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 comprises four heterologous genes from poultry pathogens: a VP2 gene from IBDV, an F gene from NDV, and gD and gI genes from ILTV. VP2 and F genes are inserted into the Us genome region of the rHVT. The gD-gI gene is inserted into a UL genome region between the UL54 gene and the LORF3 gene. It is proved that the novel rHVT-VP2-F-gD-gI is genetically stable in vitro and in vivo, and all inserted genes can be well expressed. The vaccine is also an effective vaccine for resisting severe aggressive infection of NDV, IBDV and ILTV.

Description

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

[0002] Recombinant vector viruses are a well-known method for expressing heterologous genes and delivering the proteins they encode to human or non-human animal targets. Examples include vaccinia virus vectors, measles virus vectors, or adenovirus vectors. When the heterologous gene encodes an immunogenic protein from a pathogen, this can be an effective vaccination method for the target to combat the disease caused by the pathogen. As a replicating microorganism, the vector virus can establish a productive infection in the inoculated target, express the heterologous gene and its own genes, and in this way induce a protective immune response in the target against the antigen encoded by the heterologous gene.

[0003] In veterinary vaccination, especially poultry vaccination, vector vaccines have attracted attention due to their relative ease of use and low cost. Several avian vector vaccines have long been considered, for example based on avian adenovirus, fowl pox virus, and in particular herpesvirus of turkeys (HVT), see WO87 / 04463 and WO90 / 002803. The advantage of using HVT as a vector is that it is non-pathogenic to poultry, can carry and express inserted genes, and induce immunity against Marek's disease virus serotype 1 or 2 (MDV1 or MDV2), pathogenic members of its virus family.

[0004] Over the years, genes from different poultry pathogens have been inserted into and expressed by HVT virus vectors, for example from: Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus, see: WO93 / 025665; from avian influenza virus (AIV), see WO2012 / 052384; or from the parasite Eimeria (Cronenberg et al., 1999, Acta Virol., vol. 43, p. 192-197). This has led to the development of a variety of commercial HVT vector vaccines for poultry, for example: against ND: (MSD Animal Health) and HVT-NDV (Ceva Santé Animale); for ILT: (MSD Animal Health); For IBD: HVT+IBD (Boehringer-Ingelheim; formerly Gallivac TM HVT-IBD) and IBD (Ceva Santé Animale); and for AI: AI (Ceva Santé Animale).

[0005] Inserting heterologous genes into the viral genome can burden the vector virus because it may affect its replication, expression and / or genetic stability in vitro and / or in vivo. These problems are particularly prominent when more than one heterologous gene is inserted. Such multivalent recombinant vector vaccines may be able to protect against multiple diseases after a single vaccination. However, such vector constructs must still provide good replication of the vector and its inserts both in vitro and in vivo, and effectively express all heterologous genes at sufficiently high levels for a considerable period of time to induce and maintain a protective immune response of the vaccination target against all intended pathogens.

[0006] The genetic stability of the recombinant vector virus also allows the large-scale in vitro replication required for large-scale production. In addition, this stability is to meet the extremely high safety and biostability standards that the recombinant virus must meet in vivo (as a genetically modified organism) in order to obtain marketing approval from the government or regulatory agency so that it can be introduced into the field as a commercial product.

[0007] Many multivalent HVT vector vaccines have been described for a long time, for example: WO93 / 025665 and WO96 / 005291. However, most of the multigene constructs described in these publications are only suggestive, and only some recombinant vectors with multiple inserts have actually been constructed and isolated. Few have been tested in birds. In general, no results are given on their replication stability or the level of expression of foreign genes, let alone any data on the induction of effective immune protection in the target animals. Due to challenges with genetic stability and sustained expression of the inserts, only very few multivalent HVT vector constructs have actually been licensed as commercial vaccine products. Currently these are: ND-IBD (MSD Animal Health; WO2016 / 102647), ND-ILT (MSD Animal Health; WO 2013 / 057236), IBD ND (Ceva Santé Animale; WO 2013 / 144355), HVT+IBD+ND and HVT+IBD+ILT (both Boehringer-Ingelheim; WO 2018 / 112.051).

[0008] The efficacy of ND-IBD is also documented in: M. et al., 2021, Avian Pathol., vol. 50, p. 18–30. The efficacy of ND-ILT is also 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 rHVT containing different heterologous genes: IBDV VP2 gene inserted between HVT UL45 and UL46 genes; ILTV gD and gI genes inserted between UL65 and UL66; and AIV HA gene in Us2. These rHVT were only tested in in vitro cell culture.

[0010] WO2016 / 102647 describes an HVT vector vaccine in which rHVT expresses the IBDV VP2 gene and the NDV F gene in a single expression cassette inserted into the Us2 gene in the HVT Us genomic region, each driven by a separate promoter.

[0011] WO2018 / 112051 describes an HVT vector vaccine comprising two genes selected from IBDV VP2, ILTVgD and NDV F. The heterologous gene is inserted into the IG1 locus or between SORF3 and Us2 of the HVT genome and inserted as a single expression cassette using a single promoter, whereby the two genes are separated by an IRES or P2A sequence.

[0012] WO2019 / 072964 describes a multivalent rHVT vector vaccine that provides protection against MDV, NDV, IBDV and ILTV. Inserts are placed in the UL54.5 (LORF3) gene and the Us2 gene of the HVT genome.

[0013] However, as there are a variety of methods for treating poultry diseases, there is a constant need for further and improved options for effective poultry vaccination.

[0014] Therefore, an object of the present invention is to meet the needs of the art and provide a rHVT vector vaccine that can immunize poultry against four avian pathogens (MDV, NDV, IBDV and ILTV).

[0015] Surprisingly it was found that by providing rHVT expressing IBDV VP2 and NDV F genes from the Us genomic region and ILTV gD and gl genes from the UL genomic region this objective can be met and thus one or more disadvantages of the prior art can be overcome.

[0016] The inventors sought to extend the protection already provided by known rHVT expressing IBDV-VP2- and NDV-F genes ("rHVT-VP2-F") with protection against ILTV. The additional heterologous genes selected were the ILTV gD and gI genes. Several multi-insert rHVTs failed to generate stable multivalent recombinant viruses when tested in vitro and in vivo: some were unable to replicate multivalent recombinant HVT, and some lost expression of one or more heterologous genes.

[0017] For example, additional insertion of the gD and gI genes into the site of the HVT UL39 gene (ribonucleotide reductase large subunit) was unsuccessful, either into the central part or into the 3' region of the UL39 gene. Although these rHVT constructs replicated in vitro in CEF cells, they were completely unable to replicate when inoculated into chickens.

[0018] Several constructs including the ILTV gD and gI genes inserted in the HVT UL genomic region were also ineffective: between the UL40 (ribonucleotide reductase small subunit) and UL41 genes (viral host shutoff protein), or between the UL47 gene (tegument phosphoprotein) and UL48 genes (immediate early gene transactivator): The construct with the UL40-41 insertion replicated normally in vitro (compared to the rHVT-VP2-F construct), but at reduced levels in chickens. In addition, this construct proved to be genetically unstable in vivo, as it lost expression of the F and VP2 genes. A similar situation was observed with the UL47-48 insertion construct, which replicated normally in vitro, had a reduced replication rate in vivo, and was also genetically unstable in vivo, as the expression levels of the gD and gI genes were very low and insufficient for effective vaccination against ILTV. This finding with the UL47-48 insertion construct was particularly disappointing because the UL47 and UL48 genes have been reported to be non-essential in transposon knockout studies of the HVT genome (Hall et al., 2015, Virology Journal, vol. 12, p. 130).

[0019] The inventors' observations are consistent with the common understanding in the art that more inserts cause more problems with the genetic stability of the viral vector in terms of replication and foreign gene expression. In this case, the additional expression of the ILTV gD and gI genes is obviously complicated by the fact that the parental vector already expresses two other heterologous genes. It even reached the point where successful multivalent recombinant HVT constructs could not be predicted based on prior art observations of permissible insertion sites for heterologous genes in the HVT genome. Similarly, reports of efficient in vitro replication of certain rHVT constructs (e.g., as described in Tang et al., 2020, as described above) could not be relied upon to predict in vivo properties.

[0020] Therefore, it was unexpected that the additional integration of the expression cassette with the ILTV gD and gI genes into a specific insertion site in the HVT UL genomic region (specifically, gD and gI were inserted between the UL54 and LORF3 genes of HVT) did produce a stable and effective multivalent HVT vector construct. This insertion site is referred to herein as the "UL54-LORF3" site.

[0021] The resulting multivalent rHVT vector with F and VP2 inserted in Us and gD and gI genes inserted in the UL54-LORF3 locus was found to be genetically stable, even after 15 serial passages in in vitro cell culture. The virus was subsequently used to inoculate chickens, resulting in several in vivo replication cycles. The virus was then re-isolated from the inoculated chickens at day 21 post-inoculation to determine the level of viremia and to check for continued expression of all inserted genes. The virus re-isolated 21 days post-inoculation was found to be genetically completely stable: in immunofluorescence plaque assays, all re-isolated viruses showed expression of all heterologous genes (F, VP2, gD and gI), even 3 weeks post-inoculation.

[0022] Vaccinated chickens also showed excellent seroconversion against each of the expressed heterologous antigens (F, VP2, gD and gl). The antibody levels achieved were well above those required for known in vivo protection against infection or disease; details are provided in the Examples.

[0023] Surprisingly, new rHVT vectors were also found to be even more effective vaccines compared to the rHVT constructs described as HVP412 and HVP413 in patent application number PCT / EP2021 / 087445; these rHVT vectors also have the F and VP2 genes inserted into the Us2 gene, but have the ILTV gD and gI genes inserted between the UL44 and UL45 genes or between the UL45 and UL46 genes of the HVT genome, respectively. In particular, the new rHVT vectors using the UL54-LORF3 insertion site for ILTV gD and gI provide protection against infection and / or disease caused by NDV, IBDV and ILTV that is even superior in multiple parameters to the protection induced by the rHVT vector vaccines HVP412 and HVP413 of '87445.

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

[0025] The possibility of obtaining vaccination against four major poultry diseases from a single vaccine is very beneficial as it represents a significant reduction in stress for the target animals as well as a reduction in labor and costs for the poultry keepers.

[0026] It is unclear how or why rHVT expressing VP2 and F genes can tolerate additional expression of ILTV gD and gl genes in the UL54-LORF3 insertion site, while insertions in several other sites, which at first glance seemed appropriate, failed to result in stable and efficient vector constructs.

[0027] Although the inventors do not wish to be bound by any theory or model that might explain these findings, they postulate that this effect results from a complex interaction of the various expression patterns in the multivalent rHVT (necessary when replicating and expressing in vitro and in vivo). For unknown reasons, the insertion of additional genes at this particular locus in the UL resulted in a multivalent rHVT with a perfect balance between the intensity of expression of the heterologous genes and the resulting stress on the replication capacity and genetic stability of the HVT itself, whereas other constructs (unexpectedly) did not have this balanced composition.

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

[0029] "Herpesvirus of turkeys (HVT)" is also known as MDV3, Meleagridherpesvirus 1 or turkey herpesvirus. HVT was first described in 1970 (Witter et al., 1970, Am. J. Vet. Res., vol. 31, p. 525). Well-known HVT strains 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] Turkey herpes virus, Newcastle disease virus, infectious bursal disease virus and infectious laryngotracheitis virus are all well-known veterinary-related viruses. So are mouse and human cytomegalovirus (mCMV and hCMV) and feline herpes virus (FHV). Such viruses have the characteristics of their taxa (e.g., morphological, genomic and biochemical characteristics) and biological characteristics (e.g., physiological, immunological or pathological behavior).

[0031] General information about these viruses can be obtained from, for example, reference books such as Fields Virology (LWWpubl., ISBN:9781451105636). Information about the infection and disease caused by these viruses can be obtained from reference books such as "The Merck veterinary manual" (2010, 10th ed., 2010, CMKahn edt., ISBN:091191093X) and "Diseases of poultry" (2008, 12th ed., Y.Saif ed., Iowa State Univ. press, ISBN-10:0813807182). Samples for these viruses of the present invention can be obtained from various sources, such as viruses isolated from non-human animals in humans, wild or farms or various laboratories, (preservation) institutions or (veterinary) universities. Conventional serology or molecular biology tools can be used to easily identify viruses. Extensive genetic information for all of these viruses is available in public sequence databases such as NCBI's GenBank TM , UniProt and EBI at EMBL) are available in digital form.

[0032] As is known in the art, the classification of microorganisms in a particular taxonomic group is based on a combination of their characteristics. Therefore, the present invention also includes variants of these viral species that are subclassified in any manner, such as as subspecies, strains, isolates, genotypes, variants, subtypes or subgroups, etc.

[0033] Furthermore, it will be apparent to those skilled in the art to which the present invention pertains that while a particular virus of the present invention may currently be classified as that species, this is a taxonomic classification that may change over time as new insights may lead to reclassification into a new or different taxonomic group. However, since this does not change the virus itself or its antigenic group, but only its scientific name or classification, such reclassified viruses remain within the scope of the present invention.

[0034] "Recombinant HVT" of the present invention refers to an HVT in which the genetic material has been modified relative to its parental condition by the insertion of a molecule of a nucleic acid encoding a heterologous antigen.

[0035] For the purposes of the present invention, a gene or antigen is "heterologous" if it originates from a (micro)organism other than MDV or HVT. Thus, the rHVT of the present invention is not a chimeric viral construct in which a part of the genome has been exchanged for a corresponding part from a related virus (such as is the case with the "novel avian herpesvirus" described in WO 1998 / 037216).

[0036] "VP2 protein gene" is well known in the art, and encodes the capsid protein of IBDV. The VP2 protein gene may be derived from a classical IBDV or from a variant of IBDV, or may be chimeric.

[0037] Similarly, "F protein gene" is well known, which encodes the fusion glycoprotein of NDV. For the present invention, the F protein gene can be obtained from a mild, medium or virulent type of NDV, or can be chimeric.

[0038] The term "gene" is used to refer to a nucleic acid segment capable of encoding a protein. For the present invention, this corresponds to an "open reading frame" (ORF), i.e., a protein-coding segment of DNA from the start codon to the stop codon, excluding the promoter of the gene. The gene of the present invention can encode a complete protein, or a fragment of a protein, such as only the mature form of the protein, i.e., without a "leader", "anchor" or "signal sequence". The gene can even encode a specific fragment of a protein, such as a fragment including an immunoprotective epitope.

[0039] In this regard, the "protein" of the present invention is an amino acid molecule chain. The protein can be a native or mature protein, a preprotein or proprotein, or a functional fragment of a protein. Therefore, peptides, oligopeptides and polypeptides are all included in the definition of protein as long as they still contain relevant immune epitopes and / or functional regions.

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

[0041] An "expression cassette" is a nucleic acid fragment comprising at least one heterologous gene and a promoter driving the transcription of the gene. Termination of transcription may be due to sequences provided by the genomic insertion site of the cassette in the vector genome, or the expression cassette itself may include a termination signal, such as a transcription terminator.

[0042] In such a cassette, both the promoter and (optionally) the terminator need to be in close proximity to the gene whose expression they regulate; this is referred to as being "operably linked", ie there are no significant other sequences between them that would interfere with efficient initiation and termination, respectively, of transcription.

[0043] Although the expression cassette can be present in DNA or RNA form, due to its intended use in the HVT vector, the expression cassette of the present invention is used in DNA form. It is obvious to those skilled in the art that the expression cassette is a self-contained expression module, so its orientation in the vector viral genome is generally not critical.

[0044] Optionally, the expression cassette may contain other DNA elements, for example DNA elements that facilitate construction and cloning, such as sites for restriction enzyme recognition or PCR primers.

[0045] The expression cassette is inserted as a whole into a single locus of the vector genome. Different techniques can be used to control the locus and the orientation of the insertion. For example, by using flanking fragments of the vector genome to integrate the cassette through a homologous recombination process in a specific manner (e.g., using overlapping cosmids as described in US 5,961,982). Alternatively, integration can be performed using the CRISPR / Cas9 technology described in Tang et al., 2018 (Vaccine, vol. 36, p. 716-722).

[0046] For the present invention, an "inserted" expression cassette in a vector genome refers to one that is integrated into the genomic nucleic acid of the vector so that the inserted element is transcribed and translated along with the native genes of the vector. The effect of this insertion on the vector genome varies depending on the method of insertion: the vector genome may become larger, unchanged, or smaller, depending on whether the end result on the genome is the addition, replacement, or deletion of genetic material, respectively. One skilled in the art is fully capable of selecting and implementing a certain type of insertion and making adjustments as necessary.

[0047] The construction of the expression cassette and its insertion into the HVT vector can be accomplished by well-known molecular biology techniques, including cloning, transfection, recombination, selection and amplification. These and other techniques are explained in detail in standard textbooks 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); and C. Dieffenbach & G. Dveksler: PCR primers: a laboratory manual (CSHL Press, ISBN 0879696540); and PCR protocols, J. Bartlett and D. Stirling (Humana press, ISBN: 0896036421).

[0048] For the present invention, the terms "first" and "second" with respect to the expression cassettes are used only for ease of reference 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 segment of the genome between the “internal short repeat sequence” and the “terminal short repeat sequence.” The size of HVT Us is about 8.6 kb (see Kingham et al., 2001, J. of Gen. Virol., Vol. 82, pp. 1123-1135).

[0050] Fully annotated genome sequences of several HVT strains are publicly available, for example, through GenBank, for example: the genome sequence of HVT strain FC-126 available under GenBank Accession No. AF291866, wherein nucleotides 136990-145606 form the Us region.

[0051] "ILTV" is also known as: Gallid alphaherpesvirus 1. The fully annotated genome sequences of several ILTV strains are publicly available, for example, through GenBank, for example: ILTV reference strain SA-2 under accession number NC_006623. The genes for ILTV gD and gI envelope glycoproteins are located in the Us region of the ILTV genome, such as Us6 and Us7 genes, respectively. ILTV gD and gI proteins can induce ILTV-specific and protective antibodies, and they are usually used in combination. In their natural situation, 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 gIORF. Therefore, when used in combination in the ILT virus genome or taken out from its natural environment, the gD and gI genes can be easily subcloned as a continuous fragment, for example starting from the gD gene promoter and terminating after the gI gene.

[0052] The "Unique Long (UL)" region of the HVT genome is the upstream part of the genome and is approximately 110 kb in size in HVT. In the genome sequence of HVT strain FC-126 in GenBank Accession No. AF 291866, the UL region is formed by nucleotides 5910-117777.

[0053] The term "UL54" and similar terms used herein are well-known in the art of the invention and are used to refer to a specific gene located in the herpesvirus genome, herein: in the UL genomic region of HVT, see, for example, the annotation used in Genbank Accession No.: AF291866. This nomenclature is derived from the homologous gene in the herpesvirus type species type 1 herpes simplex virus (Herpessimplex virus 1). The same (mutatis mutans) applies to other HVT gene terms used herein.

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

[0055] The HVT gene LORF3, also known as the HVT064 gene, is also known as UL54.5 and is sometimes referred to as LORF4, similar to the nomenclature for genes at this position in MDV1 and MDV2. The HVT LORF3 gene encodes a protein of unknown function that is hypothesized to be related to the avian host range of HVT and may be involved in some aspects of lymphocyte chemotaxis.

[0056] The term "between" is used to indicate that the inserted second expression cassette is located at an insertion site (i.e., locus) on the HVT genome, which is located in the middle and outside of the specified gene and is located in the so-called intergenic region. Therefore, such an insertion is not in the open reading frame of the gene to which it is related, for example not in the ORF of UL54 or LORF3. Therefore, this is an essentially different insertion compared to the intragenic insertion into the ORF, such as the insertion into the LORF3 (UL54.5) gene of HVT applied in WO2019 / 072964.

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

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

[0059] For the purposes of the present invention, a virus is "chimeric" when a region of the viral genome is replaced by a corresponding region of the genome of a related virus; for example, a chimeric HVT is an HVT in which a portion of the HVT genome is replaced by the corresponding portion of the genome of MDV1 or MDV2.

[0060] In one embodiment, the rHVT according to the invention is characterized in that the first expression cassette comprises, in the 5' to 3' direction and in the following order:

[0061] a. Murine cytomegalovirus immediate early 1 (mCMV-IE1) gene promoter,

[0062] b. IBDV VP2 gene,

[0063] c. Transcription terminator,

[0064] d. Human cytomegalovirus immediate early 1 (hCMV-IE1) gene promoter,

[0065] e. NDV F protein gene, and

[0066] f. Transcription terminator,

[0067] And wherein the promoter and terminator are operably linked to the VP2 gene and to the F gene, respectively.

[0068] 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.

[0069] The term “comprises” and variations thereof such as “comprising”, “comprise” and “comprised” used herein are intended to mean all elements and any possible combinations of the present invention that are conceivable and covered or included in the text portion, paragraph, claim, etc. in which the term is used, even if these elements or combinations are not explicitly recited; and do not exclude any such elements or combinations.

[0070] Any such text sections, paragraphs, claims etc. may therefore also refer to one or more embodiments in which the term "comprising" (or variations thereof) is replaced by terms such as "consist of / consisting of", or "consisting essentially of".

[0071] The term "in the 5' to 3' direction", also referred to as "in the downstream direction", is well known in the art. Together with the term "in the following order", it is used to indicate the relative orientation that the elements summarized thereafter need to have with respect to each other in order to function with the gene expression machinery of a host cell in which the rHVT according to the invention can be replicated and expressed. As will be appreciated by those skilled in the art, this orientation relates to the DNA strand of the double-stranded DNA genome from the HVT, i.e., the "coding strand", and it relates to the coding mRNA molecule in the "+" or "sense" orientation.

[0072] Nevertheless, and without prejudice to the above section: on the complementary strand of the rHVT ds DNA genome, the relative order of the elements listed on the "template" strand is the same, but on that DNA strand, the orientation of these elements is 3' to 5', and in the "upstream direction".

[0073] It is known that the "promoter" of the present invention is a functional region of genetic information that directs the transcription of the downstream coding region. The promoter is therefore located upstream of the gene.

[0074] The naming of promoter is usually based on the gene that it controls expression in its natural environment.For example, term " mCMV-IE1 gene promoter " used herein refers to the promoter of natural drive IE1 gene from mCMV expression, therefore is positioned at the immediate upstream of this gene in the mCMV genome.Because IE1 gene is the gene that has been fully proved and can clearly identify, and because the genome of several mCMV has been sequenced, so this promoter can be easily identified by routine techniques.For example, in basic scheme, by roughly subcloning the region between two continuous genes, for example, from the stop codon of upstream gene to the start codon of downstream gene, promoter can be obtained simply.Then promoter can be identified by standard test, for example, by using the gradually decreasing part expression marker gene of the cloned region that contains suspicious promoter.

[0075] Typically, a promoter contains many identifiable regulatory regions, such as enhancer regions, which are involved in binding regulatory factors that affect the timing, duration, conditions, and level of transcription. Although enhancer regions are usually located in the upstream portion of a promoter, a promoter can also be affected by regions further downstream toward the start codon that are involved in the binding of transcription factors and instruct the RNA polymerase itself. The downstream region of a promoter typically includes many conserved sequence elements, such as TATA boxes, CAAT boxes, and GC boxes.

[0076] A promoter that includes both the enhancer and the downstream region is called a "full" promoter; a promoter that includes only the downstream region is called a "core" promoter.

[0077] The mCMV-IE1-gene is well known in the art and can be readily obtained from various commercial sources, for example from suppliers of commercial plasmids for cloning and expression.The IE1 gene is also known as the "major IE gene" of CMV.

[0078] mCMV-IE1 protein is also called pp89. In 1985 (K. et al., 1985, PNAS, Vol. 82, p. 8325) describes the mCMVIE1 gene promoter. 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 mCMVIE locus can be obtained from, for example, GenBank accession v number L06816.1. mCMV itself can be obtained from, for example, ATCC accession number VR-1399.

[0079] 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 the core promoter region and enhancer region of the mCMV-IE1 gene. The size of the complete mCMV-IE1 gene promoter is about 1.4 kb.

[0080] The term "about" as used in the present invention means ±25% around the indicated value, preferably "about" means ±20, 15, 12, 10, 8, 6, 5, 4, 3, 2% around the indicated value, or even "about" means ±1% around the indicated value, preferably in that order.

[0081] In one embodiment, the mCMV-IE1 gene promoter of the present invention is a DNA molecule of about 1.4 kb, which includes a nucleotide sequence having at least 95% nucleotide sequence identity to the entire length of the region of nucleotides 1 to 1391 of SEQ ID NO: 1. More preferably, it has at least 96, 97, 98, or even 99% nucleotide sequence identity in the preferred order.

[0082] In one embodiment, the mCMV-IE1 gene promoter is the region of nucleotides 1-1391 of SEQ ID NO:1.

[0083] In one embodiment of the rHVT of the present invention, in the first expression cassette, the IBDV VP2 gene of the present invention encodes the VP2 protein from the classical form of IBDV. Such genes are well known and their sequence information is readily available in the prior art, see, for example, GenBank accession number D00869 (strain F52 / 70), D00499 (strain STC), or AF499929 (strain D78). Alternatively, the gene can be obtained from the genome of a typical IBDV isolated from nature using conventional techniques for manipulating bi-RNA viruses. The classical form of IBDV can be easily identified using serology or molecular biology.

[0084] Since homologs or variants of the IBDV VP2 gene have the same potency and stability, in one embodiment, the IBDV VP2 protein gene of the present invention has at least 90% nucleotide sequence identity with the entire length of the nucleotide 1423-2781 region of SEQ ID NO: 1. The preferred nucleotide sequence identity is at least 92%, 94%, 95%, 96%, 97%, 98%, or even 99% in the preferred order.

[0085] In one embodiment, the IBDV VP2 protein gene of the present invention is derived from the classical IBDV strain Faragher 52 / 70.

[0086] In one embodiment, the IBDV VP2 protein gene of the present invention is the region of nucleotides 1423-2781 of SEQ ID NO:1.

[0087] "Transcription terminator" or terminator is a regulatory DNA element that participates in the termination of transcription of a coding region into RNA. Typically, such elements encode a segment with a secondary structure, such as a hairpin, which can cause the RNA polymerase complex to stop transcription. Therefore, a transcription terminator is always located downstream of the stop codon of the region to be translated, and is therefore located in the "3' untranslated region". The terminator may also include a polyadenylation (polyA) signal. This provides polyadenylation, which occurs in most eukaryotic mRNAs and plays a role in the transport and stability of these mRNAs.

[0088] The choice of a particular type of transcription terminator is not critical to the expression cassettes of the present invention, as long as efficient termination of RNA transcription is provided.

[0089] In the first expression cassette of the present invention, the transcription terminator element c between the VP2 and F genes not only provides for the termination of VP2 gene transcription but also provides for the efficient separation of the expression of these genes by preventing possible read-through of RNA transcription.

[0090] The two terminators used in the first expression cassette may be the same or different.

[0091] In one embodiment of the rHVT according to the present invention, the first expression cassette comprises a transcription terminator, and the transcription terminator comprises a terminator region and a polyA region.

[0092] In one embodiment of the rHVT of the present invention, in the first expression cassette, the transcription terminator of the VP2 gene is derived from Simian Virus 40 (SV40), preferably from SV40 late gene.

[0093] This terminator has been available since the late 1980s via the commercial "pCMVβ" cloning plasmid (Clontech).

[0094] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the transcription terminator of the VP2 gene is derived from the SV40 late gene and is about 0.2 kb in size, and comprises a nucleotide sequence having at least 95% nucleotide sequence identity over the entire length of the region of nucleotides 2812-3021 of SEQ ID NO: 1. More preferred is at least 96, 97, 98, or even 99% nucleotide sequence identity in order of preference.

[0095] In one embodiment, the transcription terminator from the SV40 late gene is the region of nucleotides 2812-3021 of SEQ ID NO:1.

[0096] The complete form of the hCMV-IE1 gene promoter is about 1.5 kb in size and consists of an enhancer, a core promoter and an intron, whereby the promoter activity is contained in the intron region, see Koedood et al. (1995, J. of Virol., Vol. 69, pp. 2194-2207).

[0097] The hCMV-IE1 gene promoter can be obtained from the genome of the hCMV virus (which is widely available) by subcloning the genomic region before the IE1 gene using conventional molecular biological tools and methods. Alternatively, the promoter can be derived from commercial expression plasmids such as pI17 described by Cox et al. (2002, Scand. J. Immunol., Vol. 55, pp. 14-23), or from commercially available mammalian expression vectors such as pCMV (Clontech), or pCMV-MCS series (Stratagene; Genbank accession number AF369966). The genomic sequence of hCMV is obtained, for example, from GenBank accession number X 17403.

[0098] From the hCMV-IE1 gene promoter, many highly similar versions are known, for example from GenBank. Such homologues and variants are within the scope of the present invention.

[0099] 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. The size of such a core promoter is usually less than 1 kb, and preferably the size is about 0.4 kb.

[0100] In one embodiment, the hCMV-IE1 gene core promoter used in the present invention is a DNA molecule of about 0.4 kb, which includes a nucleotide sequence having at least 95% nucleotide sequence identity to the entire length of the region of nucleotides 3160-3520 of SEQ ID NO: 1. More preferably, it is at least 96, 97, 98, or even 99% nucleotide sequence identity in the preferred order.

[0101] In one embodiment, the hCMV-IE1 gene core promoter is the region of nucleotides 3160-3520 of SEQ ID NO:1.

[0102] In one embodiment of the rHVT according to the present invention, in the first expression cassette, the NDV F protein gene is from a lentogenic type of NDV.

[0103] Preferably, the NDV F protein gene from the attenuated NDV strain is from NDV strain clone 30.

[0104] NDV clone 30 is a well-known attenuated NDV that has been used as a live vaccine for many years, e.g. ND Clone 30 (MSD Animal Health).

[0105] In one embodiment, the NDV F protein gene used in the present invention has at least 90% nucleotide sequence identity over the entire length of the region of nucleotides 3545-5206 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.

[0106] In one embodiment, the NDV F protein gene used in the present invention is the region of nucleotides 3545-5206 of SEQ ID NO:1.

[0107] 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, the size of the transcription terminator is about 0.3 kb.

[0108] In one embodiment, the transcription terminator is derived from the hCMV-IE1 gene, is about 0.3 kb in size, and includes a nucleotide sequence having at least 95% nucleotide sequence identity over the entire length of the region of nucleotides 5218-5498 of SEQ ID NO: 1. More preferred is at least 96, 97, 98, or even 99% nucleotide sequence identity in order of preference.

[0109] In one embodiment of the rHVT according to the present invention, the transcription terminator derived from the hCMV-IE1 gene is the region of nucleotides 5218-5498 of SEQ ID NO:1.

[0110] In order to optimize the expression of the VP2 and / or F genes of the present invention, their coding nucleotide sequences can be codon optimized. This is well known in the art and is commonly used to increase the expression level of DNA or RNA sequences in situations where the natural source of the encoded protein is different. It involves the adaptation of the nucleotide sequence to encode the expected amino acid, but through a nucleotide sequence that matches the codon preference (tRNA repertoire) of the recombinant vector, host cell or target organism expressing the sequence. Therefore, the nucleotide mutations applied are usually silent. Such modifications are usually planned on a computer using one of many computer software programs, after which the desired nucleotide sequence can be synthesized.

[0111] Therefore, in one embodiment of the rHVT described in the present invention, the genes encoding VP2 and F proteins are codon-optimized for the codon preference of HVT virus.

[0112] In one embodiment of the rHVT according to the present invention, the first expression cassette is an expression cassette disclosed in WO2016 / 102647.

[0113] Even more preferably, the first expression cassette is the cassette used in the rHVT construct described in WO2016 / 102647 as HVP360, which can be used in commercial vaccines. ND-IBD (MSD Animal Health).

[0114] An example of the first expression cassette of the present invention is given in SEQ ID NO: 1, and its elements are described in Table 1.

[0115] Table 1: Elements of SEQ ID NO: 1

[0116]

[0117] In one embodiment of the rHVT of the present invention, the size of the first expression cassette used in the present invention is about 5.5 kb.

[0118] In one embodiment of the rHVT according to the present invention, the first expression cassette used in the present invention is a DNA molecule of about 5.5 kb, which includes a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of SEQ ID NO: 1. More preferably, it is at least 96, 97, 98, or even 99% nucleotide sequence identity in the preferred order.

[0119] In one embodiment of the rHVT of the present invention, the first expression cassette used in the present invention comprises the nucleic acid shown in SEQ ID NO: 1. Preferably, the first expression cassette is SEQ ID NO: 1.

[0120] It will be apparent to those skilled in the art from the composition of the first expression cassette used in the present invention having two heterologous genes in one large cassette, which is designed and intended for insertion into a single location in the vector viral genome.

[0121] Furthermore, since the expression cassette is the self-contained expression module, the first expression cassette of the present invention can be inserted into the Us genome region of the rHVT of the present invention at different sites and in different directions.

[0122] Several loci in the HVT Us genomic region have been shown to allow insertion of one or more heterologous genes, see for example EP 431.668 and WO 2016 / 102647, for example: in genes Us2 or Us10, or between Us10 and SORF3, or in the region between Us2 and SORF3.

[0123] When a gene is inserted, the result is that the normal coding function of the gene receiving the insertion is disturbed in the resulting rHVT, or even completely lost. For Us2 and Us10, this was found not to significantly interfere with the replication of the resulting rHVT, nor with the expression of the inserted heterologous gene.

[0124] 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.

[0125] 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.

[0126] The HVT Us2 gene, also known as the HVT088 gene, refers to the gene in the HVT virus genome that corresponds to the gene at nucleotide positions 140065 to 140913 in the genome sequence of the HVT strain FC-126 with GenBank accession number AF291866.

[0127] In one embodiment, the Us2 gene used for insertion into the first expression cassette is the Us2 gene of HVT. Preferably, the Us2 gene is from HVT strain FC-126.

[0128] 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 in WO2016 / 102647 for HVP360.

[0129] In order to facilitate the convenient construction, manipulation and insertion of the expression cassette into the HVT of the present invention, the expression cassette itself can be included in a DNA molecule, such as a vector that allows cloning or transfection, such as a plasmid, cosmid, bacmid, etc., see WO 93 / 25.665 and EP 996.738. Examples of common cloning plasmids are, for example, plasmids from the pBR322 or pUC series. These are widely commercially available.

[0130] Plasmids that include expression cassettes are often referred to as "transfer vectors," "shuttle vectors," or "donor plasmids." In this case, the plasmid includes the expression cassette with flanking sequence regions from the target insertion site of the vector genome to direct insertion.

[0131] Typically, the transfer vector used for transfection does not itself integrate into the genome of the vector, it merely facilitates the integration of the expression cassette it carries, for example by allowing insertion to occur by homologous recombination. Thus, in the case of the first expression cassette used in the present invention, the first expression cassette is preferably flanked at its 5' and 3' ends by segments of the Us2 gene of HVT, which direct the insertion of the cassette into Us2.

[0132] Therefore, in one embodiment of the rHVT according to the invention, the first expression cassette is flanked by sequences from the Us2 gene of HVT.

[0133] Preferably, the flanking Us2 gene sequence is nucleotides 140143-140541 from GenBank Accession No. AF291866 and nucleotides 140541-141059 from GenBank Accession No. AF291866.

[0134] As described above, the present rHVT vector advantageously comprises, via a second expression cassette, another set of heterologous genes which are stably maintained in the rHVT during replication and expression in vitro and in vivo.

[0135] Therefore, in one embodiment, the rHVT according to the invention is characterized in that the second expression cassette comprises, in the 5' to 3' direction and in the following order:

[0136] a. ILTV gD gene with an upstream promoter and a downstream terminator, and

[0137] b. ILTV gI gene with upstream promoter and downstream terminator,

[0138] And wherein the promoter and the terminator are operably linked to the gD gene and to the gI gene, respectively.

[0139] In one embodiment of the rHVT according to the present invention, in the second expression cassette, the gD gene with promoter and terminator and the gI gene with promoter segment of the second expression cassette are taken from the ILTV genome as a whole. As described above: due to the overlap of ILTV gD and gI genes in their natural background, the gD gene terminator and gI gene promoter are included in the gI gene and gD gene, respectively.

[0140] SEQ ID NO: 2 shows the nucleotide sequence of the second expression cassette of the present invention, which is a HindIII fragment of about 3.2 kb; its elements are described in Table 2.

[0141] Table 2: Elements of SEQ ID NO: 2

[0142]

[0143] Preferably, the gD gene with promoter and terminator and the gI gene with promoter segment are formed by a DNA molecule of about 3 kb, which includes a nucleotide sequence having at least 90% nucleotide sequence identity over the entire length of the region of nucleotides 13 to 3081 of SEQ ID NO: 2. Even more preferred is at least 92, 94, 95, 96, 97, 98, or even 99% nucleotide sequence identity in order of preference.

[0144] In one embodiment of the rHVT according to the present invention, in the second expression cassette, the segment comprising the gD gene with a promoter and a terminator and the gI gene with a promoter is formed by the region of nucleotides 13-3081 of SEQ ID NO:2.

[0145] In one embodiment of the rHVT according to the present invention, in the second expression cassette, the transcription terminator of the gI gene is derived from FHV1, preferably from the FHV1Us9 gene. The FHV1Us9 gene is disclosed in GenBank Accession No. D42113, for example.

[0146] In one embodiment of the rHVT according to the present invention, in the second expression cassette, the transcription terminator is derived from the FHV1 Us9 gene and is about 0.05 kb in size and comprises a nucleotide sequence having at least 95% nucleotide sequence identity over the entire length of the region of nucleotides 3097-3151 of SEQ ID NO: 2. More preferred is at least 96, 97, 98 or even 99% nucleotide sequence identity in order of preference.

[0147] In one embodiment, the transcription terminator from the FHV1Us9 gene is the region of nucleotides 3097-3151 of SEQ ID NO:2.

[0148] In one embodiment of the rHVT according to the present invention, the size of the second expression cassette according to the present invention is about 3.2 kb.

[0149] In one embodiment of the rHVT according to the present invention, the second expression cassette used in the present invention is a DNA molecule of about 3.2 kb, which includes a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of SEQ ID NO: 2. More preferably, it is at least 96, 97, 98 or even 99% nucleotide sequence identity in the preferred order.

[0150] In one embodiment of the rHVT according to the present invention, the second expression cassette according to the present invention comprises the nucleic acid shown in SEQ ID NO: 2. Preferably, the second expression cassette is SEQ ID NO:2.

[0151] As described above, the second expression cassette of the present invention, which includes the ILTV gD and gI genes, is inserted into the intergenic region between the ORFs of the HVT genes UL54 and LORF3. Since these two genes are in the so-called tail-to-tail orientation in the HVT genome (their respective reading directions point toward each other), 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; the size of this region in HVT is about 0.9 kb. Figure 1 These orientations are shown graphically.

[0152] Therefore, in a preferred embodiment of the rHVT according to the present invention, the second expression cassette of the present invention is inserted in the HVT genome region of nucleotides 110.383-111.302 as disclosed in GenBank Accession No. AF291866. More preferably, the second expression cassette of the present invention is inserted in the HVT genome between nucleotides 110.395 and 110.396 as disclosed in GenBank Accession No. AF291866.

[0153] The rHVT of the present invention is preferably based on a parent HVT that is an established HVT vaccine strain that replicates well and is known to be suitable for vaccination of young birds or avian embryos in eggs; for example, the HVT vaccine strains PB1 or FC-126. These are commonly 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: Marek THV Lyo.

[0154] The incorporation of the first expression cassette and the second expression cassette, both as defined herein for the present invention, does not increase the virulence or pathogenicity of the parental HVT and, on the contrary, is not expected to reverse virulence, since HVT is naturally non-pathogenic.

[0155] Therefore, in one embodiment, the parent HVT used to produce the rHVT of the present invention is an HVT vaccine strain; preferably an HVT vaccine strain of the PB1- or FC-126 strain.

[0156] The rHVT of the present invention is a live recombinant vector microorganism, or "vector" virus, which can be advantageously used for vaccination of poultry. It combines the characteristics of being a safe and effective vaccine against Marek's disease (MD) and against one or more or all of infectious bursal disease (IBD), Newcastle disease (ND) and infectious laryngotracheitis (ILT), and is additionally genetically stable.

[0157] "Genetically stable" as used herein means that the genetic composition of the rHVT according to the present invention does not change in subsequent viral replication cycles. Alternatively, an unstable construct may result in ineffective viral replication and / or result in loss of expression of one or more inserted heterologous genes. This stability can be conveniently monitored using conventional techniques, for example by subsequently passage the rHVT according to the present invention in cell culture. The virus re-isolated in these steps can be plated on a cell culture dish, covered with agar, and incubated until HVT-specific plaques become visible; all using conventional techniques. Next, plaques are stained for expression of VP2, F, or gD and gI proteins using appropriate antibody preparations in an immunofluorescence assay (IFA) protocol with appropriate positive and negative controls. Any plaques that no longer show fluorescence of a specific heterologous protein can then be recorded, whereby preferably about 100 individual plaques of a specific rHVT sample should be monitored.

[0158] Surprisingly, it was found that even after 15 consecutive cell culture passages and after 15 days of replication in vivo, the rHVT of the present invention maintained the presence and expression of VP2, F, gD and gI protein genes in all tested plaques. Details are described in the Examples.

[0159] This is a strong and highly significant improvement over the multivalent HVT vector constructs described in the prior art.

[0160] Moreover, considering that all VP2, F, gD and gI genes have been used as inserts in effective HVT vector vaccines, the fact that they are stably maintained and expressed is also convincing evidence that the rHVT according to the present invention will induce protective immune responses against these antigens as well as against MDV in poultry and will therefore be an effective multivalent vector vaccine.

[0161] Therefore, in one embodiment of the rHVT according to the present invention, one or more conditions selected from the group consisting of:

[0162] o For the first expression cassette:

[0163] -mCMV-IE1 gene promoter is a complete promoter;

[0164] -IBDV VP2 gene encodes the VP2 protein from the classical form of IBDV;

[0165] -The transcription terminator of the VP2 gene includes a terminator region and a polyA region; preferably,

[0166] The transcription terminator is derived from SV40;

[0167] -hCMV-IE1 gene promoter is the core promoter;

[0168] -NDV F gene is from a attenuated NDV strain, preferably from NDV strain clone 30;

[0169] -The transcription terminator of the F gene is derived from the hCMV-IE1 gene;

[0170] - The genes encoding VP2 and / or F proteins are codon-optimized for HVT virus codon preference;

[0171] - the first expression cassette is an expression cassette as disclosed in WO2016 / 102647; even more preferably, the first expression cassette is a cassette as used in the rHVT construct described as HVP360 in WO2016 / 102647;

[0172] - the first expression cassette of the present invention is a DNA molecule of about 5.5 kb, which comprises a nucleotide sequence having at least 95% nucleotide sequence identity to the full length of SEQ ID NO: 1; more preferably at least 96%, 97%, 98% or even 99% nucleotide sequence identity in order of preference;

[0173] - 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;

[0174] o and / or for the second expression cassette:

[0175] - The second expression cassette comprises, in the 5' to 3' direction and in the following order:

[0176] =ILTV gD gene with upstream promoter and downstream terminator, and

[0177] =ILTV gl gene with upstream promoter and downstream terminator,

[0178] and wherein the promoter and the terminator are operably linked to the gD gene and to the gI gene, respectively;

[0179] - the promoter may be a core promoter or may be a full promoter;

[0180] - The genes encoding gD and / or gI genes are codon-optimized for HVT virus codon preference;

[0181] - The promoter of the gD gene is the ILTV gD gene promoter;

[0182] - The promoter of the gI gene is the ILTV gI gene promoter;

[0183] -The terminator of the gD gene is the ILTV gD gene terminator;

[0184] The terminator of the -gI gene is the ILTV gI gene terminator;

[0185] - the second expression cassette is a DNA molecule of about 3.2 kb, which comprises 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 order of preference;

[0186] - the second expression cassette comprises the nucleic acid shown in SEQ ID NO: 2; preferably, the second expression cassette is SEQ ID NO: 2;

[0187] - the second expression cassette is inserted into the HVT genome region of nucleotides 110.383-111.302 as disclosed in GenBank Accession No. AF291866; more preferably, the second expression cassette of the present invention is inserted into the HVT genome between nucleotides 110.395 and 110.396 as disclosed in GenBank Accession No. AF291866;

[0188] o and / or for rHVT:

[0189] - 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;

[0190] - The Us2 gene used for the first expression cassette insertion is the Us2 gene of HVT; preferably, the Us2 gene is from the HVT strain FC-126;

[0191] - The parent HVT used for rHVT production is an HVT vaccine strain; preferably, the parent HVT is an HVT vaccine strain of the PB1 or FC-126 strain.

[0192] Through different cloning and transfection methods, the first expression cassette and the second expression cassette of the present invention can be used to obtain the rHVT of the present invention, which stably includes and expresses the expression cassette described herein in its genome.

[0193] Therefore, another aspect of the present invention relates to a method for constructing a rHVT according to the present invention, said method comprising inserting a first expression cassette and a second expression cassette as defined in the present invention into a region of the HVT genome as defined in the present invention.

[0194] Insertion of the expression cassette according to the invention into the HVT genome to produce the rHVT according to the invention can be performed using different methods known in the art. A convenient method is the use of transfer vectors and homologous recombination technology, as described above.

[0195] Alternatively, the rHVT described herein can be produced using CRISPR / Cas9 technology; for example, as described in Tang et al., 2018 (supra).

[0196] In particular, the rHVT-VP2-F vector virus can be used, which is available from the commercial vaccine InnovaxND-IBD since 2017. This can be further manipulated by inserting a second expression cassette as described herein into the UL 54-LORF3 locus of the genome of the rHVT as described herein using CRISPR / Cas9 technology. Specific guide RNA sequences that can be used to direct these insertions to these loci are described in the Examples.

[0197] The rHVT of the present invention can be amplified by conventional techniques, preferably by in vitro replication, for example in chicken cell culture, typically in primary chicken embryo fibroblasts (CEF). These can be prepared by trypsinization of chicken embryos, all of which are well known in the art. The CEFs are inoculated in a monolayer and infected with HVT. The method can be scaled up to industrial scale production.

[0198] rHVT is typically collected by harvesting infected host cells containing a cell-associated form of rHVT. These cells are absorbed in a suitable carrier composition to provide stability during freezing and storage. Next, the infected cells are typically 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 absorbed into a suitable dilution buffer for stability during use. In a preferred embodiment, the dilution buffer is a buffer disclosed in WO 2019 / 121888.

[0199] Although cell-associated frozen storage of HVT is preferred, in cases where the use of liquid nitrogen is not feasible, an alternative is to use freeze drying: this exploits the advantageous feature of HVT, namely that HVT can be isolated from its host cells by cell disruption, for example by French press or sonication. This can be clarified by centrifugation and then taken up in a stabilizer and freeze dried for long-term storage.

[0200] Therefore, in another aspect, the present invention relates to a host cell comprising the rHVT according to the present invention.

[0201] The "host cell" of the present invention is a cell susceptible to infection and replication of HVT. Examples of such cells are avian cells, in particular lymphocytes or fibroblasts.

[0202] Preferably, the host cell according to the present invention is a host cell maintained under in vitro conditions.

[0203] In one embodiment, the host cell according to the invention is a primary avian cell; that is, the cell is derived in vitro from a non-human animal tissue or organ, rather than from an immortalized cell line. Typically, primary cells can only undergo a small and limited number of cell divisions.

[0204] In one embodiment, the primary avian host cell of the present invention is a primary chicken embryo fibroblast (CEF).

[0205] 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 WO97 / 044443 and WO98 / 006824.

[0206] In a preferred embodiment, the immortalized avian host cell of the present invention is an immortalized CEF; preferably the immortalized CEF disclosed in WO2016 / 087560.

[0207] As mentioned above, the main advantageous use of rHVT according to the present invention is in vaccines for poultry, providing safe, stable and effective vaccination against MD, IBD, ND and / or ILT or disease symptoms related thereto, and can be administered to very young poultry.

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

[0209] Different ways of "use in a vaccine" of rHVT or host cells according to the invention have been outlined above and include use as cell-free virus or as cell-associated virus in host cells in vaccine compositions for poultry vaccination.

[0210] In addition, in another aspect, the present invention relates to a poultry vaccine, comprising the rHVT of the present invention and / or the host cell of the present invention, and a pharmaceutically acceptable carrier.

[0211] A "vaccine" is a composition comprising an immunologically active compound in a pharmaceutically acceptable carrier. An "immunologically active compound" or "antigen" is a molecule that is recognized by the immune system of a vaccinated target and induces a protective immune response from the target's humoral and / or cellular immune system.

[0212] Vaccine according to the present invention provides protection poultry against infection and / or disease caused by MDV, IBDV, NDV and / or ILT. Obtain this effect by preventing or reducing one or more of these viruses from forming or proliferating productive infections in their respective target organs. This is for example achieved by reducing viral load or shortening the duration of viral replication. This in turn results in the minimization of the quantity, intensity or severity of the pathological changes and related clinical signs of disease caused by viral infection in the target animal.

[0213] However, depending on the virulence of the MDV, IBDV, NDV or ILTV field viruses that are prevalent in certain poultry farms or certain regions, it may be necessary to add additional vaccine components of one or more of these viruses to ensure effective vaccination against pathogenic variants or serological variants of these viruses. This is well known in the art.

[0214] Determination of the effectiveness of a poultry vaccine or poultry vaccine according to the invention is well within the skill of the conventional practitioner and can be performed, for example, by monitoring the immune response after vaccination, or by testing the appearance of clinical signs or mortality after challenge infection, for example by monitoring disease signs, clinical scores, serological parameters of the target, or by re-isolating the challenge pathogen and comparing these results with the vaccination-challenge response seen in mock-vaccinated animals. Different methods for evaluating each of the four viral infections are well known in the art.

[0215] The protection against MD, IBD, ND and ILT induced by using the vaccine of the present invention or by vaccination of the present invention results in improved health and economic performance of the target of vaccination. This can be evaluated, for example, from an increase in one or more of the following parameters: survival, growth rate, feed conversion, egg laying and quantity and health of the offspring. A further effect is to reduce the cost of health care and increase the economy of operation.

[0216] In the following, various embodiments, preferences and examples of the use, vaccine or vaccination according to the invention will be outlined.

[0217] The term "poultry" as used in the present invention relates to avian species that are relevant to veterinary practice and that are susceptible to vaccination against HVT; preferred poultry species are: chickens, turkeys, geese, ducks and quail. Chickens are the most preferred species.

[0218] For the present invention, poultry can be any type, species or variety, for example: laying hens, breeders, broilers, combination species or parental lines of any of these species. Preferred types are: broilers, breeders and laying hens. Most preferred are broiler and laying hens poultry.

[0219] "Pharmaceutically acceptable carrier" is intended to help the stability and administration of the vaccine, while being harmless to the target and well tolerated. Such a carrier may be, for example, sterile water or a sterile physiological saline solution. In a more complex form, the carrier may be, for example, a buffer, which may include other additives, such as stabilizers or preservatives. Details and examples are described, for example, in well-known manuals, such as: "Remington: the science and practice of pharmacy" (2000, Lippincott, USA, ISBN: 683306472), and "Veterinary vaccinology" (P. Pastoret et al., eds., 1997, Elsevier, Amsterdam, ISBN 0444819681).

[0220] For the present invention, when the vaccine is in the form of cell-associated HVT, the pharmaceutically acceptable carrier is preferably a mixture of culture medium, about 10% v / v serum and about 6% v / v DMSO. The carrier also provides stability of rHVT-infected host cells during freezing and frozen storage. The serum can be any serum routinely used for cell culture, such as fetal bovine serum or newborn calf serum.

[0221] The vaccine of the present invention is prepared by the rHVT of the present invention by the methods described herein, which are easily applied by those skilled in the art. For example, the rHVT of the present invention is constructed by inserting the expression cassette of the present invention by transfection and recombination. Next, the desired rHVT is selected and industrially amplified in a smaller or larger volume, preferably in an in vitro cell culture, such as CEF. From these cultures, a suspension of host cells infected with rHVT is collected, either as intact infected cells or as a cell-free preparation obtained by cell disruption. The suspension is formulated into a vaccine together with a suitable pharmaceutical carrier, and the final product is packaged. The cell-associated vaccine is then stored in liquid nitrogen and the vaccine is freeze-dried at -20°C or +4°C.

[0222] The general techniques and considerations applicable to vaccine preparation under known pharmaceutical production standards are described in, for example, government directives and regulations (Pharmacopoeia, 9 CFR) and known reference books ("Veterinary vaccinology" and "Remington", both supra). Typically, such vaccines are prepared aseptically and using pharmaceutical grade excipients.

[0223] Such preparations will include microbiological testing to ensure sterility and the absence of foreign matter; and may include in vivo or in vitro studies to confirm efficacy and safety. After completing quality, quantity, sterility, safety and efficacy tests, the vaccine can be released for sale. All of these are well known to those skilled in the art.

[0224] In one embodiment, the vaccine for poultry according to the present invention is a cell-associated vaccine.

[0225] "Cell-associated" means that the rHVT of the present invention is included in the in vitro host cell of the present invention. Therefore, this type of vaccine comprises the host cell of the present invention and the rHVT.

[0226] The target animals for the vaccines of the present invention can in principle be healthy or sick, and can be positive or negative for the presence of MDV, IBDV, NDV or ILTV, or can be positive or negative for antibodies against MDV, IBDV, NDV or ILTV. The target can also be any weight, sex or age that is susceptible to vaccination. However, it is obviously advantageous to vaccinate healthy, uninfected targets, and to vaccinate as early as possible to prevent any field infection and its consequences.

[0227] Thus, the vaccines of the present invention can be used as prophylactic or therapeutic treatments, or both, since they interfere with the establishment and progression of MDV, IBDV, NDV or ILTV infection.

[0228] In this regard, another beneficial effect of reducing viral loads with vaccines according to the invention is the prevention or reduction of shedding of viruses in the wild and the resulting spread, both vertically to offspring and horizontally to or within populations, as well as within geographic regions. Thus, the use of vaccines according to the invention results in a reduction in the prevalence of MDV, IBDV, NDV or ILTV.

[0229] Therefore, other aspects of the present invention are:

[0230] - use of the vaccine for poultry according to the invention for reducing the prevalence of MDV, IBDV, NDV or ILTV in a population or a geographical area, and

[0231] - Use of the poultry vaccine according to the invention for reducing the prevalence of MDV, IBDV, NDV or ILTV in a population or a geographical area.

[0232] The vaccine according to the invention already provides multivalent immunity: against IBD, ND and ILT through the expression of the heterologous insert, and additionally against MD through the HVT vector itself. However, further combinations with additional immunologically active components may be advantageous. This could be used to enhance the immune protection already provided, or to extend it to other pathogens.

[0233] Thus, in one embodiment, the vaccine according to the invention comprises at least one additional immunologically active component.

[0234] Such "additional immunologically active components" may be antigens, immunopotentiating substances, cytokines, additional vaccines, or any combination thereof. This provides advantages in terms of cost, efficiency and animal welfare. Alternatively, the vaccine of the present invention itself may be added to the vaccine.

[0235] In one embodiment, the at least one additional immunologically active component is an immunostimulatory compound; preferably, a cytokine or an immunostimulatory oligodeoxynucleotide.

[0236] The immunostimulatory oligonucleotide is preferably an immunostimulatory non-methylated CpG-containing oligodeoxynucleotide (INO). Preferred INOs are avian Toll-like receptor (TLR) 21 agonists as described in WO 2012 / 089800 (X4 family), WO 2012 / 160183 (X43 family) or WO 2012 / 160184 (X23 family).

[0237] In one embodiment, at least one additional immunologically active component is an antigen derived from a microorganism pathogenic to poultry. The antigen may be derived in any suitable manner, for example as a "live", attenuated, inactivated or subunit antigen from a microorganism pathogenic to poultry.

[0238] The additional antigens derived from microorganisms pathogenic to poultry are preferably derived from one or more microorganisms selected from the group consisting of:

[0239] - Viruses: infectious bronchitis virus, NDV, adenovirus, avian influenza virus, egg drop syndrome virus, IBDV, chicken anemia virus, avian encephalomyelitis virus, fowl pox virus, turkey rhinotracheitis virus, duck plague virus (duck viral enteritis), pigeon pox virus, MDV, avian leukosis virus, ILTV, avian pneumovirus, and reovirus;

[0240] - Bacteria: Escherichia coli, Salmonella spp., Clostridium botulinum, Haemophilus paragallinarum, Pasteurella multocida, Erysipelothrix rhusiopathiae, Erysipelothrix spp., Mycoplasma spp., and Clostridium spp.;

[0241] - Parasites: Eimeria spp.; and

[0242] -Fungi: Aspergillus spp.

[0243] Additional antigens may also be other vector vaccines, for example based on HVT, MDV2, NDV, etc.

[0244] In one embodiment of the vaccine of the invention, the other antigen derived from a microorganism pathogenic to poultry is a "live" attenuated vaccine strain of MDV, IBDV, NDV or ILTV. This serves to improve and broaden the immunogenicity of the vaccine according to the invention, and this is advantageous in those cases or geographical areas where virulent wild strains of MDV, IBDV, NDV or ILTV are prevalent.

[0245] In this respect, combinations of HVT with MDV1, MDV2 or HVT are known; for the present invention, MDV of strain Rispens (MDV1), strain SB1 (MDV2), or HVT of strains FC-126 or PB1 are preferred as further immunologically active components.

[0246] To improve anti-ND responses, the rHVT of the present invention can be combined with an NDV vaccine strain, such as the mild live NDV vaccine strain C2.

[0247] Similarly, to improve anti-IBD responses, the rHVT of the present invention may be combined with live IBDV vaccine strains such as D78, PBG98, Cu-1, ST-12 or 89-03.

[0248] As will be understood by those skilled in the art, these "combinations" also include vaccination regimens in which the rHVT according to the present invention and the additional immunologically active components are not combined or administered simultaneously, but rather are administered in a simultaneous or sequential vaccination regimen; for example, rHVT can be administered in ovo, NDVC2 on day one, and IBDV89-03 at about day 17 of age.

[0249] Therefore, in an embodiment of the vaccine according to the invention comprising at least one additional immunologically active component, the at least one additional immunologically active component is a microorganism selected from the following vaccine strains: MDV, IBDV, NDV, or ILTV, or any combination thereof.

[0250] More preferably, the additional immunologically active component is selected from one or more of the following: MDV Rispens, MDV SB1, NDV C2, IBDV D78 and IBDV89-03.

[0251] The vaccines of the present invention can be prepared by the methods described and exemplified herein.

[0252] Therefore, another aspect of the present invention relates to a method for preparing a poultry vaccine according to the present invention, said method comprising the following steps:

[0253] a. infecting a host cell in vitro with the rHVT according to the present invention,

[0254] b. growing and harvesting infected host cells, and

[0255] c. mixing the harvested infected host cells with a pharmaceutically acceptable carrier.

[0256] Suitable host cells and pharmaceutically acceptable carriers for use in the present invention are described above. In addition, suitable in vitro methods for infection, culture and harvesting are well known in the art and are described and exemplified herein.

[0257] Thus, the various aspects and embodiments of the present invention may be advantageously used to produce a safe, stable and effective poultry vaccine as described herein.

[0258] Therefore, in another aspect, the present invention relates to the use of rHVT, the host cell according to the present invention or any combination thereof for the preparation of a vaccine for poultry.

[0259] It goes without saying that it is also within the scope of the present invention to mix other compounds such as stabilizers, carriers, adjuvants, diluents, emulsions, etc. with the vaccine of the present invention. Such additives are described in well-known reference books such as "Remington" and "Veterinary Vaccinology" (both supra).

[0260] In this way, the efficacy of the vaccine according to the invention to protect poultry from MD, IBD, ND and ILT at very young ages after a single vaccination can be further optimized, when necessary.

[0261] The vaccines according to the present invention may be prepared in a form suitable for administration to poultry targets and matched to the desired route of administration and the desired effect.

[0262] Depending on the route of administration of the vaccine of the present invention, it may be necessary to adjust the composition of the vaccine. This is well within the capabilities of those skilled in the art and generally involves fine-tuning of vaccine efficacy or safety. This can be achieved by adjusting the vaccine dose, quantity, frequency, route, by using another form or formulation of the vaccine, or by adjusting other ingredients of the vaccine (e.g., stabilizers or adjuvants).

[0263] The vaccine according to the invention can in principle be given to the target poultry by different routes of administration and at different time points in their life, provided that the vaccinated rHVT can establish a protective infection.

[0264] However, since infection with MDV, IBDV, NDV or ILTV is already established at a very young age, it is advantageous to administer the vaccine of the invention as early as possible. Thus, the vaccine of the invention can be administered, for example, on the day of hatching ("first day") or in ovo, for example, at about day 18 of embryonic development, as is well known in the art.

[0265] Thus, in one embodiment, the vaccines of the present invention are administered to poultry in ovo.

[0266] Equipment is commercially available for automated injection of the vaccine into fertilized eggs on an industrial scale. This provides protection as early as possible while minimizing labor costs. Different in ovo routes of vaccination are known, such as into the yolk sac, embryo or allantoic fluid cavity; these can be optimized as needed. In ovo vaccination with HVT is preferably such that the needle contacts the embryo.

[0267] Preferably, the vaccine according to the invention is formulated as an injectable liquid suitable for in ovo or parenteral injection; for example as: a suspension, solution, dispersion or emulsion.

[0268] In one embodiment, the vaccine according to the invention is administered parenterally, preferably intramuscularly or subcutaneously.

[0269] The exact amount of rHVT according to the invention / animal dose of the vaccine according to the invention is not as critical as for inactivated or subunit vaccines; this is because the rHVT will replicate in the target animal to a biologically sustainable level of viremia. In principle, the vaccine dose only needs to be sufficient to induce this productive infection. Higher vaccination doses can hardly shorten the time taken to achieve an optimal viremic infection in the host. Therefore, very high doses are ineffective and are also unattractive for economic reasons.

[0270] Therefore, the preferred vaccination dose is between 1x10^1 and 1x10^5 plaque forming units (pfu) of the rHVT according to the invention / animal dose, more preferably between 1x10^2 and 1x10^4 pfu / dose, even more preferably between 500 and 5000 pfu / dose; most preferably between about 1000 and about 3000 pfu / dose.

[0271] When the vaccine of the invention is cell-associated, these amounts of rHVT are contained in infected host cells. Methods for counting rHVT virus particles of the invention are well known.

[0272] The volume per animal dose of rHVT according to the invention can be optimized depending on the intended route of administration: in ovo vaccination is usually administered at a dose between about 0.01 and about 0.5 ml / egg, while parenteral injections are usually performed at a dose between about 0.1 and about 1 ml / bird.

[0273] Determining an immunologically effective amount of a vaccine of the invention, or optimizing the volume of vaccine per animal, is within the capabilities of those skilled in the art.

[0274] The administration regimen for administering the vaccine according to the invention to the target organism may be single dose or multiple doses, in a manner compatible with the vaccine formulation, and in an immunologically effective amount.

[0275] Preferably, the regimen of administering the vaccine of the present invention is integrated into the existing vaccination schedule of other vaccines that the target poultry may need to reduce stress on the animals and reduce labor costs. These other vaccines can be administered simultaneously, concurrently or sequentially in a manner compatible with their licensed use.

[0276] As described above, and as exemplified below, the vaccines of the present invention can be advantageously used to prevent or reduce infection by one or more or all of MDV, IBDV, NDV and ILTV, as well as to prevent or reduce diseases (symptoms) associated with these infections by a single vaccination at a very young age.

[0277] Therefore, other aspects of the present invention are:

[0278] - Use of the poultry vaccine according to the present invention for preventing or reducing infection by one or more of MDV, IBDV, NDV and ILTV and / or related disease symptoms thereof.

[0279] - A method for preventing or reducing infection by one or more of MDV, IBDV, NDV and ILTV and / or their associated disease symptoms, the method comprising administering the vaccine of the invention to poultry.

[0280] - A method for vaccinating poultry to prevent or reduce infection by one or more of MDV, IBDV, NDV and ILTV and / or their associated disease symptoms, said method comprising the step of vaccinating said poultry with the vaccine of the present invention.

[0281] Details of the use of the vaccine according to the invention by vaccination of poultry have been described above; in particular, vaccination of day-old chickens by intramuscular or subcutaneous inoculation and in ovo inoculation of 18-day-old embryos.

[0282] The invention will now be further described with reference to the following non-limiting examples.

[0283]

[0284]

[0285]

[0286]

[0287]

[0288] Example

[0289] Example 1: Construction and in vitro testing of multivalent rHVT vectors 1.

[0291] 1.1. Construct preparation and testing

[0292] Based on the HVT vector construct rHVT-VP2-F (HVP360; WO 2016 / 102647), a series of HVT recombinants that additionally express the ILTV gD and gI genes from the UL genomic region of the vector were prepared. HVP360 expresses the IBDV-VP2 and NDV-F genes from an expression cassette inserted into the HVT Us2 gene. Using the CRISPR / Cas9 technology described by Tang et al., 2018 (supra), another cassette expressing ILTV gD-gI was introduced into one of the different sites in the UL region of the HVP360 genome. Several constructs were prepared by inserting the second expression cassette, and it was found that one rHVT-VP2-F-gD-gI construct (rHVT according to the present invention) had excellent levels of stable replication, heterologous gene expression, and vaccination efficacy when tested in vitro and in vivo.

[0293] Some rHVT-VP2-F vector constructs with additionally inserted gD-gl expression cassettes are described herein for comparison and to illustrate the advantageous effects of the present invention. The insertion site nucleotide numbers are indicated relative to the genome of HVT strain FC-126 as disclosed in GenBank accession number AF291866:

[0294] - rHVT construct HVP412: It has a gD-gl cassette inserted between UL44 and UL45, specifically: between nt.94482-94483,

[0295] - rHVT construct HVP413: It has a gD-gl cassette inserted between UL45 and UL46, specifically: between nt.95335-95336,

[0296] - rHVT construct G7 (rHVT according to the invention): it has a gD-gl cassette inserted between UL54 and LORF3, specifically: between nt.110.395 and 110.396, and

[0297] - rHVT construct G8: It has the gD-gl cassette inserted in the ORF of the LORF3 gene, specifically: between nt.111.219 and 111.220.

[0298] Constructs HVP412, HVP413 and G8 are included herein as comparative examples; constructs HVP412 and HVP413 have been described in more detail in PCT / EP2021 / 087445.

[0299] The guide RNA sequence used for CRISPR / Cas9 directed insertion of the gD-gI cassette into the HVT UL is:

[0300] -Insertion between UL44 and UL45: 5'-ACATCGGGACGTACATCATG

[0301] -3' (SEQ ID NO: 3),

[0302] -Insertion between UL45 and UL46: 5'-CTAACGGTTACTGTGTTTTA

[0303] -3' (SEQ ID NO:4),

[0304] - Insertion between UL54 and LORF3: 5'-TTAGATTTCCGGACAGCCTG-3' (SEQ ID NO: 5),

[0305] -Insertion in LORF3: 5'-TACCACGCCGAGGCGCCTAT-3'

[0306] (SEQ ID NO:6).

[0307] SEQ ID NOs 3-6 are represented herein by DNA code because they are inserted into DNA plasmids and then transcribed to produce guide RNAs, all using standard procedures. For the guide RNA of SEQ ID NO: 4, cleavage is performed between nucleotides 3 and 4; for the guide RNAs of SEQ ID NOs: 3, 5 and 6, cleavage is performed between nucleotides 17 and 18.

[0308] Design guide RNA using the Internet website zlab.bio / guide-design-resources. One of the many alternatives is to use Geneious Procedure (Biomatters Ltd., New Zealand).

[0309] Figure 1 A schematic representation of the expression cassette used and its insertion into the HVT genome of the rHVT construct G7 of the invention is given.

[0310] Other ways of inserting the gD-gI expression cassette into the UL region of the vector construct rHVT-VP2-F have been constructed and tested for the following insertions:

[0311] - Intragenic, in UL39 - Intermediate

[0312] - Intragenic, near the 3' end in UL39

[0313] - intergenic, between UL40 and UL41, and

[0314] -Intergenic, between UL47 and UL48.

[0315] 1.2. In vitro genetic stability

[0316] The various rHVT-VP2-F-gD-gI constructs were serially passaged 15 times in vitro on CEF cells. Expression of the inserted genes in P15 plaques was monitored by IFA as follows: CEF monolayers established overnight were infected with one of the rHVT vectors at the 15th passage level. The plates were incubated for 2-3 days until CPE was clearly visible and then fixed with 96% ethanol. Expression of VP2 and F was detected with specific monoclonal antibodies; gD and gI were detected using chicken polyclonal anti-ILTV antibodies. After the primary antibody, Alexa Fluor TM The labeled conjugate was used as the secondary antibody. The plate was then read using a UV microscope. Approximately 100 plaques were counted for each recombinant to assess expression.

[0317] For different constructs with F and VP2 in Us2 and the ILTV gD-gI gene in the locus of the UL region, several plaque losses in subsequent passages or the plaques that appeared no longer showed expression of one or more heterologous gene inserts were observed. Nevertheless, for all rHVT-VP2-F-gD-gI constructs, at least one clone could be isolated that replicated well in vitro and maintained stable expression of the heterologous insert over 15 passages in cell culture.

[0318] These rHVT-VP2-F-gD-gI isolates were next tested for replication and expression in vivo.

[0319] Example 2: Characterization of multivalent rHVT vectors in vivo 2.

[0321] 2.1. Introduction

[0322] In several experiments, various multivalent rHVT vectors constructed as described in Example 1 were tested for viral replication and expression of gene inserts, as well as the induction of serological immune responses. The experimental animals were 1-day-old SPF laying hens. To determine the in vivo replication of the rHVT vector vaccine, the level of HVT viremia was determined on days 15, 21 or 25 after vaccination. To examine the expression of the heterologous gene insert and the induction of specific antibodies, blood samples were collected from the vaccinated chickens at different time points during the experiment.

[0323] 2.2. experiment

[0324] The group size was 10-15 animals, plus 5 hatchlings. Blood samples were taken from the hatchlings on the day of vaccination for serological testing to ensure that the batch of animals was negative for antibodies against NDV, IBDV, and ILTV on the day of vaccination.

[0325] rHVT vaccine virus was used at passage 15 or 16 and stored in liquid nitrogen as infected CEF. The virus titer (in infected cells) of the vaccine stock was 0.4-1.2 x 10^6 pfu / ml. The vaccine dose was 2000 PFU per animal in 0.2 ml of standard HVT / CEF diluent and inoculated subcutaneously in the neck using standard procedures.

[0326] Control groups were either not vaccinated or received the rHVT parent vector HVP360 as a vaccine. No acclimatization was required, as the chickens were placed in negative pressure isolators shortly after hatching and were tagged and vaccinated shortly thereafter.

[0327] Blood samples were collected from vaccinated chickens on appropriate days after vaccination (e.g., 15 or 25 days after vaccination). In various vaccination-challenge trials (described below), serological samples were collected shortly before challenge, i.e., at 3, 4, or 5 weeks after vaccination. Blood samples were collected from the wing vein into tubes with a clot activator and stored at room temperature until processing.

[0328] 2.2.1. Viremia

[0329] Sampling of viremia from the spleen was performed as follows: spleens were isolated from 5-6 chickens per group after sacrifice on day 15 or 21 post-inoculation. Clean forceps were used for each chicken. The spleens were collected in a tube including 5 ml of 10 mM PBS containing phenol red indicator and antibiotics and kept on ice until processing. The spleens were next homogenized, absorbed into fresh medium and counted. To determine rHVT viremia / 5.0x10^6 splenocytes, this number of cells was added to a dish with an established CEF monolayer and incubated at 38°C for 3-4 days.

[0330] Alternatively, viremia was determined using peripheral blood lymphocytes (PBL) isolated from blood samples collected from chickens 3 or 4 weeks after vaccination. 5x10^6 PBL were then added to the CEF monolayers and incubated.

[0331] If the virus is present in the cells, it produces a cytopathic effect on CEFs, which can be seen as plaques. Three plates were counted from each animal sample. The plates were fixed with 96% ethanol and immunofluorescence assay (IFA) was performed with anti-HVT antiserum combined with staining with one of the antigens VP2, F or gD-gI to stain virus-infected cells. Therefore, three separate double stainings were performed.

[0332] 2.2.2 Serology

[0333] Blood samples for testing serological responses were collected 2-5 weeks after vaccination. Samples were centrifuged, serum was collected, and complement was inactivated. The serum was used in different tests to determine the serological response of the vaccinated chickens to the expressed heterologous genes: IBDV-VP2 response was measured by virus neutralization (VN) assay using the classical form of IBDV strain D78; Serological responses against NDV-F, IBDV-VP2 and ILTV-gD-gI were measured by ELISA and expressed in units relative to standard samples.

[0334] 2.3. Results and Conclusions

[0335] 2.3.1. Viremia

[0336] rHVT viremia was detected in the spleens of six animals in each group 15 days after inoculation or in the PBLs of five animals in each group 25 days after inoculation.

[0337] The mean spleen viremia in each group receiving one of the rHVT vaccines was:

[0338] -Control vector vaccine HVP360: 93 PFU / 5 million spleen cells,

[0339] - rHVT with gD-gI insertion near the center of UL39 or the 3' end of the UL39 gene:

[0340] No HVT viremia was detected; no replication in vivo,

[0341] - rHVT score with gD-gI insertion in UL40-41 or in UL47-48:

[0342] 36, and 45 PFU / 5x10^6 respectively. This means that the viremia is lower than that of the HVP360 control, and therefore the in vivo replication is reduced.

[0343] The mean viremia of each group was measured in PBL units and expressed as PFU / 5x10^6 as follows:

[0344] -HVP360:17,

[0345] -HVP412:15,

[0346] -HVP413:9,

[0347] -G7: 19, and

[0348] -G8:8.

[0349] Conclusion: All rHVT vector constructs HVP412, HVP413 and G7 had good viremia after vaccination, comparable to HVP360; the viremia of the G8 construct was moderate. However, the viremia of the rHVT with a gD-gI insertion between UL54 and LORF3 (rHVT according to the present invention) was the best among the tested groups.

[0350] 2.3.2 In vivo genetic stability

[0351] rHVT viruses obtained from vaccinated chickens were tested for persistent expression of the heterologous gene insert. Approximately 100 rHVT plaques from all isolates from spleens at day 15 post-inoculation were analyzed by IFA.

[0352] Both rHVT vectors with gD-gI insertions in UL39 did not replicate in vivo, and therefore stability could not be determined.

[0353] The rHVT vector with gD-gI insertion in UL40-41 showed genetic instability after 15 days of in vivo replication, as 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 gD-gI insertion between UL47 and UL48 lost expression of the gD and gI genes.

[0354] For the other rHVT constructs: rHVT with gD-gI insertions within 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 in vivo replication. Among them, the G7 construct (rHVT according to the invention) showed full expression of all heterologous inserts even after 21 days of inoculation.

[0355] 2.3.3 Serology

[0356] In nature, immune responses against pathogens producing three heterologous antigens: IBDV, NDV and ILTV all rely heavily on humoral immune responses. Therefore, measurement of antibody responses generated by vaccination is a reliable correlate of in vivo protection against infection and / or disease from these pathogens.

[0357] Serological responses induced by vaccination of chickens with various rHVT vector vaccines were analyzed by ELISA. Negative controls were hatched chicks from the same batch of animals, which were tested before vaccination. Positive controls for seroprotection against NDV and IBDV were vaccinated with HVP360 vector vaccines.

[0358] All hatching chicks tested on day 1 did not have any detectable antibody titers against the pathogens NDV, IBDV or ILTV.

[0359] Use a commercial ELISA test (ID Screen TM ILT gI Indirect, from ID vet) tests anti-ILTV serum responses. In this test, a result value above 611 indicates a protective immune response.

[0360] The average ELISA score results (n=6) of different groups on specific days after vaccination are shown in Table 3.

[0361] Table 3: ELISA scores for anti-ILTV serological responses induced by vaccination with rHVT vectors

[0362]

[0363] It is clear from the ELISA results shown in Table 3 that rHVT with a gD-gI insertion between UL47-48 induced only very low levels of anti-ILT serum at each of the tested days 22, 32, and 42 after vaccination; thus, it was always below the protective level. Therefore, even though stable in vitro and in vivo, rHVT with a gD-gI insertion between UL47-48 cannot be used as a multivalent vector vaccine against ILTV infection.

[0364] However, rHVT constructs with gD-gl insertions between UL44-45 (HVP412) or UL45-46 (HVP413) scored well above the protection level at days 32 and 42 post-inoculation.

[0365] Furthermore, on all tested days, the serum responses against NDV and IBDV induced by vaccination with constructs HVP412 and HVP413 showed ELISA score values ​​very close to those induced by the parental vector HVP360. Thus, 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 gI genes into the UL region.

[0366] 2.3.4 Further serological data from comparative experiments with the rHVT-VP2-F-gD-gI construct

[0367] As described above, similar serological testing was performed on samples obtained from chickens vaccinated with one of the HVP412, HVP413, G7, and G8 constructs in several comparative vaccination-challenge experiments described in detail below.

[0368] IBDV serology

[0369] IBDV serology induced by four rHVT-VP2-F-gD-gI constructs (by ID VET ID IBDVP2 Elisa measurement) was slightly lower than the serology induced by HVP360 at 3 weeks after vaccination, but reached the level of HVP360 at 4 weeks after vaccination. The average anti-IBDV titer induced by the rHVT-VP2-F-gD-gI construct at 4 weeks after vaccination was between 11 and 12 Log2, and the titers of HVP412 and G8 were lower than those of HVP413 and G7. The protective titer was about 10.2 Log2.

[0370] NDV serology

[0371] As for IBDV, NDV serological responses induced by the four rHVT-VP2-F-gD-gI constructs tested (by ID VET ID ND Elisa measurement) was slightly delayed compared to the serological response induced by the rHVT-VP2-F (HVP360) construct itself: while HVP360 already showed an average anti-NDV titer of 10.4Log2 at 3 weeks post-vaccination, the 4 rHVT-VP2-F-gD-gI constructs only showed titers between 7 and 9Log2 at this time. However, this situation quickly recovered and all 4 rHVT-VP2-F-gD-gI constructs induced good anti-NDV titers of about 11Log 2 at 4 weeks post-vaccination, close to the titers of HVP360, while the titers from G7 and G8 vaccination were slightly lower than those induced by HVP412 and HVP413. The protective titer was about 9.7Log2.

[0372] ILTV serology

[0373] ILTV serology induced by 4 rHVT-VP2-F-gD-gI constructs (by ID VET ID ILTVElisa measurements) showed clear differences at both 3 and 4 weeks post-vaccination: HVP412 and HVP413 constructs induced anti-ILTV titers that were 1.5-2 Log points lower than those induced by G7 and G8 constructs, which were 9.1 and 9.2 Log2 at 3 weeks and 10.6 and 10.5 Log2 at 4 weeks, respectively. The fact that most titers were below the so-called protective titer (9.2 Log2) indicated by the test manufacturer is inconsistent with the excellent clinical protection observed in this experiment. Therefore, for this test, serology does not correlate well with in vivo protection.

[0374] Example 3: Vaccination-Challenge Experiment 3.

[0376] 3.1. Introduction

[0377] To expand on the in vivo serological data described in Example 2, several vaccination-challenge experiments were performed in which we compared the vaccine efficacy of the rHVT-VP2-F-gD-gI constructs HVP412, HVP413, G7 and G8, which have been shown to be stable in vivo. These experiments were all performed in SPF chickens, which were vaccinated at day old and challenged with a virulent strain of one of the pathogens NDV, IBDV or ILTV at different times after vaccination. In these experiments, multiple samples were collected and parameters of infection and vaccine efficacy were measured. These challenges were mainly performed as described above, for example, NDV or IBDV challenges described in WO 2016 / 102647, and ILTV challenges described in WO 2019 / 072964.

[0378] 3.2. Materials and methods

[0379] 3.2.1 Commonalities of Vaccination Trials

[0380] -SPF white Leghorn laying hens were hatched in isolators; each chicken was individually marked with a tag number and vaccinated on the same day via subcutaneous route in the neck with 2000 pfu (=10^3.3) of one of the rHVT-VP2-F-gD-gI constructs (HVP412, HVP413, G7 or G8), administered in the form of infected CEF at a cell passage level of 15 or 16, 0.2 ml per dose in standard dilution.

[0381] - A similar dose of one of the commercially available bivalent rHVT vector vaccines was used as a positive control: HVT-ND-IBD (HVP360, WO 2016 / 102647; ND-IBD {MSD Animal Health}) vector, or WO 2013 / 057236 ( The HVT-ND-ILT vector disclosed in ND-ILT {MSD Animal Health}).

[0382] - Negative controls were not vaccinated and their group size was 9 or 10 birds / group.

[0383] - For the different vaccination-challenge trials, the group size was 10, 11 or 15 birds / group; each group was housed in a different isolator. For each experiment, 5 or 10 hatching chicks were bled at the beginning of the experiment to confirm the seronegative status.

[0384] - Confirm vaccination, viremia and expression by testing spleen or PBL samples 2 or 3 weeks after vaccination as described in Example 2.

[0385] 3.2.2 Detailed description of the attack used

[0386] IBDV Attack

[0387] IBDV challenge was performed according to European Pharmacopoeia (Ph. Eur.) monograph 0587, with a group size of 10 birds. At 3 weeks post-vaccination, each bird received 30 chicken infectious doses 50% (CID50) of IBDV strain CS89 in 0.1 ml PBS via eye drops divided in both eyes.

[0388] After challenge, the birds were monitored for clinical signs of IBD for 10 days and each bird was assigned a clinical score daily ranging from 0 to 3 (for no signs, signs, severe signs, and death, respectively). Clinical signs of IBD are: depression, huddling, paleness, anorexia, ruffled feathers, and fecal abnormalities. Next, all remaining birds were euthanized and the bursa was scored macroscopically and samples were taken for histological analysis, which used conventional criteria such as: macroscopically: enlargement and edema; and microscopically: the percentage of lymph nodes showing lymphocyte depletion, necrosis, and heterophil influx (in units of 25%). Observations in any of these tests were also included in the total clinical score.

[0389] NDV attack

[0390] NDV attack is carried out according to Ph.Eur. monograph 0450, using 15 chickens / group. Specifically: attack by applying 5Log10 EID50 of NDV strain Herts 33 / 56 in 0.2ml PBS to each chicken after 5 weeks of inoculation, given by intramuscular injection route. One group of 9 unvaccinated chickens is used as attack control. After the attack, chickens are observed every day for up to 14 days, and the NDV clinical score of 0-3 (for no signs-signs-serious signs-death) is assigned to each chicken every day. The typical signs of NDV infection are neurological symptoms, such as: neck twisting, obvious wing drooping, uncoordinated gait, paralysis, muscle tremor and body backward curling. After inoculation 21 days (the day before the attack), blood samples are collected from 5 chickens in each group to confirm the viremia in PBL, as well as serology and heterologous gene expression.

[0391] ILTV Attack

[0392] ILTV challenge was performed according to Ph. Eur. Monograph 1068, starting with 11 birds per group. Challenge was administered 4 weeks post-vaccination to 10 birds in each group, which received 3.0 Log10 EID50 of ILTV strain 96-3 as a CAM homogenate in standard cell culture medium. ILTV challenge virus was administered intratracheally at 0.1 ml / bird by syringe into the middle of the trachea.

[0393] Chickens were observed twice daily (morning and evening) for signs of ILT within 7 days after challenge; each chicken was assigned a clinical score ranging from 0 to 3 (for no signs, signs, severe signs, and death, respectively) in the morning. Typical clinical signs of ILTV infection are: marked dyspnea, dyspnea, wheezing, hemoptysis, nasal discharge, conjunctivitis, and swollen sinuses.

[0394] On day 7 post-challenge, chickens were euthanized, tracheas were isolated, and assessed for signs of ILTV infection by inspection of redness, type and consistency of contents. Observations in any of these tests were also included in the total clinical score.

[0395] Blood samples were collected from 10 chickens per group on day 28 post-vaccination (one day before challenge) to confirm viremia, serology and heterologous gene expression.

[0396] 3.3. result

[0397] Common findings from the attack

[0398] All chickens vaccinated with HVP412, HVP413, G7 or G8 showed good rHVT vector viremia and good expression of all heterologous gene inserts within 3, 4 or 5 weeks after vaccination, as described in Example 2.

[0399] For all comparative challenge trials, hatching chicks tested on day 1 were seronegative for NDV, IBDV and ILTV antibodies.

[0400] The total clinical score used for ILTV and IBDV results was the sum of all clinical scores assigned to each group of 10 chicks during the observation period, as well as the scores derived from the macroscopic and microscopic examinations performed.

[0401] IBDV challenge infection

[0402] The effectiveness of the IBDV vaccine was tested 3 weeks after vaccination, and all four rHVT-VP2-F-gD-gI constructs tested showed excellent IBDV vaccine effectiveness, as all birds in these groups were 100% protected, even after a very intense challenge: all 10 negative control chickens showed severe signs of IBDV infection on day 3 or 4 of challenge, and all died or had to be euthanized.

[0403] Among the protected groups, slight distinctions could be made based on the very mild residual clinical symptoms observed: the total clinical score for the HVP412 vaccine group was 7; for the HVP413 group it was 5; and for the G7 group it was only 2.

[0404] Thus, all rHVT-VP2-F-gD-gI constructs were able to induce excellent protection against IBDV challenge infection, but the vector vaccine of the G7 construct (rHVT according to the invention) resulted in almost no more clinical signs, even less than those in the HVP412 and HVP413 test groups.

[0405] NDV challenge infection

[0406] Five weeks after vaccination, the protection induced by the multivalent rHVT-VP2-F-gD-gI vector vaccine against severe NDV challenge infection was tested. All unvaccinated controls developed extensive clinical signs of NDV infection within 48 hours and were all euthanized for humane reasons, confirming the severity of the challenge infection.

[0407] The HVP412 construct was only able to protect 87% of the chickens (2 deaths out of 15), while the G8 construct performed even worse, protecting only 53% of the chickens (7 deaths out of 15). In contrast, both the HVP413 and G7 constructs were able to protect 100% of the chickens from severe NDV challenge infection.

[0408] ILTV aggressive infection

[0409] Protection against ILTV challenge was tested at 4 weeks post-vaccination and showed excellent protection for all 4 rHVT-VP2-F-gD-gI vector vaccines: all 4 vaccines produced 100% protection. In contrast, in the negative control group, all 10 birds showed severe clinical signs of ILTV infection on day 4 or 5 post-challenge: 7 of 10 died, and 2 of the remaining birds had to be euthanized.

[0410] Further distinctions can be made regarding vaccine efficacy induced by the rHVT-VP2-F-gD-gI construct based on the limited clinical symptoms observed in these vaccinated groups: groups receiving HVP412 or HVP413 vaccines still had clinical scores of 20 and 37, respectively. The group vaccinated with the G7 vaccine had a clinical score of only 6.

[0411] Thus, among the rHVT-VP2-F-gD-gI constructs tested for ILTV vaccination efficacy, the G7 construct (rHVT according to the present invention) performed best, preventing almost all clinical signs of severe ILTV challenge infection. This protection against ILTV challenge is almost as good as the protection induced by the commercial vector vaccine HVT-ND-ILT, which usually leaves only a clinical score of 1 point in similar trials.

[0412] 4. in conclusion

[0413] The vaccination-challenge experiments described in Example 3 were used to confirm and extend the in vitro test results in Example 1 and the in vivo tests and serological data described in Example 2. Taken together, these examples allow for a complete characterization of the different rHVT-VP2-F-gD-gI vector vaccine constructs tested, i.e., demonstrating not only their efficient replication and heterologous gene expression in vitro and in vivo, but also their ability to induce an effective immune response, thereby effectively protecting target animals from infection and / or disease caused by severe aggressive infection with the respective pathogens. In addition, by testing them in parallel in the same experiment, their properties and respective vaccine efficacy can be carefully compared under the same conditions.

[0414] Of the many rHVT-VP2-F-gD-gI constructs that have been established and tested over time, only four were found to be genetically stable in vivo: HVP412, HVP413, G7, and G8. All of these constructs provided 100% vaccine efficacy against IBDV challenge infection 3 weeks after vaccination and against ILTV challenge infection 5 weeks after vaccination. However, for virulent NDV challenge infection, only HVP413 and G7 constructs provided 100% protection at 5 weeks after vaccination; the other two constructs provided (much) less protection than 90%, making them ineffective NDV vaccines in practice. HVP413 and G7 can be further distinguished based on the level of residual clinical signs observed after challenge: rHVT-VP2-F-gD-gI construct G7 left fewer residual clinical signs than HVP413 vector vaccines, both after IBDV challenge infection and after ILTV challenge infection, i.e., the G7 construct protected more effectively than the HVP413 construct.

[0415] Conclusion: Of the numerous rHVT-VP2-F-gD-gI constructs prepared and tested, construct G7 (i.e., the rHVT according to the present invention) had the most favorable properties overall. In most cases, G7 was even comparable to the properties and vaccine properties of commercial rHVT vector vaccines carrying only two inserted heterologous genes. This is counterintuitive and better than what would be reasonably expected for a vector virus carrying four inserted heterologous genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0416] Figure 1 : Graphical representation of an exemplary rHVT vector construct according to the present invention:

[0417] The HVT genome is shown along the top, with multiple ORFs shown with square arrows. Thin line boxes indicate repeated regions.

[0418] The middle section amplifies the HVT genomic region where the insertion was introduced (between UL54 and LORF3, and in Us2).

[0419] An example of two inserted expression cassettes is shown at the bottom.

[0420] - Lower left: gD gene promoter-gD gene (including gI gene promoter)-gI gene (including gD gene terminator)-FHV1 Us9 gene terminator.

[0421] - Lower right: mCMV-IE1 gene promoter-IBDV VP2 gene-SV40 late gene terminator-hCMV IE1 gene promoter-NDV F gene-hCMV-IE1 gene terminator.

Claims

1. A recombinant herpesvirus of turkeys (rHVT) capable of expressing 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 genome of the rHVT, characterized in that The rHVT also expresses the glycoprotein D and glycoprotein I (gD and gl) genes of infectious laryngotracheitis virus (ILTV) from a second expression cassette inserted into the unique long (UL) region of the rHVT genome between the UL54 and LORF3 genes.

2. The rHVT according to claim 1, characterized in that The first expression cassette comprises, along the 5' to 3' direction and in the following order: a. Murine cytomegalovirus immediate early 1 (mCMV-IE1) gene promoter, b. IBDV VP2 gene, c. Transcription terminator, d. Human cytomegalovirus immediate early 1 (hCMV-IE1) gene promoter, e. NDV F protein gene, and f. Transcription terminator, And wherein the promoter and terminator are operably linked to the VP2 gene and to the F gene, respectively.

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

4. The rHVT according to any one of claims 1 to 3, characterized in that The second expression cassette comprises, along the 5' to 3' direction and in the following order: a. ILTV gD gene with an upstream promoter and a downstream terminator, and b. ILTV gI gene with upstream promoter and downstream terminator, And wherein the promoter and terminator are operably linked to the gD gene and to the gI gene, respectively.

5. A method for constructing rHVT according to any one of claims 1 to 4, the method comprising inserting the first expression cassette and the second expression cassette as defined in any one of claims 1, 2 or 4 into the region of the HVT genome as defined in claims 1 and 3. A host cell comprising the rHVT according to any one of claims 1 to 4.

7. The rHVT according to any one of claims 1 to 4 and / or the host cell according to claim 6, for use in a poultry vaccine.

8. A poultry vaccine comprising the rHVT according to any one of claims 1 to 4 and / or the host cell according to claim 6, and a pharmaceutically acceptable carrier.

9. The vaccine according to claim 8, comprising at least one additional immunologically active component.

10. A method for preparing the poultry vaccine according to claim 8 or 9, the method comprising the following steps: a. infecting a host cell in vitro with rHVT according to any one of claims 1 to 4, b. culturing and harvesting the infected host cells, and c. mixing the harvested infected host cells with a pharmaceutically acceptable carrier.

11. Use of the rHVT according to any one of claims 1 to 4 and / or the host cell according to claim 6 in the preparation of a poultry vaccine.

12. Use of the poultry vaccine according to claim 8 or 9 for preventing or reducing infection by one or more of MDV, IBDV, NDV and ILTV and / or related disease symptoms thereof.

13. A method of preventing or reducing infection by one or more of MDV, IBDV, NDV and ILTV and / or their associated disease symptoms, the method comprising administering a vaccine according to any one of claims 8 or 9 to poultry.

14. A method of vaccinating poultry to prevent or reduce infection by one or more of MDV, IBDV, NDV and ILTV and / or disease symptoms associated therewith, the method comprising the step of vaccinating the poultry with the vaccine according to claim 8 or 9.

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