, or a combination thereof. Pharmaceutically or veterinary-acceptable vectors, adjuvants, mediums, or excipients are well known to those skilled in the art. For example, a pharmaceutically or veterinary acceptable carrier, adjuvant, medium, or excipient could be a Marek's disease vaccine diluent used in MD vaccines. Other pharmaceutically or veterinary acceptable carriers, adjuvants, mediums, or excipients that can be used in the methods of the present invention include, but are not limited to, 0.9% NaCl (e.g., saline) solutions or phosphate buffers, poly-(L-glutamate), or polyvinylpyrrolidone. A pharmaceutically or veterinary acceptable carrier, medium, or excipient could be any compound or combination of compounds that facilitates the administration of a carrier (or a protein expressed in vitro from a carrier of the present invention) or facilitates the transfection or infection of a carrier (or protein) and / or improves its preservation. Dosage and dose-volume are discussed herein in the general description 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.

[0064] Optionally, other compounds may be added as pharmaceutically or veterinary acceptable carriers, adjuvants, mediators, or excipients, including but not limited to alum; CpG oligonucleotides (ODNs), particularly ODNs 2006, 2007, 2059, or 2135 (Pontarollo RA et al., Vet. Immunol. Immunopath, 2002, 84: 43-59; Wernette CM et al., Vet. Immunol. Immunopath, 2002, 84: 223-236; Mutwiri G. et al., Vet. Immunol. Immunopath, 2003, 91: 89-103); polyA-polyU, dimethyl dioctadecyl ammonium bromide (DDA) (“Vaccine Design: The Subunit and Adjuvant Approach”, edited by Michael F. Powell and Mark J. Newman, Pharmaceutical). Biotechnology, 6: p.03, p.157); N,N-bis(octadecyl-N',N'-bis(2-hydroxyethyl)propanediamine (e.g.) (Source: same as above, p. 148); Carbomer, chitosan (see, for example, U.S. Patent Series No. 5,980,912).

[0065] Pharmaceutical compositions and vaccines according to the invention may contain one or more adjuvants or consist substantially of one or more adjuvants. Suitable adjuvants for carrying out the invention are (1) polymers of acrylic acid or methacrylic acid, polymers of maleic anhydride and alkenyl derivatives, (2) immunostimulatory sequences (ISS), such as oligodeoxyribonucleotide sequences having one or more unmethylated CpG units (Klinman et al., 1996; WO98 / 16247), (3) oil-in-water emulsions, such as the SPT emulsion described on page 147 of “Vaccine Design, The Subunit and Adjuvant Approach” published by M. Powell, M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of the same work; (4) cationic lipids containing quaternary ammonium salts such as DDA, (5) cytokines, (6) aluminum hydroxide or aluminum phosphate, (7) saponins, or (8) other adjuvants discussed in any literature cited and incorporated herein by reference, or (9) any combination or mixture thereof.

[0066] Another aspect of the invention relates to a method for inducing an immune response against one or more antigens or a protective response against one or more avian pathogens in an animal, said method comprising vaccinating the animal at least once with a vaccine or pharmaceutical composition of the invention. Another aspect of the invention relates to a method for inducing an immune response against one or more antigens in an animal or a protective response against one or more avian pathogens in a priming-boost regimen, said method comprising at least one initial administration and at least one booster administration using at least one commonly used polypeptide, antigen, epitope, or immunogen. The immunizing composition or vaccine used in the initial administration may be the same or may differ in nature from the immunizing composition or vaccine used as a booster.

[0067] Avian pathogens can include Newcastle disease virus (NDV), infectious bursal disease virus (i.e., IBDV or avian kidney disease virus), Marek's disease virus (MDV), infectious laryngotracheitis virus (ILTV), avian encephalomyelitis virus, avian reovirus, avian paramyxovirus, avian mepivaca virus, avian influenza virus, avian adenovirus, fowlpox virus, avian coronavirus, avian rotavirus, avian parvovirus, avian astrovirus and chicken anemia virus coccidiosis (species of Eimeria), species of Campylobacter, species of Salmonella, Mycoplasma gallisepticum, Mycoplasma synoviae, species of Pasteurella, species of Avian bacillus, species of Escherichia coli and Clostridium.

[0068] Typically, a single administration of the vaccine is given at 1 day of age via subcutaneous or intramuscular route, or in embryos aged 17-19 days via intraocular route. A second administration may be given within the first 10 days of life. Animals are preferably at least 17 days old embryos or 1 day old at the time of the first administration.

[0069] In chickens of all ages, it can be administered via various routes, such as subcutaneously, intramuscularly, intradermally, or transdermally. Intraocular inoculation can be performed in the amniotic sac and / or embryo. Commercially available intraocular and SC administration devices can be used for inoculation.

[0070] The present invention will now be further described by way of the following non-limiting embodiments. Example

[0071] DNA inserts, plasmids, and recombinant viral vectors were constructed using standard molecular biology techniques described by J. Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd edition, ColdSpring Harbor Laboratory, ColdSpring Harbor, New York, 1989).

[0072] Example 1: Construction of recombinant vHVT114 expressing NDV-F

[0073] Preparation of donor plasmid pHM103+Fopt

[0074] Digestion of the intergenic I arm containing HVT FC126 using NotI (see...) Figure 2 The plasmid pHM103 (Merial Limited) containing the SV40 promoter and SV40 poly A was dephosphorylated and a 5.6 kb fragment was extracted by gel extraction. A 1.7 kb fragment flanked by NotI from the chemically synthesized codon-optimized genotype VIId NDV-F (SEQ ID NO:1, encoding SEQ ID NO:2) was also NotI digested, followed by gel extraction of the 1.7 kb fragment. The 5.6 kb and 1.7 kb fragments were ligated to generate pHM103+Fopt. Figure 3 ).

[0075] Production of recombinant HVT viral vectors

[0076] In vitro recombination (IVR) was performed using pHM103+Fopt (as donor plasmid) and viral DNA isolated from HVT strain FC126 via co-electroporation of second-generation chicken embryo fibroblasts (2° CEF cells). 1x10⁻¹⁰ μL of Opti-MEM was used. 7 Electroporation was performed using 2° CEF cells in a 150V electroporation cup within a 2mm electroporation vessel. Transfected cells were seeded into 96-well plates and incubated for 5 days. Cells grown in the 96-well plates were then aliquoted into two separate 96-well plates. One set of 96-well plates was used for IFA with anti-NDV-F chicken polyclonal serum to identify positive wells containing recombinant cells, while the other set of 96-well plates was used to recover infected cells from the positive wells.

[0077] First, the virus was replicated in a 96-well plate and IFA selection was performed to screen wells containing the most IFA-positive spots and the fewest IFA-negative spots for recombinant virus purification. Wells meeting these criteria were then harvested and adjusted to 1 ml in DMEM + 2% FBS. 5-20 μL of the 1 ml stock solution was taken and mixed with 1x10⁻¹⁰ FBS solution. 7 One CEF was mixed in 10 ml DMEM + 2% FBS and then aliquoted into new 96-well plates so that each well contained a single HVT plaque. The absence of parental virus in the supernatant of wells containing a single plaque was tested by PCR. After 5 rounds of plaque purification, a recombinant virus named vHVT114 was isolated, and its purity was tested by IFA and PCR to confirm NDV-F expression and the absence of parental virus.

[0078] PCR analysis of recombinant vHVT114

[0079] DNA was extracted from vHVT114 by phenol / chloroform extraction, precipitated with ethanol, and then resuspended in 20 mM HEPES. PCR primers (shown in Table 1) were designed to specifically identify the presence of codon-optimized NDV-F and SV40 promoters and the purity of recombinant virus from the FC126CL2 parent virus. PCR was performed using 200 ng of DNA template with the specified primer pairs shown in Table 1. PCR cycling conditions were as follows: 2 min at 94 °C; 30 cycles (30 s at 94 °C, 30 s at 55 °C, 3 min at 68 °C); 5 min at 68 °C. Expected PCR products are shown in Table 2. PCR results are shown in... Figure 4 In the middle. For example Figure 4 As shown, the size of the PCR products after gel electrophoresis corresponds to the well with the expected size and banding pattern.

[0080] Table 1

[0081] Primers SEQ ID NO Sequence 5'-3' MB080 13 CGA ACA AAC TTC ATC GCT ATG C MB081 14 TAA CTC AAA TGC GAA GCG TTG C optF 15 ACT GAC AAC ACC CTA CAT GGC VlloptF RP 16 GCC AGC ACC AGG CTC AGG G SV40 starter F 17 AGC TTG GCT GTG GAA TGT

[0082] Table 2

[0083]

[0084]

[0085] Expression analysis of recombinant vHVT114

[0086] Immunofluorescence assays were performed using vHVT114, which had been passaged more than 10 times outside of the experimental pre-master inoculation (pre-MSV). Pre-MSV and pre-MSV+12 materials were diluted 1:100 in culture medium. 50 μL of the diluted virus was added to a volume with a pH of 1 x 10⁻⁶. 7Cells were added to 10 ml of DMEM + 2% FBS and then aliquoted into 96-well plates (100 μL / well). The plates were incubated at 37°C + 5% CO2 for 3 days until viral spots were visible. The plates were fixed with 95% ice-cold acetone for 3 minutes and washed three times with PBS. 1:1000 dilution of anti-Newcastle disease virus chicken antiserum (lot#C0139, Charles Rivers Laboratory) and 1:3000 dilution of monoclonal antibody L-78 (Merial Limited) were added, and the plates were incubated at 37°C for 1 hour. After 1 hour of incubation, the plates were washed three times with PBS, and 1:500 dilution of FITC anti-chicken (cat#F8888, Sigma) and Alexz Fluor 568 donkey anti-mouse (IgG) (cat#A 10037, Molecular Probe) were added. The plates were incubated again at 37°C for 1 hour. After 1 hour of incubation, the cells were washed three times with PBS. A small amount of PBS was added to prevent the monolayer from drying out and to induce autofluorescence. Cells were then visualized via fluorescence microscopy using a combination of tetramethylrhodamine isothiocyanate (TRITC) and fluorescein isothiocyanate (FITC) filters.

[0087] vHVT114 viral spots were visualized using TRITC and FITC filters (for double staining). The FITC assay showed NDV-F expression, and the TRITC assay showed HVT expression. Due to the small size of the 96-well plates, spots in each well were counted first using a TRITC filter, followed by a FITC filter. Over 500 spots were counted for pre-MSV and pre-MSV+12 passages. All spots on both plates were positive for both FITC and TRITC. Figure 5 )

[0088] Southern blotting analysis of recombinant vHVT114

[0089] Total genomic DNA was extracted from HVT FC126 and vHVT114 according to standard genomic DNA extraction protocols. For each restriction enzyme digestion, a total digestion volume of 20 μl was used for each sample, employing 3 μg of genomic DNA (1 ng for the donor plasmid). Genomic DNA from HVT FC126 (negative control), pHM103+Fopt donor plasmid, and vHVT114 were digested overnight at 37°C using restriction endonucleases BamHI, PstI, SphI, and NcoI. Restriction fragments of HVT FC126 (negative control), pHM103+Fopt donor plasmid, and vHVT114 genomic DNA were separated by 1% agarose gel electrophoresis and transferred to a positively charged nylon membrane. The membrane was pre-hybridized for 1 hour according to the manufacturer's instructions of the North2South Chemiluminescent Hybridization and Detection Kit (Thermo Scientific), followed by overnight hybridization with a biotinylated NDV-F probe at 55°C. After overnight hybridization, several rigorous washes were performed until the membrane was placed in a blocking buffer containing streptavidin-HRP. After rinsing the membrane to remove any unbound streptavidin-HRP, a substrate solution of luminol and peroxide was added. The membrane was then exposed to X-ray film and developed. The regions where the biotinylated probes bound to DNA were chemiluminescent and captured by the X-ray film. Table 3 shows the expected Southern blot bands produced using the NDV-F probe. The Southern blot results showed the digestion pattern as expected ( Figure 6 ).

[0090] Table 3

[0091] NDV-F probe

[0092]

[0093] Sequence analysis of the inserted region in recombinant vHVT114

[0094] Analysis of the vHVT114 genomic DNA region was performed by PCR amplification. A total of 10 primers were used to amplify the entire cassette, including the flanking BamHI-I arm used in the donor plasmid. The 4.727 kb PCR product was gel purified, and the complete fragment was sequenced using sequencing primers. The sequencing results confirmed that vHVT114 contains the correct SV40 promoter, codon-optimized NDV-F, and SV40polyA sequences, which perfectly match the sequence described in SEQ ID NO:18 for the donor plasmid pHM103+Fopt.

[0095] Western blot analysis of recombinant vHVT114

[0096] Infect 2x10 cells with vHVT114Pre-MSV at approximately 0.1 MOI. 6 Chicken fibroblasts were incubated at 37°C for 2 days. After removing the culture medium and washing with PBS, infected and uninfected cells were collected using a cell scraper. Cells were collected with 1 ml PBS and then centrifuged. The cell pellet was lysed according to the Pierce Classic IP kit (cat#26146, Thermo Scientific). 100 μl of anti-NDV-F monoclonal antibody 001C3 (Merial Limited) was used to form immune complexes. The antibody / lysate sample was added to Protein A / G Plus agarose to capture the immune complexes. The immune complexes were washed 3 times to remove unbound material, and then eluted with sample buffer under non-reducing conditions in a 50 μl volume. After boiling for 5 min, 10 μl of the sample was loaded into a 10% acrylamide gel (Invitrogen). The PAGE gel was electrophoresed in MOPS buffer (Invitrogen) at 200 V for 1 h. The gel was then transferred to a PVDF membrane.

[0097] The Protein Detector Western Blot Kit TMB System (KPL, cat#54-11-50) was used to blot PVDF membranes using reagents and following the manufacturer's instructions. After blocking the membrane at room temperature for 1 hour, it was then washed three times for 5 minutes each time with 1X wash buffer, followed by immersion in blocking buffer containing a 1:1000 dilution of chicken serum against NDV virus (Lot#C0139, Charles River Laboratories). After washing three times with wash buffer, the membrane was incubated at room temperature for 1 hour with peroxidase-labeled goat anti-chicken IgG (KPL, cat#14-24-06) at a 1:2000 dilution. The membrane was then washed three times for 5 minutes each time with 1X wash buffer. 5 ml of TMB membrane peroxidase substrate was added to the membrane, and the membrane was gently agitated for approximately 1 minute. The development reaction was terminated by placing the membrane in water.

[0098] Immunoprecipitation and Western blotting detected approximately 55 kDa protein of the expected size corresponding to the F1 fraction of the NDV-F protein in vHVT114 samples. Figure 7 ).

[0099] Example 2: Construction of recombinant NDV-F molecules vHVT110, vHVT111, vHVT112, vHVT113, and vHVT116

[0100] The HVT recombinants vHVT110, vHVT111, vHVT112, vHVT113, and vHVT116 were generated and characterized in essentially the same manner as described in Example 1 for vHVT114. Table 4 shows the unique characteristics around the expression cassette for each construct, including their respective sequences.

[0101] Table 4

[0102] Characteristics of expression cassettes of a single HVT recombinant

[0103] name Parental virus promoter F gene Poly-A locus vHVT039 HVT MDV gB Wtnm-Texas SV40 IG1 vHVT110 HVT mCMV IE Wt-VIId SV40 IG1 vHVT111 HVT SV40 Wt-VIId SV40 IG1 vHVT112 HVT MCMV IE Wt-YZCQ SV40 IG1 vHVT113 HVT MCMV IE Wt-Texas SV40 IG1 vHVT114 HVT SV40 Opt-VIId SV40 IG1 vHVT116 HVT SV40 Opt-Ca02 SV40 IG1

[0104] vHVT110

[0105] The plasmid pCD046 (Merial's proprietary material), containing the intergenic I arm of HVT FC126, the mouse CMV promoter, and SV40poly A, was digested with NotI, dephosphorylated, and a 6.6 kb fragment was extracted via gel extraction. A 1.7 kb fragment flanked by NotI-linked fragments of the chemically synthesized NDV-F gene (SEQ ID NO:3, encoding SEQ ID NO:4), containing the wild-type F sequence, was also digested with NotI, followed by gel extraction of the 1.7 kb fragment. The 6.6 kb and 1.7 kb fragments were ligated to generate the donor plasmid pCD046+NDV-F wt (SEQ ID NO:21 for vHVT110) for transfection to obtain recombinant vHVT110. Sequencing of the inserted sequence confirmed that vHVT110 contained the correct sequence of the mCMV promoter, the wild-type NDV-F gene, and SV40poly A. This sequence also perfectly matches the sequence described in EQ ID NO:21 for the donor plasmid pCD046+NDV-F wt.

[0106] vHVT111

[0107] The plasmid pHM103 (Merial proprietary material), containing the intergenic I arm of HVT FC126, the SV40 promoter, and SV40polyA, was digested with NotI, dephosphorylated, and then a 5.6 kb fragment was extracted via gel extraction. A 1.7 kb fragment flanked by NotI-linked to the chemically synthesized NDV-F gene (SEQ ID NO:3, encoding SEQ ID NO:4), containing the wild-type F sequence, was also digested with NotI, followed by gel extraction of the 1.7 kb fragment. The 5.6 kb and 1.7 kb fragments were ligated to generate the donor plasmid for transfection to obtain recombinant vHVT111 (for vHVT1110, SEQ ID NO:22). Sequencing of the inserted sequence confirmed that vHVT111 contained the correct sequence of the SV40 promoter, the wild-type NDV-F gene, and SV40polyA, as shown in the sequence of the donor plasmid pHM103+NDV-F wt (SEQ ID NO:22).

[0108] vHVT112

[0109] Using NotI, a fragment containing the synthetic NDV-FYZCQ wild-type gene (encoding SEQ ID NO:34 of SEQ ID NO:35) was excised from the pUC57NDV-F YZCQ plasmid (synthesized by GeneScript) and inserted into the same site in the pCD046 plasmid containing the mCMV promoter and SV40 polyA tail. The ligated material was transformed using the Top10Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp liquid medium, and plasmids were extracted using the Qiagens MiniSpin Prep kit, followed by selection of insert orientation. The correct donor plasmid was named pCD046+NDV-F VII YZCQ. Large-scale cultures were grown, and plasmid extraction was performed using the Qiagens Maxi Prep kit. Transient expression of the large-scale preparation was validated in chicken embryo fibroblasts (CEFs) using Fugene transfection agent and anti-NDV chicken polyclonal serum.

[0110] The plasmid pCD046+NDV-F VII YZCQ (SEQ ID NO:29) was used for transfection to generate recombinant vHVT112. Sequencing of the inserted region confirmed that vHVT112 contained the correct sequence of the mCMV promoter, the wild-type NDV-F YZCQ gene, and SV40polyA. This sequence also perfectly matched the sequence described in SEQ ID NO:29 for the donor plasmid pCD046+NDV-F VII YZCQ.

[0111] vHVT113

[0112] Using NotI, a fragment containing the synthetic NDVTexas F gene (SEQ ID NO:36 encoding SEQ ID NO:37) was excised from the UC57 NDV Texas F plasmid (synthesized by GeneScript) and inserted into the same site in the pCD046 plasmid containing the mCMV promoter and SV40 polyA tail. The ligated material was transformed using the Top10Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp liquid medium, and plasmids were extracted using the Qiagens MiniSpin Prep kit to screen for insert orientation. The correct donor plasmid was named pCD046+TexasNDV-F. Large-scale cultures were grown, and plasmids were extracted using the Qiagens Maxi Prep kit. Transient expression of the large-scale preparation was validated in chicken embryo fibroblasts (CEFs) using Fugene transfection agent and anti-NDV chicken polyclonal serum.

[0113] Plasmid pCD046+Texas NDV-F (SEQ ID NO:30) was used for transfection to generate recombinant vHVT113. Sequencing of the inserted region confirmed that vHVT113 contained the correct sequence of the mCMV promoter, the wild-type NDV-F Texas F gene, and SV40polyA. The sequence also perfectly matched the sequence described in SEQ ID NO:30 for the donor plasmid pCD046+Texas NDV-F.

[0114] vHVT039

[0115] The MDV gB promoter (SEQ ID NO:38) was amplified from DNA extracted from MDV1RB1B strain using primers HM101 (5'-CCG-GAA-TTC-CGA-TGT-TTA-GTC-ACG-ATA-GAC-3') (SEQ ID NO:44) and HM102 (5'-ATA-AGA-GCG-GCC-GCA-GTG-AGA-TGA-TCT-TAA-TGA-TG-3') (SEQ ID NO:45). The former contains an EcoRI site, and the latter contains a NotI site for ligating a 630 bp EcoRI / NotI-digested PCR product into an EcoRI / NotI-digested pCD046 plasmid. The ligation product was used to transform DH5α competent cells. Colonies were picked, and the presence of the inserted PCR fragment was screened using restriction assays with EcoRI and NotI. The resulting plasmid was named pHM102.

[0116] The rapid-type NDV Texas strain (genotype IV) was grown in 11-day-old SPF chicken embryos and semi-purified. Total RNA was extracted and RT-PCR was performed using two primers: F-ATG (5'TAT-AGC-GGC-CGC-AAG-ATG-GGC-TCC-AGA-TCT-TCT-ACC-AG3') (SEQ ID NO:46) and F-STOP (5'CGA-GGC-GGC-CGC-TCA-TAT-TTT-TGT-AGT-GGC-TCT-C3') (SEQ ID NO:47). These primers allowed for complete amplification of the NDV F gene, with NotI sites added upstream of ATG and downstream of the stop codon. A 1.7 kb PCR fragment was digested with NotI and ligated into NotI-digested pHM102. The resulting plasmid, named pHM119, was used as a donor plasmid in the in vitro recombination study. It was co-transfected with HVT parental DNA into CEF cells to generate vHVT039. Sequencing of the inserted region confirmed that vHVT039 contained the correct sequence of the MDV gB promoter, the wild-type unmodified NDV-F gene from the Texas strain (encoding SEQ ID NO:32 of SEQ ID NO:33), and the SV40 polyA (SEQ ID NO:31) shown in a partial sequence of the donor plasmid pHM119.

[0117] vHVT116

[0118] The plasmid pHM103 (Merial patented material), containing the intergenic I arm of HVT FC126, the SV40 promoter, and SV40polyA, was digested with NotI, dephosphorylated, and a 5.6 kb fragment was extracted by gel extraction. A 1.7 kb fragment flanked by NotI was chemically synthesized, codon-optimized, and the CA02 genotype V NDV-F gene (SEQ ID NO:5, encoding SEQ ID NO:6) was also digested with NotI, and a 1.7 kb fragment was extracted by gel extraction. The 5.6 and 1.7 kb fragments were ligated to generate pHM103+NDV-F CA02 (SEQ ID NO:23, for vHVT116) for transfection to obtain recombinant vHVT116. Sequencing of the inserted region confirmed that vHVT116 contains the correct sequence of the SV40 promoter, codon-optimized CA02NDV-F gene, and SV40polyA (SEQ ID NO:23) shown in the sequence of the donor plasmid pHM103+NDV-F wt.

[0119] discuss

[0120] Various boxes under mCMV or non-CMV promoters were inserted into different loci in the HVT genome (Table 4). Despite repeated attempts, the generation of constructs with mCMV and codon-optimized F sequences was unsuccessful beyond the second generation. However, when the wild-type sequence was driven by mCMV, a stable construct, vHVT110, was generated. Furthermore, the recombinant vHVT111 with a wild-type F sequence under the SV40 promoter was also stable for more than 10 in vitro passages. Surprisingly, similarly, codon-optimized F sequences under the SV40 promoter were found to be stable for more than 10 in vitro passages (e.g., vHVT114 and vHVT116). These results demonstrate the delicate balance between promoter strength and the properties of the genes they control (codon-optimized or non-optimized) in generating genetically stable HVT constructs.

[0121] Example 3: Construction of vHVT306 (a dual HVT vector expressing NDV-F and IBDV VP2)

[0122] The donor plasmid pHVT US2 SV-Fopt-synPA was constructed, which contains the SV40 promoter, the synthetic NDV F codon-optimized VII gene, the synthetic polyA tail, and the SORF3 and US2 arms of HVT FC126 flanked by the FC126.

[0123] The generation of recombinant viruses

[0124] Following standard homologous recombination, secondary CEF cells were co-electroplated using donor plasmid pHVT US2 SV-Fopt-synPA and viral DNA isolated from vHVT13 (an HVT vector expressing the IBDV VP2 gene, Merial Limited). The recombinants were generated using methods substantially as described in Example 1 for vHVT114, purified by plaque phage purification, and identified by immunofluorescence.

[0125] After purification using 5 plaques, the pure recombinant virus (vHVT306) was isolated, and the purity of vHVT306 was tested and confirmed by IFA and PCR.

[0126] PCR analysis

[0127] Viral DNA was extracted from vHVT306 motherboard pre-MSV using the QIA DNeasy Blood and Tissue Kit (Qiagen cat#69506). PCR primers were designed to identify the presence of optimized flanking linkers for NDV F, NDV F wild-type, SV40 promoter, mCMV promoter, US2HVT virus, and SB-1 virus.

[0128] PCR amplification using different primers confirmed that vHVT306 had the expected amplification pattern and amplicon.

[0129] Expression Analysis

[0130] Indirect immunofluorescence assay (IFA) was performed on vHVT306 pre-MSV stock solution. CEF inoculated with vHVT306 was fixed with ice-cold 95% acetone for 3 minutes at room temperature, followed by air drying for 10 minutes. After washing three times with PBS, two primary antibodies were added: chicken anti-Newcastle disease virus serum diluted 1:500 (Charles Rivers Laboratories cat#10100641, lot#C0117A) and anti-HVT L78 monoclonal antibody diluted 1:3000 (Merial Select, Gainesville, GA), and incubated at 37°C for 45 minutes. After washing three times with PBS, two secondary antibodies were added: goat anti-chicken IgG-fluorescein diluted 1:500 (KPL cat#.02-24-06, lot#110020) and donkey anti-mouse IgG-Alexa Fluor 568 diluted 1:300 (Molecular Probe#A10037, lot#989784). The plates were incubated at 37°C for 45 minutes, followed by washing three times with PBS. Cells were observed using fluorescence microscopy with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) filters on a Nikon Eclipse Ti inverted microscope to identify IFA-positive plaques.

[0131] Similarly, chicken anti-IBDV serum (Charles River Laboratories cat#10100610, lot#G0117) (1:500 dilution) and anti-NDV F monoclonal antibody 001C3 (Asceitic fluid, Batch 10 / 09 / 044, 02 / 11 / 2010) (1:300 dilution) were used as primary antibodies, followed by goat anti-chicken IgG-fluorescein (KPL cat#.02-24-06, lot#110020) (1:500 dilution) and donkey anti-mouse IgG-Alexa Fluor 568 (Molecular Probe#A10037, lot#989784) (1:300 dilution) as secondary antibodies. The expression of IBDVVP2 protein (SEQ ID NO:8 encoded by SEQ ID NO:7) in vHVT306 was examined by IFA.

[0132] IFA results indicate that vHVT306 expresses the NDV F gene in virus-infected CEF.

[0133] More than 400 vHVT306 plaques were counted using FITC- and TRITC-filters under a microscope. The overall expression of the NDV F gene and IBDV VP2 matched the HVT plaques (Table 5).

[0134] Table 5 Dual IFA of vHVT306

[0135]

[0136] Southern Imprint Analysis

[0137] Total genomic DNA was extracted from CEF cells infected with vHVT306pre-MSV stock solution. Southern blot analysis was performed according to standard protocol.

[0138] A total of three probes were used to confirm the preservation of the NDV F box (SV40 promoter, NDVF codon-optimized gene, synthetic poly A tail) between SORF3 and US2 of vHVT306 and the IBDV VP2 box (mCMV promoter, IBDV VP2 gene, SV40 poly A tail).

[0139] Southern blot results showed the digestion pattern expected based on Vector NTI (Invitrogen, 1600 Faraday Ave., Carlsbad, CA) mapping analysis. The NDV F box (SV40 promoter, NDV F codon-optimized gene, synthetic poly A tail) was located between SORF3 and US2, and the IBDV VP2 box (mCMV promoter, IBDV VP2 gene, SV40 poly A tail) was as complete as the parent virus (vHVT13).

[0140] Genome analysis

[0141] Genomic DNA from vHVT306pre-MSV stock solution was sequenced to verify the recombinant arm region and the sequence of the inserted gene cassette.

[0142] Primers were designed to amplify the entire inserted gene cassette (including the recombinant arm) used in the donor plasmid. Analysis of the vHVT306 genomic DNA was performed by PCR amplification and subsequent nucleotide sequencing.

[0143] vHVT306 (donor plasmid pHVTUS2 SV-Fopt-synPA), which contains the recombinant arm, SV40 promoter, and NDV F codon optimized gene, has been confirmed as correct, as shown in SEQ ID NO:20.

[0144] Western blot analysis

[0145] CEF monolayers were infected with vHVT306pre-MSV at an MOI of 0.1. After 4 days of incubation, CEF was precipitated, washed with PBS, and then lysed using the IP lysis / wash buffer from the Pierce Classic IP kit (Thermo Scientificcat #26146) according to the manufacturer's protocol. The lysate was pre-clarified and incubated with 100 μL of anti-NDV F monoclonal antibody 001C3 to prepare immune complexes. The immune complexes were captured by Protein A / G Plus agarose gel, and after removing unbound immune complexes by washing, 50 μL of sample buffer was used for elution under non-reducing conditions. Uninfected CEF was included as a control. 20 μL of the eluted sample was separated by electrophoresis on a 10% Bis-Tris gel. After electrophoresis, the separated proteins were transferred to a PVDF membrane. Following the manufacturer's protocol, the Protein Detection TMB Western Blot Kit (KPL cat#54-11-50) was used to detect NDV antigen on PVDF membranes using chicken anti-NDV serum (Charles River Laboratories Laboratories cat#10100641, lot#C0117A) and goat anti-chicken IgG-peroxidase conjugate (KPL cat#14-24-06).

[0146] NDV F protein expression in vHVT306 was confirmed using a two-step immunoassay. First, NDV F protein expression from vHVT306-infected CEF was captured by immunoprecipitation using anti-NDV F monoclonal antibody 001C3. Subsequently, Western blot analysis of anti-NDV polyclonal serum (Charles River Laboratories, CAT #10100641, LOT #C0117A) was used to detect NDV F protein in the captured sample (NDV F protein-monoclonal antibody complex). Figure 8 The 55 kDa protein in the vHVT306 pre-MSV lysate was detected using anti-NDV serum, which corresponds to the expected size of the NDV F1 fusion protein. Figure 8 ).

[0147] Example 4: Construction of dual HVT vectors vHVT301, vHVT302, vHVT303, vHVT304, and vHVT307 expressing NDV-F and IBDV VP2, and dual HVT vector vHVT202 expressing an IBDV VP2 variant.

[0148] Example 4.1 Construction of vHVT301, vHVT302, vHVT303, vHVT304 and vHVT307

[0149] The generation and characterization of the dual HVT recombinants vHVT301, vHVT302, vHVT303, vHVT304, and vHVT307 were performed in essentially the same manner as described for vHVT306 in Example 3. Table 6.1 shows the unique features around the expression cassette for each construct, including their respective sequences.

[0150] Table 6.1 Characteristics of expression cassettes of dual HVT recombinants

[0151] name Parental virus promoter NDV-F gene Poly-A locus vHVT301 vHVT13 SV40 Wt-VIId NDV-F SV40 IG2 vHVT302 vHVT13 US10 Opt-VIId NDV-F US10 US10 vHVT303 vHVT13 US10 Opt-V NDV-F US10 US10 vHVT304 vHVT13 SV40 Opt-VIId NDV-F Synthetic IG2 vHVT306 vHVT13 SV40 Opt-VIId NDV-F Synthetic SORF3-US2 vHVT307 vHVT13 SV40 Opt-V NDV-F Synthetic SORF3-US2

[0152] vHVT301

[0153] The plasmid pHVT IG2 SbfI (Merial patented material), containing the two intergenic arms of vHVT13, was digested with SmaI, followed by dephosphorylation, and a 4.3 kb fragment was extracted via gel extraction. The donor plasmid pHM103+NDV-F wt, containing the SV40 promoter, wild-type NDV-F genotype VIId, and SV40 poly A tail, was digested with EcoRI and SalI, treated with Klenow, and a 2.3 kb fragment was extracted via gel extraction. The two fragments were ligated to generate the donor plasmid pHVT IG2 SV Fwt SbfI (SEQ ID NO: 24) for transfection to obtain recombinant vHVT301.

[0154] vHVT302

[0155] The synthetic plasmid pHVT US10 cds, containing the US10 arm sequence of vHVT13, was digested with NotI, followed by dephosphorylation and gel extraction of a 4.7 kb fragment. A 1.7 kb fragment flanked by a chemically synthesized, codon-optimized NDV-F genotype VIId was notI-linked, digested with NotI, and gel extracted. The two fragments were ligated to generate the donor plasmid pHVT US10 cds F opt for transfection to obtain recombinant vHVT302. Transcription of the inserted F gene should be driven by the native US10 promoter and terminated by the native US10 polyA signal. No exogenous promoter or polyA was added to express the insert. Sequencing of the inserted region confirmed that vHVT302 contained the correct sequence of the codon-optimized VIId NDV-F gene, as shown in the sequence of the donor plasmid pHVT US10 cds F opt (SEQ ID NO: 25).

[0156] vHVT303

[0157] The synthetically produced plasmid pHVT US10 cds, containing the US10 arm sequence of vHVT13, was digested with NotI, dephosphorylated, and a 4.7 kb fragment was extracted via gel extraction. A 1.7 kb fragment flanked by a chemically synthesized, codon-optimized NDV-F genotype V was notI-linked, digested with NotI, and extracted via gel extraction. The two fragments were ligated to generate the donor plasmid pHVT US10 cds F CAO2 opt for transfection to produce recombinant vHVT303. As with vHVT302, transcription of the inserted F gene should be driven by the native US10 promoter and terminated by the native US10 polyA signal. No exogenous promoter or polyA was added to express the insert. Sequencing of the inserted region confirmed that vHVT303 contained the correct sequence of codon-optimized NDV-F genotype V, as shown in the sequence of the donor plasmid pHVT US10 cds F CA02 (SEQ ID NO: 26).

[0158] vHVT304

[0159] The donor plasmid pHVT IG2 SbfI containing the two intergenic arms of vHVT13 was digested with SbfI, dephosphorylated, and a 4.3 kb fragment was extracted by gel extraction. A synthetically produced plasmid containing the SV40 promoter flanked by SbfI, codon-optimized NDV-F genotype VIId+, and a synthetic poly A tail was digested with SbfI and a 2.3 kb fragment was extracted by gel extraction. The two fragments were ligated to generate the donor plasmid pHVT IG2 SV Fopt syn-tail for transfection to produce recombinant vHVT304. Sequencing of the insert region confirmed that vHVT304 contained the correct sequence of the SV40 promoter, codon-optimized VIId NDV-F gene, and synthetic poly A tail, as shown in the sequence of the donor plasmid pHVT IG2 SV Fopt syn-tail (SEQ ID NO:27).

[0160] vHVT307

[0161] The donor plasmid pHVT US2-SORF3, containing the US2 and SORF3 arm sequences of vHVT13, was digested with SbfI, dephosphorylated, and a 5.1 kb fragment was extracted via gel extraction. The plasmid SB-1 UL55 SV CaF syn-tailed with SbfI, containing the SV40 promoter flanked by SbfI and codon-optimized NDV-F genotype V+ with a synthetic poly A tail, was digested with SbfI and a 2.3 kb fragment was extracted via gel extraction. The two fragments were ligated to generate the donor plasmid pHVT US2 SV-FCA02 opt-synPA for transfection to produce recombinant vHVT307. Sequencing of the insert region confirmed that vHVT307 contained the correct sequence of the SV40 promoter, codon-optimized VIId NDV-F gene, and synthetic poly A tail, as shown in the sequence of the donor plasmid pHVT US2 SV-FCA02 opt-synPA (SEQ ID NO: 28).

[0162] discuss

[0163] One of the main objectives of this work is to develop multivalent avian herpesvirus-based vectors by integrating multiple protective genes of interest into a single avian herpesvirus backbone (e.g., HVT). This approach presupposes the identification of expression cassettes containing appropriate promoter-gene-plyA combinations and the evaluation of their genetic stability and ability to protect against specific diseases.

[0164] To generate an effective MD-IBD-ND trivalent vector vaccine, codon-optimized or non-optimized Newcastle disease virus (NDV)-F gene sequences were cloned into the vHVT13 backbone (HVT-IBD, an approved vaccine that protects chickens from both MD and IBD infections) under human CMV (mouse CMV has been used in vHVT13). All vHVT-IBD-F constructs under the human CMV promoter lost F-protein expression within 6 generations, regardless of whether the NDV-F sequence was codon-optimized or not, and regardless of the insertion site. When hCMV was combined with the codon-optimized F protein, F-protein expression was rapidly lost (within 2 generations) compared to the combination of hCMV and the wild-type F sequence (where F-protein expression was lost within 6 generations). Taken together, these data suggest that human CMV is not an ideal promoter for generating stable HVT recombinants expressing the NDV-F protein. Surprisingly, this example shows that the SV40 promoter and the HVT endogenous promoter (US10 promoter) produce a stable HVT recombinant expressing the NDV-F protein.

[0165] Example 4.2 Construction of vHVT202

[0166] Construction of donor plasmid HVT SORF3-US2 gpVar-Ewtsyn

[0167] A fragment containing the synthetic Varient E wild-type IBDV VP2 gene (encoding SEQ ID NO:41 of SEQ ID NO:42) was excised from the pUC57 Varient E wt plasmid (synthesized by GeneScript) using NotI and inserted into the same site in the SORF3 and US2 plasmids containing the gpCMV promoter and a synthetic polyA tail. The ligated material was transformed using the Top10 Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp liquid medium, and plasmids were extracted using the Qiagens MiniSpin Prep kit. Insert orientation was screened using SacI+HindIII digestion. The correct donor plasmid was named pHVT SORF3-US2 gpVar-Ewt Syn. Table 6.2 shows the unique characteristics surrounding the expression cassette for the construct, including their respective sequences. Large-scale cultures were grown, and plasmid extraction was performed using the Qiagens Maxi Prep kit. Transient expression of the large-scale preparation was validated in chicken embryo fibroblasts (CEFs) using Fugene transfection agent and anti-IBDV chicken polyclonal serum.

[0168] Table 6.2 Characteristics of expression cassettes of dual HVT recombinants

[0169] name Parental virus promoter IBDV VP2 gene Poly-A locus vHVT202 vHVT306 Guinea Pig CMV IBDV E VP2 Synthetic SORF3-US2

[0170] Production of recombinants

[0171] Following standard homologous recombination, secondary CEF cells were co-electropographed using the pHVTSORF3-US2 gpVar-Ewt Syn donor plasmid and viral DNA isolated from vHVT306 and digested with SbfI. To simplify the following section process, classical VP2 expressing IBDV and vHVT306 cells expressing NDV-F were selected as parents. The variant E VP2 donor plasmid was designed to replace the F gene, and recombinants were initially selected for the absence of F gene expression, followed by PCR selection for the presence of variant E VP2. 1x10 7 Co-electroporation was performed in 2° CEF (in 300 μl Opti-MEM) via electroporation at 150 volts with a capacitance of 950 in a 2 mm electroporation cup. Transfected cells were seeded into 96-well plates and incubated for 5–7 days. Cells grown in the 96-well plates were then replicated into two 96-well plates and incubated for another 5 days. One set of 96-well plates was used for IFA (Intra-Infected Cell Analysis) with anti-NDV-F chicken polyclonal serum to identify positive wells containing the vHVT30 parent, while the other set of 96-well plates was used to recover infected cells from IFA-negative wells.

[0172] First, recombinant virus purification was performed using a 96-well plate replication method and by selecting wells containing the most IFA-negative (anti-NDV-F) plaques and the fewest IFA-positive plaques using IFA selection. These wells matching the criteria were then collected and adjusted to 1 ml in DMEM + 2% FBS. 5-20 μl (depending on the number of visible plaques) was taken from the 1 ml stock solution and mixed with 1 x 10⁻⁶ ppm of the stock solution. 7 One CEF sample was mixed in 10 ml of DMEM + 2% FBS and aliquoted into a new 96-well plate to attempt to obtain a single HVT plaque in each well. After 4 days of incubation, the 96-well plate was replicated, and the wells containing plaques were tested for the presence of recombinant HVT and the absence of parental virus by IFA and PCR. Wells showing more recombinant virus and less parental virus were harvested again (by comparison with PCR banding results), adjusted to 1 ml, and aliquoted into a new 96-well plate (as before). After 5 rounds of virus-infected cell purification, recombinant HVT with two IBDV VP2 proteins was isolated, and the purity of the recombinant virus was tested by PCR to confirm the absence of parental virus.

[0173] Sequencing of the insertion region confirmed that vHVT202 contains the correct sequence of the guinea pig CMV promoter, the wild-type VP2 gene of the IBDV variant E, and the synthetic poly A tail, as shown in the sequence of the donor plasmid HVT SORF3-US2 gpVar-Ewtsyn (SEQ ID NO:39).

[0174] Recombinant analysis by PCR

[0175] DNA was extracted from the stock virus via phenol / chloroform extraction, precipitated with ethanol, and resuspended in 20 mM HEPES. PCR primers were designed to specifically identify the Varient E wt gene, promoter, polyA, and the purity of recombinant virus from the HVT parent virus. PCR was performed using 200 μg of DNA template and the specified primer pairs shown in Table 1. PCR cycling conditions were as follows (unless otherwise specified): 94 °C–2 min; 30 cycles (94 °C–30 s, 55 °C–30 s, 68 °C–3 min); 68 °C–5 min.

[0176] The purity of the recombinant virus was confirmed by PCR using primer pairs specific to the HVT flanking linker arm, gpCMV promoter, Varient E gene, and syn tail. Primers specific to SB1 and MDV serotype 2 (SB1US1.FP + SB1Sorf4.RP) were also included in the analysis. PCR results showed that the recombinant virus vHVT202 had the expected expression cassette and that the viral stock contained no detectable amounts of parental HVT virus.

[0177] Immunofluorescence staining of recombinant vHVT202 virus expressing two VP2 proteins of IBDV

[0178] For immunofluorescence assays, P3 material was diluted 1:100 in the medium. Approximately 50 μl of diluted virus was added to 10 ml containing 1 x 10⁻⁶ cells / mL. 7 Cells were inoculated with DMEM and 2% FBS, and then aliquoted into 96-well plates (100 μl / well). The plates were incubated at 37°C with 5% CO2 for 4 days until viral plaques were visible. The plates were fixed with 95% ice-cold acetone for 3 minutes and washed three times with PBS. One well was used with 1:1000 dilution of anti-Newcastle disease virus chicken antiserum (lot#C0139, Charles Rivers Laboratory), and the plate was incubated at 37°C for 1 hour. The remaining wells were used with anti-IBDV chicken antiserum (lot#G0117). After 1 hour of incubation, the plates were washed three times with PBS, and FITC anti-chicken (cat#F8888, Sigma) was added at 1:500. The plates were again incubated at 37°C for 1 hour. After 1 hour of incubation, the cells were washed three times with PBS and visualized using a fluorescence microscope with a FITC filter.

[0179] Immunofluorescence staining results showed that vHVT202 exhibited extremely strong VP2 protein expression when polyclonal sera against classical E VP2 protein and variant E VP2 protein were used.

[0180] in conclusion

[0181] Based on PCR and immunofluorescence analysis, vHVT202 is a recombinant HVT in which the VP2 gene of variant E IBDV, under the control of the gpCMV promoter, has been successfully inserted into a recombinant HVT background (which already expresses the VP2 gene of classical IBDV). Therefore, vHVT202 possesses the VP2 gene of both variant E and classical IBDV, and it does not contain any detectable parental vHVT306 virus.

[0182] Example 5: Construction of recombinants vSB1-009, vSB1-004, vSB1-006, vSB1-007, vSB1-008, and vSB1-010 expressing NDV-F

[0183] Example 5.1 Construction of vSB1-009, vSB1-004, vSB1-006, vSB1-007 and vSB1-008

[0184] The aim of this study was to construct a recombinant SB-1 viral vector vSB1-009, in which an expression cassette containing the SV40 promoter and the Newcastle disease virus fusion protein (NDV-F) was inserted to replace the UL44 coding sequence (gC) of SB-1.

[0185] A donor plasmid pSB1 44cds SV FCAopt containing the UL44 flanking linker of the SB1 virus, the SV40 promoter, and the optimized gene sequence of the NDV F codon (SEQ ID NO:5, encoding SEQ ID NO:6) was constructed.

[0186] The generation of recombinant viruses

[0187] Following standard homologous recombination, secondary CEF cells were co-electroplated using donor plasmid pSB1 44cds SV FCAopt and viral DNA isolated from CEF cells infected with SB-1 virus. The recombinant was generated using methods substantially as described in Example 1 for vHVT114, purified by plaque phage purification, and characterized by immunofluorescence.

[0188] After five rounds of plaque purification, pure recombinant virus (vSB1-009) was isolated, and the purity of vSB1-009 was tested by IFA and PCR to confirm proper insertion and absence of residual parent virus.

[0189] PCR analysis

[0190] Viral DNA was extracted from vSB1-009 pre-MSV stock solution using the QIA DNeasy Blood and Tissue Kit (Qiagen cat#69506). PCR primers were designed to identify the presence of optimized NDV F, NDV F wild-type, SV40 promoter, mCMV promoter, the UL44 flanking linker of SB-1 virus, and HVT virus. PCR amplification was performed using approximately 200 ng of DNA template and primer pairs.

[0191] PCR amplification using different primers confirmed that vSB1-009 had the expected amplification pattern and amplicon.

[0192] Expression Analysis

[0193] Indirect immunofluorescence assay (IFA) was performed on vSB1-009 pre-MSV stock solution to examine the expression of NDV F gene and SB-1 virus antigen. CEF inoculated with vSB1-009 was fixed with ice-cold 95% acetone at room temperature for 3 minutes, followed by air drying for 10 minutes. After washing the plates with PBS, two primary antibodies were added: chicken anti-Newcastle disease virus serum diluted 1:500 (Charles Rivers Laboratories cat#10100641, lot#C0117A) and anti-SB-1 virus Y5.9 monoclonal antibody diluted 1:3000 (Merial Select, Gainesville, GA), and the plates were incubated at 37°C for 45 minutes. After washing three times with PBS, two secondary antibodies were added: goat anti-chicken IgG-fluorescein diluted 1:500 (KPL cat#.02-24-06, lot#110020) and donkey anti-mouse IgG-Alexa Fluor 568 diluted 1:250 (Molecular Probe#A10037, lot#989784). The plates were incubated at 37°C for 45 minutes, followed by three washes with PBS. IFA-positive plaques in the wells were screened using a fluorescence microscope with fluorescein isothiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) filters on a Nikon EclipseTi inverted microscope. Similarly, the reactivity of vSB1-009 with NDV F Mab was examined via dual IFA using anti-MDV serum (Charles River Laboratories, cat#10100628, lot#D0111) (1:300 dilution) and anti-NDV F monoclonal antibody (1:300 dilution) as primary antibodies. Goat anti-chicken IgG-fluorescein (KPL cat#.02-24-06, lot#110020) (1:500 dilution) and donkey anti-mouse IgG-Alexa Fluor568 (Molecular Probe#A10037, lot#989784) (1:300 dilution) were used as secondary antibodies. IFA-positive plaques were identified using fluorescence microscopy observation wells with FITC- and TRITC- filters on a Nikon Eclipse Ti inverted microscope.

[0194] IFA results showed that vSB1-009 expressed NDV F protein in virus-infected CEF. NDV F protein expression and SB-1 virus-specific protein expression were counted in over 500 vSB1-009 plaques using dual IFA. NDV F protein expression perfectly matched SB-1 virus antigen expression in each viral plaque (Table 7).

[0195] Table 7 Dual IFA of vSB1-009

[0196]

[0197] NDV F Mab reactivity was confirmed using dual IFA. NDV F Mab reactivity and anti-MDV serological reactivity were examined in over 200 vSB1-009 plaques. Anti-MDV serological reactivity in each viral plaque was perfectly matched to NDV F Mab reactivity (Table 8).

[0198] Table 8 Reactivity of vSB1-009 with anti-NDV F Mab

[0199]

[0200] Southern Imprint Analysis

[0201] Total genomic DNA was extracted from CEF cells infected with vSB1-009 pre-MSV stock solution. Genomic DNA from vSB1-009, SB-1 virus (negative control), and pSB1 44 cds SV FCAopt donor plasmid was digested at 37°C using EcoRI, NcoI, and KpnI restriction endonucleases, respectively. Restriction fragments were separated by 0.8% agarose gel electrophoresis and transferred to a positively charged Nylon membrane. After transfer, the membrane was treated with 0.4M NaOH and then neutralized with 2X SSC-HCl buffer. The membrane was then air-dried and UV cross-linked.

[0202] Following the manufacturer's instructions for the North2South Chemiluminescent Hybridization and Detection Kit (Thermo Scientific CAT #89880), the membrane was pre-hybridized for 1 hour, followed by overnight hybridization with probes at 55°C. Two probes were used for hybridization: 1) the SbfI fragment of pSB1 44cds SV FCA opt as the NDV F-box probe, and 2) the SmaI-EcoRI fragment (GenScript) of pUC57 SB1 44 arm as the recombinant arm probe. After overnight hybridization, several rigorous washes were performed until the membrane was placed in blocking buffer supplemented with streptavidin-HRP. After rinsing away any unbound streptavidin-HRP from the membrane, a substrate solution of luminol and peroxide was added. The membrane was then exposed to X-ray film for development.

[0203] The Southern blot results were as expected based on Vector NTI mapping analysis. The NDV F box (SV40 promoter, NDV-F CA02 codon-optimized gene) replaced the UL44 coding sequence of the SB-1 virus.

[0204] Genome analysis

[0205] Genomic DNA analysis of vSB1-009pre-MSV stock solution was performed by determining the nucleotide sequence of the recombinant arm region and the inserted gene cassette. Primers were designed to amplify the complete NDV-F gene cassette, including the recombinant arm.

[0206] The vSB1-009 sequence (donor plasmid pSB1 44 cds SV FCAopt) containing the recombinant arm, SV40 promoter, and NDV F codon optimized gene has been confirmed to be correct, as shown in SEQ ID NO:19.

[0207] Western blot analysis

[0208] CEF monolayers were infected with vSB1-009 pre-MSV at an MOI of 0.1. After 5 days of incubation, CEF was precipitated, washed with PBS, and then lysed using Pierce Classic IP reagent (Thermo Scientificcat #26146) IP lysis / wash buffer according to the manufacturer's protocol. The lysate was pre-clarified and incubated with 100 μL of anti-NDV F monoclonal antibody to prepare immune complexes. The immune complexes were captured by Protein A / G Plus agarose gel, and after removing unbound immune complexes by a washing step, 50 μL of sample buffer was used for elution under non-reducing conditions. Uninfected CEF was included as a control. 20 μL of the eluted sample was separated by electrophoresis in a 10% Bis-Tris gel. After electrophoresis, the separated proteins were transferred to a PVDF membrane. Following the manufacturer's protocol, the Protein Detection TMB Western Blot Kit (KPL cat#54-11-50) was used to detect NDV antigen on PVDF membranes using chicken anti-NDV serum (Charles River Laboratories Laboratories cat#10100641, lot#C0117A) and goat anti-chicken IgG-peroxidase conjugate (KPL cat#14-24-06).

[0209] NDVF protein expression in vSB1-009 was confirmed using a two-step immunoassay. First, NDVF protein expression was captured by immunoprecipitation of CEF lysates from vSB1-009 infection using anti-NDVF monoclonal antibody 001C3. Subsequently, Western blot analysis of anti-NDV polyclonal serum (Charles River Laboratories CAT #10100641, LOT #C0117A) was used to detect NDVF protein in the captured sample (NDVF protein-monoclonal antibody complex). Figure 9 The protein content of approximately 55 kDa in the vSB1-007 pre-MSV lysate was detected using anti-NDV serum, which corresponds to the expected size of the NDV F1 fusion protein. Figure 9 ).

[0210] The HVT recombinants vSB1-004, vSB1-006, vSB1-007, and vSB1-008 were generated and characterized in essentially the same manner as described for vSB1-009 in this embodiment. Table 9.1 shows the unique features surrounding the expression cassette for each construct, including their respective sequences.

[0211] Table 9.1 Characteristics of expression cassettes of SB1 recombinants

[0212] name Parental virus promoter F gene locus vSB1-009 SB1 SV40 Opt-CA02 UL44(gC) vSB1-004 SB1 mCMV IE Wt-VIId US10 vSB1-006 SB1 SV40 Opt-VIId UL55 / LORF5 vSB1-007 SB1 SV40 Opt-VIId UL44(gC) vSB1-008 SB1 SV40 Opt-CA02 UL55 / LORF5

[0213] Example 5.2 Construction of the dual construct vSB1-010

[0214] Construction of donor plasmid SB1US2 gpVIIdwtsyn

[0215] The gpCMV, Varient E, and Syn tail were removed by SbfI digestion using plasmid HVT SOrf3-US2 gpVar-Ewt Syn. This fragment was ligated into the SB1US2 donor plasmid. The Varient E gene was excised by NotI digestion and replaced with NDV-F VIIdwt. The synthetic NDV-FVIId wild-type gene (SEQ ID NO:3, encoding SEQ ID NO:4) was excised from the pUC57 NDV-F VIIdwt plasmid (synthesized by GeneScript) using NotI digestion. The ligated material was transformed using the Top10Oneshot kit (cat#C404002, Invitrogen). Bacterial colonies were grown in LBamp liquid medium, and plasmids were extracted using the Qiagens MiniSpin Prep kit. The orientation of the inserts was screened by NcoI+SalI digestion. The correct donor plasmid was named pSB1 US2 gpVIIdwt Syn. Table 9.2 shows the unique characteristics of the expression cassette for the construct, including their respective sequences. Large-scale cultures were grown, and plasmids were extracted using the Qiagen's Maxi Prep kit. Transient expression in the large-scale cultures was validated in chicken embryo fibroblasts (CEFs) using Fugene transfection reagent and anti-NDV-F chicken polyclonal serum.

[0216] Production of recombinants

[0217] Following standard homologous recombination, secondary CEF cells were co-electroporated using the pSB1 US2 gpVIIdWt Syn donor plasmid and viral DNA isolated from vSB1-009 (vSB1-009 is a recombinant virus expressing the CA02F gene of NDV). Recombinants were generated essentially according to the method described in Example 1 for vHVT114, purified by plaque phage, and characterized by immunofluorescence.

[0218] After five rounds of plaque purification, pure recombinant virus (vSB1-010) was isolated, and the purity of vSB1-010 was tested by IFA and PCR to verify proper insertion and absence of residual parent virus.

[0219] Table 9.2 Characteristics of the expression box of vSB1-010

[0220] name Parental virus promoter F gene locus vSB1-010 vSB1-009 Guinea Pig CMV NDV-F VIId SORF4-US2

[0221] Sequencing of the insertion region confirmed that vSB1-010 contained the correct sequence of the guinea pig CMV promoter and the NDV-F VIId wt gene, as shown in the sequence of the donor plasmid SB1US2 gpVIIdwtsyn (SEQ ID NO:40).

[0222] Analysis of recombinants by PCR

[0223] DNA was extracted from the viral stock solution by phenol / chloroform extraction, precipitated with ethanol, and resuspended in 20 mM HEPES. PCR primers were designed to specifically identify the NDV-F VIId wt gene, promoter, polyA, and the purity of recombinant virus from the SB1 parent virus. PCR was performed using 200 μg of DNA template and the specified primer pairs shown in Table 1. PCR cycling conditions were as follows (unless otherwise specified): 94 °C–2 min; 30 cycles (94 °C–30 s, 55 °C–30 s, 68 °C–3 min); 68 °C–5 min.

[0224] The purity of the recombinant virus was confirmed by PCR using primer pairs specific to the SB1 flanking linker, gpCMV promoter, NDV-F VIId wt gene, and syn tail. Primers specific to HVT and MDV serotype 3 (MB080+MB081) were also included in the analysis. PCR results showed that the recombinant virus vSB1-010 possessed the expected expression cassette and that the viral stock contained no detectable amounts of parental SB1-009 virus.

[0225] Immunofluorescence staining of recombinant vSB1-010 virus expressing two NDV-F proteins

[0226] For immunofluorescence assays, P3 material was diluted 1:100 in the medium. Approximately 50 μl of diluted virus was added to 10 ml containing 1 x 10⁻⁶ cells / mL. 7 Cells were inoculated with DMEM and 2% FBS, and then aliquoted into 96-well plates (100 μl / well). The plates were incubated at 37°C with 5% CO2 for 5 days until viral plaques were visible. The plates were fixed with 95% ice-cold acetone for 3 minutes and washed three times with PBS. Chicken anti-Newcastle disease virus serum (lot #C0139, Charles Rivers Laboratory) was added at a 1:1000 dilution, and the plates were incubated at 37°C for 1 hour. After 1 hour of incubation, the plates were washed three times with PBS, and FITC anti-chicken serum (cat #F8888, Sigma) was added at a 1:500 dilution. The plates were again incubated at 37°C for 1 hour. After 1 hour of incubation, the cells were washed three times with PBS and visualized using a fluorescence microscope with a FITC filter.

[0227] When polyclonal sera containing anti-NDV CA02 and VIId F proteins were used, immunofluorescence staining results showed that vSB1-010 exhibited extremely strong NDV-F protein expression.

[0228] in conclusion

[0229] Based on PCR testing and immunofluorescence analysis, vSB1-010 is a recombinant SB-1 in which the VIId-F gene of NDV, under the control of the gpCMV promoter, has been successfully inserted into vSB1-009 (which already expresses the CA02-F gene of NDV). Therefore, vSB1-010 possesses the VIId and CA02F genes of the NDV genotype and does not contain any detectable parental vSB1-009.

[0230] Example 6: Efficacy of vHVT110, vHVT111, vHVT114, and vSB1-004 expressing the NDV F gene against NDV Chimalhuacan and Malaysian (MAL04-01) strains in 14-day-old SPF chickens.

[0231] The aim of this study was to evaluate the efficacy of three HVT recombinant constructs (vHVT110, vHVT111, and vHVT114) and one SB1 recombinant construct (vSB1-004) expressing the NDV F gene against Newcastle disease challenge (Chimalhuacan and Malaysian virus strains) in 14-day-old SPF chickens.

[0232] The characteristics of these five vaccine candidates are described in Table 10 below.

[0233] Table 10 Characteristics of the vectors used for attack research

[0234] name Parental virus promoter F gene Poly-A locus vHVT110 HVT mCMV IE Wt-VIId SV40 IG1 vHVT111 HVT SV40 Wt-VIId SV40 IG1 vHVT114 HVT SV40 Opt-VIId SV40 IG1 vSB1-004 SB-1 mCMV IE Wt-VIId SV40 US10

[0235] On day D0, 100 one-day-old SPF chickens were randomly assigned to 10 groups (n=10 per group). As described in Table 11 below, on day D0, chickens were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 2000 pfu. It should be noted that the titer of vSB1-004 (31600 pfu) administered to chickens in group 6 was significantly higher than the target dose. On day D14, chickens were challenged via intramuscular route with either the immediate-type ND Malaysia strain (genotype VIId) (subgroup “a”) or the virulent ND Chimalhuacan strain (genotype V) (subgroup “b”).

[0236] Table 11. Attack Study Using vHVT110, vHVT111, vHVT114, and vSB1-004

[0237]

[0238] *Number of chickens that tested positive for NDV HI / Total number of tests

[0239] Each group was monitored before and after the attack. Clinical signs were scored daily after the attack as follows: healthy / with specific symptoms (erect feathers, weakness, torticollis, tremors) / death. On day 14, serum samples were collected from each group for serological testing (Newcastle disease virus hemagglutination inhibition (HI) test).

[0240] As expected, unvaccinated animals (G1a and G1b) did not show NDV antibodies on day 14. Low-titer seroconversion (mean HI titer <0.6 log10) was obtained in each vaccinated group (subgroups “a” and “b” of G2 to G5), confirming vaccine acquisition. The number of positive chickens / total number of chickens tested was group-dependent and highest (90%) in chickens vaccinated with vHVT114 (see table above).

[0241] The percentages of protection against mortality and morbidity are reported in the table above. Complete susceptibility was observed in the control groups G1a and G1b, confirming the high severity of both challenges. The lowest levels of protection were observed in the groups vaccinated with vHVT111 or vSB1-004. The highest rates of protection against mortality and morbidity were achieved in the groups vaccinated with vHVT110 or vHVT114, regardless of the challenge strain used (homogeneous strain i.e., Malaysian genotype VIId or heterogeneous strain i.e., Chimalhuacan genotype V). A correlation was found between the percentage of chickens that tested positive for HI before challenge and the percentage of protection.

[0242] The difference in protective effect between vHVT110 and vHVT111 clearly illustrates the importance of the promoter; for transcription of the wild-type (wt) VIId F gene, the mCMV IE promoter is stronger than the SV40 promoter. The difference in protective effect between vHVT111 and vHVT114 illustrates the importance of the F gene's nucleotide sequence; the optimized sequence is stronger than the wild-type (or natural) sequence.

[0243] In summary, the results of this study demonstrate the importance of the promoter and F gene nucleotide sequences in the protective effect against ND induced by Marek's disease vector vaccines. The optimal combination of these factors for achieving the best efficacy performance of vHVT114 needs to be identified.

[0244] Example 7: Efficacy of vHVT114, vHVT116, vHVT301, vHVT302, and vHVT303 expressing the NDV F gene against challenge with NDV Texas GB strain in 14-day-old SPF chickens.

[0245] The aim of this study was to evaluate the efficacy of two single-HVT recombinant constructs expressing the NDV F gene (vHVT114 and vHVT116) and three dual-HVT recombinant constructs expressing both the NDV F and IBDV VP2 genes (vHVT-301, vHVT302, and vHVT303) against Newcastle disease challenge (Texas GB strain, genotype II) in 14-day-old SPF chickens.

[0246] The characteristics of these four vaccine candidates are described in Table 12 below.

[0247] Table 12 Characteristics of the vectors used for attack research

[0248] name Parental virus promoter F gene Poly-A locus vHVT114 HVT SV40 Opt-VIId SV40 IG1 vHVT116 HVT SV40 Opt-V SV40 IG1 vHVT301 vHVT13* SV40 Wt-VIId SV40 IG2 vHVT302 vHVT13 US10 Opt-VIId US10 US10 vHVT303 vHVT13 US10 Opt-V US10 US10

[0249] *vHVT13 is the active ingredient of the approved Vaxxitek HVT-IBD vaccine based on an HVT vector expressing the IBDV VP2 gene (see US 5,980,906 and EP 0 719 864).

[0250] On day D0, 120 one-day-old SPF chickens were randomly assigned to 6 groups (n=20 per group). As described in Table 13 below, on day D0, chickens were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 1000 pfu. On day D14, chickens were challenged intramuscularly with the 4.5 log10 EID50 immediate-type ND Texas GB (genotype II) strain.

[0251] Table 13 Results of Efficacy

[0252]

[0253] *One chicken died before the attack.

[0254] Monitor each group before and after the attack. Record NDV clinical signs and mortality rates after the attack.

[0255] The percentage of clinical protective effects is reported in the table above. Complete susceptibility was observed in the unvaccinated challenged control group G1, confirming the high severity of both attacks. Partial protection was observed for all five vaccine candidates, with the best performance observed for vHVT114 and vHVT116. Among the dual HVT recombinants, vHVT302 showed the strongest protection. It performed better than vHVT303, suggesting that the optimized genotype VIId NDV F gene may have better cross-protection against genotype II attacks than the optimized genotype V NDV F gene. A similar trend was observed for single HVT, with vHVT114 (VIId gene) performing slightly better than vHVT116 (V gene), but the difference was not significant. These results indicate that both genotype VIId and V NDV F genes inserted into the HVT vector provide cross-protection against heterologous genotype II NDV attacks; the VIId gene may potentially be more cross-protective. vHVT302 induces better protection against ND than vHVT301, thus confirming the importance of the promoter, poly-A, and inserted locus. In summary, the results of this study demonstrate excellent early ND protection induced by the tested Marek's disease vector vaccine (particularly for the tested single HVT-ND).

[0256] Example 8: Efficacy of vHVT114, vHVT116, vSB1-007, vSB1-008 (alone or together with vHVT13) and vHVT 304 against challenge with NDV ZJ1 (genotype VIId) and California / 02 (genotype V) in 21-day-old SPF chickens.

[0257] The aim of this study was to evaluate the efficacy of two single HVT recombinant constructs (vHVT114 and vHVT116) expressing the NDV F gene, two SB1 recombinant constructs (vSB1-007 and vSB1-008) expressing the NDV F gene, and a dual HVT recombinant (vHVT304) against Newcastle disease challenge in 21-day-old SPF chickens using NDV ZJ1 (genotype VIId) and California / 02 (genotype V).

[0258] The characteristics of these five vaccine candidates are described in Table 14 below.

[0259] Table 14 Characteristics of the vectors used for attack research

[0260] name Parental virus promoter F gene Poly-A locus vHVT114 HVT SV40 Opt-VIId SV40 IG1 vHVT116 HVT SV40 Opt-V SV40 IG1 vSB1-007 SB-1 SV40 Opt-VIId gC UL44(gC) vSB1-008 SB-1 SV40 Opt-V SV40 IG1 vHVT304 vHVT13* SV40 Opt-VIId Synth IG2

[0261] *vHVT13 is the active ingredient of the approved Vaxxitek HVT-IBD vaccine based on an HVT vector expressing the IBDV VP2 gene (see US 5,980,906 and EP 0 719 864).

[0262] On day D0, 158 one-day-old SPF chickens were randomly assigned to six groups of 24 each (vaccinated groups) and one group of 12 (unvaccinated control). As described in Table 15 below, on day D0, the chickens were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 1000 pfu. The chickens were then divided into two subgroups, each of which was challenged on day D21 by intramuscular administration of either 5 log10 EID50 of the NDV ZJ1 (genotype VIId) or California / 02 (genotype V) immediate-type strain.

[0263] Table 15 Results of Effectiveness

[0264]

[0265] Each group was monitored before and after challenge. Technical issues observed with the isolates resulted in a reduction in the number of chickens in groups 2 (vHVT114: 24 to 14) and 3 (vHVT116: 24 to 20). Clinical signs of NDV were recorded post-challenge. Serum was collected from blood samples taken from chickens in groups 2 and 7 prior to challenge (D21) for NDV serology using the HI test with each challenge strain as an antigen.

[0266] The mean serum HI titers in G2 and G7 before attack are shown in Figure 10 In both groups, the HI titer was higher for the ZH1 antigen. The HI titer induced by vHVT114 was higher than that induced by vHVT304.

[0267] The percentages of protection against death and morbidity are reported in the table above. Complete susceptibility was observed in the unvaccinated challenged control group G1, confirming the high severity of both challenges. All vaccines induced high levels (≥75%) of protection against both challenges. vHVT114 and vSB1-008 induced complete clinical protection against both challenges. Following a trend similar to HI titers, vHVT304 induced slightly less ND protection than vHVT114.

[0268] Shedding samples from oral and cloacal swabs collected 2 and 4 days post-challenge were evaluated by real-time RT-PCR. The percentage of positive (Ct<40) chickens for both challenges is shown in [figure missing]. Figure 11A and 11BNote that in the control group challenged with the CA / 02 isolate, all 6 chickens died at 4 dpch, while in the control group challenged with the ZJ1 isolate, only 1 chicken (1 out of 6) remained alive at 4 dpch. Shedding was detected in all control chickens. A reduced percentage of chickens testing positive for shedding was observed in all inoculated groups.

[0269] In summary, the results of this study demonstrate excellent protection against neonatal disease induced by the tested Marek's disease vector vaccine at 3 weeks of age.

[0270] Example 9: Efficacy of vHVT114, vSB1-007, vSB1-009, vHVT306, and vHVT307 vaccines against NDV Texas GB strain challenge in 28-day-old SPF chickens.

[0271] The aim of this study was to estimate the efficacy of combinations of different Marek's disease vector vaccines expressing the NDV F and / or IBDV VP2 genes against Newcastle disease challenge (Texas GB strain, genotype II) in 28-day-old SPF chickens.

[0272] The characteristics of the five recombinant vaccine candidates tested in this study are described in Table 16 below.

[0273] Table 16 Characteristics of the vectors used for attack research

[0274] name Parental virus promoter F gene Poly-A locus vHVT114 HVT SV40 Opt-VIId SV40 IG1 vSB1-007 SB-1 SV40 Opt-VIId gC UL44(gC) vSB1-009 SB-1 SV40 Opt-V gC UL44(gC) vHVT306 vHVT13 SV40 Opt-VIId Synth SORF3-US2 vHVT307 vHVT13 SV40 Opt-V Synth SORF3-US2

[0275] In some groups, Marek's disease virus serotype 1 (CVI988 (or Rispens) strain; avian herpesvirus 2) and serotype 2 (SB-1 strain; avian herpesvirus 3) vaccines were also used in combination with recombinant viruses.

[0276] On day D0, 135 one-day-old SPF chickens were randomly assigned to nine groups (n=15 per group). On D0, chickens were subcutaneously injected into the neck with a recombinant vaccine (vSB1-007, vSB1-009, vHVT13, vHVT306, vHVT307, vHVT114) containing a target dose of 2000 pfu in 0.2 ml and parental Marek's disease vaccine strains (SB-1 and CVI988) containing 1000 pfu. The design of the nine groups is shown in Table 17 below. On D28, chickens were challenged intramuscularly with a 4.0 log10 EID50 immediate-type ND TexasGB (genotype II) strain.

[0277] Table 17 Results of Effectiveness

[0278]

[0279]

[0280] Monitor each group before and after the attack. Record post-attack clinical signs of NDV.

[0281] The percentage of protective effects against mortality and morbidity is reported in the table above. Complete susceptibility was observed in the unvaccinated, attacked control group G1, confirming the high severity of the attack. Excellent levels of protection were observed in all vaccinated groups. Chickens from G3, G6, G7, and G9 received complete protection. This study shows that the vSB1-ND candidate can be co-administered with vHVT13 and CVI988 and still provides good protection against ND. Similarly, dual HVT-IBD+ND is compatible with SB-1, and vHVT-ND (vHVT114) is compatible with both vHVT13 and SB-1.

[0282] In summary, the results of this study show that there is no interference with the protective effect against ND induced by the tested Marek's disease parental and vector vaccines.

[0283] Example 10: Efficacy of vHVT114, vHVT307, vSB1-007, and vSB1-009 in combination with vHVT13 against challenge with NDV Chimalhuacan strain (genotype V) in D28SPF chickens.

[0284] The aim of this study was to evaluate the efficacy of a combination of one HVT recombinant construct (vHVT114) and two SB1 recombinant constructs (vSB1-007 and vSB1-009) expressing the NDV F gene with vHVT-IBD (vHVT13), as well as the efficacy of dual HVT vHVT307 expressing NDV F and IBDV VP2 against Newcastle disease challenge (Chimalhuacan, genotype V) in 28-day-old SPF chickens.

[0285] The characteristics of these four vaccine candidates are described in Table 18 below.

[0286] Table 18 Characteristics of the vectors used in the attack research

[0287] name Parental virus promoter F gene Poly-A locus vHVT114 HVT SV40 Opt-VIId SV40 IG1 vSB1-007 SB-1 SV40 Opt-VIId gC UL44(gC) vSB1-009 SB-1 SV40 Opt-V gC UL44(gC) vHVT307 vHVT13 SV40 Opt-V Synth SORF3-US2

[0288] On day 0, 45 one-day-old SPF chickens were randomly assigned to four groups (n=10 per group) and one group (n=5 per group, an unvaccinated control group). As described in Table 19 below, on day 0, chickens were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 2000 pfu. On day 28, chickens were challenged intramuscularly with a 5.0 log10 EID50 immediate-type Chimalhuacan (genotype V) strain.

[0289] Table 19 Results of Effectiveness

[0290]

[0291] Monitor each group before and after the attack. Record clinical signs of NDV after the attack. Obtain oropharyngeal swabs from the vaccinated groups at 5 and 7 days post-attack to assess viral load by real-time RT-PCR.

[0292] The percentages of protection against death and morbidity are reported in the table above. Complete susceptibility was observed in the unvaccinated challenged control group G1, confirming the high severity of the attack. Excellent protection was observed in all four vaccinated groups, with vHVT114+vHVT13 inducing complete clinical protection.

[0293] The percentage of positive chickens and the mean shedding titer (expressed as log10 EID50 equivalents / mL) are shown in the figure. Figure 12A and 12B Surprisingly, no shedding was detected in G2, indicating that under the tested conditions, vHVT114 still induced complete (in terms of clinical signs and shedding) ND protection, even when co-administered with vHVT13. Low shedding levels were detected in the other vaccinated group, with only slightly higher levels detected in G3 at 5 days post-infection (pi).

[0294] In summary, this embodiment further illustrates the excellent ND protection induced by the combination of dual HVT-IBD+ND recombinant virus, SB1-ND, or HVT-ND with HVT-IBD (vHVT13) recombinant virus. Contrary to the general concept in the art that a second HVT vaccine (conventional HVT vaccine or recombinant HVT vaccine) interferes with immunity to exogenous genes inserted into the first recombinant HVT vaccine, the present invention shows surprising results: vHVT114 in combination with vHVT13 provides excellent anti-NDV protection without any interference observed.

[0295] Example 11: Efficacy of vHVT306 and vSB1-008, administered via SC or intraocular route, in SPF chickens against challenge with NDV Chimalhuacan strain (genotype V) on day 28.

[0296] The aim of this study was to evaluate the efficacy of vHVT306 dual HVT expressing NDV F and IBDV VP2 genes, administered intraocularly or subcutaneously, and vSB1-008SB1 recombinant expressing NDV F gene, in combination with vHVT-IBD (vHVT13), against Newcastle disease challenge (Chimalhuacan, genotype V) in 28-day-old SPF chickens.

[0297] The characteristics of these two ND vaccine candidates are reported in Table 14 (vSB1-008) and Table 16 (vHVT306).

[0298] The design for each group is shown in Table 20. Sixty SPF chicken eggs (after approximately 18 days and 18 hours of incubation; D-3) were used for intraocular administration (20 eggs per group for G1, G2, and G3). 50 μL of vaccine containing 2000 PFU was administered intraocularly using an IntelliLab system device from AviTech LLC (Salisbury, MD, USA). Hatching and survival rates were recorded after intraocular administration. On day D0, 20 one-day-old SPF chickens were randomly assigned to two groups (G4 and G5) (n=10 per group). As described in Table 20 below, on day D0, chickens were subcutaneously (SC) injected into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 2000 pfu. On day D28, 10 chickens in each group were challenged intramuscularly with a 5.0 log10 EID50 immediate-type Chimalhuacan (genotype V) strain.

[0299] Table 20 Results of the study design and ND efficacy

[0300]

[0301]

[0302] Monitor each group before and after the attack. Record clinical signs of NDV after the attack. Collect oropharyngeal swabs in the vaccinated groups at 5 and 7 days after the attack to assess viral load by real-time RT-PCR.

[0303] Complete hatchability and viability of chickens in groups G1 and G2 were recorded until day 28 (attack day). The hatchability in group G3 was 85%, and one chicken in this group died post-hatching. The lower hatchability in this group may be attributed to problems with the embryo incubator. The body weights of males and females in G1, G2, and G3 were similar on days 1 and 28.

[0304] The percentages of protection against death and morbidity are reported in Table 20. Complete susceptibility was observed in the unvaccinated challenged control group G1, confirming the high severity of the attack. Good protection was observed in all four vaccinated groups, with vHVT306 administered via both routes inducing complete clinical protection.

[0305] The percentage of positive chickens and the mean shedding titer (expressed as log10 EID50 equivalents / mL) are shown in Table 21. No detectable shedding or only very low shedding was observed in G2 and G4 inoculated with vHVT306. Shedding levels detected in the group inoculated with vSB1-008+vHVT13 were particularly high at 5 days post-infection (pi).

[0306] Table 21 Results of the protective effect against shed skin assessed at D5 and D7 after NDV attack (percentage of chickens with detectable shed skin and average viral load expressed in log10).

[0307]

[0308] * Average real-time quantitative PCR value expressed at log10 EID50 equivalents; threshold was set at 2.7 log10.

[0309] In summary, this embodiment demonstrates excellent ND protection induced by dual HVT recombinant of vHVT306 administered via intraocular or SC route. The performance of vSB1-008+vHVT13 was slightly lower, particularly after intraocular administration, but this is likely at least partly attributable to issues with the chicken embryo incubator. Indeed, intraocular safety tests of another SB1-ND recombinant (vSB1-009) with 1000 or 4000 PFU of vHVT13 combined with 6000 PFU showed no difference in hatchability and early survival compared to the group receiving only 6000 PFU of vHVT13.

[0310] Example 12: Efficacy of vHVT304, vHVT306, vSB1-007, and vSB1-008 in combination with vHVT13 against NDV Chimalhuacan strain (genotype V) challenge in commercial broilers on day 42.

[0311] The aim of this study was to evaluate the efficacy of two dual HVTs (vHVT304 and vHVT306) expressing the NDV F and IBDV VP2 genes, and two SB1 recombinants (vSB1-007 and vSB1-008) expressing the NDV F gene in combination with vHVT-IBD (vHVT13), against Newcastle disease challenge (Chimalhuacan, genotype V) in 42-day-old commercial broilers.

[0312] The characteristics of the four ND vaccine candidates are reported in Tables 14 and 16. The group design is shown in Table 22. On D0, 55 one-day-old commercial broilers were randomly assigned to five groups (n=11 per group). As described in Table 22 below, on D0, chickens were injected subcutaneously (SC) into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 2000 pfu. On D42, 10 chickens in each group were challenged intramuscularly with a 5.0 log10 EID50 immediate-type Chimalhuacan (genotype V) strain.

[0313] Table 22 Results of the study design and ND efficacy

[0314]

[0315]

[0316] Monitor each group before and after the attack. Record NDV clinical signs during the 14-day period post-attack. Collect oropharyngeal swabs from the vaccinated groups at 5 and 7 days post-attack to assess viral load by real-time RT-PCR.

[0317] The percentages of protection against death and morbidity are reported in Table 22. Complete susceptibility was observed in the unvaccinated attacked control group G1, confirming the high severity of the attack. Good protection was observed in all four vaccinated groups, with vHVT306 and vSB1-007+vHVT13 inducing complete clinical protection.

[0318] The percentage of positive chickens and the mean shedding titer (expressed as log10 EID50 equivalents / mL) are shown in Table 23. vHVT306 and vSB1-007+vHVT13 induced the best reduction in shedding and are also the best candidates in terms of clinical protective effect.

[0319] Table 23 Results of protection against shed chickens assessed at D5 and D7 after NDV attack (pi) (with percentage of detectable shed chickens and average viral load expressed in log10).

[0320]

[0321] * Average real-time quantitative PCR value expressed at log10 EID50 equivalents; threshold was set at 2.7 log10.

[0322] The protective effect of vHVT306ND was found to be better than that of vHVT304. These two dual HVTs contain the same NDV F expression cassette, but are inserted at two different loci, while the IBDV VP2 expression cassette is inserted at the same location. Therefore, this example illustrates the importance of the inserted locus in the design of HVT recombinants. vSB1-007+vHVT13 is better than vSB1-008+vHVT13. The genome structure of vSB1-007 differs from that of vSB1-008 in several aspects: the inserted locus, promoter, polyadenylation signal, and F gene origin. The combination of these foreign sequences with the inserted locus in vSB1-007 may be responsible for its better ND protective performance.

[0323] In summary, this example illustrates the importance of the inserted locus and other regulatory sequences in the NDV expression cassette in ND protection induced by HVT and MDV serotype 2 vectors.

[0324] Example 13: Efficacy of dual HVT-ND + IBD (vHVT304 and vHVT306) or SB1-ND (vSB1-008) in combination with vHVT13 recombinant vaccine against classical IBDV isolates in SPF chickens on day 14.

[0325] The aim of this study was to assess the early IBD efficacy of dual HVT recombinants vHVT304 and vHVT306, as well as vHVT13 co-administered with the SB1-ND (vSB1-008) recombinant construct, against highly virulent infectious bursal disease virus (vIBDV) challenge (Faragher52 / 70 strain) in 14-day-old SPF chickens.

[0326] The characteristics of the dual HVT and SB1 recombinants used in this study are shown in Tables 14 and 16.

[0327] On day 0, 95 one-day-old SPF chickens were randomly assigned to 9 groups (n=10 per group) and 1 group (n=5 per group, an unvaccinated, non-challenged control group). As described in Table 24 below, on day 0, the chickens were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 300 or 1000 pfu. On day 14, the kit was used... plus IBD (Synbiotics Corp) blood samples were collected from 5 chickens in each group for serological testing. On D14, chickens were challenged by eye drops (0.05 mL / chicken) with 2.5 log10 EID50 (10 chickens in each group except for Group 7, where one chicken died before the challenge).

[0328] Table 24 Results of Study Design and IBD Efficacy

[0329]

[0330] 1 The mean IBD+ELISA titer of serum from each of the five chickens sampled on D14 before the attack is expressed as log10.

[0331] 2 Chickens that are sick for more than 2 days or are still sick on day 25 are considered sick;

[0332] 3 Protective effect against clinical signs and severe bursal damage (bursal score <3);

[0333] 4 The mucosal cyst weight / body weight ratio in the uninoculated / unattacked group was 0.0047.

[0334] Each group was monitored before and after attack. Clinical signs of IBDV were recorded post-attack for 11 days (from D15 to D25). At the end of the post-attack observation period (D33), all surviving chickens were euthanized painlessly and necropsy was performed. Body weight and mucus bursa weight were recorded. Each supracaval bursa of Fabricius (BF) was weighed and then stored in a single container filled with 4% formaldehyde for histological testing. Histological damage to the mucus bursa was scored according to the grades provided in Table 25.

[0335] Table 25 Scoring of Histological Lesions in the Bursa of Fabricius*

[0336]

[0337] *Derived from European Pharmacopoeia monograph No. 01 / 2008:0587 "Avian Infectious Bursal Disease Vaccine (Live)"

[0338] Chickens are considered to be affected if they die and / or show significant signs of disease and / or severe damage to the bursa of Fabricius (i.e., histological score ≥3).

[0339] The mean ELISA IBD+ antibody titers expressed as log10 prior to the attack are shown in Table 24. Significantly higher titers than the control group G1 were detected in all vaccinated groups. Serological titers were not dose-dependent.

[0340] Severe clinical signs were observed in all chickens in the control group G1 after attack. Seven out of ten chickens in this group died within the 11-day observation period, indicating the high severity of the attack. After attack, no severe clinical signs were observed in the inoculated chickens except for one chicken that died in G4. The percentage of protection against severe mucosal cyst damage is shown in the table above. Significant IBD protection was observed in all groups, with the best protection observed in G2 and G3 (vHVT13 alone). Co-administration of vSB1-008+vHVT13 with the dual vHVT304 and vHVT306 constructs induced similar levels of IBD protection. The protective effect was not dose-dependent on the tested dose. The mean mucosal cyst weight / body weight ratio is also shown in Table 24. The ratio in all inoculated groups was higher than the ratio described in the attacked control group.

[0341] In summary, these data indicate that the combination of the SB1-ND vector with single HVT-IBD or dual HVT expressing both NDV-F and IBDV-VP2 induces IBD antibody and early IBD protection in severe IBDV challenge models.

[0342] Example 14: Efficacy of single HVT-ND (vHVT114) or SB1-ND (vSB1-007 and vSB1-009) in combination with the vHVT13 recombinant vaccine against challenge with highly virulent IBDV isolates in commercial broilers on day 23.

[0343] The purpose of this invention is to evaluate the IBD efficacy of vHVT13 co-administered with HVT-ND (vHVT114) or SB1-ND (vSB1-007 and vSB1-009) recombinant constructs against highly virulent infectious bursal disease virus (vvIBDV) challenge (91-168 / 980702) in 23-day-old commercial broilers.

[0344] The characteristics of the four vaccine candidates are described in Tables 14 and 16. On day 0, 90 one-day-old broilers were randomly assigned to 7 groups (12 chickens per group) and 1 group (6 chickens per group, an unvaccinated, non-challenge control group). As described in Table 26 below, on day 0, the recombinant vaccine containing a target dose of 3000 pfu was administered subcutaneously to the neck of the chickens. On day 14, blood samples were collected from 5 chickens in each group for use with the kit. Serological testing was performed using IBD (Synbiotics Corp). On day 0, serum samples from 10 additional one-day-old broilers were tested using the same kit to assess IBDV maternal antibody levels. On day 23, chickens (10 chickens / group) were challenged with the vvIBDV 91-168 isolate at 4.3 log10 EID50 via eye drops (0.05 mL / chicken).

[0345] Each group was monitored before and after attack. Clinical signs of IBDV were recorded post-attack for 11 days (from D23 to D33). At the end of the post-attack observation period (D33), all surviving chickens were euthanized painlessly and necropsy was performed. Body weight and mucus bursa weight were recorded. Each supracaval bursa of Fabricius (BF) was weighed and then stored in a single container filled with 4% formaldehyde for histological testing. Histological damage to the mucus bursa was scored according to the grades provided in Table 25.

[0346] Chickens are considered to be affected if they die and / or show significant signs of disease and / or severe damage to the bursa of Fabricius (i.e., histological score ≥3).

[0347] Table 26 Study Design and Serological Results

[0348]

[0349] 1 The mean IBD+ ELISA titer expressed as log10 in the serum of 5 chickens in each group sampled on D23 before the attack;

[0350] 2 The mucosal cyst weight / body weight ratio in the uninoculated / unattacked group was 0.0047.

[0351] The mean ELISA IBD+ serological titer at day 0 was 4.36 ± 0.01 log10, indicating extremely high levels of maternal IBD antibodies at hatching. At day 23, the mean ELISA IBD+ titer remained high (3.9) in the control group (G1). There was no significant difference in the mean ELISA titer between the vaccinated groups and the control group.

[0352] No deaths or morbidity were observed in any group after the attack. The percentage of protection against severe bursal damage is shown in Table 26 above. The results showed that co-administration of vHVT114, vSB1-007, or vSB1-009 did not interfere with the vHVT13-induced IBD protection, indicating that there was no interference. Similarly, the mean bursal weight / body weight ratio was similar in the inoculated groups and was significantly higher than that in the control group, indicating no difference in IBD protection or between inoculation regimens.

[0353] In summary, the data demonstrate the compatibility of vHVT114, vSB1-007, or vSB1-009 with vHVT13 in terms of IBD protection. The absence of interference between the two HVT carriers in IBD protection is again surprising, confirming the results observed for ND protection (see Example 10).

[0354] Example 15: Efficacy of dual HVT-ND+IBD (vHVT304 and vHVT306) with or without SB-1, and SB1-ND (vSB1-007 and vSB1-008) in combination with the vHVT13 recombinant vaccine against challenge to variant E IBDV isolates in SPF chickens on day 28.

[0355] The aim of this study was to evaluate the efficacy of two dual HVT (HVT-ND+IBD: vHVT304 and vHVT306) or two vSB-1-NDV vectorized vaccines with vHVT13 (vSB1-007+vHVT13, vSB1-008+vHVT13) administered subcutaneously (SC) to 0-day-old SPF chickens against IBDV-variant (VAR-E) challenge 28 days post-vaccination.

[0356] On day 0, 105 one-day-old SPF chickens were randomly assigned to 7 groups (n=15 per group), including a challenged control group (G6) and a non-challenged control group (G7). On day 0, chickens in groups G1 through G5 were injected subcutaneously into the neck with 0.2 mL of a combination vaccine and / or SB-1 vaccine, each containing a target dose of 2000 pfu. The study design is shown in Table 27 below. On day 28, all chickens in groups G1 through G6 were challenged with eye drops of IBDV variant E isolate from the University of Delaware (USA) (0.03 mL per chicken containing 3 log10 EID50). Each group was monitored before and after challenge. Eleven days post-challenged, chickens were weighed and necropsy was performed. Mucus sacs were collected and weighed. The mucus sac weight / body weight ratio was calculated (mucus sac weight / body weight ratio x 100).

[0357] Table 27. Study Design and Results of IBD Efficacy

[0358]

[0359]

[0360] The mean mucosal sac weight / body weight ratio is shown in Table 27. Attacked control chickens exhibited severe mucosal sac atrophy compared to unattacked chickens. The vSB1-007 and vSB1-008 vaccines did not interfere with the vHVT13-induced protective effects (G4 and G5). Chickens vaccinated with double HVT (HVT-ND+IBD) had a slightly lower mucosal sac weight / body weight ratio than the unattacked control group, but a significantly higher ratio than the attacked control group. Furthermore, the SB-1 serotype 2 Marek's disease vaccine did not interfere with the vHVT304-induced IBD protective effects.

[0361] In summary, these data indicate that the combination of the SB1-ND vector with a single HVT-IBD or with dual HVTs expressing both NDV-F and IBDV-VP2 induces IBD protection in the variant E IBDV attack model.

[0362] Example 16: vHVT114, vSB1-009, and / or SB-1 do not interfere with the protective effect of the variant EIBD induced by vHVT13 in SPF chickens.

[0363] The aim of this study was to estimate the protective efficacy of vHVT13 against IBD in a D28 SPF chicken model of IBDV-variant (VAR-E) challenge when administered via SC or intraocular route in combination with vHVT114, vSB1-009 and / or SB-1.

[0364] Seventy-five 1-day-old SPF chickens and 75 SPF 18-19-day-old embryos were randomly assigned to five groups (G1-G5 and G6-G10, respectively), including one challenged control group (G4 and G9, respectively) and one unchallenged control group (G5 and G10, respectively). On day 0, chickens in groups G1-G3 were subcutaneously injected into the neck with 0.2 mL of vaccine, each containing a target dose of 3000 pfu (except for SB-1, which had a target dose of 1000 pfu). Chickens in groups G6-G8 received the same vaccine dose via the intraocular route 2-3 days before hatching (but in a volume of 0.05 mL). The study design is shown in Table 28 below. At 28 days of age, all chickens in groups G1-G4 and G6-G9 were challenged with eye drops (0.03 mL per chicken containing 3 log10 EID50) of the IBDV variant E isolate from the University of Delaware (USA). Monitor each group before and after the attack. Eleven days after the attack, weigh and necropsy the chickens. Collect the mucus sacs and weigh them. Calculate the mucus sac weight / body weight ratio (mucus sac weight / body weight ratio x 100).

[0365] Table 28. Study Design and Results of IBD Efficacy

[0366]

[0367] The mean mucosal sac weight / body weight ratio is shown in Table 28. The attacked control chickens (G4 and G9) exhibited severe mucosal sac atrophy compared to the unattacked chickens. The mucosal sac weight / body weight ratios of the inoculated groups (G1 to G3 and G6 to G8) were similar to those of the unattacked control groups (G5 and G10) and significantly higher than those of the attacked control groups (G4 and G9). It is surprising that vHVT114 did not interfere with the protective effect of vHVT13-induced IBD after SC or the intraocular route, confirming the data obtained in Examples 10 and 14.

[0368] In summary, these data clearly demonstrate the compatibility of HVT114+vSB1-009 or +SB-1 and vSB1-009 with vHVT13 in the variant E IBDV attack model when administered via SC or intraocular route.

[0369] Example 17: Efficacy of vHVT114, vHVT13, and SB1 or vSB1-009 vectors against highly virulent virus + Marek's disease challenge.

[0370] The aim of this study was to evaluate the efficacy of different combinations of vaccines, including vHVT114, vHVT13, SB-1 and / or vSB1-009, administered to one-day-old SPF chickens via the SC route, and challenged with a highly virulent + Marek's disease virus (vv+MDV) T-King isolate four days later to induce Marek's disease.

[0371] On day 0, 100 one-day-old SPF chickens were randomly assigned to five groups (n=20 per group). On day 0, chickens in groups 1 through 3 were injected subcutaneously into the neck with 0.2 mL of each vaccine containing a target dose of 2000 pfu (except for SB-1, which has a target dose of 1000 pfu). Chickens in groups 4 and 5 were unvaccinated and served as either challenged (group 4) or unchallenged (group 5) sham controls. The study design is shown in Table 29. On day 4, all chickens in groups 1 through 4 were challenged with 0.2 mL of the vv+MDV T-King isolate via intraperitoneal administration.

[0372] Table 29 Study Design and MD Protection Results

[0373]

[0374] Monitor each group daily for any adverse reactions before and after the attack. On day 49, cull all live chickens and perform necropsies to examine for gross lesions associated with Marek's disease. Chickens are classified as positive for Marek's disease if neurological signs such as paralysis, locomotive signs, and severe emaciation or depression are observed, if death directly caused by Marek's disease is observed at necropsies, or if gross lesions are observed. Lesions may include, but are not limited to, damage to the liver, heart, spleen, gonads, kidneys, and muscles.

[0375] The results of the protective effect are shown in Table 29 above. All vaccinated groups (G1 to G3) were administered equally, and as expected, partial (65%) MD protection was induced in this very severe early challenge model. These results indicate that the vector vaccine candidates maintained their ability to protect against Marek's disease.

[0376] Example 18: Efficacy of recombinant HVT and SB1 vectors against Marek's disease

[0377] Marek's disease efficacy of HVT vectorized recombinants and SB-1 vectorized recombinants, alone or in combination, was also demonstrated. Challenge strains included virulent Marek's disease (vMD) challenges such as GA22, highly virulent Marek's disease (vvMD) challenges such as RB1B, and / or highly virulent + Marek's disease (vv+MD) challenges such as T. King virus. One-day-old chickens or 18-19-day-old chicken embryos were subcutaneously inoculated with 0.2 ml or 0.05 ml of the test virus, respectively. At 5 days of age, inoculated chickens and uninoculated controls were challenged with the relevant Marek's disease challenge virus (v, vv, or vv+MDV). The challenged chickens were observed until 7 weeks of age. All chickens were culled and necropsy performed to observe gross lesions associated with Marek's disease, as described in Example 17.

[0378] Example 19: Interference of HVT with vHVT13-induced IBDV antibodies in commercial hens

[0379] The aim of this study was to determine whether co-administration of HVT and vHVT13 affected the IBDV antibody response induced by vHVT13 in commercial hens.

[0380] Eighty 0-day-old commercial brown hens were used in three isolation units. Blood samples were taken from 15 chickens at 0 days of age to test for maternally derived antibodies (MDAs) against IBD. The remaining chickens were divided into three groups as shown in Table 30. Chickens in groups 2 and 3 were inoculated via the neck and back via the SC route using commercial doses of vHVT13 (VAXXITEK HVT+IBD; Merial SAS, Lyon, France) and / or HVT cell-bound BioHVT (Merial SpA, Noventa, Italy). Blood samples were taken at 25, 35, and 45 days of age. The ELISA kit used to evaluate IBDV serological responses was the PROFLOK PLUS IBD(IBD+)Ab ELISA kit from Synbiotics (Synbiotics Corp., Kansas City, MO, USA).

[0381] Table 30 Study Design and Serological Results

[0382]

[0383] The average ELISA titers are shown in Table 30. The titer in the unvaccinated group G1 decreased from D1 to D45, corresponding to a decrease in IBDV maternal antibodies. As expected, the ELISA titer in the vHVT13 group G2 remained high until D45, indicating that maternal antibodies were gradually replaced by vHVT13-induced antibodies. The addition of HVT to vHVT13 had a clear negative effect, as antibody titers observed in G3 were similar to those in G1. These results are contrary to those observed using vHVT114+vHVT13, since vHVT114 does not reduce vHVT13-induced IBD+ ELISA titers (see Example 14, Table 26). They confirm the unexpected property that vHVT114 does not interfere with the immunogenicity of vHVT13.

[0384] In summary, contrary to what was observed with respect to vHVT114, the addition of HVT to vHVT13 had a clear negative effect on vHVT13-induced humoral immunity to IBDV.

[0385] Example 20: Interference of commercial HVT-ND with the protective effect of vHVT13-induced IBD

[0386] The aim of this study was to determine whether co-administration of commercial HVT-ND vector vaccine with vHVT13 affected the protective effect of vHVT13-induced IBD in SPF chickens.

[0387] Seventy-five SPF chickens (3 groups (G2, G3, and G4, 25 chickens per group) were vaccinated via the SC route at 1 day of age with a commercial dose of vHVT13 (VAXXITEK HVT+IBD) and with or without a commercially approved HVT-vectored ND vaccine (vHVT-ND1 and vHVT-ND2) (as shown in Table 31). Fifteen chickens were kept as unvaccinated controls (G1). Three weeks post-vaccination, chickens (20 chickens from G2, G3, and G4 and 10 chickens from G1) were challenged with at least 2.0 log10 EID50 of the IBD virus Ph / B1 strain (isolated in the Philippines) administered via the ocular route. Clinical signs or death due to IBD challenge virus were observed in all chickens for 5 days, and they were euthanized painlessly at the end of the observation period post-challenge for necropsy of IBD lesions (particularly from the bursa of Fabricius). If the mucous sacs of chickens do not show the typical mucous sac damage of IBD: mucous sac atrophy, peripapillary edema and / or hemorrhage of the mucous sac tissue, they are considered to be protected.

[0388] Table 31 Study Design and IBD Protection Data

[0389]

[0390] The results are shown in Table 31. All 10 attacked control chickens showed clinical signs, and 8 out of 10 died at 4 or 5 dpi, indicating very severe IBDV attack. All of them had severe mucosal cyst damage, including severe atrophy and hemorrhagic spots. vHVT13 alone induced complete protection, while the two combinations with vHVT-ND induced partial clinical and mucosal cyst protection.

[0391] In summary, these results clearly demonstrate that two commercially available HVT-vectored ND vaccines interfere with the protective effect of vHVT13-induced IBD.

[0392] Example 21: Efficacy of vSB1-004, vSB1-006, vSB1-007, vSB1-008, and SB1-vectored NDV vaccines, alone or in combination with vHVT13HVT-vectored IBD vaccines, as well as vHVT302 and vHVT304 vaccines, against NDV Texas GB strain challenge in SPF chickens at 14 and / or 28 days of age.

[0393] The aim of this study was to estimate the efficacy of combination vaccines expressing different Marek's disease vectors that express the NDV F and / or IBDV VP2 genes against Newcastle disease challenge (Texas GB strain, genotype II) in SPF chickens at 14 and / or 28 days of age.

[0394] The characteristics of the six NDV recombinant vaccine candidates tested in this study are described in Table 32 below.

[0395] Table 32 Characteristics of the six NDV recombinant vaccine candidates tested in this study

[0396]

[0397] On day 0, 225 one-day-old SPF chickens were randomly assigned to 9 groups (n=15 per group) (G1a to G9a challenged on day 14) and 6 groups (n=15 per group) (G1b, G3b, G4b, G5b, G8b, and G9b challenged on day 28). On day 0, chickens were subcutaneously injected into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 2000 pfu. The design of this study is shown in Table 33 below. On day 14 or day 28, chickens were challenged intramuscularly with 4.3 and 4.2 log10 EID50 (0.1 mL), respectively.

[0398] Table 33 Results of ND efficacy

[0399]

[0400] *ND = Not performed

[0401] Monitor each group before and after the attack. Record NDV clinical signs after the attack. One chicken died in G6 and G7 before the attack, reducing the number of chickens in these groups from 15 to 14.

[0402] The percentages of clinical protective effects (including protection against death and morbidity) are reported in Table 33 above. Complete susceptibility was observed in the unvaccinated challenged controls G1a and G1b, confirming the high severity of the challenge. Partial protection was observed from 13.3% to 46.6% after challenge on D14, with vSB1-008, vSB1-007, and vHVT304 inducing the highest levels of protection. Protection levels after ND challenge on D28 were significantly higher in all vaccinated groups, and also slightly higher in groups vaccinated with vSB1-008, vSB1-007, or vHVT304. These results suggest that ND protection levels depend on the date of challenge and the construct. The vSB1-008 and vSB1-007 constructs performed slightly better than vSB1-004 and vSB1-006, and vHVT304 performed slightly better than vHVT302, indicating that different characteristics of the constructs play an important role in the performance of MDV-based vector vaccines.

[0403] In summary, the results of this study show that the level of protection against ND induced by Marek's disease vectors expressing the NDV F gene can depend on various parameters, including the vector, the inserted locus, the F gene, the promoter, the polyadenylation site, and the attack conditions.

[0404] Example 22: Efficacy of dual HVT-ND+IBD vHVT304 and vHVT306 vaccines against NDV Texas GB strain challenge in 14- and / or 28-day-old SPF chickens.

[0405] The aim of this study was to evaluate the efficacy of HVT-vectored vaccines expressing both NDV F and IBDV VP2 genes against Newcastle disease challenge (Texas GB strain, genotype II) in 14- and / or 28-day-old SPF chickens.

[0406] The characteristics of the two recombinant vaccine candidates tested in this study are described in Table 34 below.

[0407] Table 34 Characteristics of the recombinant vaccine candidates used in the study

[0408] name Parental virus promoter F gene Poly-A locus vHVT304 vHVT13 SV40 Opt-VIId Synthetic IG2 vHVT306 vHVT13 SV40 Opt-VIId Synthetic SORF3-US2

[0409] On day 0, 90 one-day-old SPF chickens were randomly assigned to three groups (n=15 per group) (G1a to G3a challenged on day 14) and three groups (n=15 per group) (G1b to G3b challenged on day 28). On day 0, chickens were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 2000 pfu. The study design is shown in Table 35 below. On day 14 or day 28, chickens were challenged intramuscularly with a target dose of 4.0 log10 EID50 (0.1 mL).

[0410] Table 35 Results of ND efficacy

[0411]

[0412] Monitor each group before and after the attack. Record NDV clinical signs after the attack. One chicken in G2b died before the attack, reducing the number of chickens in that group from 15 to 14.

[0413] The percentages of clinical protective effects (including protection against death and morbidity) are reported in Table 35 above. Complete susceptibility was observed in the unvaccinated challenged controls G1a and G1b, confirming the high severity of the challenge. The level of protection after challenge on D14 was significantly lower than that obtained after challenge on D28. These vaccine candidates possess NDV F expression cassettes with the same insertion at two different loci in the vHVT13 genome. Under test conditions, they performed identically in terms of ND protection, indicating that both insertion loci (IG2 and SORF3-US2) are equally suitable for NDV F cassette insertion.

[0414] In summary, the results of this study show that the level of protection against ND induced by Marek's disease vectors expressing the NDV F gene depends on various parameters, including the vector, the inserted locus, the F gene, the promoter, the polyadenylation site, and the attack conditions.

[0415] Example 23: Early ND efficacy against rapid-onset V NDV challenge induced in 1-day-old SPF chickens by dual HVT-ND+IBD (vHVT302, vHVT303, and vHVT304) or SB1-vectors (vSB1-006 and vSB1-007).

[0416] The aim of this study was to evaluate the efficacy of three dual HVT-ND+IBD vectors (vHVT302, vHVT303, and vHVT304) and two SB1-ND vectors (vSB1-006 and vSB1-007) against immediate-type genotype V (Chimalhuacan) NDV challenge performed on day 14 in 1-day-old SPF chickens.

[0417] The characteristics of the five recombinant vaccine candidates tested in the study are described in Table 36 below.

[0418] Table 36 Characteristics of the recombinant vaccine candidates used in this study

[0419]

[0420]

[0421] Six groups (1 and 2) of 10 one-day-old specific pathogen-free (SPF) white Leghorn chickens were randomly constructed. Chickens in groups 2 through 6 were inoculated subcutaneously (neck back) with a target dose of 2000 PFU, as shown in Table 37 below. Chickens in group 1 were not inoculated and remained as controls. At 2 weeks of age, all chickens were challenged with an immediate-type NDV strain of genotype V Mexican Chimalhuacan (MexV). 10 PFU diluted in 0.2 ml of physiologically sterile water was used. 5Egg infection dose 50 (EID50) was administered via the intramuscular (IM) route. All chickens were monitored until 14 days post-infection. Post-infection, the health status of each chicken was scored daily as follows: healthy / with specific symptoms (ruffled feathers, lethargy, torticollis, trembling) / death. Any chicken exhibiting specific symptoms for more than 2 days or recorded as sick on D28 was considered for morbidity calculation.

[0422] Table 37 shows the results of early ND protection induced in SPF 0-day-old chickens by different MDV vectorization candidates expressing the NDV F gene.

[0423]

[0424] The results of protection are summarized in Table 37. All control groups died after ND challenge. Different tested vaccines induced varying levels of ND protection, ranging from 10% to 80% and 0% to 60%, respectively, against mortality and morbidity. The vHVT304 candidate induced better protection than the vHVT303 and vHVT302 candidates; this can be attributed to the exogenous SV40 promoter positioned prior to the NDV F gene. vSB1-007 performed slightly better than vSB1-006. Furthermore, the performance obtained using vHVT304 was comparable to that obtained using vSB1-007, indicating that different Marek's disease vectors can achieve the same level of ND protection.

[0425] In summary, this study shows that both the dual HVT-ND+IBD and SB1-ND vectorized vaccines can achieve significant ND protection levels in a severely affected early NDV challenge model.

[0426] Example 24: Efficacy of dual HVT-ND+IBD vHVT306 induced in 1-day-old SPF chickens via intraocular or SC route against immediate-onset genotype VNDV challenge on day 28.

[0427] The aim of this study was to evaluate the efficacy of a dual HVT-ND+IBD (vHVT306) administered to SPF chickens via intraocular or SC route against immediate-type genotype V (Chimalhuacan) NDV challenge at 28 days of age.

[0428] The characteristics of the vHVT306 recombinant vaccine candidate tested in this study are described in Table 38 below. The single HVT-IBD vector vaccine vHVT13 was used as a control.

[0429] Table 38 Characteristics of the recombinant vaccine candidates used in this study

[0430] name Parental virus promoter F gene Poly-A locus vHVT306 vHVT13 SV40 Opt-VIId Synthetic SORF3-US2

[0431] On day -3, 40 incubated SPF chicken eggs, approximately 18 days and 18 hours old, were randomly assigned to two groups (20 eggs per group). On day 0, a group of 12-day-old SPF chickens was added. The group definitions are shown in Table 39 below. Inoculation was performed on day -3 (intraocular route) or on day 0 (SC route, in the neck and back), with a target dose of 2000 PFU / chicken for vHVT306 and vHVT13. For the intraocular route, hatchability, viability (up to day 28), and growth (between hatching and day 28) were monitored.

[0432] On day 28, 10 chickens per group were challenged with the highly virulent ND Chimalhuacan strain. The solution was diluted in 0.2 ml of physiologically sterile water. 5 Egg infection dose 50 (EID50) was administered via the intramuscular (IM) route. Chickens were monitored until 14 days post-challenge. Specific clinical signs and mortality were recorded. Any chicken exhibiting specific symptoms for more than 2 days or recorded as morbid at D42 was considered for morbidity calculation. Oropharyngeal swabs were obtained from each surviving chicken at 5 and 7 days post-challenge (i.e., at D33 and D35). All swabs were analyzed by specific NDV qRT-PCR.

[0433] Table 39. ND protection results induced by vHVT306MDV vector candidates expressing both NDV F and IBDV VP2 genes administered to SPF chickens via SC or intraocular route.

[0434]

[0435] *The threshold titer for real-time RT-PCR was set at 2.7 log10 equivalent EID50.

[0436] Complete hatchability was recorded for groups 1 and 2 after intraocular inoculation, and all hatched chickens survived to day 28. No difference in body weight was detected between the two groups at days 0 and 28, confirming the perfect safety of vHVT306 upon intraocular administration. The results of protection are summarized in Table 39. All vHVT13-inoculated control chickens died 4 days after ND challenge. vHVT306 administered via both routes induced complete clinical ND protection. Furthermore, no shedding was detected after intraocular administration, while only a few chickens shed detectable amounts of challenge virus after SC administration.

[0437] In summary, this study demonstrates that dual HVT-ND+IBD vHVT306, administered via SC or intraovarian route, induces excellent levels of ND protection in a very severe heterologous NDV attack model.

[0438] Example 25: Efficacy of the dual HVT-ND+IBD (vHVT302, vHVT303, and vHVT304) recombinant vaccine against classical IBDV isolates challenged in SPF chickens on day 15.

[0439] The aim of this study was to assess the early IBD efficacy of the dual HVT recombinants vHVT302, vHVT303, and vHVT304 against a highly virulent infectious bursal disease virus (vIBDV) challenge (Faragher 52 / 70 strain) in 15-day-old SPF chickens.

[0440] The characteristics of the three dual HVT-ND+IBD recombinant vaccine candidates tested in this study are described in Table 40 below.

[0441] Table 40 Characteristics of expression cassettes for dual HVT recombinants

[0442] name Parental virus promoter F gene Poly-A locus vHVT302 vHVT13 US10 Opt-VIId US10 US10 vHVT303 vHVT13 US10 Opt-V(CA02) US10 US10 vHVT304 vHVT13 SV40 Opt-VIId Synthetic IG2

[0443] On day 0, 40 one-day-old SPF chickens were randomly assigned to four groups (n=10 per group), including a control group (G1) inoculated with vSB1-004 (an SB-1 vector expressing the NDV F gene). Five other SPF chickens remained uninoculated and unchallenged for mucus sac weight / body weight assessment. As described in Table 41 below, on day 0, the recombinant vaccine containing a target dose of 2000 pfu was administered subcutaneously to the neck of the chickens. On day 15, blood samples were collected from all chickens in each group (10 chickens per group, except for one chicken in groups 1 and 3 who died before blood sampling) for testing using a kit. Serological testing was performed using IBD (Synbiotics Corp). On day 15, chickens in all four groups were challenged with 2.5 log10 EID50 via eye drops (0.05 mL / chicken).

[0444] Table 41 Results of the study design and IBD efficacy

[0445]

[0446] 1 Chickens that show symptoms more than 2 days after the onset of illness or are still showing symptoms on day 25 are considered to be diseased. The numbers in parentheses are the number of chickens in the attacked group.

[0447] 2 Protective effect against clinical signs and severe bursal damage (bursal score <3)

[0448] 4 The mucosal cyst weight / body weight ratio in the uninoculated / unattacked group was 0.0043.

[0449] Each group was monitored before and after attack. Clinical signs of IBDV were recorded 11 days post-attack (from D15 to D25). At the end of the post-attack observation period (D25), all surviving chickens were euthanized painlessly and necropsy was performed. Body weight and mucus bursa weight were recorded. Each supracaval bursa of Fabricius (BF) was weighed and then stored in a single container filled with 4% formaldehyde for histological testing. Histological damage to the mucus bursa was scored according to the grades provided in Table 42.

[0450] Table 42 Scoring criteria for histological damage to the bursa of Fabricius*

[0451]

[0452] *Derived from European Pharmacopoeia, Monograph No. 01 / 2008:0587, "Infectious Bursal Disease Vaccine (Live) for Chickens"

[0453] Chickens are considered to be affected if they die and / or show significant signs of disease and / or severe damage to the bursa of Fabricius (i.e., histological score ≥3).

[0454] The mean ELISA IBD+ antibody titers expressed as log10 prior to the challenge are shown in Table 41. Significantly higher titers than the control group G1 were detected in all vaccinated groups. Serological titers were slightly higher in G3 (vHVT303).

[0455] Several clinical signs were observed in all nine chickens in the control group G1 after the attack, resulting in the death of one chicken. Only one inoculated chicken in G2 (vHVT302) showed clinical signs after the attack. The percentage of protection against several mucosal sac injuries is shown in Table 41 above. Significant IBD protection was observed in all inoculated groups, with complete protection observed in G3 (vHVT303). The mean mucosal sac weight / body weight ratio is also shown in Table 41. The ratio in all inoculated groups was higher than that in the attacked control group G1, and there was no significant difference compared to the uninoculated and unattacked control group.

[0456] In summary, these data demonstrate that the three dual HVT-IBD+NDs tested in this study induce IBD antibodies and early IBD protection in a severe IBDV challenge model.

[0457] Example 26: Efficacy of five different HVT-ND vaccine candidates against challenge with the rapid-type NDV ZJ1 (genotype VIId) isolate in 14-day-old SPF chickens.

[0458] The aim of this study was to evaluate the efficacy of five single HVT recombinant constructs (vHVT39, vHVT110, vHVT111, vHVT112, and vHVT113) expressing the NDV F gene against Newcastle disease challenge with an immediate-type NDV ZJ1 (genotype VIId) isolate in 14-day-old SPF chickens.

[0459] The characteristics of these five vaccine candidates are described in Table 43 below.

[0460] Table 43 Characteristics of the HVT-ND recombinant virus used in the attack study

[0461] name Parental virus promoter F gene* Poly-A locus vHVT039 HVT MDV gB Wtnm-Texas SV40 IG1 vHVT110 HVT MCMV IE Wt-VIId SV40 IG1 vHVT111 HVT SV40 Wt-VIId SV40 IG1 vHVT112 HVT MCMV IE Wt-YZCQ SV40 IG1 vHVT113 HVT MCMV IE Wt-Texas SV40 IG1

[0462] *Wt indicates that the wild-type immediate-type F gene sequence was used, but the cleavage site was modified to the cleavage site of a slow-type virus. Wtnm indicates that the cleavage site of the wild-type sequence was not modified. The Texas immediate-type strain belongs to genotype IV, and YZCQ belongs to genotype VIId.

[0463] On day 0, 72 one-day-old SPF chickens were randomly assigned to five groups (n=12 per group) (vaccinated) and one group of 12 chickens (unvaccinated control). As described in Table 44 below, on day 0, chickens were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 6000 pfu. On day 14, chickens were challenged via intramuscular route with an immediate-type NDV strain ZJ1 / 2000 (genotype VIId) of 5 log10 EID50.

[0464] Table 44 Results of ND efficacy

[0465]

[0466] Each group was monitored before and after the attack. Clinical signs and mortality of NDV were recorded post-attack. Oropharyngeal swabs were collected at 2 and 4 days post-infection (dpi) to assess viral load by real-time RT-PCR using the method described by Wise et al. (2004; Development of a Real-Time Reverse-Transcription PCR for Detection of Newcastle disease virus RNAin Clinical Samples. J Clin Microbiol 42, 329-338).

[0467] The percentages of protection against mortality and morbidity are reported in Table 44 above. Complete susceptibility was observed in the unvaccinated challenged control group G1, confirming the high severity of the challenge. The vaccines induced varying levels of protection against mortality (25-100%) or morbidity (8%-83%). The best level of protection was induced by vHVT110, while the lowest level was induced by vHVT039, with other candidates yielding intermediate results. Results of oropharyngeal shedding at 2 and 4 dpi are also shown in Table 44 above and are consistent with the clinical protective effects. These vaccine candidates differ in their promoters and F gene sequences. These results demonstrate that these parameters are crucial for the design of optimal HVT-ND vaccine candidates.

[0468] In summary, the results of this study demonstrate the importance of promoter and F gene sequence in the ND efficacy induced by HVT-vectored ND vaccine candidates.

[0469] Example 27: Evaluation of Newcastle disease efficacy induced by a dual SB1 construct expressing IBDV VP2 and NDV F.

[0470] The aim of this study was to estimate the efficacy of a dual SB1 construct expressing IBDV VP2 and NDV F against Newcastle disease challenge.

[0471] On day 0, 1-day-old SPF chickens were randomly assigned to several groups (10-20 chickens per group), including vaccinated and unvaccinated groups. On day 0, the vaccinated groups were injected subcutaneously into the neck with 0.2 mL of the recombinant vaccine containing a target dose of 1000 to 5000 pfu. Alternatively, the same dose was administered intraocularly to the eggs 2 or 3 days before hatching. At different times following vaccination: e.g., day 14, day 28, or day 42, chickens were challenged intramuscularly with approximately 4.0 log10 EID50 (0.1 mL) of an immediate-type NDV strain such as Texas GB (genotype II), ZJ1 (genotype VIId), or Chimalhuacan (genotype V) strain (at least one vaccinated group and one unvaccinated group).

[0472] Clinically monitor each group before and after challenge. Record NDV clinical signs (morbidity) and mortality after challenge. Calculate the percentage of clinical protection in all groups. At least 90% of unvaccinated challenged SPF chickens should die or become severely ill after challenge to validate the severity of the challenge. Oropharyngeal and cloacal swabs can be obtained at different times after challenge, such as 3, 5, 7, and 9 days post-challenge, and viral load can be estimated by real-time RT-PCR. The best candidates are those that induce the highest level of clinical protection and the lowest level of swab viral load. Similar studies can be conducted in broilers containing maternal NDV antibodies; however, these maternal antibodies may potentially protect unvaccinated chickens if challenged early. Dual SB1 constructs can also be tested in combination with other Marek's disease vaccines or vector vaccines.

[0473] Example 28: Evaluation of infectious bursal disease efficacy induced by a dual SB1 construct expressing IBDV VP2 and NDV F

[0474] The aim of this study was to estimate the IBD potency of dual SB1 expressing both IBDV VP2 and NDV F.

[0475] One-day-old SPF chickens were randomly assigned to several groups (10-20 chickens per group), including vaccinated and unvaccinated groups. Unvaccinated controls were divided into two subgroups: challenged and unchallenged chickens. On day 0, chickens in the vaccinated groups were injected subcutaneously into the neck with 0.2 mL of the vaccine, each containing a target dose of 1000 to 5000 pfu. Alternatively, the same dose was administered intraeggly in 0.05 mL two or three days prior to hatching. At different times post-vaccination: e.g., 14, 21, 28, or 42 days post-vaccination, all chickens from both the vaccinated and challenged controls were challenged with eye drops (0.03 mL, containing 2 to 4 log10 EID50 / chicken) of a virulent IBDV (e.g., Faragher or US standard strain), a highly virulent IBDV such as isolate 91-168, or a variant IBDV isolate such as USDelaware variant E isolate. Each group was clinically monitored before and after challenge. Post-mortem examinations can be performed on chickens 4 or 5 days post-attack to assess gross damage to the mucous cysts. Post-mortem examinations can also be performed 10 to 11 days post-attack. Gross and / or histological damage can then be assessed. Additionally, the weight of the chicken and its mucous cysts is calculated as the mucous cyst weight / body weight ratio (mucous cyst weight / body weight ratio x 100) compared to the mucous cyst weight / body weight ratio of the unvaccinated, unattacked control group. Control SPF-attacked chickens must exhibit clinical signs and / or have significant gross and / or histological damage, and / or should have a significantly lower mucous cyst weight / body weight ratio than the unvaccinated, unattacked control chickens to confirm the severity of the attack. Vaccine efficacy is assessed by comparing these parameters to the unvaccinated / attacked and unvaccinated / unattacked groups. Such studies can be conducted in broilers containing IBDV maternal antibodies; however, these maternal antibodies may potentially protect unvaccinated chickens if the attack is performed early. The dual SB1 construct can also be combined with other Marek's disease vaccines or vector vaccines for testing.

[0476] Example 29: Evaluation of the efficacy of a dual SB1 construct expressing IBDV VP2 and NDV F in inducing Marek's disease.

[0477] The aim of this study was to evaluate the efficacy of SB1 vectors expressing both IBDV VP2 and NDVF in inducing Marek's disease.

[0478] One-day-old SPF chickens were randomly assigned to several groups (20 to 50 chickens per group), including vaccinated and unvaccinated controls. The unvaccinated controls could be further divided into two subgroups: challenged and unchallenged chickens. On day 0, chickens in the vaccinated groups were injected subcutaneously into the neck with 0.2 mL of the vaccine, each containing a target dose of 1000 to 5000 pfu. Alternatively, the same dose could be administered intraperitoneally in 0.05 mL of the vaccine 2 or 3 days before hatching. At different times post-vaccination, e.g., 3 to 10 days post-vaccination, all chickens from the vaccinated and challenged controls were challenged via the intraperitoneal route with 0.2 mL of Marek's disease virus (MDV) strain. The MDV strain could be one of several pathogenic types, such as virulent MDV (vMDV), including JM or GA22 isolates, highly virulent MDV (vvMDV), such as RB-1B or Md5 isolates, and highly virulent+ (vv+MDV), such as T-King or 648A isolates. MDV challenge inoculum is prepared by infecting chickens, harvesting their blood cells, and freezing them in liquid nitrogen in the presence of a cryoprotectant such as DMSO. The infection dose 50 (CID50) for each challenge batch of chickens is determined before conducting inoculation / challenge studies. Each group is clinically monitored before and after challenge. Chickens are necropsed at least 7 weeks post-inoculation to examine each chicken for the presence of gross Marek's disease lesions. These lesions may include, but are not limited to, damage to the liver, heart, spleen, gonads, kidneys, nerves, and muscles. Such studies can be conducted in broilers containing maternal antibodies against MDV. The dual SB1 construct can also be tested in combination with other Marek's disease vaccines (e.g., HVT and / or CVI988Rispens strain) or MD vector vaccines. MD challenge can also be performed through contact between inoculated chickens and MDV-infected uninoculated SPF chickens.

[0479] ***

[0480] The preferred embodiments of the present invention have been described in detail above. It should be understood that the present invention as defined by the above embodiments is not limited to the specific contents shown in the above description, as many apparent variations are possible without departing from the spirit or scope of the present invention.

[0481] All references or documents cited or referenced herein (“Documents cited herein”), and all specifications, instructions, product specifications and product introductions of numerous manufacturers of any product mentioned herein or in any document incorporated herein by reference, are incorporated herein by reference and may be used in the practice of this invention.

Claims

1. A recombinant turkey herpesvirus vector expressing a Newcastle disease virus F polypeptide, wherein the Newcastle disease virus F polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 2, wherein a polynucleotide encoding the Newcastle disease virus F polypeptide is operably linked to an SV40 promoter and an SV40 polyA signal, and the polynucleotide encoding the Newcastle disease virus F polypeptide, the SV40 promoter, and the SV40 polyA signal are inserted within the IG1 locus of the HVT genome; the Newcastle disease virus F polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 4, wherein a polynucleotide encoding the Newcastle disease virus F polypeptide is operably linked to an SV40 promoter or an immediate early cytomegalovirus promoter and an SV40 polyA signal, and the polynucleotide encoding the Newcastle disease virus F polypeptide, the SV40 promoter or the immediate early cytomegalovirus promoter, and the SV40 polyA signal are inserted within the IG1 locus of the HVT genome; the Newcastle disease virus F polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 6, wherein a polynucleotide encoding the Newcastle disease virus F polypeptide is operably linked to an SV40 promoter and an SV40 polyA signal, and the polynucleotide encoding the Newcastle disease virus F polypeptide, the SV40 promoter, and the SV40 polyA signal are inserted within the IG1 locus of the HVT genome; or the Newcastle disease virus F polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 37, wherein a polynucleotide encoding the Newcastle disease virus F polypeptide is operably linked to an immediate early cytomegalovirus promoter and an SV40 polyA signal, and the polynucleotide encoding the Newcastle disease virus F polypeptide, the immediate early cytomegalovirus promoter, and the SV40 polyA signal are inserted within the IG1 locus of the HVT genome.

2. The recombinant turkey herpesvirus vector of claim 1, wherein the turkey herpesvirus vector is a turkey herpesvirus F126 strain.

3. The recombinant turkey herpesvirus vector of claim 2, wherein the SV40 promoter is set forth in the sequence of SEQ ID NO: 9, and the immediate early cytomegalovirus promoter is set forth in the sequence of SEQ ID NO:

10.

4. The recombinant turkey herpesvirus vector of claim 2, wherein the SV40 polyA signal is set forth in the sequence of SEQ ID NO:

11.

5. The recombinant turkey herpesvirus vector of claim 2, wherein the polynucleotide encoding the Newcastle disease virus F polypeptide consists of the nucleotide sequence set forth in SEQ ID NO: 1, 3, 5, or 36.

6. A vaccine comprising the recombinant turkey herpesvirus vector of any one of claims 1-5.

7. The vaccine of claim 6, further comprising a pharmaceutically or veterinarily acceptable carrier, excipient, vehicle, or adjuvant. ​ 8. Use of a recombinant turkey herpesvirus vector according to any one of claims 1-5 in the manufacture of a vaccine for inducing a protective response against Newcastle disease virus in avians.

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