A novel multivalent HVT vector vaccine

Abstract

The present invention relates to a novel recombinant HVT (rHVT) construct useful as a multivalent vector vaccine for poultry. The rHVT contains four heterologous genes from poultry pathogens: VP2 gene from IBDV, F gene from NDV, and gD and gI genes from ILTV. The VP2 and F genes are inserted into the Us genome region of the rHVT. The gD and gI genes are inserted into the UL genome region between the UL54 and LORF3 genes. The novel rHVT-VP2-F-gD-gI proved to be genetically stable in vitro and in vivo, and expressed all inserted genes well. It was also an effective vaccine against severe challenge infections with NDV, IBDV and ILTV.

Description

[Technical field]

[0001] The present invention relates to the field of veterinary vaccines, i.e. poultry vaccines based on recombinant herpesvirus of turkeys as viral vector vaccines. In particular, the present invention relates to recombinant herpesvirus of turkeys (rHVT), host cells containing the rHVT, medical uses of the rHVT and the host cells, vaccines containing the rHVT and / or the host cells, and methods for producing the vaccines.

[0002] Recombinant vector virus is a well-known method to express heterologous genes and deliver their encoded proteins to human or non-human animal targets.Examples are vaccinia, measles or adenovirus vectors.If the heterologous gene encodes an immunogenic protein from a pathogen, this can be an effective method of vaccination of the target against the disease caused by that pathogen.As a replicating microorganism, the vector virus can establish a productive infection in the vaccination target, express the heterologous gene together with its own genes, and thus induce a protective immune response in the target against the antigen encoded by the heterologous gene.

[0003] In veterinary vaccination, especially in poultry vaccination, vector vaccines have attracted interest due to their relative ease of use and low cost. Several avian vector vaccines have been investigated over time, for example based on chicken adenovirus, fowlpox virus, and especially herpesvirus of turkeys (HVT), see WO 87 / 04463 and WO 90 / 002803. The advantage of using HVT as a vector is that it is non-pathogenic to birds, can carry and express inserted genes, and induces immunity against Marek's disease virus serotype 1 or 2 (MDV1 or MDV2), a pathogenic member of the virus family.

[0004] Over the years, genes from various avian pathogens have been inserted into and expressed by HVT virus vectors derived from Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), and infectious bronchitis virus (see WO 93 / 025665), avian influenza virus (AIV) (see WO 2012 / 052384), or the parasite Eimeria (Cronenberg et al., 1999, Acta Virol., vol. 43, p. 192-197). This has led to the development of a variety of commercially available HVT vector vaccines for poultry, including: For example, for ND, Innovax®-ND (MSD Animal Health), and Vectormune® HVT-NDV (Ceva Sante Animale), for ILT, Innovax®-ILT (MSD Animal Health), for IBD, Vaxxitek® HVT+IBD (Boehringer-Ingelheim, formerly Gallivac™ HVT-IBD), and Vectormune® IBD (Ceva Sante Animale), and for AI, Vectormune® AI (Ceva Sante Animale).

[0005] Inserting heterologous genes into its viral genome is a burden to vector viruses, since it may affect its replication, expression and / or its genetic stability in vitro and / or in vivo. These problems are particularly pronounced when two or more heterologous genes are inserted. Such multivalent recombinant vector vaccines may be able to protect against multiple diseases after a single vaccination. However, such vector constructs still need to allow good replication of the vector and its inserts, both in vitro and in vivo, and provide effective expression of all heterologous genes at sufficiently high levels and for a significant period of time to induce and maintain protective immune responses in vaccinated targets against all intended pathogens.

[0006] Additionally, the genetic stability of recombinant vector viruses allows for the extensive rounds of replication in vitro necessary for large-scale production, and is a requirement for compliance with the very high standards of safety and biological stability that recombinant viruses (which are genetically modified organisms) must meet in vivo to receive marketing approval from governmental or regulatory authorities before they can be introduced into the field as commercial products.

[0007] Many multivalent HVT vector vaccines have been described over time, for example in WO 93 / 025665 and WO 96 / 005291. However, most of the multigene constructs described in those publications have only been suggested, and only some of the recombinant vectors with multiple inserts have actually been constructed and isolated. Only a few have been tested in birds. Overall, no data on the induction of effective immune protection in target animals, let alone results on their stability during replication or on the expression levels of foreign genes, have been presented. Due to challenges regarding the genetic stability and continued expression of the inserts, only a few multivalent HVT vector constructs have actually been licensed as commercial vaccine products. Currently, these products are Innovax® ND-IBD (MSD Animal Health; WO 2016 / 102647), Innovax® ND-ILT (MSD Animal Health; WO 2013 / 057236), ULTIFEND® IBD ND (Ceva Sante Animale; WO 2013 / 144355), Vaxxitek® HVT+IBD+ND and Vaxxitek® HVT+IBD+ILT (both Boehringer-Ingelheim; WO 2018 / 112.051).

[0008] The efficacy of Innovax® ND-IBD is also described in van Hulten, M. et al., 2021, Avian Pathol., vol. 50, p. 18-30. The efficacy of Innovax® ND-ILT is described in Gergen L., et al., 2019, Avian Pathol. vol. 48, p. 45-56.

[0009] Tang et al. (2020, Vaccines, vol. 8, p. 97) described rHVTs containing different heterologous genes: the IBDV VP2 gene inserted between the HVT UL45 and UL46 genes, and the ILTV gD and gI genes inserted between UL65 and UL66, and the AIV HA gene in Us2. These rHVTs were only tested in in vitro cell cultures.

[0010] WO 2016 / 102647 describes an HVT vector vaccine in which rHVT expresses the IBDV VP2 gene and the NDV F gene, each driven by a separate promoter, in a single expression cassette inserted into the Us2 gene of the HVT Us genomic region.

[0011] WO 2018 / 112051 describes an HVT vectored vaccine containing two genes selected from IBDV VP2, ILTV gD, and NDV F. The heterologous gene is inserted into the HVT genome either at the IG1 locus or between SORF3 and Us2, using one promoter whereby the two genes are inserted as one expression cassette separated by an IRES or P2A sequence.

[0012] WO 2019 / 072964 describes a multivalent rHVT vectored vaccine capable of protecting against MDV, NDV, IBDV and ILTV. Inserts were placed into the UL54.5 (LORF3) and Us2 genes of the HVT genome.

[0013] However, there are several methods for combating poultry diseases and there is a continuing need for further and improved options for effective vaccination of poultry.

[0014] It is therefore an object of the present invention to address the need in the field and provide a rHVT vectored vaccine that allows immunization of poultry against the four avian pathogens MDV, NDV, IBDV and ILTV.

[0015] Surprisingly, it has been found that this objective can be met, thereby overcoming one or more of the shortcomings of the prior art, by providing a rHVT that expresses the IBDV VP2 gene and the NDV F gene from the Us genomic region, and the ILTV gD gene and the ILTV gI gene from the UL genomic region.

[0016] We attempted to extend the protection already provided by a known rHVT expressing the IBDV-VP2 and NDV-F genes ("rHVT-VP2-F") with protection against ILTV. The additional heterologous genes selected were the ILTV gD and ILTV gI genes. Several multiple insert rHVTs failed to generate stable multivalent recombinant viruses when tested in vitro and in vivo. Some of them were unable to replicate multivalent recombinant HVTs, and others had no expression of one or more of the heterologous genes.

[0017] For example, further insertion of the gD and gI genes into the HVT UL39 gene (ribonucleotide reductase large subunit) locus was unsuccessful either when inserted into the central or 3' region of the UL39 gene. These rHVT constructs replicated in vitro in CEF cells but were completely defective when inoculated into chickens.

[0018] Several constructs containing the ILTV gD and ILTV gI genes inserted into the HVT UL genome region either between the UL40 (ribonucleotide reductase small subunit) and UL41 genes (virion host shutoff protein) or between the UL47 gene (tegument phosphoprotein) and UL48 genes (immediate early gene transactivator) were also ineffective. The construct with the UL40-UL41 insert replicated normally in vitro (compared to the rHVT-VP2-F construct) but at reduced levels in vivo in chicks. In addition, this construct proved to be genetically unstable in vivo since the F and VP2 genes were not expressed. A similar situation occurred with the UL47-48 insert construct, which also replicated normally in vitro and at a reduced rate in vivo, and was also not genetically stable in vivo, since expression of the gD and gI genes was only at very low levels and insufficient for effective vaccination against ILTV. This finding for the UL47-48 insert construct was particularly disappointing because the UL47 and UL48 genes have been reported to be nonessential in transposon gene knockout studies of the HVT genome (Hall et al., 2015, Virology Journal, vol. 12, p. 130).

[0019] Our observations were consistent with the general understanding in the field that more inserts cause more problems for the genetic stability of viral vectors in terms of replication and expression of foreign genes. In this case, it is clear that the parent vector already expresses two other heterologous genes, which becomes complicated when further expressing the ILTV gD gene and the ILTV gI gene. This makes it impossible to predict successful multivalent recombinant HVT constructs based on prior art observations of possible insertion sites of heterologous genes in the HVT genome. Similarly, reports that certain rHVT constructs replicate effectively in vitro (e.g., as described in Tang et al., 2020, supra) are clearly unreliable when predicting in vivo properties.

[0020] Therefore, it was unexpected that further integration of an expression cassette carrying the ILTV gD and ILTV gI genes into a specific insertion site within the HVT UL genomic region, specifically by inserting gD and gI between the UL54 and LORF3 genes of HVT, resulted in a stable and effective multivalent HVT vector construct. This insertion locus is referred to herein as the "UL54-LORF3" locus.

[0021] The resulting polyvalent rHVT vector with F and VP2 inserted in Us and gD and gI genes at the UL54-LORF3 locus was found to be genetically stable even after 15 serial passages in in vitro cell culture. The virus was then used to inoculate chickens, which led to several more replication cycles in vivo. Virus was then reisolated from vaccinated chickens at day 21 post-vaccination to determine the level of viremia and to confirm that expression of all inserted genes was maintained. Viruses reisolated at day 21 p.v were found to be completely genetically stable. Immunofluorescence plaque assay demonstrated that all reisolated viruses tested expressed all heterologous genes, F, VP2, gD and gI, even at 3 weeks post-vaccination.

[0022] Vaccinated chickens also showed excellent seroconversion to each of the expressed heterologous antigens F, VP2, gD and gI. The antibody levels achieved far exceeded those known to be necessary for in vivo protection against infection or disease. Details are provided in the Examples.

[0023] Surprisingly, the new rHVT vectors were also found to be more effective vaccines compared to the rHVT constructs described in patent application number PCT / EP2021 / 087445 as HVP412 and HVP413. These rHVT vectors also have the F gene and VP2 gene inserted into the Us2 gene, but have the ILTV gD gene and the ILTV gI gene inserted between the UL44 gene and the UL45 gene, or between the UL45 gene and the UL46 gene, respectively, of the HVT genome. Specifically, the new rHVT vectors using the UL54-LORF3 insertion site of ILTV gD and ILTV gI provided protection against infection and / or disease caused by NDV, IBDV and ILTV, which was even better in some parameters than that induced by the rHVT vector vaccines HVP412 and HVP413 described in 87445.

[0024] Thus, the novel multivalent rHVT vector viruses are useful as vaccines, indeed improved vaccines, against one or more or all of MDV, NDV, IBDV and ILTV.

[0025] The ability to obtain vaccination against the four major poultry diseases from one vaccine would be highly beneficial as it would result in a significant reduction in stress for the target animals as well as a reduction in effort and costs for the poultry farmer.

[0026] It is unclear exactly how or why rHVT expressing the VP2 and F genes could tolerate additional expression of the ILTV gD and ILTV gI genes at the UL54-LORF3 insertion locus, while insertions into several other sites that initially seemed suitable did not result in stable and efficient vector constructs.

[0027] Although the inventors do not wish to be bound by any theory or model that may explain these findings, they hypothesize that this effect arises from a complex interplay of various expression patterns in the polyvalent rHVT when required to replicate and express in vitro and in vivo. For unknown reasons, the insertion of an additional gene at this particular locus in the UL results in a polyvalent rHVT with exactly the right balance between the expression strength of the heterologous gene and the strain this imparts to the replication capacity and genetic stability of the HVT itself, whereas other constructs (although unpredictable) do not have such a balanced composition.

[0028] Thus, in one aspect, the present invention relates to a recombinant herpesvirus of turkeys (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 a first expression cassette inserted into the unique short (Us) region of the rHVT's genome, characterized in that the rHVT also expresses the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) and glycoprotein (gI) genes from a second expression cassette inserted into the unique long (UL) region of the rHVT's genome, between the UL54 and LORF3 genes.

[0029] "Herpesvirus of turkeys (HVT)" is also called MDV3, Melaegrid herpesvirus 1, or herpesvirus of turkeys. 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.

[0030] Herpesvirus of turkeys, Newcastle disease virus, infectious bursal disease virus, and infectious laryngotracheitis virus are all well-known viruses associated with veterinary medicine. The same is true for mouse and human cytomegaloviruses (mCMV and hCMV), and feline herpesvirus (FHV). Such viruses have characteristic features of their taxon, including biological characteristics such as morphological, genomic and biochemical characteristics, as well as physiological, immunological or pathological behavior.

[0031] General information on these viruses is available from reference handbooks such as Fields Virology (LWW publ., ISBN: 9781451105636). Information on infections and diseases caused by these viruses is available from handbooks such as The Merck veterinary manual (2010, 10th ed., 2010, CM Kahn 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 the present invention can be obtained from various sources, for example, from humans or as field isolates from wild or farm non-human animals, or from various laboratories, (depository) institutions, or (veterinary) universities. The viruses can be easily identified using routine serological or molecular biological means. From all these viruses, much genetic information is digitally available in public sequence databases such as NCBI's GenBank™, UniProt, and EMBL's EBI.

[0032] As is known in the art, the classification of a microorganism in a particular taxon is based on a combination of its characteristics. Thus, the present invention also includes variants of these virus species that are subclassified in any way, such as as subspecies, strains, isolates, genotypes, variants, subtypes or subgroups.

[0033] Furthermore, while a particular virus of the invention may currently be assigned to this species, it will be apparent to one of skill in the art of the invention that this is a taxonomic classification that may change over time as new insights may result in reclassification into a new or different taxonomic group. However, such reclassified viruses remain within the scope of the invention, as this does not change the virus itself or its antigenic repertoire, only its scientific name or classification.

[0034] A "recombinant HVT" of the present invention is an HVT in which the genetic material has been altered relative to its parental state by the insertion of a molecule of nucleic acid encoding a heterologous antigen.

[0035] In the present invention, a gene or antigen is "heterologous" if it is derived from a (micro)organism other than MDV or HVT. As a result, the rHVT of the present invention is not a chimeric virus construct in which any part of the genome has been replaced by a corresponding part from a related virus, as is the case, for example, with the "novel avian herpesvirus" described in WO 1998 / 037216.

[0036] The "VP2 protein gene" encoding the capsid protein of IBDV is well known in the art. The VP2 protein gene can be derived from a classical or variant IBDV, or can be a chimera.

[0037] Similarly, the "F protein gene" encoding the fusion glycoprotein of NDV is well known. In the present invention, the F protein gene can be obtained from attenuated, intermediate or highly virulent NDV, or can be chimeric.

[0038] The term "gene" is used to indicate any portion of a nucleic acid capable of encoding a protein. In the present case, this corresponds to the "open reading frame" (ORF), i.e. the protein-coding portion of DNA from the start codon to the stop codon without the promoter of the gene. A gene of the present invention may code for a complete protein, or may code for example only the mature form of the protein, i.e. any portion of the protein without the "leader", "anchor" or "signal sequence". A gene may code for a specific portion of a protein, e.g. the portion that contains an immune-protective epitope.

[0039] In this regard, a "protein" of the present invention is a molecular chain of amino acids. A protein can be a native or mature protein, a preprotein or proprotein, or a functional fragment of a protein. Thus, peptides, oligopeptides and polypeptides are included within the definition of a protein, so long as they still contain the relevant immunological epitopes and / or functional regions.

[0040] For the present invention, the term "expression" refers to the well-known principle of gene expression, whereby genetic information provides a code for producing a protein through transcription and translation.

[0041] An "expression cassette" is a nucleic acid fragment that contains at least one heterologous gene and a promoter that drives transcription of the gene. Termination of transcription can result from sequences provided by the genomic insertion site of the cassette in the vector genome, or the expression cassette itself can contain a termination signal, such as a transcription terminator.

[0042] In such a cassette, both the promoter and (optionally) the terminator must be adjacent to the gene whose expression they regulate, known as being "operably linked," such that there are no significant other sequences between them that would intervene in the efficient initiation-respective termination of transcription.

[0043] The expression cassette can be in DNA or RNA form, but for its intended use in the HVT vector, the expression cassette of the present invention is used as DNA. As will be appreciated by those skilled in the art, the expression cassette is a self-contained expression module, and therefore its orientation in the vector virus genome is generally not important.

[0044] Expression cassettes may contain additional DNA elements, eg, to aid in construction and cloning, such as sites for restriction enzyme recognition or PCR primers.

[0045] The expression cassette is inserted into one locus in the genome of the vector as a whole.Various techniques are available to control the locus and orientation of its insertion.For example, by using adjacent parts from the genome of the vector, to integrate the cassette by homologous recombination process in a certain way, for example, by using overlapping cosmids as described in U.S. Pat. No. 5,961,982.Alternatively, integration can be carried out by using the CRISPR / Cas9 technique described in Tang et al., 2018 (Vaccine, vol.36, p.716-722).

[0046] In the present invention, an "inserted" expression cassette in the genome of a vector refers to the integration into the genomic nucleic acid of the vector, so that the inserted element is transcribed and translated together with the native gene of the vector. The effect of the insertion on the genome of the vector varies depending on the method by which the insertion is performed, and depending on whether the net result on the genome is due to the addition, substitution, or deletion of genetic material, the size of the vector genome may be larger, the same, or smaller, respectively. Those skilled in the art are fully capable of selecting and implementing a particular type of insertion and making adaptations as necessary.

[0047] The construction of an expression cassette and its insertion into the HVT vector can be carried out by well-known molecular biology techniques, including cloning, transfection, recombination, selection and amplification. These and other techniques are described in great detail in standard texts such as Sambrook & Russell: "Molecular cloning: a laboratory manual" (2001, Cold Spring Harbour Laboratory Press; ISBN: 0879695773); Ausubel et al., Current Protocols in Molecular Biology (J. Wiley and Sons Inc, NY, 2003, ISBN: 047150338X); 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).

[0048] In the present context, the terms "first" and "second" in reference to expression cassettes are used for ease of reference only and do not indicate any order or preference.

[0049] It is well known that the "unique short (Us) region" of the HVT genome is the downstream region of the genome between the "short internal repeat" and the "short terminal repeat". The size of the HVT Us is about 8.6 kb (see Kingham et al., 2001, J. of Gen. Virol., vol. 82, p. 1123-1135).

[0050] Fully annotated genome sequences of several HVT strains are publicly available, for example via GenBank. The genome sequence of HVT strain FC-126 is available under GenBank accession number AF291866, with nucleotides 136990 to 145606 forming the Us region.

[0051] "ILTV" is also called Gallid alphaherpesvirus 1. Fully annotated genome sequences of several ILTV strains are publicly available, for example, via GenBank, for example, under accession number NC_006623 for the ILTV reference strain SA-2. The genes for the ILTV gD and ILTV gI envelope glycoproteins are located within the Us region of the ILTV genome, as the Us6 and Us7 genes, respectively. The ILTV gD and ILTV gI proteins can induce ILTV-specific and protective antibodies and are often used in combination. In their natural context, these genes partially overlap, whereby the gI gene promoter is located in the upstream gD open reading frame (ORF) and the gD gene terminator is located in the downstream gI ORF. As a result, when used in combination, or when obtained from their natural context in the ILT virus genome, the gD and gI genes can be conveniently subcloned as one contiguous fragment, for example starting at the gD gene promoter and ending after the gI gene.

[0052] The "unique long (UL)" region of the HVT genome is the upstream part of the genome and, in HVT, is approximately 110 kb in size. In the genome sequence of HVT strain FC-126, GenBank accession number: AF291866, the UL region is formed by nucleotides 5910 to 117777.

[0053] The designation "UL54" and similar terms used herein are well known methods in the field of the invention to refer to a specific gene located in the genome of a herpesvirus, here the UL genome region of HVT, see for example the annotation applied to GenBank accession number AF291866. The nomenclature is derived from the homologous gene of the herpesvirus species Herpes Simplex Virus 1. The same applies (mutatis mutandis) to the designations of other HVT genes used herein.

[0054] The HVT UL54 gene, also known as the HVT063 gene, encodes a post-translational gene regulatory protein that corresponds to the HSV-1 regulatory protein ICP27.

[0055] The HVT gene LORF3, also known as the HVT064 gene, is also called UL54.5 and is sometimes referred to as LORF4, similar to the nomenclature used in MDV1 and MDV2 for genes in that location. The HVT LORF3 gene encodes a protein of unknown function that is suspected to correlate with the avian host range of HVT, potentially including some lymphotropic aspects.

[0056] The term "between" serves to indicate that the inserted second expression cassette is located at an insertion site (i.e. locus) on the HVT genome between and outside the indicated genes, the so-called intergenic region. Such an insertion is therefore not within the open reading frame of the gene to which it refers, e.g. not within the ORF of UL54 or LORF3. This is therefore an essentially different insertion compared to an intragenic insertion into an ORF such as the LORF3 (UL54.5) gene of HVT, as applied in WO 2019 / 072964.

[0057] Details of embodiments and further aspects of the invention are described below.

[0058] In one embodiment of a rHVT according to the present invention, the rHVT is not a chimeric virus.

[0059] In the present invention, a virus is "chimeric" if a region of its genome is replaced with a corresponding region from the genome of a related virus. For example, a chimeric HVT can be an HVT in which a portion of the HVT genome is replaced by a corresponding portion of the genome from MDV1 or MDV2.

[0060] In one embodiment, the rHVT according to the invention comprises a first expression cassette, in the 5' to 3' direction, a. Mouse cytomegalovirus immediate early 1 gene (mCMV-IE1) promoter, b. IBDV VP2 gene, C. transcription terminator, d. human cytomegalovirus immediate early 1 gene (hCMV-IE1) promoter, e. NDV F protein gene, and f. transcription terminator, The present invention is characterized in that it comprises Thereby, the promoter and terminator are operably linked to the VP2 gene and the F gene, respectively.

[0061] In this embodiment, the promoter of element a and the terminator of element c are operably linked to the VP2 gene, and the promoter of element d and the terminator of element f are operably linked to the F gene.

[0062] As used herein, the term "comprise" (and variations such as "comprising," "comprise," and "comprised") is intended to refer to all possible elements and any possible combinations of the invention covered or included by the text section, paragraph, claim, etc. in which the term is used, even if such elements or combinations are not explicitly recited, and does not exclude any such element or elements or combinations.

[0063] Thus, any such text section, paragraph, claim, etc. may also relate to one or more embodiments in which the term "comprising" (or variations thereof) is replaced with terms such as "consist of," "consisting of," or "consist essentially of."

[0064] The term "5' to 3' direction", also known as "downstream direction", is well known in the art. Together with the term "in this order", the present invention serves to indicate the relative orientation that the subsequently assembled elements must have with respect to each other in order to function with the host cell's gene expression machinery such that the rHVT according to the present invention can be replicated and expressed. As one skilled in the art will appreciate, this orientation is relative to the DNA strand from the double-stranded DNA genome of the HVT, which is the "coding strand", and relative to the encoded mRNA molecule, which is the "+" or "sense" orientation.

[0065] Nonetheless, without prejudice to the above section, in the "template" strand, which is the complementary strand of the rHVT ds DNA genome, the relative order of the listed elements is the same, but in that DNA strand, the orientation of these elements is 3' to 5' and "upstream".

[0066] A "promoter" according to the present invention is well known to be a functional region of genetic information that directs the transcription of a downstream coding region. Thus, the promoter is located upstream of a gene.

[0067] The nomenclature of a promoter is generally based on the gene that controls its expression in its natural context. 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 the IE1 gene in the mCMV genome. Since the IE1 gene is a well-documented and clearly recognizable gene, and several mCMV genomes have been sequenced, such promoters can be easily identified by conventional techniques. For example, in a basic protocol, a promoter can be easily obtained by roughly subcloning the region between two consecutive genes, for example from the stop codon of the upstream gene to the start codon of the downstream gene. The promoter can then be identified by standard tests, for example by expression of marker genes, using smaller and smaller portions of the cloned region that contain the possible promoter.

[0068] Generally, promoters contain multiple recognizable regulatory regions, such as enhancer regions, which are involved in the binding of regulatory factors that affect the time, duration, conditions and levels of transcription.Enhancer regions are generally located in the upstream part of promoters, but promoters can also be influenced by the region downstream of the initiation codon, which is involved in the binding of transcription factors and directs RNA polymerase itself.The downstream region of promoters generally contains multiple conserved sequence elements, such as TATA box, CAAT box and GC box.

[0069] A promoter containing both the enhancer region and the downstream region is referred to as a "complete" promoter. A promoter containing only the downstream region is referred to as a "core" promoter.

[0070] The mCMV-IE1 gene is well known in the art and can be readily obtained from a variety of commercial sources, including commercial plasmid suppliers for cloning and expression. The IE1 gene is also referred to as the "major IE gene" of CMV.

[0071] The mCMV-IE1 protein is also called 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 WO 87 / 03.905 and EP 728.842. The nucleotide sequence of the complete mCMV IE locus is available, for example, from GenBank under acc.nr.L06816.1. mCMV itself is available, for example, from ATCC under acc.nr.VR-1399.

[0072] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the mCMV-IE1 gene promoter is a complete promoter including both the core promoter region and the enhancer region of the mCMV-IE1 gene. The complete mCMV-IE1 gene promoter is about 1.4 kb in size.

[0073] The term "about" as used herein means ±25% of the indicated value, preferably "about" means ±20, 15, 12, 10, 8, 6, 5, 4, 3, 2% of the indicated value, or even "about" means ±1% of the indicated value, these being in order of preference.

[0074] In one embodiment, the mCMV-IE1 gene promoter of the present invention is a DNA molecule of about 1.4 kb comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the entire length of the region from nucleotide 1 to 1391 of SEQ ID NO: 1. In order of preference, at least 96, 97, 98, or even 99% nucleotide sequence identity is more preferred.

[0075] In one embodiment, the mCMV-IE1 gene promoter is the region from nucleotides 1 to 1391 of SEQ ID NO:1.

[0076] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the IBDV VP2 gene of the present invention encodes the VP2 protein 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 strain) or AF499929 (D78 strain)). Alternatively, this gene can be obtained from the genome of classical IBDV isolated from nature using conventional techniques for manipulating birnaviruses. Classical IBDV can be easily identified using serology or molecular biology.

[0077] Homologs or variants of the IBDV VP2 gene have comparable potency and stability, and therefore, in one embodiment, the IBDV VP2 protein gene of the present invention has at least 90% nucleotide sequence identity to the entire length of the region from nucleotide 1423 to 2781 of SEQ ID NO: 1. In order of preference, at least 92, 94, 95, 96, 97, 98, or even 99% nucleotide sequence identity is preferred.

[0078] In one embodiment, the IBDV VP2 protein gene of the present invention is derived from the classical IBDV strain Faragher 52 / 70.

[0079] In one embodiment, the IBDV VP2 protein gene of the present invention is the region from nucleotides 1423 to 2781 of SEQ ID NO:1.

[0080] A "transcription terminator" or terminator is a regulatory DNA element involved in the termination of transcription of a coding region into RNA. In general, such elements code for a portion having a secondary structure, such as a hairpin, that can allow the RNA polymerase complex to terminate transcription. Thus, a transcription terminator is always located downstream of the stop codon of the region to be translated, and thus in the "3' untranslated region" of the ORF. A terminator can also contain a polyadenylation (polyA) signal. This results in polyadenylation, which occurs in most eukaryotic mRNAs and plays a role in the transport and stability of such mRNAs.

[0081] In the expression cassettes of the present invention, the selection of a particular type of transcription terminator is not critical, so long as it provides for efficient termination of RNA transcription.

[0082] In the first expression cassette for the present invention, the transcription terminator element c between the VP2 gene and the F gene not only provides for the termination of transcription of the VP2 gene, but also for effective isolation of the expression of these genes by preventing possible read-through of RNA transcription.

[0083] The two terminators shown for the first expression cassette may be the same or different.

[0084] In one embodiment of a rHVT according to the present invention, the first expression cassette comprises a transcription terminator that includes both a terminator region and a polyA region.

[0085] In one embodiment of the rHVT according to the invention, in the first expression cassette, the transcription terminator of the VP2 gene is derived from Simian Virus 40 (SV40), preferably from the SV40 late gene.

[0086] This terminator has been available since the late 1980s via the commercially available "pCMVβ" cloning plasmid (Clontech).

[0087] In one embodiment of the rHVT according to the invention, in the first expression cassette, the transcription terminator of the VP2 gene is derived from an SV40 late gene, is about 0.2 kb in size, and comprises a nucleotide sequence having at least 95% nucleotide sequence identity to the entire length of the region from nucleotide 2812 to 3021 of SEQ ID NO: 1. In order of preference, at least 96, 97, 98, or even 99% nucleotide sequence identity is more preferred.

[0088] In one embodiment, the transcription terminator from the SV40 late gene is the region from nucleotides 2812 to 3021 of SEQ ID NO:1.

[0089] The complete hCMV-IE1 gene promoter is approximately 1.5 kb in size and consists of an enhancer, a core promoter and an intron, whereby promoter activity proceeds into the intron region (e.g., Koedood et al. 1995, J. of Virol., vol. 69, p. 2194-2207).

[0090] The hCMV-IE1 gene promoter can be obtained from the genome of the hCMV virus (widely available) by subcloning the genomic region preceding the IE1 gene using conventional molecular biology means and methods. Alternatively, the promoter can be derived from a commercially available expression plasmid, such as pI17 described by Cox et al. (2002, Scand. J. Immunol., vol. 55, p. 14-23), or from a commercially available mammalian expression vector, such as pCMV (Clontech) or pCMV-MCS series (Stratagene; GenBank acc.nr. AF369966). The genome sequence of hCMV is available, for example, from GenBank acc.nr. X17403.

[0091] Many highly similar versions of the hCMV-IE1 gene promoter are known, for example from GenBank. Such homologs and variants are within the scope of the present invention.

[0092] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the hCMV-IE1 gene promoter is a core promoter. Such a core promoter is typically less than 1 kb in size, preferably about 0.4 kb in size.

[0093] In one embodiment, the hCMV-IE1 gene core promoter of the present invention is a DNA molecule of about 0.4 kb comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the entire length of the region from nucleotide 3160 to 3520 of SEQ ID NO: 1. In order of preference, at least 96, 97, 98, or even 99% nucleotide sequence identity is more preferred.

[0094] In one embodiment, the hCMV-IE1 gene core promoter is the region from nucleotides 3160 to 3520 of SEQ ID NO:1.

[0095] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the NDV F protein gene is derived from an attenuated NDV.

[0096] Preferably, the NDV F protein gene from the attenuated NDV strain is from NDV strain clone 30.

[0097] NDV clone 30 is a well-known attenuated NDV that has been used as a live vaccine for many years, for example, Nobilis® ND Clone 30 (MSD Animal Health).

[0098] In one embodiment, the NDV F protein gene of the present invention has at least 90% nucleotide sequence identity to the entire length of the region from nucleotide 3545 to 5206 of SEQ ID NO: 1. Preferably, the nucleotide sequence identity is at least 92, 94, 95, 96, 97, 98, or even 99%, in order of preference.

[0099] In one embodiment, the NDV F protein gene of the present invention is the region from nucleotides 3545 to 5206 of SEQ ID NO:1.

[0100] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the transcription terminator of the F gene is derived from the hCMV-IE1 gene. Preferably, this transcription terminator is about 0.3 kb in size.

[0101] In one embodiment, the transcription terminator is derived from the hCMV-IE1 gene, is about 0.3 kb in size, and comprises a nucleotide sequence having at least 95% nucleotide sequence identity to the entire length of the region from nucleotide 5218 to 5498 of SEQ ID NO: 1. In order of preference, at least 96, 97, 98, or even 99% nucleotide sequence identity is more preferred.

[0102] In one embodiment of a rHVT according to the present invention, the transcription terminator derived from the hCMV-IE1 gene is the region from nucleotides 5218 to 5498 of SEQ ID NO:1.

[0103] To optimize the expression of VP2 and / or F genes for the present invention, their encoding nucleotide sequences can be subjected to codon optimization. This is well known in the art and is generally applied to improve the expression level of DNA or RNA sequences in a context different from the expression level of the natural origin of the encoded protein. This involves adapting the nucleotide sequence to code for the intended amino acid, but with the nucleotide sequence matching the codon preference (tRNA repertoire) of the recombinant vector, host cell or target organism in which the sequence is expressed. As a result, the applied nucleotide mutations are generally silent. Such modifications are generally planned in silico by using one of many computer software programs, after which the desired nucleotide sequence can be synthesized.

[0104] Thus, in one embodiment of a rHVT according to the present invention, the genes encoding the VP2 and / or F proteins are codon-optimized for HVT viral codon preference.

[0105] In one embodiment of the rHVT according to the invention, the first expression cassette is an expression cassette disclosed in WO 2016 / 102647.

[0106] Even more preferably, the first expression cassette is the cassette used in the rHVT construct described in WO 2016 / 102647 as HVP360, available in the commercial vaccine Innovax® ND-IBD (MSD Animal Health).

[0107] A first exemplary expression cassette for the present invention is represented by SEQ ID NO:1 and its elements are set forth in Table 1. [Table 1]

[0108] In one embodiment of a rHVT according to the invention, the first expression cassette of the invention is approximately 5.5 kb in size.

[0109] In one embodiment of a rHVT according to the present invention, the first expression cassette of the present invention is an approximately 5.5 kb DNA molecule comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of SEQ ID NO: 1. In order of preference, at least 96, 97, 98, or even 99% nucleotide sequence identity is more preferred.

[0110] In one embodiment of the rHVT according to the present invention, the first expression cassette of the present invention comprises the nucleic acid shown in SEQ ID NO: 1. Preferably, the first expression cassette is SEQ ID NO: 1.

[0111] As will be apparent to one of skill in the art from the composition of the first expression cassette for use in the present invention, which has two heterologous genes in one large cassette, the first expression cassette is designed and intended for insertion at a single location within the genome of the vector virus.

[0112] Also, as described, since the expression cassette is a self-contained expression module, the first expression cassette of the present invention can be inserted in different orientations at different loci within the Us genome region of the rHVT according to the present invention.

[0113] It has been demonstrated that some loci in the HVT Us genome region allow the insertion of one or more heterologous genes (see, for example, EP 431.668 and WO 2016 / 102647). For example, genes Us2 or Us10, or the region between Us10 and SORF3, or the region between Us2 and SORF3.

[0114] When inserted into a gene, the result is that the normal coding function of the receiving gene is disrupted or completely destroyed in the resulting rHVT. For Us2 and Us10, this was found not to significantly disrupt either the replication of the resulting rHVT or the expression of the inserted heterologous gene.

[0115] Therefore, in a preferred embodiment, the rHVT according to the invention is characterized in that the first expression cassette is inserted into the Us2 gene or the Us10 gene.

[0116] In a further preferred embodiment, the rHVT according to the invention is characterized in that the first expression cassette is inserted into the Us2 gene.

[0117] The HVT Us2 gene, also known as the HVT088 gene, refers to a gene in the genome of the HVT virus that corresponds to the gene located at nucleotides 140065 to 140913 in the genome sequence of HVT strain FC-126, GenBank accession number AF291866.

[0118] In one embodiment, the Us2 gene for inserting the first expression cassette is the Us2 gene of HVT. Preferably, the Us2 gene is derived from the HVT strain FC-126.

[0119] The insertion "into the Us2 gene" of the present invention disrupts the function of the Us2 gene. In one embodiment, the insertion in Us2 deletes at least 25, 50, or even at least 75% of the Us2 gene, as described for HVP360 in WO 2016 / 102647.

[0120] For the present invention, the expression cassette itself can be included in a DNA molecule, such as a cloning or transfection vehicle, such as a plasmid, cosmid, bacmid, etc., to facilitate easy construction, manipulation, and insertion of the cassette into HVT (see WO 93 / 025665 and EP 996.738). Examples of common cloning plasmids are, for example, plasmids from the pBR322 or pUC series. These are widely available commercially.

[0121] A plasmid containing an expression cassette is commonly referred to as a "transfer vector," "shuttle vector," or "donor plasmid." In this context, the plasmid contains the expression cassette with flanking sequence regions from the target insertion locus of the vector's genome to direct insertion.

[0122] Typically, the transfection vector used for transfection does not itself integrate into the genome of the vector, but only facilitates the integration of the expression cassette it carries, for example by allowing insertion to occur by homologous recombination. Thus, in the case of a first expression cassette for use in the present invention, the first expression cassette is preferably flanked at its 5' and its 3' ends by portions of the Us2 gene of the HVT that direct the process of inserting the cassette into Us2.

[0123] Thus, in one embodiment of a rHVT according to the invention, the first expression cassette is flanked by sequences from the Us2 gene of HVT.

[0124] Preferably, the flanking Us2 gene sequences are nucleotides 140143 to 140541 from GenBank accession nr. AF291866 and nucleotides 140541 to 141059 from GenBank accession nr. AF291866.

[0125] As described above, the rHVT vector of the present invention advantageously contains a set of additional heterologous genes by means of a second expression cassette, which genes are stably maintained within the rHVT during replication and expression both in vitro and in vivo.

[0126] Thus, in one embodiment, the rHVT according to the invention comprises a second expression cassette, in the 5' to 3' direction, in this order: a. the ILTV gD gene with an upstream promoter and a downstream terminator, and b. It is characterized in that it contains the ILTV gI gene with an upstream promoter and a downstream terminator, This results in the promoter and terminator being operably linked to the gD gene and the gI gene, respectively.

[0127] In one embodiment of the rHVT according to the invention, in the second expression cassette, the portion of the second cassette having the gD gene with its promoter and terminator, and the gI gene with its promoter are entirely removed from the ILTV genome. As described above, the ILTV gD gene and the ILTV gI gene overlap in the natural context, so that the gD gene terminator and the gI gene promoter are contained in the gI gene and the gD gene, respectively.

[0128] SEQ ID NO:2 shows an example of the nucleotide sequence of a second expression cassette for the present invention as a Hind3 fragment of approximately 3.2 kb, the elements of which are set forth in Table 2. [Table 2]

[0129] Preferably, the gD gene with its promoter and terminator, and the portion of the gI gene with its promoter are formed by a DNA molecule of about 3 kb containing a nucleotide sequence having at least 90% nucleotide sequence identity to the entire length of the region from nucleotide 13 to 3081 of SEQ ID NO: 2. In order of preference, a nucleotide sequence identity of at least 92, 94, 95, 96, 97, 98, or even 99% is more preferred.

[0130] In one embodiment of the rHVT according to the present invention, in the second expression cassette, the portion containing the gD gene having a promoter and a terminator and the gI gene having a promoter is formed by the region from nucleotides 13 to 3081 of SEQ ID NO:2.

[0131] In one embodiment of the rHVT according to the invention, in the second expression cassette, the transcription terminator of the gI gene is derived from FHV1, preferably from the FHV1 Us9 gene, which is disclosed, for example, in GenBank Accession No. D42113.

[0132] In one embodiment of the rHVT according to the invention, in the second expression cassette, the transcription terminator is derived from the FHV1 Us9 gene, is about 0.05 kb in size, and comprises a nucleotide sequence having at least 95% nucleotide sequence identity to the entire length of the region from nucleotide 3097 to 3151 of SEQ ID NO: 2. In order of preference, at least 96, 97, 98, or even 99% nucleotide sequence identity is more preferred.

[0133] In one embodiment, the transcription terminator from the FHV1 Us9 gene is the region from nucleotides 3097 to 3151 of SEQ ID NO:2.

[0134] In one embodiment of a rHVT according to the invention, the second expression cassette of the invention is approximately 3.2 kb in size.

[0135] In one embodiment of a rHVT according to the invention, the second expression cassette of the invention is an approximately 3.2 kb DNA molecule comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of SEQ ID NO: 2. In order of preference, at least 96, 97, 98, or even 99% nucleotide sequence identity is more preferred.

[0136] In one embodiment of the rHVT according to the present invention, the second expression cassette of the present invention comprises the nucleic acid shown in SEQ ID NO: 2. Preferably, the second expression cassette is SEQ ID NO: 2.

[0137] As described, the second expression cassette of the invention, comprising the ILTV gD gene and the ILTV gI gene, is inserted into the intergenic region between the ORFs of UL54 and LORF3 of the HVT genome. These two genes in the HVT genome are located in a so-called tail-to-tail orientation (each reading orientation directed toward each other), so that the intergenic region between UL54 and LORF3 in HVT is formed by the region downstream of the UL54 stop codon and downstream of the LORF3 stop codon. This region in HVT is approximately 0.9 kb in size. Figure 1 herein illustrates these orientations diagrammatically.

[0138] Thus, in a preferred embodiment of the rHVT according to the invention, the second expression cassette of the invention is inserted into the HVT genome region from nucleotides 110.383 to 111.302, as published in GenBank Accession No. AF291866. More preferably, the second expression cassette of the invention is inserted into the HVT genome between nucleotides 110.395 and 110.396, as published in GenBank Accession No. AF291866.

[0139] The rHVT according to the present invention is preferably based on a parent HVT, which is an established HVT vaccine strain, known to replicate well and suitable for inovo inoculation of young birds or avian embryos, such as HVT vaccine strains PB1 or FC-126. These are publicly available: FC-126 from ATCC: VR#584-C, and PB1 is commercially available as a live vaccine in frozen infected cells, for example from MSD Animal Health as Nobilis® Marek THV Lyo.

[0140] The incorporation of the first and second expression cassettes, both defined herein for the present invention, does not increase the virulence or pathogenicity of the parent HVT (to the contrary), and since HVT is naturally non-pathogenic, reversion to virulence is not expected.

[0141] Thus, in one embodiment, the parent HVT used in the generation of the rHVT according to the present invention is an HVT vaccine strain, preferably an HVT vaccine strain of the PB1 or FC-126 strain.

[0142] The rHVT according to the present invention is a live recombinant carrier microorganism or "vector" virus that can be advantageously used for vaccination of poultry, combining the features of being a safe and effective vaccine against Marek's Disease (MD) and one or more or all of Infectious Bursal Disease (IBD), Newcastle Disease (ND) and Infectious Laryngotracheitis (ILT), as well as being genetically stable.

[0143] "Genetically stable" in the context of the present invention 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 inefficient viral replication and / or loss of expression of one or more of the inserted heterologous genes, which may result in overreplication of defective viral mutants. This stability can be conveniently observed, for example, by subjecting the rHVT according to the present invention to subsequent passages in cell culture, using conventional techniques. Viruses reisolated during these steps can be plated on cell culture dishes, covered with agar, and incubated until HVT-specific plaques become visible, all using conventional techniques. The plaques can then be stained for expression of VP2, F or gD and gI proteins, with appropriate positive and negative controls, using suitable antibody preparations in an immunofluorescence assay (IFA) protocol. Any plaques that do not further show fluorescence for a particular heterologous protein can then be scored, whereby preferably about 100 individual plaques from a particular rHVT sample should be observed.

[0144] Surprisingly, it was found that the rHVT according to the present invention maintained the presence and expression of the VP2, F, gD and gI protein genes, respectively, in all plaques tested, even after 15 serial cell culture passages and subsequent replication in vivo for 21 days, as described in detail in the Examples.

[0145] This is a powerful and highly significant improvement over many purported multivalent HVT vector constructs that have been described in the prior art but whose stability in vitro and in vivo has not been demonstrated.

[0146] Moreover, considering that the VP2, F, gD and gI genes are all already used as inserts in effective HVT vector vaccines, the fact that they are stably maintained and expressed is also a reliable proof that the rHVT according to the present invention will induce protective immune responses in poultry against the pathogens from which these antigens are derived as well as MDV, and thus will be an effective multivalent vector vaccine.

[0147] Thus, in one embodiment of the rHVT according to the present invention, one or more conditions selected from the group consisting of the following apply:

[0148] o For the first expression cassette: - the mCMV-IE1 gene promoter is a complete promoter; - The IBDV VP2 gene encodes the VP2 protein from classical IBDV. the transcription terminator of the VP2 gene comprises both a terminator region and a polyA region, preferably the transcription terminator is derived from SV40; - the hCMV-IE 1 gene promoter is a core promoter; - the NDV F gene is derived from an attenuated NDV strain, preferably from the NDV strain clone 30; The transcription terminator of the -F gene is derived from the hCMV-IE1 gene. - the genes encoding the VP2 and / or F proteins are codon-optimized towards the codon preference of the HVT virus, - the first expression cassette is an expression cassette as disclosed in WO 2016 / 102647, even more preferably the first expression cassette is the cassette used in the rHVT construct described in WO 2016 / 102647 as HVP360, the first expression cassette of the invention is a DNA molecule of about 5.5 kb comprising a nucleotide sequence having at least 95% nucleotide sequence identity with the full length of SEQ ID NO:1, with at least 96, 97, 98 or even 99% nucleotide sequence identity being more preferred, in order of preference; - the first expression cassette for the present invention comprises the nucleic acid shown in SEQ ID NO: 1, preferably the first expression cassette is SEQ ID NO: 1, o and / or for the second expression cassette: The second expression cassette comprises, in the 5' to 3' direction, = the ILTV gD gene with an upstream promoter and downstream terminator, and = comprising the ILTV gI gene with an upstream promoter and a downstream terminator, Thereby, the promoter and terminator are operably linked to the gD gene and the gI gene, respectively. - the promoter may be a core promoter or a complete promoter, The genes encoding gD and / or gI genes are codon-optimized toward the HVT virus codon preference; - the promoter of the gD gene is the ILTV gD gene promoter, - the promoter of the gI gene is the ILTV gI gene promoter, - the terminator of the gD gene is the ILTV gD gene terminator, - the terminator of the gI gene is the ILTV gI gene terminator, the second expression cassette is a DNA molecule of about 3.2 kb comprising a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of SEQ ID NO:2, more preferably at least 96, 97, 98 or even 99% nucleotide sequence identity, in that order of preference; - the second expression cassette comprises the nucleic acid shown in SEQ ID NO: 2, preferably the second expression cassette is SEQ ID NO: 2; - the second expression cassette is inserted into the HVT genome region from nucleotides 110.383 to 111.302 as published in GenBank accession number AF291866, more preferably the second expression cassette for the present invention is inserted into the HVT genome between nucleotides 110.395 and 110.396 as published in GenBank accession number AF291866.

[0149] For o and / or rHVT: - the first expression cassette is inserted into the Us2 gene or the Us10 gene, preferably the first expression cassette is inserted into the Us2 gene; - the Us2 gene for the insertion of the first expression cassette is the Us2 gene of HVT, preferably the Us2 gene is from the HVT strain FC-126; The parent HVT used to generate the rHVT is an HVT vaccine strain, preferably the parent HVT is an HVT vaccine strain of the PB1 or FC-126 strain.

[0150] By different cloning and transfection methods, the first and second expression cassettes of the present invention can be used to obtain an rHVT according to the present invention, which stably contains and expresses the expression cassettes in its genome as described herein.

[0151] Therefore, a further aspect of the present invention relates to a method for constructing a rHVT according to the present invention, comprising inserting a first and a second expression cassette as defined in the present invention into a region of the genome of a HVT as defined in the present invention.

[0152] The insertion of the expression cassette according to the invention into the HVT genome to generate the rHVT according to the invention can be carried out in different ways, all known in the art. One convenient way is to use a transfer vector and homologous recombination techniques, as described above.

[0153] Alternatively, rHVTs according to the present invention can be generated using CRISPR / Cas9 technology, for example as described in Tang et al., 2018 (supra).

[0154] In particular, the rHVT-VP2-F vector virus available since 2017 in the commercial vaccine Innovax ND-IBD can be used. This can be further engineered by inserting a second expression cassette as described herein into the UL54-LORF3 locus of the genome of the rHVT described herein using CRISPR / Cas9 technology. The specific guide RNA sequence that can be used to target this insert to this locus is described in the Examples.

[0155] The rHVT according to the present invention can be amplified by common techniques, preferably in vitro, for example by replicating in cultures of avian cells, typically primary chicken embryo fibroblasts (CEFs). These can be prepared by trypsinization of chicken embryos, all of which are well known in the art. The CEFs are plated in monolayers and infected with the rHVTs. This process can be scaled up to industrial scale production.

[0156] Generally, rHVT is collected by harvesting infected host cells that contain rHVT in cell-associated form. These cells are then incorporated into a suitable carrier composition to provide stabilization during freezing and storage. The infected cells are then generally filled into glass ampoules, which are sealed, frozen, and stored in liquid nitrogen. When used for vaccination, the ampoules are thawed, and the infected cells are incorporated into a suitable dilution buffer to stabilize them during use. In a preferred embodiment, the dilution buffer is the buffer disclosed in WO2019 / 121888.

[0157] Although cell-associated frozen storage of HVT is preferred, in situations where the use of liquid nitrogen is not feasible, an alternative is to use freeze-drying. This method takes advantage of the favorable feature of HVT, which can be isolated from its host cells by cell disruption, e.g., French press or sonicator, using whole cultures, which can be clarified by centrifugation and then taken up in a stabilizer and freeze-dried for long-term storage.

[0158] Thus, in a further aspect, the present invention relates to a host cell comprising a rHVT according to the invention.

[0159] A "host cell" of the present invention is a cell that is susceptible to infection and replication by HVT. Examples of such cells are avian cells, particularly lymphocytes or fibroblasts.

[0160] Preferably, the host cell according to the invention is a host cell maintained under in vitro conditions.

[0161] In one embodiment, the host cells according to the invention are primary avian cells, i.e., cells derived from non-human animal tissues or organs in vitro, rather than from an immortalized cell line. Typically, primary cells are only capable of undergoing a small and limited number of cell divisions.

[0162] In one embodiment, the primary avian host cells of the invention are primary chicken embryo fibroblasts (CEF).

[0163] In one embodiment, the host cell according to the invention is an immortalized avian cell. Several immortalized avian cell lines are described, for example, in WO 97 / 044443 and WO 98 / 006824.

[0164] In a preferred embodiment, the immortalized avian host cell according to the invention is an immortalized CEF, preferably an immortalized CEF as disclosed in WO 2016 / 087560.

[0165] As described, a major advantageous use of the rHVT according to the present invention is in a poultry vaccine that provides a safe, stable and effective vaccination against MD, IBD, ND and / or ILT or related disease indications, and can be administered to poultry at a very young age.

[0166] Thus, a further aspect of the present invention relates to an rHVT according to the invention and / or a host cell according to the invention for use in a poultry vaccine.

[0167] Various methods of "use in vaccines" of rHVT or host cells, both according to the invention, are outlined above, including use as cell-free virus or cell-associated virus in host cells in vaccine compositions for inoculating poultry.

[0168] In yet a further aspect, the present invention relates to a vaccine for poultry comprising an rHVT according to the invention and / or a host cell according to the invention and a pharma- ceutically acceptable carrier.

[0169] It is well known that a "vaccine" is a composition containing an immunologically active compound in a pharma- ceutically acceptable carrier. An "immunologically active compound" or "antigen" is a molecule that is recognized by the immune system of an inoculated target and induces a protective immune response from the humoral and / or cellular immune system of the target.

[0170] The vaccine according to the present invention provides protection of poultry against infection and / or disease caused by MDV, IBDV, NDV and / or ILT. This effect is obtained by preventing or reducing the establishment or growth of productive infection by one or more of these viruses in the respective target organs. This is achieved, for example, by reducing the viral load or shortening the period 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 viral infection in the target animals.

[0171] However, depending on the virulence of the MDV, IBDV, NDV, or ILTV field viruses prevalent on a particular farm or in a particular region, it may be necessary to add additional vaccine components of one or more of these viruses to ensure effective vaccination against pathogenic or serological variants of these viruses, all of which are well known in the art.

[0172] Determining the effectiveness of a poultry vaccine or use as a poultry vaccine, both according to the invention, is well within the skill of the routine practitioner and can be done, for example, by monitoring the immunological response after vaccination, or by examining the appearance of clinical symptoms or death after challenge infection, for example by monitoring target signs of disease, clinical scores, serological parameters, or by re-isolating the challenge pathogen, and comparing these results with the vaccination-challenge response seen in non-vaccinated animals. Various methods for assessing each of the four viral infections are well known in the art.

[0173] The protection against MD, IBD, ND and ILT induced by the vaccine, use of vaccine or vaccination according to the present invention is the target of vaccination in improving health and economic performance.This can be evaluated from parameters such as, for example, increasing one or more of survival, growth rate, feed conversion, egg production, and number and health of offspring.Another effect is the reduction of medical expenses and improvement of operational economy.

[0174] Various embodiments, preferences and examples of vaccines, uses of vaccines or vaccinations, all according to the invention, are outlined below.

[0175] The term "poultry" in the present invention relates to avian species that are relevant to veterinary practice and susceptible to inoculation with HVT. Preferred poultry species are chickens, turkeys, geese, ducks and quails. Chickens are the most preferred species.

[0176] For the present invention, poultry can be of any species, breed, or type, such as layers, breeders, meat chickens, cross breeds, or parent lines of any of these species. Preferred species are breeders, meat chickens, and layers. Meat chickens and layer breeds of poultry are most preferred.

[0177] A "pharmaceutical acceptable carrier" is intended to aid in the stabilization and administration of the vaccine while being harmless and well tolerated by the target. Such a carrier may be, for example, sterile water or sterile saline. In more complex forms, the carrier may be, for example, a buffer, and may contain further additives such as stabilizers or preservatives. Details and examples are described in well-known handbooks, such as, for example, "Remington: the science and practice of pharmacy" (2000, Lippincott, USA, ISBN: 683306472) and "Veterinary vaccinology" (P.Pastoret et al. ed., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0178] In the present invention, when the vaccine is in the form of cell-associated HVT, the pharma- ceutically acceptable carrier is preferably a mixture of culture medium, about 10% v / v serum, and about 6% v / v DMSO. This carrier also provides stabilization of rHVT-infected host cells during freezing and frozen storage. The serum can be any serum normally used for cell culture, such as fetal or newborn calf serum.

[0179] Vaccines according to the present invention are prepared from rHVT according to the present invention by the methods described herein that can be easily applied by those skilled in the art. For example, rHVT according to the present invention is constructed by transfection and insertion of an expression cassette described in the present invention by recombination. The desired rHVT is then selected and industrially amplified in smaller or larger volumes, preferably in in vitro cell cultures, for example in CEF. From such cultures, a suspension of host cells infected with rHVT is taken, either as whole infected cells or as a cell-free preparation obtained by cell disruption. This suspension is formulated into a vaccine with a suitable pharmaceutical carrier, and the final product is packaged. The cell-associated vaccine is then stored in liquid nitrogen and freeze-dried at -20°C or +4°C.

[0180] General techniques and considerations to be applied in the manufacture of vaccines under well-known standards for pharmaceutical manufacturing are described, for example, in government directives and regulations (pharmacopoeias, 9 CFR) and well-known handbooks (Veterinary vaccinology and Remington, both supra). Generally, such vaccines are prepared sterile and using pharmaceutical quality grade excipients.

[0181] Such preparations may incorporate microbiological testing for sterility and the absence of adventitious agents, and may include in vivo or in vitro testing to ensure efficacy and safety. After completion of testing for quality, quantity, sterility, safety and efficacy, the vaccine may be released for sale, all of which are well known to those skilled in the art.

[0182] In one embodiment, the poultry vaccine according to the invention is a cell-associated vaccine.

[0183] "Cell-associated" means that the rHVT according to the invention is contained in a host cell in vitro according to the invention, such that a vaccine of this kind contains both a host cell and a rHVT according to the invention.

[0184] The target animals of the vaccine according to the present invention can in principle be healthy or diseased, and can be positive or negative for the presence of MDV, IBDV, NDV or ILTV, or for antibodies against MDV, IBDV, NDV or ILTV. Also, the target can be of any weight, sex or age that is susceptible to vaccination. However, it is clearly preferred to vaccinate healthy uninfected targets, and vaccinate as early as possible to prevent any field infection and its consequences.

[0185] Thus, a vaccine according to the invention prevents both the establishment and progression of infection by MDV, IBDV, NDV or ILTV and can be used as a prophylactic or therapeutic treatment, or both.

[0186] In this regard, a further advantageous effect of reducing viral load with a vaccine according to the invention is to prevent or reduce the transmission or spread of field viruses vertically to offspring, and horizontally within a flock or population, and within a geographical area, resulting in a reduction in the prevalence of MDV, IBDV, NDV or ILTV using a vaccine according to the invention.

[0187] Thus, a further aspect of the present invention is - use of a poultry vaccine according to the invention to reduce the prevalence of MDV, IBDV, NDV or ILTV in a population or geographic area, and - a poultry vaccine according to the invention for reducing the prevalence of MDV, IBDV, NDV or ILTV in a population or geographical area.

[0188] The vaccine according to the present invention already provides multivalent immunity against IBD, ND and ILT by expression of heterologous inserts, and also against MD by the HVT vector itself. Nevertheless, it may be advantageous to carry out further combinations with further immunoactive components. This may serve to boost the immune protection already provided or to extend it to other pathogens.

[0189] Thus, in one embodiment, the vaccine according to the invention comprises at least one additional immunoactive component.

[0190] Such "additional immunoactive 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 invention may itself be added to the vaccine.

[0191] In one embodiment, the at least one additional immunoactive component is an immunostimulatory compound, preferably a cytokine or an immunostimulatory oligodeoxynucleotide.

[0192] The immunostimulatory oligodeoxynucleotide is preferably an immunostimulatory unmethylated CpG-containing oligodeoxynucleotide (INO).Preferred INO is an avian Toll-like receptor (TLR) 21 agonist, such as those described in WO2012 / 089800 (X4 family), WO2012 / 160183 (X43 family) or WO2012 / 160184 (X23 family).

[0193] In one embodiment, the at least one additional immunoactive component is an antigen derived from a microorganism pathogenic to poultry. This antigen may be "derived" in any suitable manner, for example as a "live" attenuated antigen, an inactivated antigen, or a subunit antigen from a microorganism pathogenic to poultry.

[0194] The additional antigen derived from a microorganism pathogenic to poultry is preferably derived from one or more microorganisms selected from the group consisting of:

[0195] -Viruses: infectious bronchitis virus, NDV, adenovirus, avian influenza virus, egg drop syndrome virus, IBDV, chicken anemia virus, avian encephalomyelitis, fowl pox virus, turkey rhinotracheitis virus, duck plague virus (duck viral enteritis), swinepox virus, MDV, avian leukosis virus, ILTV, avian pneumonia virus, and reovirus; - Bacteria: Escherichia coli, Salmonella, Ornitobacterium rhinotracheale, Haemophilus paragallinarum, Pasteurella multocida, Erysipelothrix rhusiopathiae, Erysipelas, Mycoplasma and Clostridium; Parasites: Eimeria; and Fungus: Aspergillus.

[0196] The further antigen may also be a further vector vaccine, such as, for example, HVT-based, MDV2-based, NDV-based, etc.

[0197] In one embodiment of the vaccine according to the invention, the additional antigen derived from a microorganism pathogenic to poultry is a "live" attenuated vaccine strain of MDV, IBDV, NDV or ILTV, which serves to improve and extend the immunogenicity of the vaccine according to the invention, which is advantageous in cases or geographical areas where highly virulent field strains of MDV, IBDV, NDV or ILTV are prevalent.

[0198] In this regard, combinations of HVT with MDV1, MDV2 or HVT are known. For the present invention, MDV of the Rispens strain (MDV1) or SB1 strain (MDV2) or HVT of the FC-126 or PB1 strains are preferred as additional immunoactive components.

[0199] 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.

[0200] Similarly, to improve the response against IBD, the rHVT according to the present invention can be combined with a live IBDV vaccine strain such as D78, PBG98, Cu-1, ST-12 or 89-03.

[0201] As will be appreciated by those skilled in the art, these "combinations" also include vaccination schedules in which the rHVT according to the present invention and additional immunoactive components are not applied in combination or simultaneously, but in a simultaneous or sequential vaccination schedule. For example, the rHVT can be applied in ovo, NDV C2 on day 1, and IBDV89-03 at about day 17 of age.

[0202] Thus, in one embodiment of the vaccine according to the invention comprising at least one additional immunoactive component, the at least one additional immunoactive component is a microorganism selected from the group consisting of a vaccine strain from MDV, IBDV, NDV, or ILTV, or any combination thereof.

[0203] 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.

[0204] Vaccines according to the invention may be prepared by the methods described and exemplified herein.

[0205] Thus, a further aspect of the invention is a method for preparing a poultry vaccine according to the invention, comprising the steps of: a. infecting a host cell in vitro with the rHVT of the present invention; b. culturing and harvesting the infected host cells; c. mixing the harvested infected host cells with a pharma- ceutically acceptable carrier.

[0206] Suitable host cells and pharma- ceutically acceptable carriers for the present invention are described above, and suitable in vitro methods for infection, culture and harvesting are well known in the art and are described and exemplified herein.

[0207] Thus, different aspects and embodiments of the present invention may be advantageously used to produce a safe, stable and effective vaccine for poultry according to the present invention.

[0208] Therefore, in a further aspect, the present invention relates to the use of both the rHVT and / or the host cell according to the invention for producing a vaccine for poultry.

[0209] It is, of course, within the scope of the present invention to incorporate other compounds into the vaccines according to the present invention, such as stabilizers, carriers, adjuvants, diluents, emulsions, etc. Such additives are described in well-known handbooks such as "Remington" and "Veterinary Vaccinology" (both cited above).

[0210] In this way, the efficacy of the vaccine according to the invention to protect poultry against MD, IBD, ND and ILT following a single vaccination at a very young age can be further optimized if necessary.

[0211] Vaccines according to the invention are suitable for administration to poultry targets and can be prepared in a form consistent with the desired route of application and the desired effect.

[0212] Depending on the application route of the vaccine according to the invention, it may be necessary to adapt the composition of the vaccine. This is well within the capabilities of a person 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, 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).

[0213] The vaccine according to the invention can in principle be given to the target poultry by different application routes and at different times during its life, as long as the inoculated rHVT is capable of establishing a protective infection.

[0214] However, since infection with MDV, IBDV, NDV or ILTV can become established at a very young age, it is advantageous to apply the vaccine according to the invention as early as possible. Thus, the vaccine according to the invention can be applied, for example, at the day of hatch ("day 1"), or in ovo, at about day 18 of embryonic development, all of which are well known in the art.

[0215] Thus, in one embodiment, the vaccine according to the invention is administered to poultry in ovo.

[0216] Devices for automated industrial-scale injection of vaccines into fertilized eggs are commercially available, for example from Embrex™ or Vinovotm™. This provides the earliest possible protection while minimizing labor costs. Different routes of in ovo inoculation are known, such as in the yolk sac, in the embryo, or in the allantoic cavity. These can be optimized as needed. Preferably, in ovo inoculation of HVT is performed so that the needle touches the embryo.

[0217] Preferably, the vaccines according to the invention are formulated as injectable liquids, for example as suspensions, solutions, dispersions or emulsions, suitable for either in ovo or parenteral injection.

[0218] In one embodiment, the vaccine according to the invention is administered by parenteral route, preferably by intramuscular or subcutaneous route.

[0219] The exact amount of rHVT according to the invention per animal dose of vaccine according to the invention is not as important as in the case of inactivated or subunit vaccines. This is because rHVT replicates in target animals to a level of biologically sustainable viremia. In principle, the vaccine dose only needs to be sufficient to initiate such a productive infection. A high inoculum hardly shortens the time required to reach optimal viremia infection in the host. Therefore, a very high dose does not increase efficacy, and is also not attractive for economic reasons.

[0220] Therefore, the preferred inoculation dose is 1x10^1 to 1x10^5 plaque forming units (pfu) of the rHVT according to the present invention per animal dose, more preferably 1x10^2 to 1x10^4 pfu / dose, even more preferably 500 pfu / dose to 5000 pfu / dose, and most preferably about 1000 to about 3000 pfu / dose.

[0221] When the vaccine according to the invention is cell-associated, these amounts of rHVT are contained in infected host cells.Methods for counting viral particles of rHVT according to the invention are well known.

[0222] The volume per animal dose of the rHVT according to the invention can be optimized according to the intended route of application: in ovo inoculation is generally applied at a dose of about 0.01 to about 0.5 ml / egg, whereas parenteral injection is generally performed at a dose of about 0.1 to about 1 ml / bird.

[0223] Determining an immunologically effective amount of a vaccine according to the invention or optimizing the volume of vaccine per animal dose is both well within the capabilities of one skilled in the art.

[0224] The administration regimen for applying the vaccine according to the invention to the target organism may be single or multiple administrations in a manner compatible with the formulation of the vaccine and in such amount as is immunologically effective.

[0225] Preferably, the regimen for administering the vaccine according to the invention is incorporated into an existing vaccination schedule of other vaccines that the target poultry may require to reduce stress on the animals and reduce labor costs. These other vaccines may be administered simultaneously, during the same period, or sequentially in a manner compatible with their licensed use.

[0226] As described above and exemplified below, the vaccine according to the invention can be advantageously used by a single vaccination at a very young age to prevent or reduce infection with one, more or all of MDV, IBDV, NDV and ILTV, as well as to prevent or reduce (the symptoms of) disease associated with such infection.

[0227] Thus, further aspects of the invention are as follows.

[0228] - Use of a poultry vaccine according to the invention for preventing or reducing the symptoms of infection with and / or diseases associated with one or more of MDV, IBDV, NDV and ILTV.

[0229] - A method for preventing or alleviating the symptoms of infection with and / or diseases associated with one or more of MDV, IBDV, NDV and ILTV, comprising administering to poultry a vaccine according to the present invention.

[0230] - A method of vaccinating poultry to prevent or reduce the symptoms of infection with and / or diseases associated with one or more of MDV, IBDV, NDV and ILTV, comprising the step of vaccinating poultry with a vaccine according to the present invention.

[0231] Details regarding the use of the vaccine according to the invention by inoculation of poultry, in particular by intramuscular or subcutaneous inoculation of one-day-old chicks and by in ovo inoculation of 18-day-old embryos, have been described above.

[0232] The invention will now be further described with reference to the following non-limiting examples. [Sequence table] Sequence listing in ST25 format Sequence Listing TIFF2025515050000003.tif225153TIFF2025515050000004.tif229153TIFF2025515050000005.tif229150T IFF2025515050000006.tif229150TIFF2025515050000007.tif230150TIFF2025515050000008.tif230151TI FF2025515050000009.tif229150TIFF2025515050000010.tif231149TIFF2025515050000011.tif229151TIFF2025515050000012.tif230152TIFF2025515050000013.tif229152TIFF2025515050000014.tif231153[Example] Example 1: Construction and in vitro testing of multivalent rHVT vectors 1.1. Constructs created and tested Based on the HVT vector construct rHVT-VP2-F (HVP360; WO 2016 / 102647), a series of HVT recombinants were generated that further express the ILTV gD and ILTV gI genes from the UL genome region of the vector. HVP360 expresses the IBDV-VP2 and NDV-F genes from one expression cassette inserted into the HVT Us2 gene. Using the CRISPR / Cas9 technology described by Tang et al. 2018 (supra), an additional cassette expressing ILTV gD-gI was introduced into one of the different sites in the UL region of the HVP360 genome. Several constructs were generated by inserting a second expression cassette, and one rHVT-VP2-F-gD-gI construct (rHVT according to the present invention) was found to have excellent levels of stable replication, heterologous gene expression and vaccination efficacy when tested in vitro and in vivo. To be compared and to illustrate the advantageous effects of the present invention, several rHVT-VP2-F vector constructs with additional inserts of gD-gI expression cassettes are described herein. Insertion site nucleotide numbers are shown relative to the genome of HVT strain FC-126, as published in GenBank Accession No. AF291866. - rHVT construct HVP412: a gD-gI cassette is inserted between UL44 and UL45, specifically between nt.94482 and 94483; - rHVT construct HVP413: a gD-gI cassette is inserted between UL45 and UL46, specifically between nt.95335 and 95336; - rHVT construct G7 (rHVT according to the invention): a gD-gI cassette is inserted between UL54 and LORF3, specifically between nt 110.395 and 110.396, and - rHVT construct G8: The gD-gI cassette is inserted into the LORF3 gene ORF, specifically between nt 111.219 and 111.220. Constructs HVP412, HVP413 and G8 are included herein as comparative examples. Constructs HVP412 and HVP413 are described in more detail in PCT / EP2021 / 087445. The guide RNA sequences used for CRISPR / Cas9-directed insertion of the gD-gI cassette in HVT UL are as follows: Insertion between UL44 and UL45: 5'-ACATCGGGACGTACATCATG-3' (SEQ ID NO:3); Insertion between UL45 and UL46: 5'-CTAACGGTTACTGTGTTTTA-3' (SEQ ID NO: 4); Insertion between UL54 and LORF3: 5'-TTAGATTTCCGGACAGCCTG-3' (SEQ ID NO:5), Insertion into LORF3: 5'-TACCACGCCGAGGCGCCTAT-3' (SEQ ID NO: 6). SEQ ID NOs: 3-6 are shown here in DNA code, as they are all inserted into a DNA plasmid and then transcribed to produce guide RNAs using standard procedures. In the guide RNA of SEQ ID NO: 4, cleavage occurs between nucleotides 3 and 4. In the guide RNAs of SEQ ID NOs: 3, 5 and 6, cleavage occurs between nucleotides 17 and 18 thereof. Guide RNAs were designed using the internet website zlab.bio / guide-design-resources. One of many alternatives is to use the program Geneious Prime® (Biomatters Ltd., New Zealand). A diagram of the expression cassettes used and their insertion into the HVT genome of the rHVT construct G7 of the present invention is shown in FIG. Another insertion of the gD-gI expression cassette into the UL region of the vector construct rHVT-VP2-F was constructed and previously tested with the following insertion: - Intragenic - Central Within the UL39 gene, near the 3' end between genes UL40 and UL41, and Intergenic between UL47 and UL48. 1.2.In vitro genetic stability The various rHVT-VP2-F-gD-gI constructs were serially passaged in vitro 15 times on CEF cells. P15 plaques were observed for expression of the inserted genes by IFA as follows: Overnight established CEF monolayers were infected with one of the rHVT vectors at the 15th passage level. Plates were incubated for 2-3 days until CPE was clearly visible and then fixed with 96% ethanol. VP2 and F expression was detected using specific monoclonal antibodies. gD and gI were detected using chicken polyclonal anti-ILTV antibodies. After the first antibody, Alexa™-labeled conjugates were used as secondary antibodies. Plates were then read by UV microscopy. Approximately 100 plaques were counted for each of the recombinants to assess expression. For the different rHVT constructs carrying the F and VP2 genes in Us2 and the ILTV gD-gI genes at a locus in the UL region, it was observed that plaques were lost several times in later passages or that plaques appeared that no longer showed expression of one or more of the heterologous gene inserts. Nevertheless, for all rHVT-VP2-F-gD-gI constructs, it was possible to isolate at least one clone that was able to replicate well in vitro and maintained stable expression of the heterologous insert for 15 passages in cell culture. These rHVT-VP2-F-gD-gI isolates were then tested for their replication and expression in vivo. Example 2: Characterization of multivalent rHVT vectors in vivo 2.1. Introduction In several in vivo experiments, various multivalent rHVT vectors constructed as described in Example 1 were tested for viral replication, expression of gene inserts, and induction of serological immune responses. Experimental animals were 1-day-old SPF layer hens. To determine the in vivo replication of rHVT vector vaccines, HVT viremia levels were determined 15, 21, or 25 days after vaccination. To confirm the expression of heterologous gene inserts and the induction of specific antibodies, blood samples were taken from inoculated chickens at different times during the study. 2.2.Experiment Group sizes were 10-15 animals and 5 hatchery chickens. Blood samples taken from hatchery chickens on the day of vaccination were serologically tested to confirm that the batch of animals was negative for antibodies to NDV, IBDV and ILTV on the day of vaccination. rHVT vaccine viruses were used at cell passage levels of 15 or 16 and stored as infected CEF in liquid nitrogen. Viral titers (in infected cells) of vaccine stocks ranged from 0.4 to 1.2 x 10^6 pfu / ml. Vaccine doses were 2000 PFU / animal in 0.2 ml of standard HVT / CEF diluent and inoculated subcutaneously in the neck using standard procedures. Control groups were either unvaccinated or received the rHVT parent vector HVP360 as the vaccine. No acclimation was required as chicks were placed in negative pressure isolators immediately after hatching and were subsequently marked and vaccinated. Blood samples were taken from vaccinated chicks on a convenient day after vaccination, e.g., p.v. day 15 or day 25. In the various vaccination-challenge studies (described below), samples for serology were taken just prior to challenge, thus at p.v. weeks 3, 4, or 5. Blood samples were collected from the wing vein into tubes containing clot activator and kept at ambient temperature until processing. Viremia Viremia sampling from spleens was performed as follows: Spleens were isolated postmortem from 5-6 chicks / group on p.v. day 15 or 21. Clean forceps were used for each chick. Spleens were collected in tubes containing 5 ml of 10 mM PBS with phenol red indicator and antibiotics and kept on ice until processing. Spleens were then homogenized, taken up in fresh medium, and cells were counted. To determine rHVT viremia per 5.0 x 10^6 spleen cells, that number of cells was added to a culture dish containing an established CEF monolayer and incubated at 38°C for 3-4 days. Alternatively, viremia was determined from peripheral blood lymphocytes (PBLs) isolated from blood samples taken from vaccinated chicks at p.v. 3 or 4 weeks. 5x10^6 PBLs were then added to CEF monolayers and incubated. When rHVT virus was present intracellularly, it caused a cytopathogenic effect on CEF, which was visible as plaques. Three plates per animal sample were counted. Plates were fixed using 96% ethanol and an immunofluorescence assay (IFA) was applied to stain virus-infected cells with anti-HVT antiserum in combination with staining for one of the antigens VP2, F or gD-gI. As a result, three separate double stainings were performed. Serology Blood samples for testing serological responses were taken at p.v. 2-5 weeks. Samples were centrifuged, serum was collected and complement was inactivated. Serum was used in various tests to determine the seroresponse of vaccinated chickens to the expressed heterologous genes. IBDV-VP2 responses were measured by virus neutralization (VN) assay using classical IBDV virus strain D78. Serological responses to NDV-F, IBDV-VP2 and ILTV-gD-gI were measured by ELISA and expressed in units relative to the results of standard samples. 2.3. Results and Conclusions Viremia rHVT viremia was detected in the spleens of 6 animals / group on day p.v. 15 or in the PBL of 5 animals / group on day p.v. 25. The mean splenic viremia counts per group receiving one of the rHVT vaccines were as follows: - control vector vaccine HVP360: 93 PFU / 5 million spleen cells; -rHVT with gD-gI inserts in the UL39 center or near the 3' end of the UL39 gene. HVT viremia was undetectable, no replication in vivo, -rHVT containing gD-gI inserts of UL40-41 or UL47-48, score: 36, 45 PFU / 5x10^6 respectively. This means less viremia than the HVP360 control and therefore reduced replication in vivo. The mean viremia per group, measured in PBLs and expressed as PFU / 5x10^6 PBLs, was: -HVP360:17, -HVP 412:15, -HVP 413:9, -G7:19, and -G8:8. Conclusion: All rHVT vector constructs HVP412, HVP413 and G7 had good viremia after vaccination comparable to that of HVP360, while the viremia of the G8 construct was moderate. However, the viremia of the rHVT with the gD-gI insert between UL54 and LORF3 (the rHVT according to the present invention) was the best among the tested groups. 2.3.2.In vitro genetic stability The continued expression of the heterologous gene insert was tested in rHVT viruses obtained from vaccinated chickens. Approximately 100 rHVT plaques from all isolates from spleens on day pv15 were analyzed by IFA. Both rHVT vectors carrying the gD-gI insert in UL39 did not replicate in vivo and therefore stability could not be determined. The rHVT vector with the gD-gI insert in UL40-41 showed genetic instability after 15 days of in vivo replication, since only 75% of the tested plaques showed expression of the NDV-F gene and only half of the plaques showed expression of the IBDV-VP2 gene. Similarly, the rHVT with the gD-gI insert between UL47 and UL48 showed plaques without expression of the gD and gI genes. In other rHVT constructs: rHVTs with gD-gI inserts in UL44-45 (HVP412), UL45-46 (HVP413), UL54-LORF3 (G7), or LORF3 (G8), all plaques maintained expression of all heterologous genes: IBDV-VP2, NDV-F, and ILTV-gD and gI after 15 days of replication in vivo. Of these, the G7 construct (rHVT according to the invention) showed full expression of all heterologous inserts even at day 21 of pv. Serology In nature, immune responses to the pathogens from which the three heterologous antigens IBDV, NDV, and ILTV are derived are all highly dependent on humoral immune responses, and as a result, measurement of antibody responses generated by vaccination is a reliable correlate of in vivo protection against infection and / or disease from these pathogens. The serological responses induced by vaccination of chickens with the various rHVT vectors were analyzed by ELISA. Negative controls were hatchery chickens from the same batch of animals tested before vaccination. Positive controls for seroprotection against NDV and IBDV were vaccination with the HVP360 vector. None of the hatched chickens tested on day 1 had detectable antibody titers to one of the pathogens NDV, IBDV, or ILTV. Anti-ILTV serological responses were tested using a commercially available ELISA test (IDScreen™ ILT gI Indirect, from IDvet), where a result value above 611 indicates a protective immune response. The results of the mean ELISA scores (n=6) of the different groups on specific days after vaccination are shown in Table 3. [Table 3] As is evident from the ELISA results shown in Table 3, rHVT with gD-gI inserted between UL47-48 induced only very low levels of anti-ILT antisera at each of the tested pv22, 32 and 42 days, and thus always below protective levels. As a result, rHVT with gD-gI insert between UL47-48 is not useful as a multivalent vector vaccine against ILTV infection, even though it is stable in vitro and in vivo. However, rHVT constructs with gD-gI inserted between UL44-45 (HVP412) or UL45-46 (HVP413) scored well above protective levels on days 32 and 42 pv. Furthermore, the serologic responses against NDV and IBDV induced by vaccination with constructs HVP412 and HVP413 showed ELISA score values ​​very close to those induced by the parent vector HVP360 on all test days. As a result, in these constructs HVP412 and HVP413, the expression and delivery of the NDV-F and IBDV-VP2 genes were not affected by the additional insertion of the ILTV gD and ILTV gI genes into the UL region. 2.3.4. Further serological data from comparative experiments with the rHVT-VP2-F-gD-gI construct As mentioned above, similar serological tests were performed on samples obtained from chicks vaccinated with one of the constructs: HVP412, HVP413, G7 and G8, in the context of several comparative vaccination-priming experiments described in detail below. IBDV serology IBDV serology (measured by ID VET ID Screen® IBD VP2 Elisa) induced by the four rHVT-VP2-F-gD-gI constructs was slightly lower than that induced by HVP360 at week 3 pv, but reached the level of HVP360 at week 4 pv. The rHVT-VP2-F-gD-gI constructs induced mean anti-IBDV titers of 11 Log2 to 12 Log2 at week 4 pv, with titers of HVP412 and G8 being lower than those of HVP413 and G7. Protective titers are approximately 10.2 Log2. NDV serology Similar to IBDV, the NDV serological responses (measured by ID VET ID Screen® ND Elisa) induced by the four tested rHVT-VP2-F-gD-gI constructs were slightly delayed compared to those induced by the rHVT-VP2-F (HVP360) construct itself. Meanwhile, HVP360 already showed a mean anti-NDV titer of 10.4 Log2 at week 3 pv, whereas the four rHVT-VP2-F-gD-gI constructs showed only titers of 7-9 Log2 at that time. However, this recovered quickly and at week 4 pv, all four rHVT-VP2-F-gD-gI constructs induced good anti-NDV titers of about 11 Log2, close to HVP360, whereby the anti-NDV titers of G7 and G8 vaccinations were slightly lower than those induced by HVP412 and HVP413. The protective titer is about 9.7 Log2. ILTV serology ILTV serology (measured by ID VET ID Screen® ILTV Elisa) induced by the four rHVT-VP2-F-gD-gI constructs showed a clear split at both pv3 and pv4 weeks. Anti-ILTV titers induced by HVP412 and HVP413 constructs were 1.5-2 Log points lower than those induced by G7 and G8 constructs, 9.1 Log2 and 9.2 Log2 at pv3 week and 10.6 Log2 and 10.5 Log2 at pv4 week, respectively. That most titers were below the 9.2 Log2 claimed protective titer given by the test manufacturer is not consistent with the excellent clinical protection observed in this study. As a result, serology did not correlate well with in vivo protection in this study. Example 3: Vaccination-challenge study Introduction To further investigate the in vivo serological data described in Example 2, several vaccination-challenge tests were performed, in which the inventors compared in parallel the vaccine efficacy of rHVT-VP2-F-gD-gI constructs found to be stable in vivo: HVP412, HVP413, G7 and G8. All these tests were performed in SPF chickens vaccinated at 1 day of age and challenged with a virulent strain of one of the pathogens NDV, IBDV or ILTV at different times of pv. During these experiments, several samples were taken and parameters of infection and vaccine efficacy were measured. These challenges were mainly performed as previously described, for example, NDV challenge or IBDV challenge described in WO 2016 / 102647, and ILTV challenge described in WO 2019 / 072964. 3.2. Materials and Methods 3.2.1. Commonalities in vaccination trials -SPF White Leghorn layer chickens were hatched in an isolator. Each chick was individually tagged and vaccinated on the same day by subcutaneous route in the neck with 2000 pfu (=10^3.3) of rHVT-VP2-F-gD-gI constructs of cell passage level 15 or 16 administered as infected CEF in 0.2 ml / dose in standard diluent: HVP412, HVP413, G7 or G8. As positive controls, a similar dose of one of the commercially available bivalent rHVT vector vaccines was used: either the HVT-ND-IBD (HVP360, WO 2016 / 102647; Innovax® ND-IBD {MSD Animal Health}) vector, or the HVT-ND-ILT vector (Innovax® ND-ILT {MSD Animal Health}) disclosed in WO 2013 / 057236. - Negative controls were not vaccinated and had group sizes of 9 or 10 birds / group. - Group sizes for the various vaccination-challenge studies were 10, 11, or 15 birds / group. Each group was housed in a different isolator. For each experiment, 5 or 10 hatchlings were bled to confirm seronegative status at the start of the experiment. - Inoculation, viremia and expression were verified by testing spleen or PBL samples at pv2 or 3 weeks as described in Example 2. 3.2.2. Details of the antigenic stimulation applied: IBDV antigen stimulation IBDV challenge was performed in group sizes of 10 chicks according to European Pharmacopoeia monograph 0587. At week 3 pv, each chick was administered 30 chicken infectious doses 50% (CID50) of IBDV strain CS89 by eye drop in 0.1 ml PBS, divided into both eyes. After challenge, chicks were observed for 10 days for clinical signs of IBD, and a clinical score was assigned to each chick daily on a scale of 0 to -3 for no signs, some signs, severe signs, and death, respectively. Clinical signs of IBD are depression, gregariousness, pallor, anorexia, ruffled feathers, and fecal abnormalities. All remaining birds were then euthanized and cysts were scored macroscopically and sampled for histological analysis using common criteria such as: grossly enlarged and edematous, microscopically lymphocyte depletion, necrosis, and percentage of follicles showing heterophyllic influx (25% block). Observations in any of these tests were also included as part of the total clinical score. NDV antigen stimulation NDV challenge was performed according to the European Pharmacopoeia monograph 0450 using 15 chicks / group. Specifically, challenge was performed at week 5 pv by administering 5 Log10 EID50 of NDV strain Herts 33 / 56 in 0.2 ml PBS given by intramuscular route to one chick. A group of 9 non-vaccinated chicks served as challenge controls. Chicks were observed daily for up to 14 days after challenge and an NDV clinical score was assigned to each chick daily on a scale of 0 to -3 for no signs-some signs-severe signs-death, respectively. Typical signs of NDV infection are neurological symptoms such as twisted neck, obviously droopy wings, uncoordinated gait, paralysis, muscle tremors, and body twisted backwards. On day pv21 (the day before challenge), blood samples were taken from 5 chicks / group to confirm viremia as well as serology and expression of the heterologous gene in PBL. ILTV antigen stimulation ILTV challenge was performed according to European Pharmacopoeia monograph 1068, starting with 11 chicks / group. Challenge was performed on 10 chicks / group at week 4 pv with 3.0 Log10 EID50 of ILTV strain 96-3 as CAM homogenate in standard cell culture medium. ILTV challenge virus was administered intratracheally via syringe at 0.1 ml / chick into the mid-part of the trachea. Chicks were observed twice daily (morning and evening) for signs of ILT for 7 days after challenge. Each morning, a clinical score was assigned on a scale of 0 to 3 for no signs, some signs, severe signs, and death, respectively. Clinical signs typical of ILTV infection are marked difficulty in breathing, labored breathing, gasping, expectoration of blood, nasal discharge, conjunctivitis, and swelling of the sinuses. Seven days after challenge, chicks were euthanized and the trachea was isolated and scored for signs of ILTV infection by noting redness and the type and consistency of contents. Observations in any of these tests were also included as part of the total clinical score. On day pv28 (the day before challenge), blood samples were taken from 10 chicks / group to check viremia, serology and expression of the heterologous gene. 3.3.Results Common findings from antigen stimulation All vaccinated chicks that received the HVP412, HVP413, G7 or G8 vaccines showed good viremia of the rHVT vectors and good expression of all heterologous gene inserts at 3, 4 or 5 weeks post-vaccination, as described in Example 2. For all comparative challenge studies, hatchlings tested on day 1 were seronegative for antibodies to NDV, IBDV and ILTV. The total clinical score used for ILTV and IBDV results was the sum of all clinical scores assigned to all 10 chicks / group over the observation period, as well as the scores derived from gross and microscopic examinations performed. IBDV antigen-stimulated infection IBDV vaccination efficacy was tested at week 3 pv and all four rHVT-VP2-F-gD-gI constructs tested showed excellent IBDV vaccine efficacy as all chicks in these groups were 100% protected, even though the challenge was severe; all ten negative control chicks showed severe signs of IBDV infection on days 3 or 4 post-challenge and all died or had to be euthanized. Within the protected groups, few distinctions could be made based on the very faint residual clinical signs observed, with the HVP412 vaccine group having a total clinical score of 7, the HVP413 group 5, and the G7 group only a total clinical score of 2. As a result, all rHVT-VP2-F-gD-gI constructs induced excellent protection against IBDV antigen-challenged infection, while the vector vaccine of the G7 construct (rHVT according to the present invention) hardly caused any clinical signs anymore, even lower than the HVP412 and HVP413 test groups. NDV antigen-stimulated infection Protection induced by the multivalent rHVT-VP2-F-gD-gI vector vaccine against severe NDV challenge was tested 5 weeks after vaccination. The severity of the challenge was confirmed by the fact that all non-vaccinated controls showed extensive clinical signs of NDV infection within 48 hours and all had to be euthanized for humane reasons. The HVP412 construct was able to protect only 87% of the chicks (2 of 15 deaths) and the G8 construct performed even worse, protecting only 53% of the chicks (7 of 15 deaths). In contrast, both the HVP413 and G7 constructs were able to protect 100% of the chicks against the consequences of severe NDV challenge infection. ILTV antigen-stimulated infection Protection against ILTV challenge was tested at week 4 pv and all four rHVT-VP2-F-gD-gI vector vaccines tested provided excellent protection, with all four inducing 100% protection. In contrast, in the negative control group, all ten chicks showed severe clinical signs of ILTV infection on days 4 or 5 post-challenge, seven of the ten chicks died, and two of the remaining chicks had to be euthanized. Based on the limited clinical signs observed in these vaccinated groups, some further distinctions can be made regarding the vaccine efficacy induced by the rHVT-VP2-F-gD-gI construct, while groups receiving the HVP412 or HVP413 vaccines still had a clinical score of 20, respectively.37 The G7 vaccinated group had a clinical score of only 6. As a result, among the rHVT-VP2-F-gD-gI constructs tested for ILTV vaccination efficacy, the G7 construct (rHVT according to the invention) performed best, preventing almost all clinical signs of severe ILTV challenge infection, a protection against ILTV challenge that was almost as good as that induced by the commercial vector vaccine HVT-ND-ILT, which typically only achieved a clinical score of 1 in similar studies. 4. Conclusion The vaccination-priming experiments described in Example 3 serve to confirm and elaborate on the in vitro results of Example 1, as well as the in vivo and serological data described in Example 2. Together, these examples provide a complete characterization of the various rHVT-VP2-F-gD-gI vector vaccine constructs tested, i.e., not only their efficient replication and heterologous gene expression in vitro and in vivo, but also their ability to induce effective immune responses that effectively protect target animals from infection and / or disease caused by severe challenge infection with the respective pathogens. Also, by testing them side by side in the same study, their properties and respective vaccine potencies could be carefully compared under the same conditions. Of the many rHVT-VP2-F-gD-gI constructs that were created and tested over time, only four were found to be genetically stable in vivo: HVP412, HVP413, G7, and G8. All of them provided 100% vaccination efficacy against challenge with IBDV at week 3 pv and ILTV at week 5 pv. However, against challenge with virulent NDV, only constructs HVP413 and G7 provided 100% protection at week 5 pv, while the other two provided (much) less than 90% protection, making them practically ineffective NDV vaccines. Among HVP413 and G7, a further distinction could be made based on the level of remaining clinical signs observed after challenge, with the rHVT-VP2-F-gD-gI construct G7 showing fewer remaining clinical signs than the HVP413 vector vaccine after both IBDV and ILTV challenge infections, i.e., the G7 construct provided more effective protection than the HVP413 construct. In conclusion, of the many rHVT-VP2-F-gD-gI constructs made and tested, the rHVT according to the invention, construct G7, had the most advantageous properties when considered overall. In most circumstances, G7 even matched the characteristics and vaccine properties of a commercial rHVT vector vaccine carrying only two inserted heterologous genes. This is counterintuitive and better than could reasonably be expected for a vector virus carrying four inserted heterologous genes. [Brief description of the drawings]

[0233] [Figure 1]1 is a diagram of an exemplary rHVT vector construct according to the invention. At the top, the HVT genome is shown with many of its ORFs indicated with block arrows. Thin line boxes indicate repeat regions. In the middle, between UL54 and LORF3, and in Us2, are enlarged regions of the HVT genome where inserts were introduced. At the bottom, examples of two inserted expression cassettes are displayed. - Bottom left: gD gene promoter-gD gene (including gI gene promoter)-gI gene (including gD gene terminator)-FHV1 Us9 gene terminator. - Bottom right: mCMV-IE1 gene promoter-IBDV VP2 gene-SV 40 late gene terminator-hCMV IE1 gene promoter-NDV F gene-hCMV-IE1 gene terminator.

Claims

1. Recombinant turkey herpesvirus (rHVT) is characterized in that the rHVT can express the infectious bursal disease virus (IBDV) viral protein 2 (VP2) gene and the Newcastle disease virus (NDV) fusion (F) protein gene from a first expression cassette inserted into a unique short (Us) region of the rHVT genome, and also expresses the infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) and glycoprotein (gI) genes from a second expression cassette inserted into a unique long (UL) region of the rHVT genome between the UL54 gene and the LORF3 gene.

2. The rHVT according to claim 1, wherein the first expression cassette is in the 5' to 3' direction, in this order, a. Mouse cytomegalovirus early stage 1 (mCMV-IE1) gene promoter, b. IBDV VP2 gene, c. Transfer terminator, d. Human cytomegalovirus early stage 1 (hCMV-IE1) gene promoter, e. NDV F protein gene, and f. Transfer terminator, Includes, As a result, the promoter and the terminator are operably linked to the VP2 gene and the F gene, respectively, in an rHVT.

3. The rHVT according to claim 1 or 2, characterized in that the first expression cassette is inserted into the Us2 gene.

4. The second expression cassette is in the 5' to 3' direction, in this order. a. ILTV gD gene having an upstream promoter and a downstream terminator, and b. Characterized by containing an ILTV gI gene having an upstream promoter and a downstream terminator, The rHVT according to claim 1 or 2, wherein the promoter and the terminator are operably linked to the gI gene, respectively, to the gD gene.

5. A method for constructing an rHVT according to claim 1 or 2, comprising inserting the first and second expression cassettes according to claim 1 or 2 into the region of the genome of the HVT according to claim 1.

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

7. rHVT according to claim 1 or 2, for use in poultry vaccines.

8. The host cell according to claim 6, for use in a poultry vaccine.

9. A poultry vaccine comprising the rHVT described in claim 1 and a pharmaceutically acceptable carrier.

10. A poultry vaccine comprising the host cells described in claim 6 and a pharmaceutically acceptable carrier.

11. The vaccine according to claim 9 or 10, comprising at least one additional immunoactive ingredient.

12. A method for preparing a poultry vaccine according to claim 9 or 10, a. A step of infecting host cells in vitro with the rHVT described in claim 1 or 2, b. The process of culturing and collecting the infected host cells, c. A method comprising the step of mixing the collected infected host cells with a pharmaceutically acceptable carrier.

13. Use of rHVT according to claim 1 or 2 for manufacturing a poultry vaccine.

14. Use of the poultry vaccine according to claim 9 or 10 for preventing or mitigating symptoms of infection by one or more of MDV, IBDV, NDV, and ILTV, and / or related diseases.

15. A method for preventing or mitigating symptoms of infection with one or more of MDV, IBDV, NDV, and ILTV, and / or related diseases, comprising administering the vaccine according to claim 9 or 10 to poultry.

16. A method for vaccinating poultry to prevent or reduce signs of infection with one or more of MDV, IBDV, NDV, and ILTV, and / or related diseases, comprising administering the vaccine according to claim 9 or 10 to the poultry.

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