Multivalent HVT vector vaccine

Abstract

The present invention describes a recombinant herpesvirus of turkeys (rHVT) that can be used as a vector vaccine for poultry against infection and disease caused by multiple poultry pathogens. Specifically, the rHVT expresses the infectious bursal disease virus (IBDV) viral protein 2 (VP2) gene and the Newcastle disease virus (NDV) fusion (F) protein gene from first and second expression cassettes inserted into the unique short (Us) region of the rHVT, and expresses the avian influenza virus (AIV) hemagglutinin (HA) gene from a third expression cassette inserted either between the UL40 and UL41 genes or between the UL44 and UL45 genes in the unique long (UL) region of the rHVT. This rHVT can be used to vaccinate poultry against MDV, IBDV, NDV, and AIV.

Description

Technical Field

[0001] The present invention relates to the field of veterinary vaccines, namely poultry vaccines based on recombinant turkey herpesvirus as a viral vector vaccine. In particular, the present invention relates to recombinant turkey herpesvirus (rHVT), host cells containing rHVT, the medical use of rHVT and host cells, vaccines containing rHVT and / or host cells, and methods for producing said vaccines.

Background Art

[0002] Recombinant vector viruses are well-known methods for expressing heterologous genes and delivering their protein products to human or non-human animal targets. Examples are vaccinia virus vectors or adenovirus vectors. When the heterologous gene encodes an immunogenic protein derived from a pathogen, this can be an effective method of vaccinating the target against the disease caused by that pathogen. As a replicating microorganism, the vector virus can establish a productive infection in the vaccinated target, co-express the heterologous gene and its own genes, and thus stimulate the immune system of the target.

[0003] In veterinary vaccination, particularly in poultry vaccination, vector vaccines have attracted interest due to their relatively easy use and low cost. For example, several avian vector vaccines based on avian adenovirus, fowlpox virus, particularly turkey herpesvirus (HVT) have been investigated for many years (see WO 87 / 04463 and WO 90 / 002803). The advantage of using HVT as a vector is that it is non-pathogenic to birds but induces immunity against pathogenic members of its viral family: Marek's disease virus 1 or 2 (MDV1 or MDV2).

[0004] For many years, genes derived from various avian pathogens have been expressed in HVT vectors derived from, for example, Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), and infectious bronchitis virus (see International Publication No. 93 / 025665); avian influenza virus (AIV) (see International Publication No. 2012 / 052384); or the parasite Eimeria (Cronenberg et al., 1999, Acta Virol., vol. 43, pp. 192-197). This led to the development of various commercially available HVT vector vaccines for poultry, such as: for ND: Innovax(registered trademark)-ND (MSD Animal Health) and Vectormune(trademark) HVT-NDV (Ceva Sante Animale); for ILT: Innovax(registered trademark)-ILT (MSD Animal Health); for IBD: Vaxxitek(trademark) HVT+IBD (Boehringer-Ingelheim; formerly known as Gallivac(trademark) HVT-IBD), and Vectormune(registered trademark) ND Ceva Sante Animale; and for AI: Vectormune(registered trademark) AI (Ceva Sante Animale).

[0005] The insertion of heterologous genes into the viral genome is a burden on the vector virus. This can affect its replication, expression, and / or genetic stability in vitro and / or in vivo. These problems are particularly pronounced when more than one heterologous gene is inserted. Such multivalent recombinant vector vaccines may potentially protect against multiple diseases after a single dose. However, such vector constructs must still provide good replication of the vector and its inserts both in vitro and in vivo, as well as effective expression of all heterologous genes at sufficiently high levels and for a significant duration, in order to induce and maintain a protective immune response against all intended pathogens in the vaccinated target.

[0006] This replication and expression stability also enables large-scale in vitro replication necessary for large-scale production. Furthermore, such stability is a requirement for recombinant viruses (genetically modified organisms) to meet the very high standards of safety and biological stability that they must meet in vivo in order to obtain sales authorization from government or regulatory authorities before they can be introduced into the field as commercial products.

[0007] For example, many multivalent HVT vector vaccines have been described over the years, such as in International Publication No. 93 / 025665 and International Publication No. 96 / 005291. However, most of the multigene constructs described in such publications remain only suggestions, and only a fraction of recombinant vectors with multiple inserts have actually been constructed and isolated. Very few have been tested in birds. Overall, no results have been shown regarding their stability during replication or the expression levels of exogenous genes, let alone data on the induction of effective immune defense in target animals. Due to such challenges regarding the genetic stability and sustained expression of inserts, only a small number of multivalent HVT vector constructs have actually become approved commercial vaccine products. Currently, these include Innovax® ND-IBD (MSD Animal Health; International Publication No. 2016 / 102647), Innovax® ND-ILT (MSD Animal Health; International Publication No. 2013 / 057236), ULTIFEND® IBD ND (Ceva Sante Animale; International Publication No. 2013 / 144355), and Vaxxitek® HVT+IBD+ND (Boehringer-Ingelheim; International Publication No. 2018 / 112.051). International Publication No. 2019 / 072964 also describes a multivalent rHVT vector vaccine that provides protection against MDV, NDV, IBDV, and ILTV.

[0008] However, there is a continued need for more multivalent vector vaccines due to the presence of many more prominent poultry diseases that require effective vaccination.

[0009] International Publication No. 1999 / 018215 describes multivalent HVT vector constructs containing inserts of the NDV-F or HN gene and the IBDV-VP2 gene. Several insertable sites are described, one of which is between the HVT UL40 and UL41 genes. However, the only recombinant HVT (rHVT) constructs actually produced have inserts between the HVT genes UL44 and UL45, or between UL45 and UL46. No data on genetic stability is provided for any of the described vectors.

[0010] International Publication No. 2012 / 052384 describes rHVT expressing the AIV HA gene, driven by the mammalian herpesvirus-derived glycoprotein B (gB) gene promoter, from a unique small (Us) genome region locus.

[0011] International Publication No. 2016 / 102647 describes a polyvalent rHVT that expresses the NDV F gene and the IBDV VP2 gene from a single expression cassette inserted into the Us genome region. [Prior art documents] [Patent Documents]

[0012] [Patent Document 1] International Publication No. 87 / 04463 [Patent Document 2] International Publication No. 90 / 002803 [Patent Document 3] International Publication No. 93 / 025665 [Patent Document 4] International Publication No. 2012 / 052384 [Patent Document 5] International Publication No. 96 / 005291 [Patent Document 6] International Publication No. 2016 / 102647 [Patent Document 7] International Publication No. 2013 / 057236 [Patent Document 8] International Publication No. 2013 / 144355 [Patent Document 9] International Publication No. 2018 / 112051 [Patent Document 10] International Publication No. 2019 / 072964 [Patent Document 11] International Publication No. 1999 / 018215 [Non-Patent Document]

[0013] [Non-Patent Document 1] Cronenberg et al., 1999, Acta Virol., vol. 43, p. 192-197 [Summary of the Invention] [Problems to be Solved by the Invention]

[0014] An object of the present invention is to provide for the first time an rHVT vector vaccine that adaptively meets this need in situ and enables vaccination of poultry against four avian pathogens: MDV, NDV, IBDV, and AIV from a single vaccination.

[0015] Surprisingly, this object can be achieved by providing an rHVT that expresses the NDV F and IBDV VP2 genes from its Us genomic region, and the AIV HA gene from its UL genomic region, and as a result, it has been found that one or more drawbacks of the prior art can be overcome. [Means for Solving the Problems]

[0016] The inventors attempted to expand the number of heterologous gene inserts in an existing rHVT (“rHVT-VP2-F”) that already contained two heterologous genes: NDV F and IBDV VP2, both expressed from the Us region of the virus. The additional heterologous gene selected was the AIV HA gene. The inventors tested a series of constructs in which the HA gene was inserted at different insertion sites in the HVT genome. Unfortunately, several of the tested insertion sites were found to be unable to generate HVTs that stably replicated and expressed the three inserted heterologous genes, so they were unable to generate stable multivalent recombinant viruses. For example, insertion of the HA gene between the UL47 gene and the UL48 gene, or between the UL54 gene and the LORF4 gene, within the UL genomic region of rHVT-VP2-F was unsuccessful. When tested after 16 passages in cell culture, both of these constructs showed several plaques that had lost the expression of one or two of the heterologous genes. This was notable because these genes are not known to be essential for HVT. In fact, UL47 and UL48 had been reported to be non-essential in transposon gene knockout studies of the HVT genome (Hall et al., 2015, Virology Journal, vol. 12, p. 130).

[0017] Clearly, the fact that the parental vector was already expressing two other heterologous genes complicated the ability to predict, based on prior art observations, what the acceptable insertion sites for additional heterologous genes in the HVT genome might be.

[0018] Thus, it was unexpected that two other insertion sites within the HVT UL genomic region, namely: between the UL40 gene and the UL41 gene of HVT, or between the UL44 gene and the UL45 gene of HVT (referred to herein as the “UL40-41” and “UL44-45” loci, respectively), could be used for the insertion of additional heterologous genes.

[0019] The resulting multivalent rHVT vectors containing the HA gene at the UL40-41 or UL44-45 locus were found to be genetically stable even after 16 consecutive passages in in vitro cell culture. The 16th passaged virus was then used for chicken vaccination, although several further replication cycles in vivo were considered. Subsequently, the virus was re-isolated from vaccinated chickens at 11 and 32 days post-vaccination, and the maintenance of expression of the inserted genes was analyzed. From both time points, the re-isolated viruses were found to be completely genetically stable: in immunofluorescence plaque assays, all re-isolated viruses studied demonstrated expression of all three heterologous genes: F, VP2, and HA. No non-fluorescent plaques were observed for any one of the heterologous inserts.

[0020] Vaccinated chickens showed excellent seroconversion to each of the expressed antigens: F, VP2, and HA. The antibody levels achieved for each of the three antigens were far above the levels known to be necessary for in vivo protection against the challenge. Further details are provided in the examples.

[0021] Therefore, these new multivalent rHVT vector viruses are stable and useful as vaccines against one or more, or all, of MDV, NDV, IBDV, and AIV.

[0022] The possibility of obtaining vaccination against four major poultry diseases from a single vaccination is highly beneficial because it represents a significant reduction in stress on the target animals, as well as a reduction in labor and costs for poultry farmers.

[0023] It is not precisely known how or why rHVT expressing VP2 and F genes can tolerate additional AIV HA genes at the UL40-41 or UL44-45 insertion sites, while insertions at other sites that initially appeared suitable did not result in stable and effective vector constructs.

[0024] While the inventors do not wish to be bound by any theory or model that could explain these findings, they surmise that this effect is due to the complex interaction of various expression patterns in polyvalent rHVT when replication and expression are required in vitro and in vivo. For unknown reasons, inserting an additional gene at a specific site results in a polyvalent rHVT with just the right balance between the strengths of heterologous gene expression, in which the strain exhibits the replication ability of HVT itself, but other constructs do not (unpredictably).

[0025] Accordingly, in one embodiment, the present invention relates to a recombinant turkey herpesvirus (rHVT) that expresses the infectious bursal disease virus (IBDV) viral protein 2 (VP2) gene and the Newcastle disease virus (NDV) fusion (F) protein gene from first and second expression cassettes inserted into the unique short (Us) region of the rHVT genome, wherein the rHVT also expresses the avian influenza virus (AIV) hemagglutinin (HA) gene from a third expression cassette inserted either between the UL40 gene and the UL41 gene or between the UL44 gene and the UL45 gene in the unique long (UL) region of the rHVT genome.

[0026] "Recombinant" refers to nucleic acid molecules or microorganisms whose genetic material has been altered from its original or innate state, thereby resulting in a genetic makeup that it did not originally possess.

[0027] Turkey herpesvirus (HVT) is also known as MDV3, melagrid herpesvirus 1, or simply turkey herpesvirus. HVT was first described in 1970 (Witter et al., 1970, Am.J.Vet.Res., vol.31, p.525). Well-known strains of HVT, such as PB1 or FC-126, have long been used as live vaccines for poultry against Marek's disease caused by MDV1 or MDV2.

[0028] Turkey herpesvirus, Newcastle disease virus, bursal disease virus, and avian influenza virus are all well-known viruses of veterinary relevance. The same is true for cytomegalovirus (CMV), Simian virus 40 (SV40), feline herpesvirus (FHV), and pseudorabies virus (PRV). Such viruses possess taxonomic characteristics such as morphological, genomic, and biochemical features, as well as biological characteristics such as physiological, immunological, or pathological behavior.

[0029] General information on these viruses is available from reference handbooks such as Fields Virology (LWW publ., ISBN: 9781451105636). Information on diseases caused by these viruses is available from handbooks such as 'The Merck veterinary manual' (2010, 10th ed., 2010, CMKahn edt., ISBN: 091191093X) and 'Diseases of poultry' (2008, 12th ed., Y. Saif ed., Iowa State Univ. Press, ISBN-10: 0813807182). Samples of these viruses for use in this invention can be obtained from various sources, e.g., from humans, or as field isolates from wild or farm-raised non-human animals, or from various laboratories, (depository) institutions, or (veterinary) universities. Viruses can be easily identified using routine serological or molecular biological tools. From all of these viruses, much genetic information is digitally available in public sequence databases such as NCBL's GenBank and EMBL's EBI.

[0030] As is well known in the art, the classification of microorganisms in a particular taxonomic group is based on a combination of their characteristics. Accordingly, the present invention also includes variants of viral species that are subclassified in some way therefrom, such as subspecies, strain, isolate, genotype, variant, subtype, or subgroup.

[0031] Furthermore, while certain viruses of the present invention can now be assigned to this species, it will be apparent to those skilled in the art of the present invention that this is a taxonomic classification that may change over time, as new insights may lead to reclassification into new or different taxa. However, since this does not change the virus itself or its antigenic repertoire, but only its scientific name or classification, such reclassified viruses remain within the scope of the present invention.

[0032] The "VP2 protein gene," which encodes the IBDV capsid protein, is well known. The VP2 protein gene may originate from classical or mutant IBDV, or it may be a chimeric gene.

[0033] Similarly, the "F protein gene" encoding the immunodominant fusion glycoprotein of NDV is also well known in the art. In the present invention, the F protein gene can be obtained from long-latency, subpathogenic, or short-latency NDV, or it may be a chimeric gene.

[0034] The term "express" refers to the well-known principle of gene expression, in which genetic information provides the code for protein production through transcription and translation.

[0035] The term “gene” is used to refer to a portion of nucleic acid that can code for a protein. In the present invention, this corresponds to an “open reading frame” (ORF), i.e., the portion of DNA between the start codon and stop codon that does not contain the gene’s promoter. The gene in the present invention may code for a complete protein, or it may code for a portion of a protein that does not contain a “leader,” “anchor,” or “signal sequence,” i.e., only the mature form of the protein. The gene may further code for a specific portion of a protein, such as a portion containing an immunodefense epitope.

[0036] In this regard, the “protein” of the present invention is a molecular chain of amino acids. A protein may be a native or mature protein, a preprotein or proprotein, or a functional fragment of a protein. Therefore, peptides, oligopeptides, and polypeptides are included within the definition of a protein, insofar as they still contain relevant immunological epitopes and / or functional regions.

[0037] In this invention, if a gene is not present in the parental HVT used to construct the rHVT vector, that gene is "heterogeneous" to the rHVT vector that carries it.

[0038] An "expression cassette" is a nucleic acid fragment containing at least one heterologous gene and a promoter that drives the transcription of that gene. Transcription termination may be provided by a sequence provided by the genomic insertion site of the cassette, or the expression cassette itself may contain a termination signal such as a transcription terminator.

[0039] In such a cassette, both the promoter and terminator must be in close proximity to the genes they regulate, a condition known as "operably linked," so that no other significant sequences interfering with the effective start or termination of transcription are present between them.

[0040] The expression cassette can exist in DNA or RNA form for its intended use in the HVT vector, and therefore, the expression cassette of the present invention is used as DNA. As will be apparent to those skilled in the art, the expression cassette is a self-contained expression module, and therefore, its orientation in the vector viral genome is generally not important.

[0041] The expression cassette may contain additional DNA elements, such as sites for restriction enzyme recognition or PCR primers, to aid in construction and cloning.

[0042] The expression cassette as a whole is inserted into a single locus within the vector's genome. Various techniques are available to control the locus and orientation of this insertion. For example, the cassette can be incorporated by homologous recombination using a suitable adjacent section from the vector's genome, for example, by using a duplicated cosmid as described in U.S. Patent No. 5,961,982. Alternatively, the integration can be carried out using CRISPR / Cas9 technology as described below.

[0043] In the present invention, an “insert” or “inserted” expression cassette within the vector’s genome refers to the integration of the vector into the vector’s genomic nucleic acid such that the inserted element is transcribed and translated together with the vector’s native genes. The effect of the vector on the genome varies depending on how the insertion is performed, and the size of the vector genome can be larger, the same, or smaller, depending on whether the final result on the genome is the addition, substitution, or deletion of genetic material, respectively. Those skilled in the art are fully capable of selecting and performing specific types of insertions and making adjustments as necessary.

[0044] The construction of expression cassettes and their insertion into HVT vectors can be carried out by well-known molecular biological techniques, including cloning, transfection, recombination, selection, and amplification. These and other techniques are described in great detail in standard textbooks such as Sambrook & Russell: "Molecular cloning: a laboratory manual" (2001, Cold Spring Harbour Laboratory Press; ISBN: 0879695773); Ausubel et al., in: Current Protocols in Molecular Biology (J. Wiley and Sons Inc, NY, 2003, ISBN: 047150338X); and C. Dieffenbach & G. Dveksler: "PCR primers: a laboratory manual" (CSHL Press, ISBN 0879696540); and "PCR protocols", by: J. Bartlett and D. Stirling (Humana press, ISBN: 0896036421).

[0045] In relation to the present invention, the terms “first,” “second,” and “third” with respect to expression cassettes are used solely for the purpose of facilitating reference and do not indicate any order or priority.

[0046] It is well known that the "unique short (Us) region" of the HVT genome is the downstream portion of the genome between the "internal repeat short sequence" and the "terminal repeat short sequence." The HVT Us is approximately 8.6 kb in size (see Kingham et al., 2001, J. of Gen. Virol., vol. 82, pp. 1123-1135).

[0047] The fully annotated genome sequence of the well-known HVT strain FC-126 is available, for example, as GenBank accession number: AF291866, where nucleotides 5910-117777 are the UL region and nucleotides 136990-145606 are the Us region.

[0048] The hemagglutinin (HA) protein gene encodes the main antigen of AIV. The HA protein can be any of the currently known serotype variants: H1-H18, or it can be a chimeric variant. Highly pathogenic AIV has HA from the H5 or H7 serotype.

[0049] The "unique length (UL)" region of the HVT genome is the upstream part of the genome and is approximately 110kb in size in HVT.

[0050] The designation "UL40" is well known in the field of this invention to refer to a specific gene located in the UL genomic region; see, for example, GenBank accession number: AF291866. The same applies to the designations "UL41," "UL44," and "UL45" (with necessary modifications).

[0051] The term "between" is used to indicate that the inserted expression cassette is located at an insertion site (i.e., a locus) on the HVT genome outside the coding sequence of the indicated gene, and therefore not within the open reading frames of UL40, 41, 44, or 45. Such regions are also called inter-gene regions. [Modes for carrying out the invention]

[0052] Details of embodiments and further aspects of the present invention are described below.

[0053] Preferably, the cassette expressing the IBDV VP2 gene has specific elements.

[0054] In one embodiment, the rHVT according to the present invention is such that the IBDV VP2 gene is located in the 5' to 3' direction. a. Promoter of mouse cytomegalovirus pre-early 1 gene (mCMV-IE1), b. IBDV VP2 gene, and c. Transfer terminator, These are expressed from the first expression cassette containing them in this order. This is characterized by the fact that the promoter and terminator of the expression cassette are operablely linked to the VP2 gene.

[0055] As used herein, the terms “comprising” (and variations such as “comprises,” “comprise,” and “comprised”) are intended to refer to all conceivable elements and any possible combinations relating to the Invention that are covered or included by the text section, paragraph, claim, etc., in which the terms are used, and are not intended to exclude any such elements or combinations even if they are not expressly enumerated.

[0056] Therefore, any such text section, paragraph, claim, etc., may also relate to one or more embodiments in which the term “comprising” (or a variation thereof) is replaced by terms such as “consist of,” “consisting of,” or “consist essentially of.”

[0057] The term “5' to 3' direction,” also known as “downstream,” is well known in the art. The addition of the term “in this order” helps to indicate the relative orientation that the elements subsequently summed up must have relative to each other in order to function with the gene expression mechanism of the host cell in which the rHVT according to the present invention, including the expression cassette, can be replicated and expressed. As those skilled in the art will understand, this orientation relates to the DNA strand of the HVT's double-stranded DNA genome, which is the “coding strand,” and to the encoding mRNA molecule which is in a “+” or “sense” orientation.

[0058] Nevertheless, without any preconceived notions about the above section, in the "template" strand, which is the complementary strand of the HVT ds DNA genome, the relative order of the enumerated elements is the same, but in that DNA strand, the orientation of these elements is 3' to 5'.

[0059] The "promoter" of the present invention is well known to be a functional region of genetic information that directs the transcription of the downstream coding region. Therefore, the promoter is located upstream of the gene.

[0060] Promoter nomenclature is generally based on the gene whose expression it controls. For example, the term “mCMV-IE1 gene promoter” as used herein refers to a promoter that essentially drives the expression of the IE1 gene from mCMV and is therefore located immediately upstream of that gene in the mCMV genome. Since the IE1 gene is thus a well-documented and clearly recognizable gene, and the genomes of several mCMVs have been sequenced, such promoters can be easily identified by routine techniques. For example, in a basic protocol, a promoter can be easily obtained by roughly subcloning a region between two consecutive genes, e.g., from the polyA signal of the upstream gene to the transcription initiation signal of the downstream gene. The promoter can then be identified by standard testing, for example, by the expression of a marker gene using progressively smaller fragments of the nucleic acid region containing the suspected promoter.

[0061] Generally, a promoter contains several recognizable regulatory regions, such as enhancer regions, which are involved in the binding of regulatory factors that affect the time, duration, condition, and level of transcription. While enhancer regions are generally located upstream of the promoter, the promoter can also be influenced by regions further downstream toward the start codon, which are involved in the binding of transcription factors and the orientation of RNA polymerase itself. Such regions generally contain several conserved promoter sequence elements, such as the TATA box, CAAT box, and GC box.

[0062] A promoter that includes both the enhancer region and the downstream region is called a "complete" promoter, while a promoter that includes only the downstream region is called a "core" promoter.

[0063] A "transcriptional terminator" is a regulatory DNA element involved in the termination of transcription of a coding region into RNA. Generally, such elements encode a portion containing a secondary structure, such as a hairpin, that can cause the RNA polymerase complex to stop transcription (strop). Therefore, transcriptional terminators are always located in the "3' untranslated region" downstream of the stop codon in the region being translated. Terminators can also contain polyadenylation (poly-A) signals. This occurs in most eukaryotic mRNAs and induces polyadenylation, which is related to the transport and stability of the mRNA molecule.

[0064] The mCMV-IE1 gene is well-known in the art and readily available from various commercial sources, such as suppliers of commercially available plasmids for cloning and expression. The IE1 gene is also known as the "major IE gene" of CMV.

[0065] The mCMV-IE1 protein is also known as pp89. The mCMV IE1 gene promoter was described in 1985 (K. Dorsch-Hasler, et al., 1985, PNAS, vol. 82, p. 8325). The use of this promoter in heterologous expression is described in International Publication No. 87 / 03.905 and European Patent No. 728.842. The complete nucleotide sequence of the mCMV IE locus is available from GenBank under acc.nr.L06816.1 (since March 2004). mCMV itself is available from ATCC under acc.nr.VR-1399.

[0066] In one embodiment of rHVT according to the present invention, in the first expression cassette, the mCMV-IE1 gene promoter is a complete promoter that includes both the core promoter region and the enhancer region of the mCMV-IE1 gene. The complete mCMV-IE1 gene promoter is approximately 1.4 kb in size.

[0067] In this invention, the term "approximately" means ±25% of the indicated value, preferably ±20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, or 2% of the indicated value, or even more preferably ±1% of the indicated value.

[0068] In one embodiment, the mCMV-IE1 gene promoter of the present invention is a DNA molecule of about 1.4 kb containing a nucleotide sequence having at least 95% nucleotide sequence identity with respect to the entire length of the region of nucleotides 1 to 1391 of SEQ ID NO: 1. More preferably, and in that order, 96%, 97%, 98%, or even 99% nucleotide sequence identity is preferred.

[0069] In one embodiment, the mCMV-IE1 gene promoter is the region of nucleotides 1-1391 in SEQ ID NO: 1.

[0070] In one embodiment of the rHVT according to the present invention, in a first expression cassette, the IBDV VP2 gene of the present invention encodes a VP2 protein derived from classical IBDV. Such genes are well known, and their sequence information is readily available in the prior art (see, for example, GenBank acc.nr:D00869 (F52 / 70 strain), D00499 (STC), or AF499929 (D78)). Alternatively, this gene can be obtained from the genome of classical IBDV isolated from nature using routine techniques for manipulating birnavirus. Classical IBDV can be readily identified using serology or molecular biology.

[0071] Since homologs or variants of the IBDV VP2 gene may have equivalent potency and stability, in one embodiment, the IBDV VP2 protein gene of the present invention has at least 90% nucleotide sequence identity to the full length of the region nucleotides 1423-2781 of SEQ ID NO: 1. At least 92, 94, 95, 96, 97, 98, or even 99% nucleotide sequence identity is preferred, in that order more preferred.

[0072] In one embodiment, the IBDV VP2 protein gene of the present invention is derived from the classical IBDV Faragher 52 / 70 strain.

[0073] In one embodiment, the IBDV VP2 protein gene of the present invention is the region of nucleotides 1423 to 2781 of SEQ ID NO: 1.

[0074] In the expression cassette of the present invention, the selection of a particular type of transcription terminator is not important, as long as effective termination of RNA transcription is provided.

[0075] In one embodiment of rHVT according to the present invention, the first, second, and / or third expression cassette of the present invention includes a transcription terminator that includes both a terminator region and a poly-A region.

[0076] In one embodiment of rHVT according to the present invention, in the first expression cassette, the transcription terminator is derived from Simian virus 40 (SV40), preferably the late SV40 gene.

[0077] This terminator and its use in heterologous expression have been available since the late 1980s via the commercially available "pCMVβ" cloning plasmid (Clontech).

[0078] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the transcription terminator is derived from the late SV40 gene and is approximately 0.2 kb in size and contains a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of the nucleotide region 2812-3021 of SEQ ID NO: 1. More preferably, and in that order, 96%, 97%, 98%, or even 99% nucleotide sequence identity is preferred.

[0079] In one embodiment, the transcription terminator derived from the SV40 late gene is the region of nucleotides 2812-3021 of SEQ ID NO: 1.

[0080] In one embodiment of rHVT according to the present invention, one or more or all of the following conditions are applied to the first expression cassette: - The mCMV-IE1 gene promoter is a complete promoter; - The IBDV VP2 gene encodes the VP2 protein derived from classical IBDV; and - The first transfer terminator includes both a terminator region and a poly-A region, and preferably the transfer terminator is derived from SV40.

[0081] In one embodiment of rHVT according to the present invention, the first expression cassette is the region of nucleotides 1 to 3021 of SEQ ID NO: 1.

[0082] Preferably, the cassette expressing the NDV F gene has a specific element.

[0083] In one embodiment, the rHVT according to the present invention has the NDV F gene in the 5' to 3' direction, a. Promoter of the human cytomegalovirus pre-early 1 gene (hCMV-IE1), b. NDV F protein gene, and c. Transfer terminator, It is expressed from a second expression cassette containing these in this order, This is characterized by the fact that the promoter and terminator of the expression cassette are operablely linked to the F gene.

[0084] The complete version of the hCMV-IE1 gene promoter is approximately 1.5 kb in size and consists of an enhancer, core promoter, and introns, thereby enabling promoter activity to proceed to the intron region. See Koedood et al. (1995, J. of Virol., vol. 69, p. 2194-2207).

[0085] The hCMV-IE1 gene promoter can be obtained from the genome of the hCMV virus (which is widely available) by subcloning the genomic region preceding the IE1 gene using routine molecular biological tools and methods. Alternatively, the promoter can be derived from commercially available expression plasmids, such as pI17 described by Cox et al. (2002, Scand. J. Immunol., vol. 55, p. 14-23), or from commercially available mammalian expression vectors, such as pCMV (Clontech) or the pCMV-MCS series (Stratagene; GenBank® acc. nr. AF369966). The hCMV genome sequence is available, for example, from GenBank accession number X17403.

[0086] Many very similar versions derived from the hCMV-IE1 gene promoter are known, for example, from GenBank. Such homologs and variants are within the scope of this invention.

[0087] In one embodiment of rHVT according to the present invention, the hCMV-IE1 gene promoter is the core promoter in the second expression cassette of the present invention. Such a core promoter is typically less than 1 kb in size, preferably about 0.4 kb in size.

[0088] In one embodiment, the hCMV-IE1 gene core promoter of the present invention is a DNA molecule of about 0.4 kb containing a nucleotide sequence having at least 95% nucleotide sequence identity with respect to the entire length of the region nucleotides 3160-3520 of SEQ ID NO: 1. More preferably, and in that order, 96%, 97%, 98%, or even 99% nucleotide sequence identity is preferred.

[0089] In one embodiment, the hCMV-IE1 gene core promoter is the region of nucleotides 3160-3520 of SEQ ID NO: 1.

[0090] In one embodiment of rHVT according to the present invention, the NDV F protein gene in the second expression cassette of the present invention is derived from a long-latency type of NDV.

[0091] Preferably, the NDV F protein gene derived from the long-latency NDV strain is derived from NDV strain clone 30. NDV clone 30 is a well-known long-latency NDV that has been used for many years as a live vaccine, such as Nobilis® ND Clone 30 (MSD Animal Health).

[0092] In one embodiment, the NDV F protein gene of the present invention has at least 90% nucleotide sequence identity with respect to the entire length of the region nucleotides 3545-5206 of SEQ ID NO: 1. Preferably, it has at least 92, 94, 95, 96, 97, 98, or even 99% nucleotide sequence identity, in that order of increasing preference.

[0093] In one embodiment, the NDV F protein gene of the present invention is the region of nucleotides 3545-5206 of SEQ ID NO: 1.

[0094] In one embodiment of rHVT according to the present invention, the transcription terminator in the second expression cassette of the present invention is derived from the hCMV-IE1 gene. Preferably, this transcription terminator is about 0.3 kb in size.

[0095] In one embodiment, the transcription terminator is derived from the hCMV-IE1 gene, is about 0.3 kb in size, and contains a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of the region nucleotides 5218-5498 of SEQ ID NO: 1. More preferably, and in that order, 96, 97, 98, or even 99% nucleotide sequence identity is preferred.

[0096] In one embodiment of rHVT according to the present invention, the transcription terminator derived from the hCMV-IE1 gene is the region of nucleotides 5218-5498 of SEQ ID NO: 1.

[0097] In one embodiment of rHVT according to the present invention, one or more or all of the following conditions are applied to the second expression cassette: - The hCMV-IE1 gene promoter is the core promoter; - The NDV F gene is derived from a long-latency NDV strain, preferably NDV strain clone 30; and - The transcription terminator is derived from the hCMV-IE1 gene.

[0098] In one embodiment of rHVT according to the present invention, the second expression cassette is the region of nucleotides 3160 to 5498 of SEQ ID NO: 1.

[0099] Since the expression cassette is a self-contained expression module, as described above, the first and second expression cassettes of the present invention can be inserted into the Us genomic region of rHVT according to the present invention at different loci and in different orientations. These loci may be the same or different from each other.

[0100] In one embodiment, the rHVT according to the present invention is characterized in that the first and second expression cassettes are inserted into the same or different loci in the Us region of the rHVT genome.

[0101] Several loci in the HVT Us genomic region have been demonstrated to allow insertion of one or more heterologous genes (see, for example, European Patent No. 431.668 and International Publication No. 2016 / 102647). Examples include Us2, Us10, the region between Us10 and SORF3, and the region between Us2 and SORF3.

[0102] Therefore, in one embodiment, the rHVT according to the present invention is characterized in that both the first and second expression cassettes are inserted into the Us2 gene, or both are inserted into the Us10 gene, or one is inserted into the Us2 gene and the other is inserted into the Us10 gene.

[0103] As a result of such insertions, the normal coding function of the Us2 or Us10 gene is disrupted or completely lost in the resulting rHVT.

[0104] To facilitate the assembly of rHVT according to the present invention, the first and second expression cassettes of the present invention can be combined into a single expression cassette. This can be conveniently inserted into the locus of the HVT Us genomic region.

[0105] Therefore, in one embodiment, the rHVT according to the present invention is characterized in that the first and second expression cassettes are combined within a single expression cassette.

[0106] Since the first and second cassettes of the present invention contain their own promoter and terminator elements, in a combined single-expression cassette of the present invention, they can be oriented relative to each other in different ways. Specifically, these orientations can be "head to head," "tail to tail," and "tail to head" of the two orientations, all of which are well known in the art of the present invention.

[0107] In a preferred embodiment of the rHVT according to the present invention, in which the first and second expression cassettes are combined into a single expression cassette, the first and second expression cassettes are oriented tail-to-head, thereby positioning the VP2 gene upstream of the F gene.

[0108] In a more preferred embodiment, the combined single expression cassette is the expression cassette disclosed in International Publication No. 2016 / 102647.

[0109] More preferably, the combined single-expression cassette is the cassette used in the rHVT construct described as HVP360 in International Publication No. 2016 / 102647, which is commercially available as Innovax® ND-IBD (MSD Animal Health).

[0110] Therefore, in one embodiment of rHVT according to the present invention, the combined single expression cassette is in the 5' to 3' direction, a.mCMV-IE1 promoter, b. IBDV VP2 gene, c. Transfer terminator, d.hCMV-IE1 promoter, e.NDV F protein gene, and f. Transfer terminator, It includes them in this order, This allows promoters and terminators to be operably linked to their corresponding genes.

[0111] In the combined single expression cassette of the present invention, the transcriptional terminator element c. between the VP2 gene and the F gene provides effective separation of their expressions by preventing the possibility of RNA transcription read-through.

[0112] The two terminators shown for a combined single-expression cassette may be the same or different.

[0113] "Promoters and terminators are operably ligated to their corresponding genes" means that promoter a. and terminator c. are operably ligated to gene b., and similarly, promoter d. and terminator f. are operably ligated to gene e.

[0114] An example of a combined single-expression cassette of the present invention is shown in Sequence ID No. 1, and its elements are listed in Table 1. [Table 1]

[0115] In one embodiment, the single combination expression cassette of the present invention is approximately 5.5 kb in size.

[0116] In one embodiment of the rHVT according to the present invention, the combined single expression cassette of the present invention is a DNA molecule of about 5.5 kb containing a nucleotide sequence having at least 95% nucleotide sequence identity with respect to the full length of SEQ ID NO: 1. More preferably, and in that order, 96%, 97%, 98%, or even 99% nucleotide sequence identity is preferred.

[0117] In one embodiment, the combined single-expression cassette of the present invention is Sequence ID No. 1.

[0118] As will be apparent to those skilled in the art from the composition of the combined single-expression cassette for use in the present invention, which has two heterologous genes in one large cassette, it is designed and intended for insertion into a single position in the genome of a vector virus.

[0119] In one embodiment, the rHVT according to the present invention is characterized in that the combined single expression cassette defined for the present invention is inserted into the Us2 gene or the Us10 gene in the Us region of the rHVT genome.

[0120] The stable and effective rHVT vector of the present invention can be constructed by using the Us2 gene of the HVT genome as the gene insertion site of a combined single-expression cassette.

[0121] In one embodiment, the rHVT according to the present invention is characterized in that a combined single expression cassette, as defined for the present invention, is inserted into the Us2 gene.

[0122] To facilitate the simple construction, manipulation, and use of the expression cassette of the present invention, it may itself contain a DNA molecule, for example, a vehicle that enables cloning or transfection, such as a plasmid, cosmid, bacmid, etc. (see International Publication No. 93 / 25.665 and European Patent No. 996.738). Examples of common cloning plasmids are, for example, the pBR322 or pUC series plasmids, which are widely available commercially.

[0123] Plasmids containing an expression cassette are commonly referred to as "transfer vectors," "shuttle vectors," or "donor plasmids." In this context, the plasmid contains an expression cassette with a sequence region adjacent to the target insertion locus in the vector's genome to direct the insertion.

[0124] Typically, the transfer vectors used for transfection are not themselves integrated into the vector's genome, but merely facilitate the integration of their own expression cassettes by inducing insertions, for example, through homologous recombination.

[0125] In one embodiment of rHVT according to the present invention, the AIV HA gene in the third expression cassette is driven by a promoter derived from the mammalian herpesvirus glycoprotein B(gB) gene.

[0126] Therefore, in one embodiment, the rHVT according to the present invention is such that the AIV HA gene is located in the 5' to 3' direction. a. Mammalian herpesvirus-derived glycoprotein B(gB) gene promoter, b. AIV HA protein gene, and c. Transfer terminator, These are expressed from a third expression cassette containing them in this order. This is characterized by the fact that the promoter and terminator of the expression cassette are operablely linked to the HA gene.

[0127] A “mammalian herpesvirus-derived gB gene promoter” refers to a promoter that drives the expression of the herpesvirus gB gene and is located immediately upstream of the gB gene in the genome of a mammalian herpesvirus. In normal herpesvirus replication, the gB protein is involved in cell entry and cell spread. Since the gB gene is thus a well-documented and clearly recognizable gene, and the genomes of many herpesviridae have been sequenced (in whole or in part), those skilled in the art can readily identify and obtain such promoters by routine techniques. Embodiments thereof are disclosed in International Publication No. 2012 / 052384.

[0128] A review of herpesvirus gB proteins has been presented by Perreira (1994, Infect. Agents Dis., vol.3, pp.9-28). The promoter of the HSV1 gB gene was studied in detail by Pederson et al. (1992, J. of Virol., vol.66, pp.6226-6232).

[0129] The “mammalian herpesviruses” of the present invention relate to herpesviruses that commonly infect and replicate in mammalian species. Preferably, these viruses are derived from the taxonomic subfamily of the family Alphaherpesviridae. For example: human herpesvirus 1 (herpes simplex virus 1), bovine herpesvirus 1, feline herpesvirus 1, equine herpesvirus 1 (EHV), or pseudorabies virus (PRV, also known as porcine herpesvirus 1). gB gene promoters derived from such mammalian herpesviruses are advantageously used in the present invention.

[0130] In one embodiment of the rHVT according to the present invention, in the third expression cassette, the mammalian herpesvirus-derived gB gene promoter of the present invention is derived from PRV or EHV. More preferably, the gB gene promoter is derived from PRV.

[0131] PRV is also known as porcine alphaherpesvirus 1, and the gB gene is also known as gII, gp14, or UL27. The first publication of the PRV gB(gII) gene and its promoter was European Patent No. 353809 of 1990. The use of the PRV gB promoter to drive heterologous genes in HVT vectors is described in International Publication No. 2012 / 052384.

[0132] Such promoters draw from prior art, for example, GenBank, for example: - From PRV, GenBank acc.nr:BK001744, region 20139~19596 (the PRV gB gene is Ul27 or gII), or - From EHV, GenBank acc.nr::AY 665713, region 60709~61570 (the EHV 1 gB gene is ORF 33) It can be obtained easily.

[0133] Furthermore, the GenBank accession number pfam00606 conveniently represents a cluster of herpesvirus gB proteins.

[0134] To further improve the effectiveness of the mammalian herpesvirus-derived gB gene promoter of the present invention, the promoter was adapted while maintaining its stability. The adaptation involved extending the promoter sequence; therefore, the promoter sequence was not terminated before the A+1 codon of the gB gene, but extended downstream of A+1 to the coding region of the gB gene that is normally translated into protein.

[0135] As a result, the elongated promoter now contained one or more ATG codons, i.e., the original start codon and other methionine coding triplet candidates. Such an ATG codon at this position downstream of the TATA box within the promoter could be interpreted as a start codon by the cellular transcription mechanism of rHVT, leading to undesirable premature translation initiation. Therefore, the ATG codon downstream of the TATA box in the gB gene promoter, which was included in the elongated promoter sequence here, was modified by mutation to render such an ATG nonfunctional as a potential start codon. This allowed the gB promoter of the present invention to incorporate nucleotides downstream of the native gB gene start codon and elongate into the translational region of the gB gene, but these additional nucleotides could not be translated and acted as an elongated leader sequence.

[0136] As a result, we constructed a gB gene promoter sequence containing nucleotides from the gB coding region downstream of the original start codon.

[0137] Therefore, in a more preferred embodiment, the gB gene promoter derived from mammalian herpesvirus includes a nucleotide sequence derived from the coding region of the gB gene, where an arbitrary ATG nucleotide sequence is modified.

[0138] The "modification" of the ATG nucleotide sequence in the extended PRV gB gene promoter of the present invention is preferably carried out by mutation. The ATG nucleotide sequence can, in principle, be changed to any other triplet, as long as it does not reduce replication stability or expression from the vector construct.

[0139] Preferably, the change is by a single nucleotide, preferably from ATG to TTG.

[0140] Therefore, to improve its effectiveness, the PRV gB gene promoter for use in the present invention may include a nucleotide sequence extended downstream of the native PRV gB gene start codon. In this case, all ATG codons in that region of PRV gB are mutated to prevent false initiation. Preferably, the PRV gB gene promoter is extended by 129 nt before A+1.

[0141] The number of downstream nucleotides of ATG included in the extended gB promoter of the present invention is at least 10, preferably at least 20, 30, 50, 75, or 100, in that order of increasing preference. In practice, the number of downstream nucleotides of A+1 incorporated into the extended promoter of the present invention is, conveniently, the sequence from A+1 to the next downstream ATG codon, but does not include the next downstream ATG codon. In that case, only one ATG sequence (the ATG sequence of the start codon) needs to be changed by mutation.

[0142] The third expression cassette construct used in this invention contains a PRV gB gene promoter that is extended by 129 nt prior to A+1. The only ATG sequence in the extended sequence is from the original start codon, which was changed to TTG by mutation. This cassette showed similar efficacy and stability in vitro as the unadapted PRV gB gene promoter, but its efficacy in vivo was significantly improved.

[0143] In one embodiment of the rHVT according to the present invention, in the third expression cassette of the present invention, the PRV gB gene promoter of the present invention is a DNA molecule of about 0.7 kb containing a nucleotide sequence having at least 95% nucleotide sequence identity with respect to the entire length of the region of nucleotides 20-701 of SEQ ID NO: 2. More preferably, and in that order more preferably, 96, 97, 98, or even 99% nucleotide sequence identity.

[0144] In one embodiment of rHVT according to the present invention, in the third expression cassette of the present invention, the PRV gB gene promoter corresponds to nucleotides 20-701 of SEQ ID NO: 2.

[0145] In one embodiment of rHVT according to the present invention, the HA protein gene is derived from avian influenza A virus.

[0146] Currently, the AIV serotypes most relevant to poultry health are serotypes H5, H7, and H9. H5 and H7 are associated with highly pathogenic pathological forms that may even be zoonotic, while H9 is the most prevalent serotype worldwide, particularly in the Middle East and Asia.

[0147] Therefore, in one embodiment of rHVT according to the present invention, the AIV HA protein gene encodes an HA protein of a serotype selected from H5, H7, and H9.

[0148] Determining whether the HA protein is of a specific serotype can be easily done, for example, using standard antisera available from the International Reference Laboratory for AIV, whose list is published by the WHO.

[0149] In a preferred embodiment of the rHVT according to the present invention, the AIV HA gene is the codon-optimized HP AIV HA gene for serotype H5 or H7 disclosed in SEQ ID NOs. 3 and 5 of International Publication No. 2012 / 052384.

[0150] The specific HA H9 gene sequence used in this invention was a synthetic sequence based on a consensus sequence derived from recently published HA H9 sequences of AIV isolates.

[0151] Furthermore, to further improve the efficacy of the rHVT vector according to the present invention in poultry vaccines, the HA gene contained in this vector can be subjected to codon optimization. The codon optimization process is well known in the art and involves fitting nucleotide sequences to encode the intended amino acids, but using nucleotide sequences that utilize the codon priority of the (microorganism) used to express the gene. As a result, mutations are essentially silent. This improves the level at which the coding sequence is expressed in a context different from the origin of the expressed gene.

[0152] In this invention, the coding sequence of the AIV HA gene used in this invention was optimized for expression according to the codon priority of HVT.

[0153] In one embodiment of the rHVT according to the present invention, in the third expression cassette of the present invention, the AIV HA gene of the present invention is a DNA molecule of about 1.7 kb containing a nucleotide sequence having at least 90% nucleotide sequence identity with respect to the entire length of the region from nucleotides 713 to 2395 of SEQ ID NO: 2. More preferably, and in that order more preferably, 96%, 97%, 98%, or even 99% nucleotide sequence identity.

[0154] In one embodiment of rHVT according to the present invention, in the third expression cassette of the present invention, the AIV HA gene corresponds to nucleotides 713 to 2395 of SEQ ID NO: 2.

[0155] In one embodiment of rHVT according to the present invention, in the third expression cassette, the transcriptional terminator is derived from the FHV1 gene, preferably the FHV1 Us9 gene. The FHV1 U gene is disclosed, for example, under GenBank accession number D42113.

[0156] In one embodiment of rHVT according to the present invention, in the third expression cassette, the transcription terminator is derived from the FHV1 Us9 gene, is about 0.05 kb in size, and contains a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of the region nucleotides 2404-2458 of SEQ ID NO: 2. More preferably, and in that order, 96%, 97%, 98%, or even 99% nucleotide sequence identity is preferred.

[0157] In one embodiment, the transcription terminator derived from the FHV1 Us9 gene is the region of nucleotides 2404-2458 in SEQ ID NO: 2.

[0158] In a preferred embodiment, the third expression cassette is the cassette disclosed in International Publication No. 2012 / 052384.

[0159] More preferably, the third expression cassette is a cassette used in the rHVT construct described as HVP310 in International Publication No. 2012 / 052384.

[0160] In one embodiment of the rHVT according to the present invention, the third expression cassette of the present invention comprises an extended gB gene promoter derived from PRV and a codon-optimized AIV HA gene of the H9 serotype.

[0161] As described above for the first and second expression cassettes, in the third expression cassette, its promoter and terminator are "operably linked" to the AIV HA gene. [Table 2]

[0162] In nature, the AIV HA protein is expressed as the HA0 (HA zero) protein, which is post-translationally cleaved by a protease produced in tissues where AIV replicates. This also activates the infectivity of AIV. The resulting HA1 and HA2 proteins interact to form a heterodimer, with HA1 being the "head" portion and HA2 being the "stem" portion.

[0163] In Sequence ID No. 2, the HA1 region of the HA gene is formed by nucleotides 713-1660, and the HA2 region is formed by nucleotides 1661-2395.

[0164] In one embodiment of rHVT according to the present invention, the AIV HA protein gene encodes either the HA1 or HA2 subunit protein.

[0165] Since HA2 has been reported to induce a broad cross-protective immune response (see Chiu et al., 2013, Ann. NY Acad. Sci., vol. 1283, pp. 13-21), therefore, in a preferred embodiment, the AIV HA protein gene encodes the HA2 subunit protein.

[0166] In one embodiment of the rHVT according to the present invention, in the third expression cassette of the present invention, the AIV HA gene of the present invention is a DNA molecule of about 0.75 kb containing a nucleotide sequence that encodes the HA2 portion of the HA protein and has at least 90% nucleotide sequence identity to the full length of the region nucleotides 1661-2395 of SEQ ID NO: 2. More preferably, and in that order, 96%, 97%, 98%, or even 99% nucleotide sequence identity is preferred.

[0167] In one embodiment of rHVT according to the present invention, in the third expression cassette of the present invention, the AIV HA gene corresponds to nucleotides 1661-2395 of SEQ ID NO: 2.

[0168] The rHVT according to the present invention is preferably based on a parent HVT, such as the HVT vaccine strain PB1 or FC-126, which is an established HVT vaccine strain known to replicate well and be suitable for inoculation of young birds or bird embryos in eggs. These are generally available: FC-126 is from ATCC:VR#584-C, and PB1 is commercially available as a live vaccine in frozen infected cells, for example, from MSD Animal Health.

[0169] The incorporation of any of the first, second, and third expression cassettes, as defined herein, and / or combined single expression cassettes, does not increase (on the contrary) the pathogenicity or virulence of the parental HVT, and since HVT is naturally non-pathogenic, a return to pathogenicity is not expected.

[0170] Therefore, in one embodiment, the parent HVT used to generate rHVT according to the present invention is an HVT vaccine strain, preferably the PB1 strain or the FC-126 strain HVT vaccine strain.

[0171] The rHVT according to the present invention is a live recombinant carrier microorganism or “vector” virus that can be advantageously used for vaccinating poultry. It combines the characteristics of being a safe and effective vaccine against Marek’s disease (MD) and one or more of infectious bursal disease (IBD), Newcastle disease (ND), and avian influenza (AI), and is also genetically stable.

[0172] To say that the present invention is "genetically stable" means that the genetic makeup of the rHVT according to the present invention does not change in subsequent rounds of viral replication. Alternatively, an unstable construct may result in the loss of expression of one or more inserted heterologous genes(s). This stability can be easily monitored using routine techniques, for example, by subjecting the rHVT according to the present invention to subsequent passaging in cell culture. The viruses re-isolated during these steps can be plated onto cell culture dishes, covered with agar, and incubated until HVT-specific plaques become visible, all using routine techniques. The plaques can then be stained for the expression of VP2 protein, F protein, or HA protein using suitable antibody preparations in an immunofluorescence assay (IFA) protocol, as well as appropriate positive and negative controls. Any plaques that no longer show fluorescence can be recorded, thereby preferably monitoring at least 100 individual plaques of a particular rHVT sample.

[0173] Remarkably, the rHVT according to the present invention was found to maintain the presence and expression of the VP2, F, and HA protein genes, respectively, in all tested plaques, even after 16 consecutive cell culture passages and even through several weeks of in vivo replication. Further details are shown in the examples.

[0174] This represents a powerful and significant improvement over the claimed multivalent HVT vector constructs described in the prior art.

[0175] Furthermore, given that VP2, F, and HA have all been used as inserts in effective HVT vector vaccines, the fact that they are stably maintained and expressed is also reliable proof that the rHVT according to the present invention induces a protective immune response against these antigens as well as MDV in poultry, and is therefore an effective tetravalent vector vaccine.

[0176] The rHVT according to the present invention can be amplified by conventional techniques, primarily in vitro, by replication in cultures of chicken cells, typically primary chicken embryo fibroblasts (CEFs). These can all be prepared by trypsin treatment of chicken embryos, which is well known in the art. CEFs are plated in a monolayer and infected with HVT. This process can be scaled up to industrial-scale production.

[0177] Generally, rHVT is collected by recovering infected host cells containing rHVT in a cell-associated form. These cells are lysed in a suitable carrier composition to provide stabilization during freezing and storage. The infected cells are then generally filled into glass ampoules, sealed, frozen, and stored in liquid nitrogen. For use in vaccination, the ampoules are thawed and the infected cells are lysed in a suitable dilution buffer for stabilization during use. In a preferred embodiment, the dilution buffer is the buffer disclosed in International Publication No. 2019 / 121888.

[0178] Cell-associated cryopreservation of HVT is preferred, but in situations where the use of liquid nitrogen is not feasible, an alternative is to use lyophilization: this takes advantage of the favorable characteristic of HVT that the entire culture can be isolated from its host cells by cell disruption, e.g., French press or sonication. This can then be clarified by centrifugation, dissolved in a stabilizer, and lyophilized for long-term storage.

[0179] Therefore, in a further embodiment, the present invention relates to a host cell comprising rHVT according to the present invention.

[0180] The “host cell” of this invention is a cell that is susceptible to infection and replication by HVT. Examples of such cells are avian cells, particularly lymphocytes or fibroblasts.

[0181] In one embodiment, the host cells according to the present invention are primary avian cells maintained in vitro; that is, cells directly derived from the tissues or organs of non-human animals and not from immortalized cell lines. Typically, primary cells can only undergo a limited number of cell divisions.

[0182] In one embodiment, the primary avian host cells of the present invention are primary chicken embryo fibroblasts (CEFs) maintained in vitro.

[0183] In one embodiment, the host cells according to the present invention are immortalized avian cells maintained in vitro. Several immortalized avian cell lines are described, for example, in International Publication No. 97 / 044443 and International Publication No. 98 / 006824.

[0184] In a preferred embodiment, the immortalized avian host cell according to the present invention is an immortalized CEF maintained in vitro, preferably an immortalized CEF disclosed in International Publication No. 2016 / 087560.

[0185] By various cloning and transfection methods, the first, second, and third expression cassettes of the present invention, and / or combined single expression cassettes, can be used to obtain rHVT according to the present invention, which stably contains and expresses the expression cassettes described herein in its genome.

[0186] Accordingly, a further aspect of the present invention relates to a method for constructing rHVT according to the present invention, the method comprising inserting the first, second, and third expression cassettes of the present invention, and / or a combined single expression cassette, into a region of the HVT genome described in the present invention.

[0187] The insertion of the expression cassette according to the present invention into the HVT genome for generating rHVT according to the present invention can be carried out by various methods known in the art. One simple method is to use a transfector and homologous recombination techniques.

[0188] Alternatively, the rHVT according to the present invention can be generated, for example, using the CRISPR / Cas9 technology described by Tang et al, 2018 (Vaccine, vol.36, pp.716-722).

[0189] In particular, the rHVT-VP2-F vector virus can be used and has been available since 2017 in the commercially available vaccine Innovax ND-IBD. This can be further manipulated by using CRISPR / Cas9 technology to insert the third expression cassette described herein into the UL40-41 or UL44-45 loci of the rHVT genome described herein. Specific guide RNA sequences that can be used for these insertions into these loci are described in the examples.

[0190] As described, the primary advantageous use of rHVT according to the present invention is to provide a safe, stable, and effective vaccine for MD, IBD, ND and / or AI or related disease symptoms in poultry vaccines that can be administered to poultry at a very young age.

[0191] Therefore, a further aspect of the present invention relates to rHVT and / or host cells according to the present invention for use in poultry vaccines.

[0192] Various aspects and embodiments of the “vaccination use” of rHVT according to the present invention have been outlined above, and include its use as a cell-free or cell-associated virus in vaccine compositions for inoculation of poultry.

[0193] In a further embodiment, the present invention relates to a poultry vaccine comprising rHVT and / or host cells according to the present invention and a pharmaceutically acceptable carrier.

[0194] It is well known that a "vaccine" is a composition containing an immunologically active compound in a pharmaceutically acceptable carrier. The "immunologically active compound" or "antigen" is a molecule that is recognized by the immune system of the inoculated target and induces a protective immune response from the target's humoral and / or cellular immune system.

[0195] The vaccine according to the present invention provides protection for chickens against infections and / or diseases caused by MDV, IBDV, NDV, and / or AIV. This effect is achieved by preventing or reducing the establishment or proliferation of one or more of these viruses in their respective target organs. This is achieved, for example, by reducing the viral load or shortening the duration of viral replication. This then results in a reduction in the number, intensity, or severity of lesions and associated clinical signs of disease caused by the viral infection in the target animals.

[0196] However, depending on the pathogenicity of the MDV, IBDV, NDV, or AIV field viruses prevalent in a particular poultry farm or region, it may be necessary to add additional vaccine components against one or more of these viruses to ensure effective vaccination against the most pathogenic variants of these viruses. This is all well known in the art.

[0197] Determining the efficacy of the poultry vaccine according to the present invention is well within the scope of routine general practice skills and can be done, for example, by monitoring the immunological response after vaccination, or by testing the appearance of clinical symptoms or mortality after challenge infection, for example, by monitoring target disease signs, clinical scores, serological parameters, or by re-isolating the challenge pathogen, and by comparing these results with the vaccination-challenge response observed in pseudovaccinated animals. Different methods for evaluating each of the four viral infections are well known in the art.

[0198] The vaccine or vaccination-induced prevention of MD, IBD, ND, and AI according to the present invention results in improved health and economics of the vaccinated target. This can be evaluated by parameters such as increased survival rates, growth rates, feed conversion, and egg production, as well as reduced healthcare costs.

[0199] Various embodiments, preferred ones, and examples of the vaccine according to the present invention are outlined below.

[0200] The term "poultry" in this invention relates to veterinary practice and to bird species susceptible to HVT vaccination; preferred poultry species are chickens, turkeys, and quail. Chickens are the most preferred species.

[0201] In the present invention, poultry can be any type, breed, or variant, such as laying hens, breeding stock, broilers, hybrids, or parent lines of any of such breeds. Preferred types are broilers, breeding stock, and laying hens. Broilers and laying hens are most preferred.

[0202] A "pharmaceutically acceptable carrier" is intended to assist in the stabilization and administration of a vaccine, while being harmless and well-tolerated to the target. Such carriers may, for example, be sterile water or sterile saline. In more complex forms, the carrier may be a buffer solution that may contain further additives, such as stabilizers or preservatives. Details and examples are found in well-known handbooks such as "Remington: The Science and Practice of Pharmacy" (2000, Lippincott, USA, ISBN: 683306472) and "Veterinary Vaccineology" (P. Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0203] In this invention, when the vaccine is in the form of cell-associated HVT, the pharmaceutically acceptable carrier is preferably a mixture of culture medium, about 10% serum, and about 6% DMSO. This carrier also provides stabilization of rHVT-infected host cells during freezing and cryopreservation. The serum may be any serum routinely used in cell culture, such as fetal bovine serum or neonatal bovine serum.

[0204] The vaccine according to the present invention is prepared from rHVT according to the present invention by the method described herein, which is readily applicable to those skilled in the art. For example, rHVT according to the present invention is constructed by transfection and recombination insertion of the expression cassette described in the present invention. Next, the desired rHVT is selected and industrially amplified in small or large volumes, preferably in in vitro cell cultures, e.g., CEF. From such cultures, a suspension containing the virus is recovered as a cell-free preparation obtained by whole infected cells or by cell disruption. This suspension is formulated into a vaccine, and the final product is packaged. The cell-associated vaccine is then stored in liquid nitrogen at -20°C or +4°C, and as a lyophilized vaccine.

[0205] General techniques and considerations applicable to the manufacture of vaccines under well-known standards for pharmaceutical manufacturing are described, for example, in government directives and regulations (Pharmacopoeia, 9th CFR) and well-known handbooks ("Veterinary Vaccineology" and "Remington," both cited above). Generally, such vaccines are prepared aseptically and using pharmaceutical-grade excipients.

[0206] Such preparations incorporate microbiological testing for sterility and the absence of exogenous drugs, and may include in vivo or in vitro testing to confirm efficacy and safety. After the completion of testing for quality, quantity, sterility, safety, and efficacy, the vaccine can be marketed. All of this is well known to those skilled in the art.

[0207] In one embodiment, the vaccine according to the present invention is a cell-associated vaccine.

[0208] "Cell association" means that the rHVT according to the present invention is included in the host cells according to the present invention. As a result, this type of vaccine contains both host cells and rHVT according to the present invention.

[0209] The target animals of the vaccine according to the present invention may, in principle, be healthy or diseased, and may be positive or negative for the presence of MDV, IBDV, NDV, or AIV, or for antibodies against MDV, IBDV, NDV, or AIV. Furthermore, the target may be of any weight, sex, or age that is easily vaccinated. However, it is clearly preferable to vaccinate healthy, uninfected targets as early as possible to prevent any field infection and its consequences.

[0210] Therefore, since the vaccine according to the present invention prevents both the establishment and progression of infection by MDV, IBDV, NDV, or AIV, it can be used as a preventive or therapeutic measure, or both.

[0211] In this regard, a further advantageous effect of the reduction in viral load by the vaccine according to the present invention is the prevention or reduction of shedding and the resulting spread of the field virus, both vertically to offspring and horizontally both within herds or populations and within geographical areas. As a result, the use of the vaccine according to the present invention leads to a reduction in the prevalence of MDV, IBDV, NDV, or AIV.

[0212] Therefore, further aspects of the present invention are: - Use of vaccines according to the present invention to reduce the prevalence of MDV, IBDV, NDV, or AIV in populations or geographical areas, and - A vaccine according to the present invention for reducing the prevalence of MDV, IBDV, NDV, or AIV in a population or geographical area. That is the case.

[0213] The vaccine according to the present invention already provides multivalent immunity against IBD, ND, and AI through the expression of a heterologous insert, as well as against MD through the HVT vector itself.

[0214] Nevertheless, further combinations with additional immunoactive components may be beneficial. This could help enhance already provided immune defenses or expand immune defenses against other pathogens.

[0215] Therefore, in one embodiment, the vaccine according to the present invention comprises at least one additional immunoactive component.

[0216] Such “additional immune-activating components” may be antigens, immune enhancers, cytokines, further vaccines, or any combination thereof. This offers advantages in terms of cost, efficiency, and animal welfare. Alternatively, the vaccine according to the present invention can be added to the vaccine itself.

[0217] In one embodiment, at least one additional immunoactive component is an immunostimulant compound, preferably a cytokine or an immunostimulant oligodeoxynucleotide.

[0218] The immunostimulant oligodeoxynucleotide is preferably an immunostimulant unmethylated CpG-containing oligodeoxynucleotide (INO). Preferred INOs are avian Toll-like receptor (TLR) 21 agonists described in International Publication No. 2012 / 089800 (X4 family), International Publication No. 2012 / 160183 (X43 family), or International Publication No. 2012 / 160184 (X23 family).

[0219] In one embodiment, at least one additional immunoactive component is an antigen derived from a pathogenic microorganism to poultry. This antigen can be “induced” in any suitable way, for example, as a “live” attenuated antigen, inactivated antigen, or subunit antigen derived from that pathogenic microorganism to poultry.

[0220] Additional antigens derived from pathogenic microorganisms against poultry are preferably derived from one or more microorganisms selected from the following group: -Viruses: Infectious bronchitis virus, NDV, adenovirus, AIV, Egg drop syndrome virus, IBDV, chicken anemia virus, avian encephalomyelitis virus, fowlpox virus, turkey rhinotracheitis virus, duck plague virus (duck viral enteritis), pigeonpox virus, MDV, avian leukemia virus, ILTV, tripneumovirus, and reovirus; - Bacteria: Escherichia coli, Salmonella, Ornitobacterium rhinotracheale, Haemophilus paragallinarum, Pasteurella multocida, Erysipelothrix rhusiopathiae, Erysipelas, Mycoplasma, and Clostridium; - Parasites: Genus Eimeria; and -Fungus: Aspergillus genus.

[0221] Additional antigens could also be further vector vaccines based on, for example, HVT, MDV2, NDV, etc.

[0222] In one embodiment of the vaccine according to the present invention, the additional antigen derived from a pathogenic microorganism to poultry is a “live” attenuated MDV, IBDV, or NDV vaccine strain. This helps to improve and expand the immunogenicity of the vaccine according to the present invention, which is advantageous in situations where highly virulent field strains of MDV, IBDV, or NDV are prevalent or in geographical areas.

[0223] In this regard, combinations of HVT with MDV1, MDV2, or HVT itself are known, and for the purposes of the present invention, MDV from the Rispens strain (MDV1), SB1 strain (MDV2), or FC-126 or PB1 strain (HVT) are preferred as additional immunoactive components.

[0224] To improve the response to ND, the rHVT according to the present invention can be combined with an NDV vaccine strain such as the mild live NDV vaccine strain C2.

[0225] Similarly, to improve the response to IBD, the rHVT according to the present invention can be combined with live IBDV vaccine strains such as D78, PBG98, Cu-1, ST-12, or 89-03.

[0226] As those skilled in the art will understand, these “combinations” also include vaccination schedules in which rHVT and additional immunoactive components according to the present invention are applied simultaneously or sequentially, rather than at the same time, for example, rHVT may be applied in the egg, NDV C2 on day 1, and IBDV 89-03 on day 17.

[0227] Therefore, in one embodiment of the vaccine according to the present invention comprising at least one additional immunoactive component, the at least one additional immunoactive component is a microorganism selected from the group consisting of vaccine strains from MDV, IBDV, NDV, or AIV, or any combination thereof.

[0228] More preferably, the additional immunoactive component is one or more selected from the group consisting of MDV Rispens, MDV SB1, NDV C2, IBDV D78, and IBDV 89-03.

[0229] The vaccine according to the present invention can be prepared by the methods described and illustrated herein.

[0230] Therefore, a further aspect of the present invention is a method for preparing a poultry vaccine according to the present invention, the method being: a. A step of infecting host cells with rHVT according to the present invention in vitro. b. A step of collecting infected host cells, and c. A step of mixing the recovered infected host cells with a pharmaceutically acceptable carrier. Includes.

[0231] Suitable host cells and pharmaceutically acceptable carriers for the present invention are described above. Appropriate in vitro methods for infection, culture, and recovery are well known in the art and are described and illustrated herein.

[0232] Therefore, different aspects and embodiments of the present invention can be advantageously used to produce safe, stable, and effective vaccines for poultry.

[0233] Therefore, in a further embodiment, the present invention relates to the use of rHVT, or host cells, or any combination thereof, according to the present invention for the production of poultry vaccines.

[0234] It goes without saying that the vaccine according to the present invention may also be mixed with other compounds such as stabilizers, carriers, adjuvants, diluents, and emulsions. Such additives are described in well-known handbooks such as "Remington" and "Veterinary Vaccinology" (both mentioned above).

[0235] In this way, a single dose administered at a very young age can further optimize, as needed, the effectiveness of the vaccine according to the present invention for protecting poultry from MD, IBD, ND, and AI.

[0236] The vaccine according to the present invention is suitable for administration to poultry targets and can be prepared in a form that matches the desired route of application and desired effect.

[0237] Depending on the route of application of the vaccine according to the present invention, it may be necessary to adapt the composition of the vaccine. This is well within the capabilities of those skilled in the art and generally involves fine-tuning the efficacy or safety of the vaccine. This can be done by adapting the dose, amount, frequency, or route of the vaccine, by using a different form or formulation of the vaccine, or by adapting other components of the vaccine (e.g., stabilizers or adjuvants).

[0238] In principle, the vaccine according to the present invention can be administered to target poultry via different routes of application and at different points in their lifespan, as long as the vaccinated rHVT can establish protective infection.

[0239] However, since infection with MDV, IBDV, NDV, or AIV can already be established at a very young age, it is advantageous to apply the vaccine according to the present invention as early as possible. Accordingly, the vaccine according to the present invention can be applied, for example, on the day of incubation ("day 1"), or in the egg, for example, on day 18 of embryonic development, all of which are well known in the art.

[0240] Therefore, in one embodiment, the vaccine according to the present invention is administered to poultry in the egg.

[0241] Devices for automated injection of vaccines into fertilized eggs on an industrial scale are commercially available. This provides the earliest possible protection while minimizing labor costs. Different intraocular inoculation routes are known, such as into the yolk sac, embryo, or allantoalanthin space, and these can be optimized as needed. Preferably, intraocular inoculation of HVT is performed so that the needle is in contact with the embryo.

[0242] Preferably, the vaccine according to the present invention is formulated as an injectable solution suitable for either intraocular injection or parenteral injection, for example, as a suspension, solution, dispersion, or emulsion.

[0243] In one embodiment, the vaccine according to the present invention is administered by a parenteral route. Preferably, it is administered by an intramuscular or subcutaneous route.

[0244] The precise amount of rHVT according to the present invention per animal dose of the vaccine according to the present invention is not as important as in the case of inactivated vaccines; this is because rHVT replicates in the target animal to a level of biologically sustainable vireamia. In principle, the vaccine dose should be sufficient to initiate such a proliferative infection. Higher doses do not significantly shorten the time it takes to reach an optimal vireamic infection in the host. Therefore, very high doses are not necessarily more effective and are not attractive for economic reasons.

[0245] Therefore, the preferred dose is 1 × 10 ∧ 1 to 1 x 10 ∧ 5. rHVT / animal dose of plaque-forming units (pfu) according to the present invention, more preferably 1 × 10⁻⁶ ∧ 2~1×10 ∧ The dose is between 4 pfu / dose, more preferably between 500 and 5000 pfu / dose, and most preferably between approximately 1000 and 3000 pfu / dose.

[0246] If the vaccine according to the present invention is cell-contingent, these amounts of rHVT are contained in the infected host cells.

[0247] The method for counting rHVT virus particles according to the present invention is well known.

[0248] The volume per animal dose of rHVT according to the present invention can be optimized according to the intended route of application: intraovo administration is generally performed at a dose of about 0.01 to about 0.5 ml / egg, and parenteral injection is generally performed at a dose of about 0.1 to about 1 ml / bird.

[0249] Determining the immunological effective dose of the vaccine according to the present invention, or optimizing the volume of vaccine per dose, is both well within the capabilities of those skilled in the art.

[0250] The administration regimen for applying the vaccine according to the present invention to a target organism can be administered in a single or multiple doses in an immunologically effective amount, in a manner compatible with the vaccine formulation.

[0251] Preferably, the vaccine administration regimen according to the present invention is integrated into an existing vaccination schedule for other vaccines that may be required by the target poultry in order to reduce stress on the animals and lower labor costs. These other vaccines can be administered concurrently, in combination, or sequentially in a manner that is appropriate for their authorized use.

[0252] As described above and illustrated below, the vaccines according to the present invention can be advantageously used by a single dose at a very young age to prevent or reduce infection with one or more, or all, of the MDV, IBDV, NDV, and AIV, and to prevent or reduce the disease (or its symptoms) associated with such infections.

[0253] Therefore, further aspects of the present invention are: - Use of the vaccine according to the present invention to prevent or mitigate the signs of infection by MDV, IBDV, NDV and / or AIV, or related diseases. - A method for preventing or mitigating the signs of infection by MDV, IBDV, NDV and / or AIV or related diseases, the method comprising administering the vaccine according to the present invention to poultry, - A method for vaccinating poultry to prevent or mitigate infection with MDV, IBDV, NDV and / or AIV or associated disease symptoms, the method comprising the step of inoculating poultry with the vaccine according to the present invention. That is the case.

[0254] Details regarding the use of the vaccine according to the present invention by inoculation of poultry are described above, specifically by intramuscular or subcutaneous injection into 1-day-old chicks, and intraocular injection into 18-day-old embryos.

[0255] The present invention will be further described here with reference to the following non-limiting embodiments.

[0256] [Examples] [Example 1] Construction and testing of multivalent rHVT vectors 1.1. Constructions fabricated and tested Based on the HVT vector construct HVP360 (International Publication No. 2016 / 102647), a series of HVT recombinants further expressing the AIV HA gene were constructed. HVP360 expresses the NDV-F and IBDV-VP2 genes from the Us2 locus. Using the CRISPR / Cas9 technology described by Tang et al., 2018 (previously cited), an external cassette expressing AIV-HA H9 was introduced into different sites in the UL region of the HVP360 genome. Several constructs were constructed by inserting the HA gene expression cassette, and four constructs were evaluated in more detail; the insertion sites are shown relative to GenBank accession number AF291866.

[0257] -HVP400: The HA gene is inserted between UL40 and UL41 of HVP360, between nt.88054~88055.

[0258] -HVP401: Insert HA between UL44 and UL45, between nt.94482 and 94483 -HVP402: Insert HA between UL47 and UL48, between nt.99588 and 99589 -HVP403: Insert HA between UL54 and LORF4, between nt:110395 and 110396.

[0259] The guide RNA sequences used for CRISPR / Cas9-directed insertion are as follows: - Insertion between UL40 and UL41: 5’-ACCTAAAGTACACGTGAATC-3’ (SEQ ID NO: 3) - Insertion between UL44 and UL45: 5’-ACATCGGGACGTACATCATG-3’ (SEQ ID NO: 4) - Insertion between UL47 and UL48: 5’-TGGCGGTTACAATTTCCACG-3’ (SEQ ID NO: 5) - Insertion between UL54 and LORF4: 5’-TTAGATTTCCGGACAGCCTG-3’ (SEQ ID NO: 6).

[0260] NB: SEQ ID NOs: 3-6 are shown here in DNA code because they are inserted into a DNA plasmid and then transcribed to generate the guide RNA.

[0261] The guide RNA was designed using the internet website: zlab.bio / guide-design-resources.

[0262] 1.2. Genetic stability in vitro These four rHVT vector constructs were passaged 16 times in CEF cells in vitro. P16 plaques were monitored for expression of the inserted genes by IFA as follows: An established CEF monolayer overnight was infected with one of the four HVT recombinants at the 16th passage level. The plates were incubated for 2 - 3 days until CPE was clearly visible and then fixed with 96% ethanol. Expression of IBDV-VP2, NDV-F, or AIV-H9 was detected using a monoclonal antibody specific for each antigen as the primary reagent and an Alexa(trademark) labeled conjugate as the secondary antibody. Next, the plates were read by a UV microscope. Approximately 100 plaques for each recombinant were counted to evaluate the expression.

[0263] All plaques tested for HVP400 and HVP401 showed complete expression of the VP2, F, and HA genes. This confirmed the functional and stable expression of the three genes (at least) up to cell passage level 16. Plaques tested for HVP402 and HVP403 showed a slight loss of expression of VP2 and / or F. This is summarized in Table 3. [Table 3]

[0264] Unfortunately, even slight instability is not tolerated in the expression of one of the heterologous genes. Under conditions of high selection pressure, such as in the production of large amounts of virus or in vivo replication for several weeks in vaccinated targets, such mutants have a replication advantage and rapidly outgrow their closely related species that express all the heterologous genes. This is thought to result in loss of expression in the increasing portion of the vector virus. As a result, only constructs showing completely stable expression for all of the heterologous genes, here HVP400 and HVP401, were approved for further vaccine development.

[0265] [Example 2] Vaccination and In Vivo Passage 2.1. Introduction In this experiment, the replication and serological efficacy of rHVT vector constructs HVP400 and HVP401 were tested in chickens by subcutaneous vaccination of 1-day-old SPF laying hen chicks. The group size consisted of 12 animals and 5 hatchmates.

[0266] To determine in vivo replication of the vector vaccine, HVT viremia levels were determined in the spleen (day 11) and peripheral blood lymphocytes (day 32) of vaccinated animals. Blood samples were collected periodically, and serum antibody levels could be determined using specific serological tests.

[0267] 2.2. Experimental Design HVP 400: This is an rHVT virus containing a gB-AIV / HA / H9 insert in the intergenetic region UL40-41, with mIE1-IBDV / VP2+hIE1-NDV / F inserted in Us2. The virus was from the 16th cell passage and was stored in infected CEF cells in liquid nitrogen. The viral titer (in infected cells) was 1.2 × 10⁻⁶. ∧ The concentration was 6 pfu / ml.

[0268] HVP401 This is an rHVT virus containing a gB-AIV / HA / H9 insert in the intergenetic region UL45-46, with mIE1-IBDV / VP2+hIE1-NDV / F inserted in Us2. The virus was from the 16th cell passage and was stored in infected CEF cells in liquid nitrogen. The viral titer (in infected cells) was 1.0 × 10⁶. ∧ The concentration was 6 pfu / ml.

[0269] The dose was 0.2 ml / chicken, administered subcutaneously to the neck at approximately 2000 pfu / chicken in a standard HVT / CEF diluent.

[0270] The chicks were placed in a negative pressure isolation chamber immediately after hatching, and then tagged and vaccinated shortly thereafter, so no acclimatization was applied.

[0271] Blood samples were collected from five hatchmates on day 1, and these samples were serologically tested to confirm that all animals were negative for antibodies against NDV, IBDV, and AIV on the day of vaccination.

[0272] Blood samples were collected from vaccinated chicks 14, 21, 28, and 42 days after vaccination. The blood samples were collected from the subwing vein in a tube containing a coagulation activator and maintained at ambient temperature.

[0273] Viremia: Viremia sampling in the spleen and peripheral blood lymphocytes (PBL) was performed as follows: On day 11 of PV, spleens were isolated postmortem from 5 chicks per group. Clean forceps were used for each chick.

[0274] The spleens were collected in tubes containing 5 ml of 10 mM PBS with phenol red indicator and antibiotics, and kept on ice until processing. The spleens were then homogenized, dissolved in fresh medium, and counted.

[0275] Blood samples for testing for viremia in PBL were collected from the subwing vein on day 32 of pv into heparinized tubes, kept on ice until centrifugation, dissolved in fresh culture medium, and counted.

[0276] For each sample, approximately 2 × 10^6 cells were inoculated into a pre-established CEF monolayer, incubated for 3-4 days, then the plate was fixed and stained with IFA.

[0277] Serology A blood sample for testing the serological response was centrifuged, serum was collected, and the complement was inactivated. These samples were used in various tests to determine the serum response of vaccinated chickens to the expressed heterologous genes: the IBDV-VP response was measured by a virus neutralization (VN) assay using the classical IBDV virus strain D78; the NDV-F response was measured by ELISA and expressed in units relative to a standard sample; and the AIV-HA response was determined by a hemagglutination inhibition assay using HA H9 antigen.

[0278] 2.3. Results and conclusions Viremia rHVT viremia was detected in the spleen and PBL on days 11 and 32 post-vaccination (dpv), respectively. Five animal isolates were tested from each rHVT and each time point. Details of the mean viremia are shown in Table 4. [Table 4]

[0279] As is clear from these results, the multivalent rHVT according to the present invention replicates and seeds in the target animals. However, their replication is relatively slow. This is undoubtedly the result of maintaining and sustaining the expression of the three heterologous gene inserts.

[0280] Genetic stability in vivo The rHVT virus obtained in the viremia assay was tested for continuous expression of the heterologous genes. 100 plaques were analyzed by IFA from all 3 + 2 isolates from the spleen and PBL of the spleen and HVP400, as well as 5 isolates from the spleen and 5 isolates from the PBL of HVP401.

[0281] In all cases, all analyzed plaques of HVP400 and HVP40l maintained the expression of all three heterologous genes: IBDV-VP2, NDV-F and AIV-HA, after replication in vivo.

[0282] The genetic stability of the rHVT constructs HVP400 and HVP401 in vivo was concluded to be excellent.

[0283] Serology Table 5 shows the results of serological responses induced by vaccination of chickens with rHVT vectors HVP400 and HVP401. "Control" refers to hatchmates tested on day 1 of the study.

[0284] In nature, immune responses to pathogens derived from three heterologous antigens—IBDV, NDV, and AIV—are all highly dependent on humoral immunity. Consequently, measurements of the antibody response produced have a strong correlation with in vivo protection against infection.

[0285] Therefore, we selected a type of serological diagnostic test to be applied to detect the immune response and measured this protective antibody response. This correlation is self-evident for viral neutralization of IBDV and HI of AIV as indicators of viral capture by specific antibodies. This also applies to Elisa titers against NDV-F: from prior art and previous experience, it is known that titers exceeding approximately 1000 Elisa units in this test indicate protection against NDV challenge infection.

[0286] In addition, since the vector viruses for the HVP400 and HVP401 constructs are still functional HVT viruses, their protective ability against Marek's disease is intrinsic and unchanging.

[0287] Therefore, it can be concluded that vaccination with rHVT HVP400 or HVP401, respectively, induces a protective immune response against IBDV, NDV, AIV, and MDV in target animals. To confirm this, vaccination-challenge experiments with each of these antigens are underway.

[0288] Regarding the differences between constructs HVP400 and HVP401: HVP400 appeared to induce slightly lower viremia than HVP401, but the serological responses induced by both of these vectors were still very similar. [Table 5]

[0289] [Example 3] Vaccination Challenge Experiment As described in Example 2 above, a vaccination-challenge test was performed to confirm the protective ability of the recombinant HVT vector according to the present invention against the serological response to avian influenza virus.

[0290] 3.1. Setup The animals used were 1-day-old chicks that were either SPF or MDA+ for H9N2 AIV. These were vaccinated at 1 day of age with either HVT vector vaccine HVP400 or HVP401 (n=16 in both groups), which was at the 16th cell passage level, essentially via the subcutaneous route described above. The dose of HVT vector vaccine was approximately 800 PFU / chicken. Next, the chicks were challenged with a heterologous LPAI H9N2 strain; SPF chicks were challenged at 3 weeks post-vitro, and MDA+ chicks were challenged at 6 weeks post-vitro. The challenge virus used was LPAI A / chicken / Egypt / V1527 / 2018(H9N2), which was administered intranasally at 10^6 EID50 in 0.2 ml.

[0291] The negative control group (n=16) was vaccinated with non-recombinant HVT, while the positive control group (n=11) was vaccinated with the classic inactivated H9N2 vaccine (NOBILIS® INFLUENZA H9N2+ND). Five SPF hatchmates were tested for serology to confirm that SPF animals were negative for HA antibodies. Similarly, ten MDA+ animals were tested for serology to confirm their MDA+ H9 HA status.

[0292] Five chicks from all HVT-vaccinated groups were euthanized on day 15 of the challenge, and their spleens were tested for HVT viremia to confirm ingestion and replication of the HVT vector vaccine. To monitor challenge virus shedding, posterior nasal swabs were collected 1, 3, and 6 days after the challenge. Blood samples were collected at regular intervals to measure serum antibody levels. Clinical signs were monitored from 1 day after the challenge until all chicks in a group no longer showed AI symptoms.

[0293] The daily clinical scoring system used the following point system: 0 No clinical signs 1. Mild clinical signs 2. Moderate clinical signs 3. Severe clinical signs.

[0294] 3.2.Results The HVT vaccine was well replicated: spleens tested from all HVT-vaccinated groups were all positive for vaccine administration on day 15. This persisted throughout the experiment: viremia tests of PBLs collected on day 42 (SPF) or day 63 (MDA+) were also all positive for HVT.

[0295] To determine the in vivo genetic stability of HVP400 and HVP401 vector viruses, viremia plaques of the virus on day 15 of pv were stained with specific antisera against NDV-F, IBDV-VP2, or AIV-HA H9. All plaques showed stable expression of all inserted genes. Clear positive expression of the NDV F and IBDV-VP2 genes was also detected by Elisa in serum samples collected on days 28, 42, and 63 of pv in the group administered one of the vector vaccines. Negative controls (non-recombinant HVT vaccine) were negative for heterologous antigens at all time points.

[0296] AIV serology, detected by indirect Elisa and HI tests, showed that MDA+ chicks had high anti-HA H9 titers at baseline. MDA+ chicks administered with the vector vaccine had clearly positive anti-HA antibodies on day 14 of the pv, but these titers decreased to background levels on days 28 and 42 of the pv, similar to pseudo-HVT-vaccinated MDA+ chicks. SPF chicks administered with the vector vaccine showed an increase in anti-H9 titers on day 21 of the pv.

[0297] It is noteworthy that, despite the HVT vector vaccine according to the present invention expressing the HA H9 antigen, the prior presence of H9 HA MDA in chicks did not affect the HVT vector vaccine according to the present invention. Vector replication and dissemination in the vaccinated chicks were good, and heterologous gene expression, including the expression of the H9 HA antigen, was also good.

[0298] H9 N2 challenge virus replication was detected by collecting posterior nostril swabs on days 1, 3, and 6 after challenge. Swabs were analyzed by qPCR using FLU(brand name)PCR (BioChek). The peak of AIV challenge virus replication was found on day 3 after challenge. To obtain traces of the reduced level of challenge virus replication induced by vector vaccine, the qPCR scores of vector-vaccinated chicks on that day were compared to scores from pseudo-HVT-vaccinated, challenged chicks. Given the field conditions in which all chicks were born from AIV-vaccinated mothers and therefore HA antibody-positive, the results for the MDA+ group are reasonable.

[0299] The results were as follows: In the MDA+ chick group, the relative amount of H9 N2 challenge virus (relative to 100% set for the pseudo-HVT vaccine), as measured by qPCR and re-isolated on day 3 of pc, was 53.2% for the HVP400 vector vaccine; 61.4% for the HVP401 vector vaccine; and 77.7% for the H9 N2 inactivated vaccine. This indicates that the reduction of AIV challenge virus replication by the HVT vector vaccine according to the present invention was at least as good as, and slightly better than, the reduction by the classical inactivated AIV vaccine.

[0300] A similar pattern was observed from the clinical scores observed after the challenge. These were measured up to week 3 of the pc, but no scores were observed after day 14 of the pc. Table 6 shows the average daily clinical scores after the challenge for various groups of vaccinated MDA+ chicks, as well as the total clinical scores per group from day 1 to day 14 of the pc.

[0301] Interestingly, the HVT vector vaccine according to the present invention prevented clinical signs of challenge infection better than classical inactivated vaccines. [Table 6]

[0302] 3.3. Conclusion: The HVP400 and HVP401 vector vaccines were genetically stable in vivo at pv day 15 and fully replicated and expressed all three heterologous genes, even in chicks that were MDA+ for one of the expressed antigens. Both vectors induced distinct antibody titers against each of the three heterologous antigens in vaccinated chicks.

[0303] Furthermore, we were able to significantly prevent infections and diseases resulting from challenge infections with heterologous LPAI H9N2 viruses: challenge virus replication was reduced, and clinical signs were almost completely prevented.

[0304] The relatively low levels of HA-specific antibodies induced in MDA+ animals vaccinated with the HVT vector are noteworthy. This suggests that the humoral immune response plays a limited role in the immune defense against AIV infection induced by the HVT vector vaccine according to the present invention.

Claims

1. Recombinant turkey herpesvirus (rHVT) expressing the infectious bursal disease virus (IBDV) viral protein 2 (VP2) gene and the Newcastle disease virus (NDV) fusion (F) protein gene from first and second expression cassettes inserted into the unique short (Us) region of the rHVT genome, The rHVT is characterized in that it also expresses the avian influenza virus (AIV) hemagglutinin (HA) gene from a third expression cassette inserted either between the UL40 gene and the UL41 gene, or between the UL44 gene and the UL45 gene, in the unique length (UL) region of the rHVT genome; Here, the IBDV VP2 gene is located in the 5' to 3' direction. a. Promoter of mouse cytomegalovirus pre-early 1 gene (mCMV-IE1), b. IBDV VP2 gene, and c. Transfer terminator, These are expressed from a first expression cassette containing them in this order. This is characterized in that the promoter and terminator of the expression cassette are operably linked to the VP2 gene, and, The NDV F gene is located in the 5' to 3' direction. a. Promoter of the human cytomegalovirus pre-early 1 gene (hCMV-IE1), b. NDV F protein gene, and c. Transfer terminator, These are expressed from a second expression cassette containing them in this order. This is characterized in that the promoter and terminator of the expression cassette are operably linked to the F gene. and, The first and second expression cassettes are inserted into the same or different loci in the Us region of the rHVT genome, and the first and second expression cassettes are combined within a single expression cassette. This is characterized by the insertion of the combined single expression cassette into the Us2 gene. In addition, The AIV HA gene is located in the direction from 5' to 3'. a. Mammalian herpesvirus-derived glycoprotein B (gB) gene promoter, b. AIV HA protein gene, and c. Transfer terminator, These are expressed from a third expression cassette containing them in this order. This is characterized in that the promoter and terminator of the expression cassette are operably linked to the HA gene, and, The AIV HA protein gene is characterized in that it encodes an HA protein of a serotype selected from H5, H7, and H9. Recombinant turkey herpesvirus (rHVT).

2. A host cell containing rHVT as described in claim 1.

3. rHVT according to claim 1 or host cell according to claim 2, for use in poultry vaccines.

4. A poultry vaccine comprising the rHVT described in claim 1 and / or the host cell described in claim 2, and a pharmaceutically acceptable carrier.

5. The vaccine according to claim 4, comprising at least one additional immunoactive ingredient.

6. A method for preparing a poultry vaccine according to claim 4 or 5, The method is, a. A step of infecting host cells in vitro with the rHVT described in claim 1. b. A step of collecting the infected host cells, and c. A step of mixing the recovered infected host cells with a pharmaceutically acceptable carrier. A method that includes this.

7. Use of the rHVT according to claim 1, the host cell according to claim 2, or any combination thereof, for the manufacture of a poultry vaccine.

8. Use of the vaccine according to claim 4 or 5 in poultry to prevent or mitigate signs of infection by MDV, IBDV, NDV and / or AIV, or related diseases.

9. A method for preventing or mitigating signs of infection with MDV, IBDV, NDV, and / or AIV or related diseases, comprising administering the vaccine according to any one of claims 4 or 5 to poultry.

10. A method for vaccinating poultry to prevent or mitigate infection with MDV, IBDV, NDV, and / or AIV or associated disease symptoms, comprising the step of vaccinating the poultry with the vaccine according to claim 4 or 5.

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