Recombinant non-pathogenic Marek's disease virus constructs encoding multiple heterologous antigens

Abstract

The present invention discloses novel recombinant multivalent non-pathogenic Marek's Disease virus constructs that encode and express foreign antigens from three or more avian viruses and methods for using multivalent poultry virus vaccines. [Selected Figure] Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 571,524, filed October 12, 2017, and U.S. Provisional Patent Application No. 62 / 729,673, filed September 11, 2018, the contents of which are incorporated by reference herein in their entireties.

[0002] FIELD OF THE INVENTION The present invention relates to novel recombinant, multivalent, non-pathogenic Marek's Disease virus constructs that encode and express foreign antigens from three or more avian viruses, as well as methods for using these multivalent, recombinant, non-pathogenic Marek's Disease virus constructs in poultry vaccines. [Background technology]

[0003] Pathogenic poultry viruses not only debilitate chickens but also cause great losses to poultry farmers because most of the resulting diseases are contagious and the poultry industry is heavily dependent on large-scale confinement breeding facilities. Vaccination of young chickens is often the only practical means to combat these viruses. While attenuated or killed poultry virus vaccines remain important in the market, in recent years considerable resources have been devoted to the development of vaccines containing recombinant viral constructs expressing pathogenic viral protein antigens. Furthermore, stable and effective multivalent recombinant avirulent Marek's disease virus (rMDV) expressing foreign genes from multiple viral pathogens has been developed. np Considerable efforts have been made to construct recombinant monovalent rMDV vectors (abbreviated as rMDVs). Such multivalent vaccines would help minimize the number of injections administered to chickens, thereby reducing discomfort and stress for the vaccinated chickens and significantly reducing labor and material costs. Vaccination with such a single multivalent construct would allow for the delivery of multiple recombinant monovalent rMDV vectors. np Alternative multivalent rMDV containing constructs npThese alternative vaccines would be preferable to monovalent rMDV vaccines because, at least at present, these alternative vaccines only provide protection against a single viral pathogen. The failure of such alternative vaccines is likely due to the lack of protection against monovalent rMDV. np One of the constructs is another monovalent rMDV np These other monovalent rMDV constructs were not propagated and did not induce significant immune responses. np In either case, a stable and effective multivalent recombinant rMDV expressing foreign genes from multiple viral pathogens was obtained. np Despite considerable past efforts to construct vectors (indeed, such vaccines were suggested more than 20 years ago [see, e.g., US 5,965,138]), only recently have multivalent vaccines containing recombinant herpesvirus of turkeys (abbreviated as rHVT) encoding antigens from two or more other pathogens been shown to be stable and effective.

[0004] One poultry viral disease that can be controlled by vaccination is Marek's disease, a pathogenic disease that affects chickens worldwide. Marek's disease occurs primarily in young chickens aged 2 to 5 months. Clinical signs include progressive paralysis of one or more limbs, loss of coordination due to leg paralysis, drooping limbs due to wing involvement, and drooping head position due to neck muscle involvement. In acute cases, severe functional decline can occur. Bursal and thymic atrophy can also occur.

[0005] The causative agent of Marek's disease is Marek's disease virus serotype 1 (abbreviated as MDV1), a cell-associated virus with a double-stranded DNA genome. MDV1 is a lymphotropic avian alphaherpesvirus that causes both (i) infection of B cells, which can cause cell lysis, and (ii) latent infection of T cells, which can induce T-cell lymphoma. Closely related to the virulent MDV1 strain, Marek's disease virus serotype 2 (abbreviated as MDV2), previously known as avian herpesvirus 3, is a naturally attenuated MDV strain that has been shown to have little or no pathogenicity in chickens (Petherbridge et al., J. Virological Methods 158:11-17 (2009)). SB-1 is a specific MDV2 strain that has been shown to be useful in vaccines against MDV1 [see, eg, Murthy and Calnek, Infection and Immunity 26(2)547-553 (1979)].

[0006] Another closely related alphaherpesvirus, Marek's disease virus serotype 3 (abbreviated as MDV3), more commonly known as turkey herpesvirus (abbreviated as HVT), is a nonpathogenic virus of domestic turkeys (see, e.g., Kingham et al., J. of General Virology 82:1123-1135 (2001)). Two commonly used HVT strains are PB1 and FC126. HVT is also nonpathogenic in chickens, but it induces a long-lasting protective immune response against MDV1 in chickens. Therefore, HVT has been used for many years in poultry vaccines against virulent MDV1, typically in combination with SB-1, which is more viremic than HVT but is considered less safe. Alternatively, HVT can be combined with Rispen's vaccine when challenging bird populations with a particularly virulent MDV1 strain. The Rispen vaccine is an isolate derived from the low-virulence MDV1 strain and then further attenuated by cell passage, but the Rispen strain retains some virulence against highly susceptible strains of chicken.

[0007] The complete genome sequence of HVT has been published [Afonso et al., J. Virology 75(2):971-978 (2001)], and like most alphaherpesviruses, HVT has a significant number of potential non-essential insertion sites [see, e.g., US 5,187,087; US 5,830,745; US 5,834,305; US 5,853,733; US 5,928,648; US 5,961,982; US 6,121,043; US 6,299,882 B1]. HVT has also been shown to be amenable to genetic modification and has therefore been used as a recombinant vector for many years [WO 87 / 04463]. Thus, recombinant HVT vectors have been reported to express foreign genes encoding antigens derived from, for example, Newcastle disease virus (NDV) [Sondermeijer et al., Vaccine, 11:349-358 (1993); Reddy et al., Vaccine, 14:469-477 (1996)], infectious bursal disease virus (IBDV) [Darteil et al., Virology, 211:481-490 (1995); Tsukamoto et al., J. of Virology 76(11):5637-5645 (2002)], and infectious laryngotracheitis virus (ILTV) [Johnson et al., Avian Disease, 54(4):1251-1259 (2010); WO 92 / 03554; US 6,875,856]. The complete genome sequence of MDV2 is also known (see GenBank acc.nr:AB049735.1 and Petherbridge et al., supra). The genome organization of MDV2 is very similar to that of HVT, and in particular the US region is identical to that of HVT (see Kingham et al., supra).

[0008] Also, recombinant chimeric viruses known as novel avian herpesviruses (NAHVs) have been constructed in which specific regions of the HVT genome have been replaced by corresponding regions of the MDV1 genome. NAHVs have also been used to express foreign genes encoding antigens from other poultry viruses [US 5,965,138; US 6,913,751].

[0009] Similar to MDV, infectious laryngotracheitis virus (ILTV or ILT) is an alphaherpesvirus that affects chickens worldwide [Fuchs et al., Veterinary Research 38:261-279 (2007)]. ILTV causes acute respiratory disease in chickens characterized by respiratory depression, shortness of breath, and expectoration of bloody exudates. Viral replication is restricted to cells of the respiratory tract, where infection causes tissue erosion and bleeding within the trachea.

[0010] Newcastle disease is another highly contagious and debilitating disease of chickens. The causative agent of Newcastle disease is Newcastle disease virus (NDV). NDV belongs to the order Mononegavirales and the family Paramyxoviridae. Newcastle disease virus has a non-segmented, negative-sense, single-stranded RNA genome. NDV is classified into three different pathotypes according to their virulence. Infection of poultry with avirulent lentogenic strains of NDV is virtually asymptomatic. In stark contrast, mesogenic (intermediate pathogenic) and lentogenic (highly pathogenic) NDV strains cause severe disease that can be fatal. Most types of NDV infect the respiratory and / or nervous system, which can cause shortness of breath and torticollis.

[0011] Infectious bursal disease virus (IBDV or IBD for short), also known as Gumboro disease virus, is the causative agent of infectious bursal disease. IBDV causes an acute, highly contagious viral infection of the lymphoid tissues of chickens, the primary target of which is the bursa of Fabricius, an essential immune organ in birds. Morbidity in susceptible bird populations is high, with rapid weight loss and moderate to severe mortality. Chickens that recover from the disease may exhibit immunodeficiency due to the destruction of (part of) the bursa of Fabricius, making them particularly susceptible to secondary infections.

[0012] IBDV is a member of the Birnaviridae family. Viruses in this family have a genome consisting of two segments (A and B) of double-stranded RNA. Two IBDV serotypes (serotypes 1 and 2) exist, which can be distinguished by virus neutralization (VN) tests. Serotype 1 viruses have been shown to be pathogenic for chickens, while serotype 2 viruses only cause subacute disease in turkeys. Historically, IBDV serotype 1 viruses consisted of only one type, now known as the "classical" IBD virus. Subsequently, so-called "mutant" IBDV strains emerged. Classical and mutant strains of IBDV can be identified and distinguished by virus neutralization tests using a panel of monoclonal antibodies or by RT-PCR [Wu et al., Avian Diseases, 51:515-526 (2007)]. Well-known classical IBDV strains include D78, Faragher 52 / 70, and STC, while 89 / 03 is a well-known variant. Numerous live or inactivated IBDV vaccines are commercially available, for example, live vaccines such as NOBILIS® Gumboro D78 (MSD Animal Health).

[0013] As mentioned above, because HVT can function as both an antigen and a recombinant vector to provide significant protection against Marek's disease, it is currently used as a platform vector for multivalent vaccines such as Innovax®-ILT (sold by Merck Animal Health), which protects against ILTV; and Innovax®-ND-SB (sold by Merck Animal Health), which both protect against NDV; Vectormune® HVT-NDV (sold by Ceva); and Vaxxitek® HVT+IBD (Merial; formerly Gallivac™ HVT-IBD) and Vectormune™ HVT-IBD (Ceva), which both protect against IBDV. Notably, Innovax®-ILT contains two foreign genes, i.e., ILTV gD and ILTV gI, which have been shown to be safe, effective, and stable. However, these two foreign genes are derived from the same pathogen, and furthermore, they are naturally overlapping and need to be co-expressed to enable adequate immunization against ILTV. More recently, a safe, effective, and stable recombinant multivalent vaccine containing HVT-ILTV-NDV has been disclosed [US 8,932,604 B2 and US 9,409,954 B2 (incorporated herein by reference in their entireties)]. An early HVT-NDV-IBDV was also disclosed; however, one of the lead constructs, HVP309, did not demonstrate sufficient genetic stability or sustained expression of the heterologous insert during long-term testing during product development [WO 2013 / 057,235]. Subsequently, a more stable and efficient construct was developed [WO 2016 / 102647]. Other recombinant HVT constructs have also been described [see, e.g., US 9,114,108, US 9,555,016, US 9,555,096, and US 2018 / 0163230 A1].

[0014] However, stable multivalent recombinant MDVs that can efficiently express heterologous antigens from three or more different pathogens have not been developed. npDespite the obvious advantages of the constructs, and the considerable efforts to design them, none have been achieved so far. np The failure of past attempts to construct a construct was due to the inclusion of foreign antigens from three or more different viral pathogens, including MDV. np This has led to a general consensus in the art that insertions into the construct place undue strain on the construct, resulting in the observed lack of stability. np The suitability of the recombinant MDV as a vaccine is determined by incorporating a combination of heterologous antigens derived from a unique combination of three or more poultry viruses into the recombinant MDV. np Therefore, novel stable recombinant MDVs that can be used in multivalent vaccines as the sole active ingredient to protect against three or more different non-MDV1 poultry viral pathogens remain unpredictable at best. np There is a clear need to overcome the accumulated industrial failures by constructing vectors.

[0015] The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] US5,965,138 [Patent Document 2] US5,187,087 [Patent Document 3] US5,830,745 [Patent Document 4] US5,834,305 [Patent Document 5] US5,853,733 [Patent Document 6] US5,928,648 [Patent Document 7] US5,961,982

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Non-licensed literature

[0017] [Non-licensed document 1] Petherbridge, J. Virological Methods 158:11-17(2009) [Non-licensed document 2] Murthy Calnek,Infection and Immunity 26(2)547-553(1979) [Non-licensed document 3] Kingham, J. of General Virology 82:1123-1135(2001)

Non-licensed Document 4

[0018] Overview of the invention Accordingly, the present invention provides a novel multivalent recombinant non-pathogenic Marek's disease virus (rMDV) vector for use as a vector to express foreign genes from multiple viral pathogens. np In certain embodiments, the rMDV np is a recombinant herpesvirus of turkeys (rHVT). In another embodiment, rMDV np is a recombinant Marek's disease virus serotype 2 (rMDV2). The rMDV of the present invention np The recombinant non-pathogenic Marek's disease virus (rMDV), e.g., rHVT or rMDV2, can be used in a safe and effective multivalent vaccine against pathogenic poultry viruses. Thus, the present invention provides recombinant non-pathogenic Marek's disease virus (rMDV), which encodes and expresses antigens from three or more exogenous chicken viral pathogens. np) vectors (including HVT vectors). In certain embodiments, rMDV np encodes one or more antigens from laryngotracheitis virus (ILTV), one or more antigens from infectious bursal disease virus (IBDV) and one or more antigens from Newcastle disease virus (NDV). In more particular embodiments, such rMDV np The vectors aid in vaccinating chickens to protect them from clinical signs resulting from infection with pathogenic MDV, pathogenic IBDV, pathogenic NDV and / or pathogenic ILTV. The vaccine is preferably effective for vaccination of healthy animals on embryonic day 18-19 and from day 1 of age onwards.

[0019] In certain embodiments, recombinant avirulent Marek's disease virus (rMDV) np ) comprises, at one or more non-essential sites of its genome, a first heterologous nucleotide sequence encoding one or more antigens from a first chicken pathogen, a second heterologous nucleotide sequence encoding one or more antigens from a second chicken pathogen, and a third heterologous nucleotide sequence encoding one or more antigens from a third chicken pathogen. In a specific embodiment, the first chicken pathogen, the second chicken pathogen, and the third chicken pathogen are all avian (bird) viruses. In a more particular embodiment, the first chicken pathogen, the second chicken pathogen, and the third chicken pathogen are all different viral species from each other and from Marek's disease virus. In a specific embodiment of this type, the first chicken pathogen is infectious bursal disease virus (IBDV), the second chicken pathogen is infectious laryngotracheitis virus (ILTV), and the third chicken pathogen is Newcastle disease virus (NDV).

[0020] Specific rMDV npIn this embodiment, the first heterologous nucleotide sequence encodes infectious bursal disease virus viral protein 2 (IBDV VP2), and / or the second heterologous nucleotide sequence encodes infectious laryngotracheitis virus glycoprotein D (ILTV gD), infectious laryngotracheitis virus glycoprotein I (ILTV gI), or both ILTV gI and ILTV gD, and / or the third heterologous nucleotide sequence encodes Newcastle disease virus fusion protein (NDV F). np In this embodiment, the first heterologous nucleotide sequence encodes infectious bursal disease virus viral protein 2 (IBDV VP2), the second heterologous nucleotide sequence encodes infectious laryngotracheitis virus glycoprotein D (ILTV gD) and infectious laryngotracheitis virus glycoprotein I (ILTV gI), and the third heterologous nucleotide sequence encodes Newcastle disease virus fusion protein (NDV F).

[0021] Therefore, the specific rMDV of the present invention np In this embodiment, the first heterologous nucleotide sequence, the second heterologous nucleotide sequence, and the third heterologous nucleotide sequence are rMDV. np The non-essential sites are located at three different non-essential sites in the genome. In certain embodiments, the three different sites are individually selected from the group consisting of the US2 site, the UL54.5 site, the UL7 / 8 site, the UL40 site, the UL43 site, the UL45 / 46 site, the UL55 site, the US10 site, the region between US10 and SORF3, the region between US2 and SORF3, the IG1 site, the IG2 site, and the IG3 site. In certain embodiments of this type, the first non-essential site is the US2 site, the second non-essential site is the UL54.5 site, and the third non-essential site is the UL45 / 46 site.

[0022] In another embodiment, the first heterologous nucleotide sequence, the second heterologous nucleotide sequence, and the third heterologous nucleotide sequence are rMDV. np First nonessential site in the genome or rMDV npIt is located at a second non-essential site in the genome. In particular embodiments of this type, the first and second non-essential sites are identical (i.e., there is only one non-essential insertion site). In particular embodiments of this type, the only non-essential insertion site is the US2 site. In other embodiments of this type, the only non-essential insertion site is the UL54.5 site. In yet other embodiments of this type, the only non-essential insertion site is the UL7 / 8 site. In yet other embodiments of this type, the only non-essential insertion site is the UL40 site. In yet other embodiments of this type, the only non-essential insertion site is the UL45 / 46 site. In yet other embodiments of this type, the only non-essential insertion site is the UL55 site. In yet other embodiments of this type, the only non-essential insertion site is the US10 site. In yet other embodiments of this type, the only non-essential insertion site is the region between US10 and SORF3. In yet other embodiments of this type, the only non-essential insertion site is the region between US2 and SORF3. In yet other embodiments of this type, the only non-essential insertion site is the IG1 site. In yet other embodiments of this type, the only non-essential insertion site is the IG2 site. In yet other embodiments of this type, the only non-essential insertion site is the IG3 site. In yet other embodiments of this type, the only non-essential insertion site is the UL43 site.

[0023] In yet another type of embodiment, the first and second non-essential sites are different. The two different sites are individually selected from the group consisting of the US2 site, the UL54.5 site, the UL7 / 8 site, the UL40 site, the UL43 site, the UL45 / 46 site, the UL55 site, the US10 site, the region between US10 and SORF3, the region between US2 and SORF3, the IG1 site, the IG2 site, and the IG3 site. In certain embodiments, the first and second heterologous nucleotide sequences are located at the first non-essential site, and the third heterologous nucleotide sequence is located at the second non-essential site. In other embodiments, the first and third heterologous nucleotide sequences are located at the first non-essential site, and the second heterologous nucleotide sequence is located at the second non-essential site. In yet other embodiments, the second and third heterologous nucleotide sequences are located at the first non-essential site, and the first heterologous nucleotide sequence is located at the second non-essential site. In preferred embodiments of this type, the first heterologous nucleotide sequence encodes infectious bursal disease virus viral protein 2 (IBDV VP2), the second heterologous nucleotide sequence encodes infectious laryngotracheitis virus glycoprotein D (ILTV gD), infectious laryngotracheitis virus glycoprotein I (ILTV gI) or both ILTV gI and ILTV gD, and the third heterologous nucleotide sequence encodes Newcastle disease virus fusion protein (NDV F).

[0024] In one embodiment of this type, a rMDV comprises a heterologous nucleotide sequence encoding an ILTV gD protein, an ILTV gI protein, and an IBDV VP2 protein in a first non-essential site, and a heterologous nucleotide sequence encoding an NDV F protein in a second non-essential site. np is constructed such that the heterologous nucleotide sequence encoding the IBDV VP2 protein is 5' to the heterologous nucleotide sequences encoding the ILTV gD protein and the ILTV gI protein. npis constructed such that the heterologous nucleotide sequence encoding the ILTV gD protein and the ILTV gI protein is 5' to the heterologous nucleotide sequence encoding the IBDV VP2 protein.

[0025] In an alternative embodiment, the rMDV comprises a heterologous nucleotide sequence encoding an NDV F protein and an IBDV VP2 protein in a first non-essential site, and a heterologous nucleotide sequence encoding an ILTV gD protein and an ILTV gI protein in a second non-essential site. np is constructed such that the heterologous nucleotide sequence encoding the NDV F protein is 5' to the heterologous nucleotide sequence encoding the IBDV VP2 protein. np is constructed such that the heterologous nucleotide sequence encoding the IBDV VP2 protein is 5' to the heterologous nucleotide sequence encoding the NDV F protein.

[0026] In yet another alternative embodiment, a rMDV comprising a heterologous nucleotide sequence encoding an NDV F protein, an ILTV gD protein, and an ILTV gI protein in a first non-essential site, and a heterologous nucleotide sequence encoding an IBDV VP2 protein in a second non-essential site. np is constructed such that the heterologous nucleotide sequence encoding the NDV F protein is 5' to the heterologous nucleotide sequences encoding the ILTV gD protein and the ILTV gI protein. np is constructed such that the heterologous nucleotide sequence encoding the ILTV gD protein and the ILTV gI protein is 5' to the heterologous nucleotide sequence encoding the NDV F protein.

[0027] In particular embodiments of this type, the first non-essential site is a US2 site and the second non-essential site is a UL54.5 site. In alternative embodiments, the first non-essential site is a UL54.5 site and the second non-essential site is a US2 site. In yet another embodiment of this type, the first non-essential site is a US2 site and the second non-essential site is a UL45 / 46 site. In yet another embodiment of this type, the first non-essential site is a UL45 / 46 site and the second non-essential site is a US2 site. In yet another embodiment of this type, the first non-essential site is a UL54.5 site and the second non-essential site is a UL45 / 46 site. In yet another embodiment of this type, the first non-essential site is a UL45 / 46 site and the second non-essential site is a UL54.5 site. In yet another embodiment of this type, the first non-essential site is a US2 site and the second non-essential site is a UL55 site. In yet another embodiment of this type, the first non-essential site is a UL55 site and the second non-essential site is a US2 site.

[0028] Therefore, the rMDV of the present invention np A vector can contain heterologous nucleotide sequences encoding any combination of these foreign protein antigens. In a specific embodiment, the ILTV gD protein comprises the amino acid sequence of SEQ ID NO: 2. In another embodiment, the ILTV gI protein comprises the amino acid sequence of SEQ ID NO: 4. In yet another embodiment, the IBDV VP2 protein comprises the amino acid sequence of SEQ ID NO: 6. In yet another embodiment, the NDV F protein comprises the amino acid sequence of SEQ ID NO: 8. In yet another embodiment, the NDV F protein comprises the amino acid sequence of SEQ ID NO: 10. Furthermore, the present invention also provides rMDVnp vectors comprising any combination of nucleotide sequences encoding one or more of these amino acid sequences, including specific embodiments encoding all of them.

[0029] rMDV npIn yet other embodiments, the ILTV gD protein comprises the amino acid sequence of SEQ ID NO:2, and the ILTV gI protein comprises the amino acid sequence of SEQ ID NO:4. In certain embodiments of this type, the ILTV gD protein comprises the amino acid sequence of SEQ ID NO:2, the ILTV gI protein comprises the amino acid sequence of SEQ ID NO:4, and the IBDV VP2 protein comprises the amino acid sequence of SEQ ID NO:6. In related embodiments, the ILTV gD protein comprises the amino acid sequence of SEQ ID NO:2, the ILTV gI protein comprises the amino acid sequence of SEQ ID NO:4, and the NDV F protein comprises the amino acid sequence of SEQ ID NO:8. In similar embodiments, the ILTV gD protein comprises the amino acid sequence of SEQ ID NO:2, the ILTV gI protein comprises the amino acid sequence of SEQ ID NO:4, and the NDV F protein comprises the amino acid sequence of SEQ ID NO:10. In more particular embodiments, the ILTV gD protein comprises the amino acid sequence of SEQ ID NO:2, the ILTV gI protein comprises the amino acid sequence of SEQ ID NO:4, the IBDV VP2 protein comprises the amino acid sequence of SEQ ID NO:6, and the NDV F protein comprises either the amino acid sequence of SEQ ID NO:8 or the amino acid sequence of SEQ ID NO:10. In specific embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0030] In a related embodiment, the ILTV gD protein is encoded by the nucleotide sequence of SEQ ID NO: 1. In another embodiment, the ILTV gI protein is encoded by the nucleotide sequence of SEQ ID NO: 3. In yet another embodiment, the IBDV VP2 protein is encoded by the nucleotide sequence of SEQ ID NO: 5. In yet another embodiment, the NDV F protein is encoded by the nucleotide sequence of SEQ ID NO: 7. In yet another embodiment, the NDV F protein is encoded by the nucleotide sequence of SEQ ID NO: 9.

[0031] Similarly, the rMDV of the present invention npA vector can contain heterologous nucleic acid comprising any combination of such heterologous nucleotide sequences. np is rMDV np a first heterologous nucleic acid located at a first non-essential site in the genome of the rMDV; np and a second heterologous nucleic acid located at a second non-essential site within the genome, wherein the first heterologous nucleic acid comprises both the first heterologous nucleotide sequence and the second heterologous nucleotide sequence, while the second heterologous nucleic acid comprises a third heterologous nucleotide sequence.

[0032] In certain embodiments of this type, the first heterologous nucleic acid comprises a heterologous nucleotide sequence encoding an infectious laryngotracheitis virus (ILTV) glycoprotein D (gD) protein, an infectious laryngotracheitis virus (ILTV) glycoprotein I (gI) protein, and an infectious bursal disease virus (IBDV) viral protein 2 (VP2), while the second heterologous nucleic acid comprises a heterologous nucleotide sequence encoding a Newcastle disease virus (NDV) F protein. In certain embodiments of this type, the first heterologous nucleic acid is constructed such that the heterologous nucleotide sequence encoding the IBDV VP2 protein is 5' to the heterologous nucleotide sequence encoding the ILTV gD protein and the ILTV gI protein, and / or rMDV. np In other embodiments of this type, the first heterologous nucleic acid is constructed such that the heterologous nucleotide sequence encoding the ILTV gD protein and the ILTV gI protein is 5' to the heterologous nucleotide sequence encoding the IBDV VP2 protein, and / or the rMDV npIn an alternative embodiment, the first heterologous nucleic acid comprises a heterologous nucleotide sequence encoding an NDV F protein and an IBDV VP2 protein, while the second heterologous nucleic acid comprises a heterologous nucleotide sequence encoding an ILTV gD protein and an ILTV gI protein. In certain embodiments of this type, the first heterologous nucleic acid is constructed such that the heterologous nucleotide sequence encoding the NDV F protein is 5' to the heterologous nucleotide sequence encoding the IBDV VP2 protein, and / or np In an alternative embodiment of this type, the first heterologous nucleic acid is constructed such that the heterologous nucleotide sequence encoding the IBDV VP2 protein is 5' to the heterologous nucleotide sequence encoding the NDV F protein, and / or the rMDV np It is inserted into the genome.

[0033] In yet another alternative embodiment, the first heterologous nucleic acid comprises a heterologous nucleotide sequence encoding an NDV F protein, an ILTV gD protein, and an ILTV gI protein, while the second heterologous nucleic acid comprises a heterologous nucleotide sequence encoding an IBDV VP2 protein. In certain embodiments of this type, the first heterologous nucleic acid is constructed such that the heterologous nucleotide sequence encoding the NDV F protein is 5' to the heterologous nucleotide sequence encoding the ILTV gD protein and the ILTV gI protein, and / or np In alternative embodiments of this type, the first heterologous nucleic acid is constructed such that the heterologous nucleotide sequences encoding the ILTV gD protein and the ILTV gI protein are 5' to the heterologous nucleotide sequence encoding the NDV F protein, and / or the rMDV np It is inserted into the genome.

[0034] Furthermore, the present invention also provides rMDVs encoding any combination of these nucleotide sequences. npVectors are provided, and in certain embodiments encompassed therein, a first heterologous nucleic acid encodes the nucleotide sequences of SEQ ID NOs: 1, 3, and 5, and a second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 7. In alternative embodiments, a first heterologous nucleic acid encodes the nucleotide sequences of SEQ ID NOs: 1, 3, and 5, and a second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 9. In certain embodiments, an rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0035] In certain embodiments, rMDV np The two different non-essential sites are individually selected from the group consisting of the US2 site, the UL54.5 site, the UL7 / 8 site, the UL40 site, the UL43 site, the UL45 / 46 site, the UL55 site, the US10 site, the region between US10 and SORF3, the region between US2 and SORF3, the intergenic region 1 (IG1) site, the intergenic region 2 (IG2) site and the intergenic region (IG3).

[0036] In one embodiment, the rMDV np The first non-essential site of rMDV is the US2 site. np The second non-essential site is a non-essential site other than the US2 site. np The first non-essential site of rMDV is the UL54.5 site. npand the second non-essential site is a non-essential site other than the UL54.5 site. In particular embodiments of this type, the first non-essential site is a US2 site and the second non-essential site is a UL54.5 site. In alternative embodiments, the first non-essential site is a UL54.5 site and the second non-essential site is a US2 site. In yet another embodiment of this type, the first non-essential site is a US2 site and the second non-essential site is a UL45 / 46 site. In yet another embodiment of this type, the first non-essential site is a UL45 / 46 site and the second non-essential site is a US2 site. In yet another embodiment of this type, the first non-essential site is a UL54.5 site and the second non-essential site is a UL45 / 46 site. In yet another embodiment of this type, the first non-essential site is a UL45 / 46 site and the second non-essential site is a UL54.5 site. In yet another embodiment of this type, the first non-essential site is a US2 site and the second non-essential site is a UL55 site. In yet another embodiment of this type, the first non-essential site is a UL55 site and the second non-essential site is a US2 site. In a related embodiment, np The first nonessential site of rMDV is UL54.5. np In yet another embodiment, the second non-essential site is the UL7 / 8 site. np The first nonessential site of rMDV is UL54.5. np In a related embodiment, the second non-essential site is the US10 site. np The second nonessential site in rMDV is the US2 site. np In yet another embodiment, the first non-essential site is the UL7 / 8 site. np The second nonessential site is the US2 site, np The first non-essential site is the US10 site. np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0037] In certain embodiments of this type, the first non-essential site and the second non-essential site are the same (i.e., there is only one non-essential insertion site). In certain embodiments of this type, the only non-essential insertion site is the US2 site. In other embodiments of this type, the only non-essential insertion site is the UL54.5 site. In still other embodiments of this type, the only non-essential insertion site is the UL7 / 8 site. In still other embodiments of this type, the only non-essential insertion site is the UL40 site. In still other embodiments of this type, the only non-essential insertion site is the UL45 / 46 site. In still other embodiments of this type, the only non-essential insertion site is the UL55 site. In still other embodiments of this type, the only non-essential insertion site is the US10 site. In still other embodiments of this type, the only non-essential insertion site is the region between US10 and SORF3. In still other embodiments of this type, the only non-essential insertion site is the region between US2 and SORF3. In still other embodiments of this type, the only non-essential insertion site is the IG1 site. In still other embodiments of this type, the only non-essential insertion site is the IG2 site. In still other embodiments of this type, the only non-essential insertion site is the IG3 site. In still other embodiments of this type, the only non-essential insertion site is the UL43 site. In certain embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0038] The nucleotide sequences encoding the ILTV gD protein, the ILTV gI protein, and the IBDV VP2 protein are expressed by an exogenous promoter, i.e., MDV npIn certain embodiments, the three nucleotide sequences may be operably present under the control of a promoter not naturally found in rMDV. In certain embodiments, these three nucleotide sequences may be operably present under the control of different promoters. That is, the nucleotide sequence encoding the ILTV gD protein is operably present under the control of a first promoter, the nucleotide sequence encoding the ILTV gI protein is operably present under the control of a second promoter, and the nucleotide sequence encoding the IBDV VP2 protein is operably present under the control of a third promoter, wherein the first promoter, second promoter, and third promoter are all different. In certain embodiments, the promoter for the nucleotide sequence encoding the ILTV gD protein is the endogenous ILTV gD promoter (i.e., endogenous to ILTV). In certain embodiments, the promoter for the nucleotide sequence encoding the ILTV gI protein is the endogenous ILTV gI promoter. In certain embodiments of this type, the promoter for the nucleotide sequence encoding the ILTV gD protein is the endogenous ILTV gD promoter, and the promoter for the nucleotide sequence encoding the ILTV gI protein is the endogenous ILTV gI promoter. In certain embodiments ...I protein is the endogenous ILTV gI promoter. np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0039] In certain embodiments, at least one of the promoters operably linked to the nucleotide sequence encoding the NDV F protein, ILTV gD protein, ILTV gI protein, or IBDV VP2 protein is a murine cytomegalovirus immediate-early (mCMV IE) promoter. In a related embodiment, at least one of the promoters operably linked to the nucleotide sequence encoding the NDV F protein, ILTV gD protein, ILTV gI protein, or IBDV VP2 protein is a human cytomegalovirus immediate-early (hCMV IE) promoter or a derivative thereof (e.g., from strain AD169). In other embodiments, at least one of the promoters operably linked to the nucleotide sequence encoding the NDV F protein, ILTV gD protein, ILTV gI protein, or IBDV VP2 protein is a guinea pig cytomegalovirus immediate-early promoter. In other embodiments, at least one of the promoters operably linked to the nucleotide sequence encoding the NDV F protein, ILTV gD protein, ILTV gI protein, or IBDV VP2 protein is a chicken β-actin promoter. In yet other embodiments, at least one of the promoters operably linked to a nucleotide sequence encoding the NDV F protein, ILTV gD protein, ILTV gI protein, or IBDV VP2 protein is a pseudorabies virus (PRV) gpX promoter.

[0040] In a specific embodiment, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is the mCMV IE promoter. In a related embodiment, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is the human cytomegalovirus immediate-early (hCMV IE) promoter or a derivative thereof (e.g., from strain AD169). In another embodiment, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is the guinea pig cytomegalovirus immediate-early promoter. In yet another embodiment, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is the chicken beta-actin gene promoter.

[0041] In some embodiments, the promoter operably linked to the nucleotide sequence encoding the NDV F protein is the human cytomegalovirus immediate-early (hCMV IE) promoter. In other embodiments, the promoter operably linked to the nucleotide sequence encoding the NDV F protein is the pseudorabies virus (PRV) gpX promoter. In related embodiments, the promoter operably linked to the nucleotide sequence encoding the NDV F protein is the chicken beta-actin gene promoter. In yet other embodiments, the promoter operably linked to the nucleotide sequence encoding the NDV F protein is the simian virus 40 (SV40) promoter.

[0042] In more particular embodiments, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is the mCMV IE promoter, the promoter for the nucleotide sequence encoding the ILTV gD protein is the endogenous ILTV gD promoter, the promoter for the nucleotide sequence encoding the ILTV gI protein is the endogenous ILTV gI promoter, and the promoter for the nucleotide sequence encoding the NDV F protein is the hCMV IE promoter. In other specific embodiments, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is the hCMV IE promoter (or a derivative thereof), the promoter for the nucleotide sequence encoding the ILTV gD protein is the endogenous ILTV gD promoter, the promoter for the nucleotide sequence encoding the ILTV gI protein is the endogenous ILTV gI promoter, and the promoter for the nucleotide sequence encoding the NDV F protein is the hCMV IE promoter. In yet another specific embodiment, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is a chicken β-actin promoter, the promoter for the nucleotide sequence encoding the ILTV gD protein is an endogenous ILTV gD promoter, the promoter for the nucleotide sequence encoding the ILTV gI protein is an endogenous ILTV gI promoter, and the promoter for the nucleotide sequence encoding the NDV F protein is an hCMV IE promoter.

[0043] In one embodiment, the rMDV of the invention comprises an insertion of nucleotide sequences encoding an ILTV gD protein, an ILTV gI protein, and an IBDV VP2 protein. npalso contain one or more exogenous transcription termination sequences. In particular embodiments of this type, a transcription termination sequence is present downstream of the nucleotide sequence encoding the IBDV VP2 protein. In particular embodiments, the nucleotide sequences encoding the ILTV gD protein and the ILTV gI protein share one transcription termination sequence, and the nucleotide sequence encoding the IBDV VP2 protein has another. In more particular embodiments, at least one of the transcription termination sequences comprises a feline herpesvirus US-9 (FHV US-9) polyadenylation sequence. In even more particular embodiments, at least one of the transcription termination sequences comprises a simian virus 40 (SV40) polyadenylation sequence.

[0044] In certain embodiments, the NDV F protein also comprises one or more exogenous transcription termination sequences. In particular embodiments of this type, the transcription termination sequences are present downstream of the nucleotide sequence encoding the NDV F protein. In related embodiments, at least one of the transcription termination sequences comprises a herpes simplex virus thymidine kinase (HSV TK) polyadenylation sequence. In alternative embodiments, at least one of the transcription termination sequences comprises a human cytomegalovirus immediate early (hCMV IE) polyadenylation sequence. In particular embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0045] The present invention also provides a vector comprising (i) an mCMV IE promoter, a chicken beta-actin gene promoter, or an hCMV promoter, (ii) a coding sequence for an IBDV VP2 protein, (iii) a transcription termination sequence, (iv) an ILTV gD promoter, (v) a coding sequence for an ILTV gD protein, (vi) an ILTV gI promoter, and (vii) a coding sequence for an ILTV gI protein (rMDV np rMDV containing np In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 21 is npIn certain embodiments, the rMDV is contained within the UL54.5 region of the genome. np further comprises (i) an hCMV IE promoter, (ii) a coding sequence for the NDV F protein, and (iii) an hCMV IE transcription termination sequence (at the US2 site of its genome). In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 22 is np It is contained within the US2 locus of the genome.

[0046] The present invention further provides a vector comprising (i) an mCMV IE promoter, a chicken beta-actin gene promoter, or an hCMV promoter, (ii) a coding sequence for an IBDV VP2 protein, (iii) a transcription termination sequence, (iv) an ILTV gD promoter, (v) a coding sequence for an ILTV gD protein, (vi) an ILTV gI promoter, and (vii) a coding sequence for an ILTV gI protein (rMDV np rMDV containing np In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 24 is np In yet another embodiment of this type, the nucleotide sequence of SEQ ID NO: 25 is contained within the US2 region of the genome. np In a more particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 23 is contained within the US2 region of the genome. np In certain embodiments, the rMDV is contained within the US2 region of the genome. np further comprises (i) an hCMV IE promoter, (ii) a coding sequence for the NDV F protein, and (iii) an hCMV IE transcription termination sequence (at the UL54.5 site of its genome). In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 26 is np It is contained within the UL54.5 locus of the genome.

[0047] Furthermore, the present invention also provides a method for the production of a NDV F protein comprising the steps of: (i) an ILTV gD promoter; (ii) a coding sequence for an ILTV gD protein; (iii) an ILTV gI promoter; (iv) a coding sequence for an ILTV gI protein; (v) an hCMV IE promoter; (vi) a coding sequence for an NDV F protein; and (vii) an hCMV IE transcription termination sequence (rMDV). np rMDV containing np In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 31 is np In certain embodiments, the rMDV is contained within the UL54.5 region of the genome. np further comprising (i) the mCMV IE promoter, (ii) a coding sequence for the IBDV VP2 protein, and (iii) a transcription termination sequence (rMDV np In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 32 is np It is contained within the US2 locus of the genome.

[0048] The present invention also provides (i) an ILTV gD promoter, (ii) a coding sequence for an ILTV gD protein, (iii) an ILTV gI promoter, (iv) a coding sequence for an ILTV gI protein, (v) an hCMV IE promoter, (vi) a coding sequence for an NDV F protein, (vii) an hCMV IE transcription termination sequence (rMDV np rMDV containing np In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 28 is np In certain embodiments, the rMDV is contained within the US2 region of the genome. np further comprising (i) the mCMV IE promoter, (ii) a coding sequence for the IBDV VP2 protein, and (iii) a transcription termination sequence (rMDV np In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 27 is np It is contained within the UL54.5 locus of the genome.

[0049] The present invention further provides a vector comprising (i) an mCMV IE promoter, (ii) a coding sequence for an IBDV VP2 protein, (iii) a transcription termination sequence, (iv) an hCMV IE promoter, (v) a coding sequence for an NDV F protein, and (vi) an hCMV IE transcription termination sequence (rMDV). np rMDV containing np In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 30 is np In certain embodiments, the rMDV is contained within the US2 region of the genome. np further comprises (i) an ILTV gD promoter, (ii) a coding sequence for an ILTV gD protein, (iii) an ILTV gI promoter, and (iv) a coding sequence for an ILTV gI protein (contained within the UL54.5 locus of its genome). In a particular embodiment of this type, the nucleotide sequence of SEQ ID NO: 29 is np It is contained within the UL54.5 locus of the genome.

[0050] The present invention also provides a method for the preparation of a medicament ... np rMDV containing np In certain embodiments, the rMDV np further comprises (i) an ILTV gD promoter, (ii) a coding sequence for the ILTV gD protein, (iii) an ILTV gI promoter, and (iv) a coding sequence for the ILTV gI protein (contained within the US2 region of its genome).

[0051] The present invention also provides a method for the preparation of a medicament ... np in the US2 region of its genome), and a rMDV comprising (i) an ILTV gD promoter, (ii) a coding sequence for the ILTV gD protein, (iii) an ILTV gI promoter, and (iv) a coding sequence for the ILTV gI protein (contained within the UL7 / 8 region of its genome). np to provide.

[0052] In more specific embodiments, the present invention provides a rHVT comprising a first heterologous nucleic acid and a second heterologous nucleic acid. The first heterologous nucleic acid comprises (i) a murine cytomegalovirus immediate-early (mCMV IE) promoter, (ii) a coding sequence for the IBDV VP2 protein, (iii) a transcription termination sequence, (iv) an ILTV gD promoter, (v) a coding sequence for the ILTV gD protein, (vi) an ILTV gI promoter, and (vii) a coding sequence for the ILTV gI protein. In particular embodiments of this type, the specific 5' to 3' order of the nucleotide sequence of the recombinant nucleic acid is (i) to (vii). In more specific embodiments, the transcription termination sequence comprises an SV40 polyadenylation sequence. In even more specific embodiments of this type, the first heterologous nucleic acid is present in the US2 region of the rHVT genome. The second heterologous nucleic acid comprises a recombinant nucleic acid comprising, in the following order from 5' to 3', (i) a human cytomegalovirus immediate-early (hCMV IE) promoter, (ii) a coding sequence for the NDV F protein, and (iii) a transcription termination sequence. In more particular embodiments, the transcription termination sequence comprises a human cytomegalovirus immediate-early (hCMV IE) polyadenylation sequence. In more particular embodiments of this type, the second heterologous nucleic acid is located at the UL54.5 site of the rHVT genome.

[0053] Thus, the present invention includes a recombinant HVT (rHVT) comprising two heterologous nucleic acids, each inserted into a separate, non-essential site in the HVT genome. In certain embodiments, the first heterologous nucleic acid comprises (i) a murine cytomegalovirus immediate-early (mCMV IE) promoter, (ii) a coding sequence for the IBDV VP2 protein, (iii) a transcription termination sequence, (iv) an ILTV gD promoter, (v) a coding sequence for the ILTV gD protein, (vi) an ILTV gI promoter, and (vii) a coding sequence for the ILTV gI protein. In particular embodiments of this type, the specific 5' to 3' order of the nucleotide sequence of this recombinant nucleic acid is (i) to (vii). The second heterologous nucleic acid comprises, in the following 5' to 3' order: (i) a human cytomegalovirus immediate-early (hCMV IE) promoter, (ii) a coding sequence for the NDV F protein, and (iii) a transcription termination sequence. In certain embodiments, the first heterologous nucleic acid is inserted within the US2 site and the second heterologous nucleic acid is inserted within the UL54.5 site. In certain embodiments of this type, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:23 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:26. In alternative embodiments, the second heterologous nucleic acid is inserted within the US2 site and the first heterologous nucleic acid is inserted within the UL54.5 site. In some embodiments of this type, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:21 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:22.

[0054] In other embodiments, the first heterologous nucleic acid comprises (i) a murine cytomegalovirus immediate-early (mCMV IE) promoter, (ii) a coding sequence for the IBDV VP2 protein, (iii) a transcription termination sequence, (iv) a human cytomegalovirus immediate-early (hCMV IE) promoter, (v) a coding sequence for the NDV F protein, and (vi) a transcription termination sequence. In particular embodiments of this type, the particular 5' to 3' order of the nucleotide sequence of the recombinant nucleic acid is (i) to (vi). The second heterologous nucleic acid comprises, in 5' to 3' order, the following: (i) an ILTV gD promoter, (ii) a coding sequence for the ILTV gD protein, (iii) an ILTV gI promoter, and (iv) a coding sequence for the ILTV gI protein. In particular embodiments, the first heterologous nucleic acid is inserted into the US2 site and the second heterologous nucleic acid is inserted into the UL54.5 site. In a particular embodiment, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 30 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 29. In an alternative embodiment, the second heterologous nucleic acid is inserted within the US2 site and the first heterologous nucleic acid is inserted within the UL54.5 site.

[0055] In yet other embodiments, the first heterologous nucleic acid comprises (i) an ILTV gD promoter, (ii) a coding sequence for an ILTV gD protein, (iii) an ILTV gI promoter, (iv) a coding sequence for an ILTV gI protein, (v) a human cytomegalovirus immediate-early (hCMV IE) promoter, (vi) a coding sequence for an NDV F protein, and (vii) a transcription termination sequence. In particular embodiments of this type, the particular 5' to 3' order of the nucleotide sequence of the recombinant nucleic acid is (i) to (vii). The second heterologous nucleic acid comprises, in 5' to 3' order, the following: (i) a murine cytomegalovirus immediate-early (mCMV IE) promoter, (ii) a coding sequence for an IBDV VP2 protein, and (iii) a transcription termination sequence. In particular embodiments, the first heterologous nucleic acid is inserted into the US2 site and the second heterologous nucleic acid is inserted into the UL54.5 site. In particular embodiments of this type, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 28 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 27. In alternative embodiments, the second heterologous nucleic acid is inserted within the US2 site and the first heterologous nucleic acid is inserted within the UL54.5 site. In particular embodiments of this type, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 31 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 32.

[0056] The present invention further provides any rMDV of the present invention. np (e.g., rHVT). In certain embodiments, a first heterologous nucleic acid is constructed to include a nucleotide sequence encoding an ILTV gD protein, a nucleotide sequence encoding an ILTV gI protein, and a nucleotide sequence encoding an IBDV VP2 protein. In particular embodiments of this type, the promoters for the nucleotide sequences encoding the ILTV gD protein and the ILTV gI protein are their respective endogenous promoters. In related embodiments, the promoter for the nucleotide sequence encoding the IBDV VP2 protein is an mCMV IE promoter, a chicken beta-actin gene promoter, or an hCMV promoter.

[0057] The first heterologous nucleic acid is then introduced into the rMDV of the invention. np In certain embodiments, the first heterologous nucleic acid is an expression cassette. In particular embodiments of this type, the expression cassette comprises the nucleotide sequence of SEQ ID NO:21.

[0058] The method further comprises constructing a second heterologous nucleic acid, similar to the rMDV. np In certain embodiments, the second heterologous nucleic acid comprises a human cytomegalovirus immediate early (hCMV IE) promoter, a coding sequence for the NDV F protein, and a transcription termination sequence. In some embodiments, the second heterologous nucleic acid is an expression cassette. In certain embodiments of this type, the expression cassette comprises the nucleotide sequence of SEQ ID NO: 22. In certain embodiments, the first heterologous nucleic acid is a rMDV np and the second heterologous nucleic acid is inserted within the first non-essential site of the rMDV. np In one embodiment, the rMDV is inserted into the second non-essential site of np In a related embodiment, the first non-essential site is the UL54.5 site. np The second non-essential site is the US2 site. np The first non-essential site of rMDV is the US2 site. np The second non-essential site is the UL54.5 site. In particular embodiments of this type, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:23 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:26. In other embodiments of this type, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:24 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:26. In yet other embodiments of this type, the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:25 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:26. In certain embodiments, the rMDV np The method for producing rHVT is a method for producing rMDV. np The manufacturing method is the same as that of rMDV2.

[0059] Thus, in one aspect, the present invention provides a rMDV of the present invention. np (e.g., rHVT). In certain embodiments, these immunogenic compositions and / or vaccines are stable, safe, and have relatively strong antigen expression and / or efficacy. Alternatively, or in addition, the rMDV of the present invention np The immunogenic composition and / or vaccine comprising helps protect chickens against disease caused by ILTV and / or IBDV and / or NDV and / or MDV1 after administration of the immunogenic composition and / or vaccine to chickens.

[0060] The present invention further provides any of the rMDVs of the present invention further combined with additional IBDV, ILTV, NDV and / or MDV antigens to improve and increase the immunogenicity conferred. np (e.g., rHVT). In particular embodiments of this type, the antigen is an attenuated or mild live mutant IBDV (e.g., IBDV 89 / 03). In another particular embodiment of this type, the antigen is an attenuated (or mild live) Newcastle Disease Virus (NDV), e.g., NDV C2. In yet another particular embodiment of this type, the antigen is an attenuated Marek's Disease Virus, e.g., SB1. Furthermore, the present invention also provides any rMDV of the invention that further encodes an antigen for a pathogen other than MDV, ILTV, or NDV. np The present invention provides an immunogenic composition and / or vaccine comprising:

[0061] The present invention also provides methods for helping to protect poultry subjects (e.g., chickens) against disease caused by ILTV and / or IBDV and / or NDV and / or MDV1 by administering such vaccines and / or immunogenic compositions to the poultry. In particular embodiments of this type, the vaccines of the invention are administered subcutaneously. In other embodiments, the vaccines of the invention are administered in ovo. These and other aspects of the present invention will be better understood with reference to the following drawings and detailed description.

[0062] Detailed Description of the Invention The present invention provides a single rMDV that encodes and expresses antigens from three or more foreign pathogenic chicken viruses. np In certain embodiments, the rMDV vectors of the present invention are capable of being constructed in a manner that overcomes past failures. np encodes and expresses foreign antigenic proteins from three or more avian viruses. In a specific embodiment, the avian viruses are Newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), and infectious bursal disease virus (IBDV). Such rMDVs np The vectors may be used in vaccines and / or immunogenic compositions to help protect against Marek's disease, infectious bursal disease (Gumboro disease), infectious laryngotracheitis virus, and / or Newcastle disease virus. np is rHVT. In an alternative embodiment, rMDV np The present invention further provides any rMDV of the present invention in combination with additional IBDV, ILTV, NDV and / or MDV antigens and / or one or more antigens from chicken pathogens other than MDV, ILTV, NDV or IBDV. np In a completely different embodiment, the recombinant vector encoding and expressing a foreign antigen from NDV, ILTV, IBDV is rMDV. npRather, it is a chimeric Marek's disease virus, which contains identified genomic sequences from MDV1 that replace their counterparts in the HVT vector, e.g., novel avian herpesvirus (NAHV) [see, e.g., US 6,913,751].

[0063] Prior to the present invention, an HVT vector containing an NDV gene inserted within the US10 region had already been constructed. This HVT-NDV vector was shown to be stable and to express sufficient levels of the corresponding NDV gene product, the NDV F protein, to protect vaccinated chickens against virulent (virulent, pathogenic) NDV challenge. Also, an HVT vector containing a pair of ILTV genes inserted within the HVT UL54.5 region had already been constructed. This HVT-ILTV vector was shown to be stable and to express sufficient levels of the corresponding ILTV gene product, the ILTV gI and gD proteins, to protect vaccinated chickens against virulent ILTV challenge virus. More recently, other multivalent constructs have also been reported.

[0064] More specifically, based on the success of a construct comprising the insertion of the NDV-F gene into the US10 site and the insertion of the ILTV gD and gI genes into the UL54.5 site in separate constructs, a multivalent rHVT construct for protection against both NDV and ILTV was designed [see US 8,932,604 B2]. However, unexpectedly, after passage of this multivalent construct in tissue culture, the recombinant virus lost its ability to express the ILTVgD, ILTVgI, and NDV F proteins. This proved to be the case for several double recombinant rHVT constructs. In fact, these recombinant viruses were unstable and unsuitable for further development as vaccines. These findings indicate that designing a single multivalent rHVT vector capable of stably expressing both the NDV F protein and the ILTVgD and ILTVgI proteins was not a straightforward process as could be deduced from existing information. In fact, if such stable and effective multivalent rHVT vectors were possible, their design would have had to be premised on an unpredictable combination of complex interactions, including at least the relationship between the insertion site used and the foreign gene to be inserted. Therefore, the design of rHVT constructs remains unpredictable from the prior art. This is because rMDV vectors encoding heterologous antigens from three or more avian viral pathogens are np In this case, it seems to be an even bigger problem.

[0065] Despite the obvious challenges outlined above, and the incorporation of foreign antigens from more than three different viral pathogens into MDV np Despite the general consensus that inserting genes into a construct would overburden the construct and lead to a lack of stability, the present invention surprisingly demonstrates that two genes from ILTV, one gene from IBDV and one gene from NDV are integrated into a single MDV construct. np Stable recombinant MDV inserted into np Therefore, such a single rMDV vector is provided. np The construct can be used as the sole active ingredient in a vaccine to help protect against the four major pathogenic poultry viruses.

[0066] In a specific embodiment of the present invention, nucleotide sequences encoding four foreign antigens are inserted into one or more nonessential regions of the genome of a single HVT. Therefore, such a recombinant HVT vector could be used to provide protection against MDV, NDV, IBDV, and ILTV infection. Previously, protection against these four viruses required multiple different rHVT vectors, which could cause antigenic interference with each other.

[0067] Thus, the present invention provides a single recombinant MDV np This is advantageous compared to current methods because it provides simultaneous protection against MDV, NDV, IBDV, and ILTV infection by inoculating poultry and / or poultry eggs with only one vector. In particular, since there is a limit to the volume that can be injected into an egg, the present invention allows additional vaccines to be administered by the in ovo route, and furthermore, the present invention reduces production costs because only one vaccine is required instead of two vectors.

[0068] Furthermore, the present invention provides for the use of different rMDVs in the same vaccine and / or immunogenic composition. npIn certain embodiments of this type, the vaccine and / or immunogenic composition includes both an rMDV2 and an rHVT, each of which encodes one or more foreign antigens. Indeed, unlike the combination of two rHVTs, which inevitably results in significant overgrowth of one construct relative to the other, it has been reported that the combination of an rHVT and an rMDV2 does not result in such significant overgrowth. Thus, in certain embodiments, the vaccine of the present invention comprises an rHVT encoding an ILTV gD protein, an ILTV gI protein, an IBDV VP2 protein, and an NDV F protein, together with an rMDV2 encoding an additional poultry virus antigen. To date, no rMDV has been shown to encode and express foreign antigens from three different poultry viruses and yet remain stable and capable of expressing the corresponding antigens at sufficient levels to protect vaccinated chickens against virulent challenge with the corresponding three viruses and against virulent MDV. np was not shown at all.

[0069] Thus, the present invention provides any rMDV of the present invention. np In certain embodiments, the present invention provides immunogenic compositions and / or vaccines comprising rMDV. np is rHVT. In an alternative embodiment, rMDV np is rMDV2. The present invention also provides methods for assisting in the protection of poultry against (and in certain embodiments protecting poultry against) disease caused by ILTV and / or IBDV and / or NDV and / or MDV1 by administering such vaccines and / or immunogenic compositions of the present invention. In particular embodiments, the poultry subject is a chicken. In particular embodiments of this type, the vaccine of the present invention is administered subcutaneously. In other embodiments, the vaccine of the present invention is administered in ovo. In preferred embodiments, the rMDV of the present invention np The vaccine is safe, stable and effective.

[0070] In order that this invention may be more fully understood, the following definitions are set forth.

[0071] The use of singular terms for convenience of description is not intended to be so limiting. Thus, for example, reference to a composition containing a "polypeptide" includes reference to one or more of such polypeptides.

[0072] As used herein, "non-pathogenic Marek's disease virus" or "MDV" np " or "npMDV" is a virus of the MDV family that shows little or no pathogenicity in poultry. np The term "MDV" includes naturally occurring MDV that have been passaged or similarly manipulated, but does not include viral constructs in which specific regions of the genome of one MDV serotype have been replaced with the corresponding region of a different MDV serotype to generate chimeric viruses, such as novel avian herpesviruses (NAHVs). In certain embodiments, the MDV np is HVT. In another embodiment, MDV np is MDV2. In particular embodiments of this type, MDV2 is SB1.

[0073] As used herein, an MDV that has been genetically modified to encode a heterologous nucleotide sequence (e.g., a foreign gene). np "Recombinant MDV np " or "rMDV np " is defined as "rMDV np The term " includes naturally occurring MDV nps that have been genetically modified to encode heterologous nucleotide sequences, but does not include viral constructs in which specific regions of the genome of one MDV serotype have been replaced with the corresponding regions of a different MDV serotype to generate chimeric viruses, such as novel avian herpesviruses (NAHVs).

[0074] As used herein, a "novel avian herpesvirus" ("NAHV") is a recombinant chimeric virus comprising a unique long viral genomic region naturally occurring in herpesvirus of turkeys (HVT) and a unique short viral genomic region naturally occurring in Marek's disease virus 1 (MDV1) [see US 5,965,138, US 6,183,753, US 6,913,751 B2]. In a preferred embodiment, the NAHV comprises a unique long viral genomic region naturally occurring in herpesvirus of turkeys (HVT), a unique short viral genomic region naturally occurring in Marek's disease virus 1 (MDV1), and a repeated viral region of HVT [see US 6,913,751 B2].

[0075] As used herein, a "non-essential site" means that insertion of a heterologous nucleotide sequence into the site will result in MDV replication in the host cell. np (or NAVH) does not prevent the MDV from replicating np A non-essential site is a site within a genome (or within a NAVH genome). A non-essential site is generally identified by the open reading frame in which it is located (e.g., the US2 site) or by the region between two open reading frames (e.g., the UL7 / 8 site). The use of the term "non-essential site" is intended to be used in the context of MDV. np (or NAVH) maintains its ability to replicate in a host cell.

[0076] As used herein, rMDV np (or NAHV) but rMDV np When a given nucleic acid is described as being "inserted" into a non-essential site of a genome (or NAHV genome), it means that the given nucleic acid is np (or NAHV) in a non-essential site thereof.np a first nucleic acid inserted within a first non-essential site of the genome; and np and a second nucleic acid inserted into a second non-essential site of the genome. np is rMDV np a first heterologous nucleic acid located at a first non-essential site in the genome; and np and a second heterologous nucleic acid located at a second non-essential site in the genome. np is equivalent to (and vice versa).

[0077] As used herein, the term "poultry" may include chickens, turkeys, ducks, geese, quail, and pheasants.

[0078] As used herein, a "vaccine" is a composition suitable for application to animals (including humans in some embodiments, although other embodiments are specifically directed to non-humans), typically comprising one or more antigens in combination with a pharmaceutically acceptable carrier, such as, for example, a water-containing liquid, which, when administered to an animal, induces an immune response strong enough to at least assist in protection against clinical disease resulting from infection with a wild-type microorganism, i.e., to assist in the prevention of the disease and / or to prevent, ameliorate, or treat the disease.

[0079] As used herein, the term "aiding in protection" does not require complete protection from any manifestation of infection. For example, "aiding in protection" can mean that the protection is sufficient such that after challenge, the symptoms of the underlying infection are at least alleviated and / or one or more of the underlying cellular, physiological, or biochemical causes or mechanisms that cause the symptoms are alleviated and / or eliminated. It is understood that "alleviation" as used in this context refers to the state of infection, including not only the physiological state of the infection, but also the molecular state of the infection.

[0080] The vaccine of the present invention comprises at least one stable rMDV of the present invention. np Contains rMDV npis considered phenotypically stable if, after at least 10 tissue culture passages from the original strain (stock) or after re-isolation of the virus from vaccinated birds, at least 90% of the virus plaques examined are positive for expression of the inserted foreign antigen, as indicated by binding of antibodies specific for the expressed protein in an immunofluorescence assay.

[0081] The vaccines of the present invention are also effective, preferably providing at least 70% protection against NDV and / or at least 70% protection against IBDV and / or at least 70% protection against ILTV and / or at least 60% protection against MDV from disease-associated clinical signs or lesions. More preferably, the vaccine preferably provides at least 80% protection against NDV from disease-associated clinical signs or lesions, at least 80% protection against IBDV, at least 80% protection against ILTV, and at least 70% protection against MDV. Even more preferably, the vaccine follows guidelines established by the USDA in Title 9 Code of Federal Regulations, part 113 (9 CFR 113), "Standard requirements for Animal Products," which state that to be licensed, a live virus vaccine must provide at least 90% protection from disease-associated clinical signs or lesions in the case of NDV, IBDV, and ILTV, and at least 80% protection in the case of MDV.

[0082] As used herein, a "multivalent vaccine" is a vaccine that contains two or more different antigens. In certain embodiments of this type, the multivalent vaccine stimulates the recipient's immune system against two or more different pathogens.

[0083] As used herein, an "adjuvant" is a substance that can accelerate or enhance the cascade of immune events, ultimately leading to a better immune response, i.e., an integrated bodily response to an antigen. Adjuvants are generally not required for the production of an immune response, but they accelerate or enhance this response.

[0084] As used herein, the term "pharmaceutically acceptable" is used adjectively to denote that the modified noun is suitable for use in a pharmaceutical product. When it is used to refer to an excipient in, for example, a pharmaceutical vaccine, it characterizes the excipient as being compatible with the other ingredients of the composition and not harmful to the intended recipient.

[0085] As used herein, "systemic administration" refers to administration into the body's circulatory system (including the cardiovascular and lymphatic systems), affecting the entire body rather than to a specific site, such as the gastrointestinal tract (e.g., by oral or rectal administration) and respiratory system (e.g., by intranasal administration). Systemic administration can be achieved, for example, by administration into muscle tissue (intramuscular), the dermis (intradermal or transdermal), under the skin (subcutaneous), under the mucous membrane (submucosal), into a vein (intravenous), etc.

[0086] The term "parenteral administration" as used herein includes subcutaneous injections, submucosal injections, intravenous injections, intramuscular injections, intradermal injections and infusion.

[0087] The term "approximately" is used interchangeably with the term "about" and means that a value is within 25% of the stated value, i.e., a peptide containing "approximately" 100 amino acid residues may contain 75-125 amino acid residues.

[0088] As used herein, the term "polypeptide" is used interchangeably with the terms "protein" and "peptide" to refer to a polymer comprising two or more amino acids linked by peptide bonds. As used herein, the term "polypeptide" encompasses significant fragments or segments, including stretches of amino acid residues of at least about 8 amino acids, generally at least about 12 amino acids, typically at least about 16 amino acids, preferably at least about 20 amino acids, and in particularly preferred embodiments, at least about 30 amino acids or more (e.g., 35, 40, 45, 50, etc.). Such fragments may have ends starting and / or terminating at substantially any position in all practical combinations, e.g., starting at residue 1, 2, 3, etc., and terminating at, e.g., 155, 154, 153, etc.

[0089] Optionally, the polypeptide can lack certain amino acid residues encoded by the gene or by the mRNA. For example, the gene or mRNA molecule may encode a sequence of amino acid residues at the N-terminus of the polypeptide that are cleaved from and do not become part of the final protein (i.e., a signal sequence).

[0090] The term "antigenic fragment," as used herein with respect to a particular protein (e.g., a protein antigen), refers to a fragment of that protein (including larger fragments lacking only a single amino acid from the full-length protein) that is antigenic, i.e., capable of specifically interacting with an antigen-recognizing molecule of the immune system, such as an immunoglobulin (antibody) or a T-cell antigen receptor. For example, an antigenic fragment of the IBDV VP2 protein is a fragment of the VP2 protein that is antigenic. Preferably, antigenic fragments of the present invention are immunodominant with respect to antibody and / or T-cell receptor recognition. In certain embodiments, an antigenic fragment with respect to a given protein antigen is a fragment of that protein that retains at least 25% of the antigenicity of the full-length protein. In preferred embodiments, an antigenic fragment retains at least 50% of the antigenicity of the full-length protein. In more preferred embodiments, it retains at least 75% of the antigenicity of the full-length protein. Antigenic fragments can be as small as 5-10 amino acids, or, conversely, larger fragments that lack only a single amino acid from the full-length protein. In certain embodiments, an antigenic fragment comprises 25-100 amino acid residues.

[0091] As used herein, an amino acid sequence is 100% "homologous" to another amino acid sequence if the two amino acid sequences are identical and / or differ only by neutral or conservative substitutions, as defined below. Thus, an amino acid sequence is about 80% "homologous" to another amino acid sequence if about 80% of the two amino acid sequences are identical and / or differ only by neutral or conservative substitutions.

[0092] Residues within a sequence can often be substituted with a functionally equivalent amino acid residue, resulting in conservative amino acid substitutions. Such changes are the meaning of the term "conservative substitution" as used herein. For example, one or more amino acid residues within a sequence can be substituted with another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent change. Substitutes for amino acids within a sequence can be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Amino acids containing aromatic ring structures include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such changes would not be expected to affect the apparent molecular weight as determined by polyacrylamide gel electrophoresis or isoelectric point.

[0093] Particularly preferred conservative substitutions include Arg to Lys and vice versa, which maintain a positive charge; Asp to Glu and vice versa, which maintain a negative charge; Thr to Ser, which maintains a free -OH; and Asn to Gln, which maintains a free NH2. Amino acids can also be substituted with the following analogous groups: (1) proline, alanine, glycine, serine, and threonine; (2) glutamine, asparagine, glutamic acid, and aspartic acid; (3) histidine, lysine, and arginine; (4) cysteine; (5) valine, leucine, isoleucine, and methionine; and (6) phenylalanine, tyrosine, and tryptophan.

[0094] In related embodiments, two highly homologous DNA sequences can be identified by their own homology or by the homology of the amino acids they encode. Such sequence comparisons can be performed using standard software available in sequence data banks. In particular embodiments, two highly homologous DNA sequences encode amino acid sequences that share about 80% identity, more preferably about 90% identity, and even more preferably about 95% identity. More specifically, two highly homologous amino acid sequences share about 80% identity, even more preferably about 90% identity, and even more preferably about 95% identity.

[0095] As used herein, percent identity of protein and DNA sequences can be determined using software such as MacVector v9 and the Clustal W algorithm, commercially available from Accelrys (Burlington, Massachusetts), with default parameters for alignment and identity. See, e.g., Thompson et al., 1994. Nucleic Acids Res. 22:4673-4680. ClustalW is available for free download for DoS, Macintosh, and Unix platforms from, for example, EMBLI (European Bioinformatics Institute). The download link can be found at http: / / www.ebi.ac.uk / clustalw / . These and other available programs can also be used to determine sequence similarity using the same or similar default parameters.

[0096] As used herein, the terms "polynucleotide" or "nucleic acid" or "nucleic acid molecule" are used interchangeably and refer to molecules containing nucleotides, including, but not limited to, RNA, cDNA, genomic DNA, and even synthetic DNA sequences. These terms are also intended to include nucleic acid molecules containing any of the art-known base analogs of DNA and RNA.

[0097] A nucleic acid "coding sequence" or "sequence encoding" a particular protein or peptide is a nucleotide sequence that is transcribed and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory elements.

[0098] The boundaries of a coding sequence are determined by a start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., avian) DNA, and even synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0099] "Operably linked" refers to an arrangement of elements in which the components so described are positioned so as to perform their usual function. Thus, control elements operably linked to a coding sequence are capable of effecting expression of the coding sequence. The control element need not be contiguous with the coding sequence, so long as it functions to direct the expression of the coding sequence. Thus, for example, an intervening sequence that is not translated but is transcribed can be present between the promoter and coding sequence and the promoter would still be considered to be "operably linked" to the coding sequence.

[0100] As used herein, the term "transcription termination sequence" is used interchangeably with "polyadenylation regulatory element" and refers to a sequence that is generally downstream of a DNA coding region and may be required for complete termination of transcription of that DNA coding sequence. A transcription terminator is a regulatory DNA element involved in terminating transcription of a coding region into RNA. Generally, such elements encode a fragment, such as a hairpin structure, that has a secondary structure that directs the RNA polymerase complex to terminate transcription. Thus, transcription terminators are always located downstream of the stop codon from the region to be translated, i.e., in the 3' untranslated region.

[0101] As used herein, an "expression cassette" is a recombinant nucleic acid that includes at least a promoter and a heterologous coding sequence operably linked to the promoter. In many such embodiments, the expression cassette further includes a transcription termination sequence. Thus, rMDV np Insertion of an expression cassette into a non-essential site in the genome of rMDV np In certain embodiments, the rMDV may result in expression of a heterologous coding sequence. np is a rHVT. In another embodiment, np is rMDV2.

[0102] As used herein, a "heterologous nucleotide sequence" is a nucleotide sequence that is added to a nucleotide sequence of the invention by recombinant methods to obtain a nucleic acid that does not occur in nature. In certain embodiments, a "heterologous nucleotide sequence" of the invention is a protein antigen (e.g., a rMDV of the invention). np The heterologous nucleotide sequence may encode a "foreign gene" (encoded by a "foreign gene" to the vector), such as the IBDV VP2 protein, the ILTV gI protein, the ILTV gD protein, and / or the NDV F protein. The heterologous nucleotide sequence may also encode a fusion (e.g., chimeric) protein. In this case, such a protein antigen may be referred to as a "foreign antigen" or, more specifically, a "foreign protein antigen."

[0103] Heterologous nucleotide sequences may also encode fusion (e.g., chimeric) proteins. Heterologous nucleotide sequences may also encode peptides and / or proteins containing regulatory and / or structural features. In other such embodiments, heterologous nucleotide sequences may encode proteins or peptides that function as a means of detecting the protein or peptide encoded by the nucleotide sequence of the invention after the recombinant nucleic acid is expressed. In yet another embodiment, heterologous nucleotide sequences may function as a means of detecting the nucleotide sequence of the invention. Heterologous nucleotide sequences may contain non-coding sequences, including restriction sites, regulatory sites, promoters, etc. "Heterologous nucleic acid" includes heterologous nucleotide sequences.

[0104] of nucleic acids encoding the antigens of the present invention, np Insertion into a vector is easily accomplished when both the nucleic acid and the vector termini contain compatible restriction sites. If this is not possible, it may be necessary to modify the nucleotide sequence and / or the vector termini by redigesting single-stranded nucleic acid overhangs (e.g., DNA overhangs) resulting from restriction endonuclease cleavage to obtain blunt ends, or by filling in the single-stranded ends with an appropriate polymerase to achieve the same result. Alternatively, desired sites can be generated, for example, by ligating nucleotide sequences (linkers) onto the termini. Such linkers can contain specific oligonucleotide sequences that define the desired restriction sites. Restriction sites can also be generated using the polymerase chain reaction (PCR) [see, e.g., Saiki et al., Science 239:487-491 (1988)]. The cleaved vector and the DNA fragment can also be modified, if desired, by homopolymeric tail joining. Alternatively, recombinant nucleotide sequences can be synthesized de novo.

[0105] Protein antigens and nucleic acids encoding the protein antigens The ILTV gD gene appears to encode a 434 amino acid long glycoprotein with a molecular weight of 48,477 daltons, although some have suggested that a downstream initiation codon is the actual codon, resulting in an ILTV gD protein containing only 377 amino acid residues [Wild et al., Virus Genes 12:104-116 (1996)]. The ILTV gI gene encodes a 362 amino acid long glycoprotein with a molecular weight of 39,753 daltons [US Pat. No. 6,875,856, incorporated herein by reference]. Nucleic acids encoding naturally occurring and / or laboratory-derived variants of ILTV gD and ILTV gI can be used in place of those exemplified herein.

[0106] In certain embodiments of the present invention, rMDV npcomprises a recombinant nucleic acid (e.g., an expression cassette) encoding an ILTV gD protein or antigenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 2. In a related embodiment, the rMDV np comprises a recombinant nucleic acid encoding an ILTV gD protein comprising an amino acid sequence having greater than 90%, and / or greater than 95%, and / or greater than 98%, and / or greater than 99% identity to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the ILTV gD protein is encoded by the nucleotide sequence of SEQ ID NO: 1. In certain embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0107] In one embodiment of the present invention, the rMDV np comprises a recombinant nucleic acid (e.g., an expression cassette) encoding an ILTV gI protein or antigenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 4. In a related embodiment, the rMDV np comprises a recombinant nucleic acid encoding an ILTV gI protein comprising an amino acid sequence having greater than 90%, and / or greater than 95%, and / or greater than 98%, and / or greater than 99% identity to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the ILTV gI protein is encoded by the nucleotide sequence of SEQ ID NO: 3. In certain embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0108] As mentioned above, IBDV is a member of the Birnaviridae family and has a genome consisting of two segments (A and B) of double-stranded RNA. The larger segment, A, encodes a 110-kDa polyprotein, which is subsequently autoproteolytically cleaved to generate the mature viral proteins VP2, VP3, and VP4. Of these, VP2 and VP3 are structural capsid proteins of the virion, and the VP2 protein is the primary host defense immunogen. In the case of IBDV, two serotypes (serotypes 1 and 2) exist, which can be distinguished by virus neutralization (VN) tests. Serotype 1 viruses have been shown to be pathogenic in chickens, while serotype 2 IBDV only causes subacute disease in turkeys. Historically, IBDV serotype 1 viruses consisted of only one type, now known as the "classical" IBD virus; however, so-called "mutant" IBDV strains have subsequently emerged. In a specific embodiment of the present invention, the IBDV VP2 gene encodes the VP2 protein from a classical IBDV. Such genes are well known, and their sequence information is readily available (see, for example, GenBank acc.nr: D00869 (F52 / 70), D00499 (STC), or AF499929 (D78)). Alternatively, this gene can be obtained from the genome of a classical IBDV isolated from nature using conventional techniques for manipulating birnaviruses. Classical IBDVs can be easily identified using serological or molecular biological techniques.

[0109] In certain embodiments of the present invention, rMDV np comprises a recombinant nucleic acid (e.g., an expression cassette) encoding an IBDV VP2 protein or antigenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 6. In a related embodiment, the rMDV npcomprises a recombinant nucleic acid encoding an IBDV VP2 protein comprising an amino acid sequence having greater than 90%, and / or greater than 95%, and / or greater than 98%, and / or greater than 99% identity to the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the IBDV VP2 protein is encoded by the nucleotide sequence of SEQ ID NO: 5. In certain embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0110] Conventional vaccination against IBDV is performed as early as possible in the life of poultry using attenuated IBDV strains, but these are only applicable once the level of MDA against IBDV has been sufficiently reduced, which is generally about 15-20 days after hatch. Numerous "live" or inactivated IBDV vaccines are commercially available, for example, "live" vaccines such as Nobilis™ Gumboro D78 (Merck Animal Health).

[0111] NDV has a non-segmented, negative-sense, single-stranded RNA genome, which is approximately 15 kb in size and contains six genes, one of which is the NDV F protein gene, which encodes the so-called "fusion" glycoprotein (F protein). The F protein is involved in the attachment and entry of NDV into host cells, and as an immunodominant protein, it is the basis for an effective immune response to NDV. The NDV F protein is expressed as a native F0 protein, which is activated when cleaved by extracellular peptidases.

[0112] The NDV F protein gene can be derived, for example, from NDV clone 30, a common lentogenic NDV vaccine strain. np comprises a recombinant nucleic acid (e.g., an expression cassette) encoding an NDV F protein or antigenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 8. In a related embodiment, the rMDV npcomprises a recombinant nucleic acid encoding an NDV F protein comprising an amino acid sequence having greater than 90%, and / or greater than 95%, and / or greater than 98%, and / or greater than 99% identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the NDV F protein is encoded by the nucleotide sequence of SEQ ID NO: 7. In certain embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0113] In a related embodiment of the invention, rMDV np comprises a recombinant nucleic acid (e.g., an expression cassette) encoding an NDV F protein or antigenic fragment thereof comprising the amino acid sequence of SEQ ID NO: 10. np includes a recombinant nucleic acid encoding an NDV F protein comprising an amino acid sequence having greater than 90%, and / or greater than 95%, and / or greater than 98%, and / or greater than 99% identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the NDV F protein is encoded by the nucleotide sequence of SEQ ID NO: 9. In certain embodiments, the rMDV np is rHVT. In an alternative embodiment, rMDV np is rMDV2.

[0114] Nucleic acids encoding natural and / or laboratory-derived variants of the F protein gene may be equally applicable to lentogenic, mesogenic, or velogenic NDV, since the F protein gene sequence itself is highly conserved among these various NDV pathotypes.

[0115] The rMDV of the present invention np The nucleotide and / or protein sequences of chicken pathogen protein antigens encoded by may also be found in publicly available databases such as GenBank or the Protein Information Resource.

[0116] Promoter and polyadenylation regulatory elements A promoter is a functional region in an organism's genome that directs the transcription of downstream coding regions. Therefore, promoters are located upstream of the coding region of a gene. Promoter-directed mRNA synthesis begins at a "transcription start site" (TSS). The resulting mRNA is then translated into a protein starting at the gene's start codon, which is the first ATG sequence in the open reading frame (the first AUG in the mRNA). Typically, the TSS is located 30-40 nucleotides upstream of the start codon. The TSS can be determined by sequencing the 5' end of the gene's mRNA, for example, by RACE technology. Generally, promoters are found approximately 1000 nucleotides upstream of the A position of the start codon (commonly designated A+1), with most promoters located between nucleotides -500 and A+1.

[0117] The nomenclature of a promoter is generally based on the name of the gene whose expression it controls. For example, the term "mCMV-IE1 gene promoter" refers to a promoter that essentially drives the expression of the early 1 gene (IE1 gene) from mCMV and is therefore located immediately upstream of that gene. Because the IE1 gene is a well-described and clearly recognizable gene, and because several mCMV genomes have been sequenced (in whole or in part), such promoters can be easily identified using standard techniques. For example, in a basic protocol, a promoter can be easily obtained by roughly subcloning the region between two consecutive genes (e.g., from the poly(A) signal) of the upstream gene to the TSS of the downstream gene). The promoter can then be identified by standard tests, such as by expressing a marker gene with successively smaller segments of the suspected promoter.

[0118] Generally, promoters contain several recognizable regulatory regions, such as enhancer regions, which are involved in the binding of regulatory factors that affect the time, duration, conditions and level of transcription.Enhancer regions are generally located upstream, but promoters also contain downstream regions that are involved in the binding of transcription factors and the direction of RNA polymerase itself.This downstream region generally contains several conserved promoter sequence elements, such as TATA box, CAAT box and GC box.

[0119] A promoter containing both an enhancer region and a downstream region is referred to as a "complete" promoter, whereas a promoter containing only a downstream region is referred to as a "core" promoter.

[0120] A promoter for the expression of a (heterologous) gene in a (viral) vector must be able to effectively drive the transcription of its downstream coding sequence. This is generally referred to as a promoter "operably linked" to a coding sequence, in which case the gene is "under the control" of or "driven by" the promoter. This generally means that in an expression cassette, the promoter and the gene coding sequence are found on the same nucleic acid in effective proximity, with no signals or sequences between them that would prevent effective transcription of the coding sequence.

[0121] The mCMV-IE1 gene promoter is well known in the art and readily available from various commercial sources, such as suppliers of commercially available plasmids for cloning and expression. The IE1 gene is also referred to as the "major IE gene." The mCMV-IE1 protein is also referred to as pp89. Dorsch-Hasler et al. [Proc. Nat. Acad. Sci., 82:8325-8329 (1985)] described the mCMV IE1 gene promoter in 1985, and the use of this promoter in heterologous expression has also been described in WO 87 / 03.905 and EP 728.842. The nucleotide sequence of the complete mCMV IE locus is available from GenBank under accession number L06816.1 (as of March 2004). mCMV itself was originally available from the ATCC under accession number VR-194, which has more recently been continued under accession number VR-1399.

[0122] In one embodiment of the present invention, the mCMV-IE1 gene promoter is a complete promoter containing both the enhancer and core promoter regions of the mCMV-IE1 gene. The size of the complete mCMV-IE1 gene promoter is approximately 1.4 kb. However, the present invention allows for some variation in the length of not only the mCMV-IE1 gene promoter but also the other elements constituting the recombinant DNA expression cassette used in the present invention. This may result from differences in the exact conditions used for cloning and construction. For example, this variation may result from using different restriction enzyme sites, PCR cloning primers, or different conditions for matching the ends of the cloning molecules used. As a result, some variation in the length of the components (smaller or larger) may occur without affecting the stability and relatively strong antigen expression and / or efficacy of the overall expression cassette. In this case, these length differences are not significant and are within the scope of the present invention. Therefore, an "approximately 1.4 kb" mCMV-IE1 gene promoter is 1.4 kb ± about 25%. In a specific embodiment, the promoter is 1.4 kb ± about 20%. In still other embodiments, the variation (variance) may be 1.4kb ± about 15%, 1.4kb ± about 12%, 1.4kb ± about 10%, 1.4kb ± about 8%, 1.4kb ± about 6%, 1.4kb ± about 5%, 1.4kb ± about 4%, 1.4kb ± about 3%, 1.4kb ± about 2% or even 1.4kb ± about 1%.

[0123] Similarly, homologs or variants of promoter elements that are equally effective and stable can be used. Thus, in certain embodiments, the mCMV-IE1 gene promoter of the present invention can be an approximately 1.4 kb DNA molecule comprising a nucleotide sequence having at least 95%, 96%, 97%, 98%, or even 99% nucleotide sequence identity to the nucleotide sequence of SEQ ID NO: 13. In a specific embodiment, the mCMV-IE1 gene promoter consists of the nucleotide sequence of SEQ ID NO: 13.

[0124] The rMDV of the present invention npA number of alternative promoters can be used to direct the expression of heterologous genes encoding protein antigens or antigenic fragments thereof in the vector. Specific examples include the pseudorabies virus (PRV) gpX promoter [see WO 87 / 04463], the Rous sarcoma virus LTR promoter, the SV40 early gene promoter, the chicken beta-actin gene promoter comprising the nucleotide sequence of SEQ ID NO: 17, the Towne Strain hCMV IE promoter SEQ ID NO: 16, a derivative of the hCMV IE promoter (from the AD169 strain) comprising the nucleotide sequence of SEQ ID NO: 14, the ILTV gD promoter comprising the nucleotide sequence of SEQ ID NO: 11, and the ILTV gI promoter comprising the nucleotide sequence of SEQ ID NO: 12 [see, e.g., US Pat. No. 6,183,753 B1], the human cytomegalovirus immediate early 1 (hCMV IE1) gene promoter [US Pat. No. 5,830,745; US Pat. No. 5,980,906], and the chicken beta-actin gene promoter [EP 1 298 139 B1]. A particular heterologous promoter for the IBDV VP2 gene is the murine (mCMV IE1) cytomegalovirus promoter. In particular embodiments of this type, the mCMV IE1 comprises the nucleotide sequence of SEQ ID NO: 13 [see, e.g., EP 728,842; PCT / EP2015 / 081121].

[0125] The inclusion of a polyadenylation regulatory element downstream of a DNA coding region is often necessary to terminate transcription of the coding DNA sequence. Accordingly, many genes contain polyadenylation regulatory elements at the downstream end of their coding sequences. Many such regulatory elements have been identified, and the rMDV of the present invention npSpecific examples of polyadenylation regulatory elements exemplified herein include the feline herpesvirus (FHV) US-9 polyadenylation signal comprising the nucleotide sequence of SEQ ID NO: 18, and the human herpes simplex virus (HSV) thymidine kinase polyadenylation signal comprising the nucleotide sequence of SEQ ID NO: 19. Terminator sequences (termination sequences) and polyadenylation sequences may also be derived from the glycoprotein B (gB) gene of feline herpesvirus (FHV), the immediate early (IE) gene of human cytomegalovirus (hCMV), strain AD 169, or simian virus 40 (SV40).

[0126] Vaccines and Immunogenic Compositions The present invention relates to a recombinant MDV. np Use of the MDV np The present invention also relates to nucleic acid molecules used to construct the multivalent recombinant MDV of the present invention, or host cells for propagating the same, or any combination thereof, all of which are used in the present invention for the production of poultry vaccines. np The present invention provides a vaccine and / or immunogenic composition comprising: (a) a vaccine comprising: (a) a human ovarian tumor suppressor antibody (HCV-1) or a human ovarian tumor suppressor antibody (HCV-1) ...

[0127] The recombinant MDV of the present invention np can be propagated by a number of means currently practiced in the art. For example, the recombinant MDV of the present invention np can be propagated by the use of in vitro culture of primary chicken cells. See, for example, the examples below using chicken embryo fibroblasts (CEFs). CEFs can be prepared by trypsinization of chicken embryos. Alternatively, CEFs can be plated in monolayers and then transfected with MDV.np This particular method can be easily scaled up for industrial scale production.

[0128] Therefore, another aspect of the present invention is the recombinant MDV of the present invention. np The present invention relates to a method for producing the vaccine of the present invention, comprising the steps of infecting host cells with a vector, harvesting the infected host cells, and then combining the harvested infected host cells with a pharmaceutically acceptable carrier. Suitable methods for infection, culture, and harvesting are well known in the art and are described and exemplified herein.

[0129] Typically, infected host cells are harvested while still intact and the recombinant MDV is then introduced. np are obtained in their cell-associated form. These cells can be incorporated into a suitable carrier composition to stabilize them for storage and freezing. The infected cells can be filled into glass ampoules, which are sealed, frozen, and stored in liquid nitrogen. Thus, in certain embodiments of the invention, the vaccine and / or immunogenic compositions of the invention are cryopreserved and therefore include a cryoprotectant, such as dimethyl sulfoxide (DMSO), to protect the frozen infected cells.

[0130] Alternatively, recombinant MDV np If is a recombinant HVT, it can be isolated from its host cells, for example, by sonication at the end of culture, then incorporated into a stabilizer and lyophilized for stable storage, or subjected to a liquid volume reduction for storage and then reconstituted (reconstituted) in a liquid diluent before or at the time of administration. Such reconstitution can be accomplished, for example, using vaccine-grade water. In certain embodiments, the lyophilized portion of the multivalent vaccine can contain one or more antigens, and the diluent can contain one or more other antigens.

[0131] In certain embodiments, the vaccines (or portions thereof) of the present invention may be in lyophilized form as tablets and / or spheres, for example, prepared by the methods described in WO 2010 / 125084, which is incorporated herein by reference in its entirety. See in particular the Examples on page 15, line 28 to page 27, line 9 of WO 2010 / 125084, which describe methods for preparing such rapidly disintegrating tablets / spheres. Such lyophilized forms may be readily dissolved in a diluent to allow systemic administration of the vaccine.

[0132] Vaccines and immunogenic compositions may, but do not necessarily, include physiologically acceptable buffers, saline solutions, and the like, as well as pharmaceutically acceptable adjuvants. Adjuvants may be useful for improving immune responses and / or enhancing the stability of vaccine formulations. While adjuvants are typically described as non-specific stimulators of the immune system, they may also be useful for targeting specific parts of the immune system. One or more compounds with this activity may be added to the vaccine. Accordingly, certain vaccines of the present invention may further include an adjuvant. Examples of compounds that can be used as adjuvants include, but are not limited to, aluminum compounds (e.g., aluminum hydroxide), metabolizable and non-metabolizable oils, mineral oils such as mannide oleate derivatives in mineral oil solutions (e.g., MONTANIDE ISA 70 from Seppic SA, France), and light mineral oils such as DRAKEOL 6VR, block polymers, ISCOMs (immunostimulating complexes), vitamins and minerals (including, but not limited to, vitamin E, vitamin A, selenium, and vitamin B12), and CARBOPOL®.

[0133] Other suitable adjuvants, sometimes referred to as immune stimulants, include, but are not limited to, cytokines, growth factors, chemokines, supernatants from cell cultures of monocytes, lymphocytes, cells from lymphoid organs, cell preparations and / or extracts from plants, bacteria or parasites (such as Staphylococcus aureus or lipopolysaccharide preparations), and mitogens. Generally, the adjuvant is administered simultaneously with the antigen of the invention. However, in addition or instead, the adjuvant can be administered up to two weeks prior to vaccination and / or for a period of time after vaccination (i.e., when the antigen, e.g., the recombinant MDV of the invention, is administered). np The drug may be administered in the presence of a steroid or other active ingredient (as long as the active ingredient remains in the tissue).

[0134] The vaccine and / or immunogenic compositions of the present invention may be administered by any route, for example, by parenteral administration, including in ovo, intramuscular injection, subcutaneous injection, intravenous injection, intradermal injection, by scarification, by oral administration, or by any combination thereof.

[0135] Furthermore, the multivalent recombinant MDV of the present invention np may be combined and / or used in conjunction with additional IBDV, ILTV, NDV and / or MDV antigens to improve and enhance the immunogenicity conferred, and / or antigens of other pathogens to provide immune protection against such other pathogens. These additional antigens may be live or killed whole microorganisms, other recombinant vectors, cell homogenates, extracts, proteins or any other such derivatives that do not negatively affect the safety, stability with relatively strong antigen expression and efficacy of the vaccine of the present invention.

[0136] The multivalent recombinant MDV of the present invention np and additional MDV, IBDV, NDV and / or ILTV antigens may be advantageous when highly virulent field strains of MDV, IBDV, NDV or ILTV are widespread, for example, within a particular geographic region. npand MDV1, MDV2, or HVT include strains Rispens (MDV1), SB1 (MDV2), FC-126 (HVT), and / or PB1 (HVT). np may be combined in a polyvalent vaccine with an IBDV vaccine strain, such as a mild live IBDV vaccine strain, such as D78 (a cloned intermediate strain), PBG98, Cu-1, ST-12 (an intermediate strain), or 89 / 03 (a live Delaware variant).

[0137] The multivalent recombinant MDV of the present invention np Examples of other microorganisms that can be used as antigens in conjunction with the above include: (i) viruses, such as infectious bronchitis virus, avian influenza virus, adenovirus, egg-laying drop syndrome virus, infectious bursal disease virus, chicken anemia virus, chicken encephalomyelitis virus, fowl pox virus, turkey rhinotracheitis virus, duck plague virus (duck viral enteritis), pigeon pox virus, avian leukosis virus, avian pneumovirus, and reovirus; (ii) bacteria, such as Escherichia coli, Salmonella spec., Ornitobacterium rhinotracheale, Haemophilus paragallinarum, Pasteurella multocida, Erysipelothrix rhusiopathiae, and the like. rhusiopathiae, Erysipelas spec., Mycoplasma spec. and Clostridium spec., (iii) parasites, such as Eimeria spec., and (iv) fungi, such as Aspergillus spec. In a particular embodiment of the invention, the recombinant MDV of the invention npcan be combined with mild live NDV vaccine strains, such as vaccine strain C2. Many such strains are used in commercial vaccines.

[0138] The combination vaccine (mixed vaccine) comprises the recombinant MDV of the present invention. np The vaccines may be prepared in a variety of ways, including by combining a preparation of a virus, or a bacterium, or a fungus, or a parasite, or a host cell, or a mixture of any and / or all of these. In certain embodiments, the components of such combination vaccines are conveniently prepared separately and then combined and packaged in the same vaccine container.

[0139] As noted above, the vaccines of the present invention can be advantageously used to provide safe and effective immune protection to chickens against, for example, one or more poultry diseases by a single inoculation at a very young age or in ovo. Alternatively, as will be apparent to anyone skilled in the art of poultry vaccines, the above combinations can be used in combination with the multivalent recombinant MDV of the present invention. np and vaccination regimes in which additional antigens are not administered at the same time. For example, recombinant MDV np could be applied in ovo, and NDV C2 and / or IBDV strains (e.g., 89 / 03) could be applied at later times / days.

[0140] Thus, the vaccines of the present invention can be administered to avian subjects in a single dose or multiple doses. For example, the vaccines of the present invention can be applied in ovo at the day of hatch and / or 16-18 days (embryonic day) ED. When multiple doses are administered, they can be administered simultaneously or sequentially in immunologically effective amounts in a manner and at a time appropriate for the vaccine formulation. Thus, the vaccines of the present invention can function effectively as a primary vaccination, which can be followed by a booster vaccination with the same vaccine or a different vaccine formulation (e.g., a classical inactivated adjuvanted whole virus vaccine) to provide an immune boost. Alternatively, the vaccines of the present invention can be administered alone to avian subjects as a booster vaccination.

[0141] The volume per dose of the vaccine of the present invention can be optimized according to the intended route of administration. In ovo vaccination is generally administered in a volume of 0.05 to 0.5 ml / egg, while parenteral injection is generally administered in a volume of 0.1 to 1 ml / bird. In either case, optimizing the vaccine dose volume is well within the capabilities of those skilled in the art. [Table 1] TIFF0007721269000002.tif99161 [Brief explanation of the drawings]

[0142] [Figure 1] Figure 1 is a schematic diagram of the insert fragments for generating the HVT / IBDV / ILTV / NDV construct described in Example 2 below. Briefly, this is a schematic diagram of the HVT genome, consisting of two unique regions, each flanked by a repeat region, and the cloned fragments necessary to reconstruct the HVT / IBDV / ILTV / NDV 670-14 virus. The orientation of the inserted gene cassettes (mIE-IBDV-vp2 and ILTV-gD / gI or hIE-NDV-F) relative to the interrupted gene (UL54.5 or US2) and flanking genes is shown in the magnified view. Legend: TRL: terminal repeat long region; UL: unique long region; IRL: internal repeat long region; IRS: internal repeat short region; US: unique short region; TRS: terminal repeat short region. [Figure 2] Figure 2 is a schematic diagram of the inserts used to generate HVT / IBDV / ILT / NDV construct #2. The two HVT insertion sites are UL54.5 and US2. [See also the description of Figure 1 above]. [Figure 3] Figure 3 is a schematic diagram of the inserts used to generate HVT / IBDV / ILT / NDV construct #3. The two HVT insertion sites are UL54.5 and US2. [See also the description of Figure 1 above]. [Figure 4] Figure 4 is a schematic diagram of the inserts used to generate HVT / IBDV / ILT / NDV construct #4. The two HVT insertion sites are UL54.5 and US2. [See also the description of Figure 1 above]. [Figure 5] Figure 5 is a schematic diagram of the inserts used to generate HVT / IBDV / ILT / NDV construct #5. The two HVT insertion sites are UL54.5 and US2. [See also the description of Figure 1 above].

[0143] The present invention may be better understood by reference to the following non-limiting examples, which are set forth as exemplary of the invention. The following examples are presented in order to more fully illustrate embodiments of the invention and should not be construed in any way as limiting the broad scope of the invention.

[0144] Example Example 1 Construction of recombinant HVT / ILTV / IBDV / NDV virus vectors A recombinant multivalent non-pathogenic Marek's disease virus construct has been produced that encodes and expresses (i) two infectious laryngotracheitis virus protein antigens, (ii) an infectious bursal disease virus protein antigen, and (iii) a Newcastle disease virus protein antigen. The present invention overcomes the problem of vaccine interference observed when two recombinant HVT vaccines, such as Innovax®-ILT (sold by Merck Animal Health) and Vaxxitek® (sold by Merial), are administered to the same animal. Furthermore, the present invention provides the first recombinant non-pathogenic Marek's disease virus (rMDV) that encodes antigens from three different viral pathogens other than MDV. np ) is provided independently.

[0145] Recombinant herpesvirus of turkeys (HVT) constructs were generated. In this case, antigen donor material from three poultry pathogens, infectious laryngotracheitis virus (ILTV), Newcastle disease virus (NDV), and infectious bursal disease virus (IBDV), was inserted into the HVT vector (see also US 8,932,604 B2, WO 2013 / 057,235, WO 2016 / 102647, and US Serial No. 62 / 351,471, filed June 17, 2016, which are incorporated by reference in their entireties). The donor material included the following: a 3.563 kb SalI-HindIII fragment from ILTV, the NVSL challenge strain, lot #83-2 [nucleotides 10532-14094; Wild et al., Virus Genes 12:104-116 (1996): Acc. #U28832] encoding the full-length genes for glycoprotein D (gD) and glycoprotein I (gI) and partial coding regions from glycoprotein E (amino acids 1-101) and ORF5 (amino acids 734-985); NDV, clone 30 strain, expression cassette consisting of the coding region of the fusion protein (F) gene (nucleotides 4544-6205; Romer-Oberdorfer et al. (1999); Acc. #Y18898) (driven by a viral promoter) followed by a termination sequence; and IBDV, Faragher, type F52 / 70 strain, expression cassette consisting of the coding region of the viral protein 2 (vp2) gene (driven by a viral promoter) followed by a termination sequence. In the exemplified embodiment, the promoter driving IBDV VP2 expression is derived from the immediate-early (IE) gene of murine cytomegalovirus (mCMV) strain ATCC VR-194, while the promoter for NDV F expression is derived from the immediate-early (IE) gene of human cytomegalovirus (hCMV), strain AD169. The termination and polyadenylation sequences for IBDV VP2 are derived from simian virus 40 (SV40), while the termination and polyadenylation sequences for NDV F are derived from the immediate-early (IE) gene of human cytomegalovirus (hCMV).The donor material for the first heterologous nucleic acid was inserted into the UL54.5 site (positions 111240 / 111241, Afonso et al., J. Virology 75(2):971-978 (2001); Acc. #AF291866, between amino acid residues 21 and 22), while the donor material for the second heterologous nucleic acid was inserted into the US2 site (positions 140540 / 140541, Afonso et al. (2001) supra; Acc. #AF291866, between amino acid residues 124 and 125) [see Figure 1].

[0146] Genetic and phenotypic stability are key elements of the safety and relatively strong antigen expression and / or efficacy profile of any novel recombinant viral vaccine candidate. The IBDV / ILTV and NDV expression cassettes inserted into the HVT backbone are not essentially required for viral replication and, therefore, can be lost by mutation during amplification of viral stocks in tissue culture passage. A satisfactory vaccine candidate should not readily undergo mutations that result in loss of expression of the foreign gene insert. A vaccine candidate is considered stable if it can be demonstrated that at least 90% of viral plaques express the inserted foreign antigen protein after 10 or more passages in tissue culture.

[0147] The feasibility of generating herpesviruses using the cosmid rearrangement method was previously demonstrated for pseudorabies virus [van Zijl et al., J. Virology 62:2191-2195 (1988)]. This method was later used to construct recombinant HVT vectors [see U.S. Pat. No. 5,853,733, incorporated herein by reference, for methods disclosed for the construction of recombinant HVT vectors] and was used to construct the recombinant HVT / IBDV / ILTV / NDV vectors of the present invention. In this method, the entire HVT genome is cloned into a bacterial vector as several large, overlapping subgenomic fragments constructed using standard recombinant DNA techniques [Maniatis et al., (1982) Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1982); and Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)]. A cosmid library of HVT strain FC126 was derived from sheared viral DNA cloned into the cosmid vector pWE15 (Stratagene; now Agilent Technologies, Santa Clara, CA). Several large genomic DNA fragments were also isolated by restriction digestion with the enzyme BamHI and cloned into pWE15 or the plasmid vector pSP64 (Promega, Madison, WI). As described in U.S. Pat. No. 5,853,733, cotransfection of these fragments into chicken embryo fibroblast (CEF) cells resulted in the regeneration of the HVT genome by homologous recombination across the overlapping regions of the fragments. When an insertion was directly engineered into one or more of the subgenomic fragments prior to cotransfection, this method yielded a high frequency of viruses containing the insertion. Five overlapping subgenomic clones were required to obtain FC126 HVT, which served as the basis for the generation of all HVT / IBDV / ILTV / NDV recombinant viruses.

[0148] Construction of HVT / IBDV / ILTV / NDV 670-14.1-1A1 or A2 The triple recombinant HVT vector virus HVT / IBDV / ILTV / NDV 670-14.1-1 contains an IBDV / ILTV expression cassette inserted into the HVT UL54.5 site and an NDV expression cassette inserted into the HVT US2 site. Cosmid regeneration of HVT / IBDV / ILTV / NDV 670-14.1-1 was performed essentially as described in US 5,853,733 [e.g., Figure 8 of US 5,853,733]. To allow integration into the UL54.5 region of the FC126 HVT genome, the region covered by cosmid number 407-31.1C1 in US 5,853,733 was obtained from three smaller plasmids: 672-01.A40 and 672-07.C40, and one transfer plasmid (484-1050-2641-10859) that overlaps these two and contains an IBDV / ILTV expression cassette within the UL54.5 locus. To allow integration into the US region of the FC126 HVT genome, the region covered by cosmid number 378-50 in US 5,853,733 was obtained from three smaller plasmids: pSY640 and 556-60.6, and one transfer plasmid (1322-48.1) that overlaps these two and contains an NDV expression cassette within the US2 locus.

[0149] The entire set of nine linearized constructs (two cosmids and seven plasmids) was transfected together into chicken embryo fibroblasts (CEFs) using standard CaCl transfection methods, and the resulting virus stock was plaque-purified twice.

[0150] Description of the subgenomic fragments used to generate FC126 HVT Subgenomic clone 407-32.2C3 Cosmid 407-32.2C3 contains an approximately 40,170 base pair region of genomic HVT DNA (left end -position 39,754; Afonso et al., 2001, supra; Acc. #AF291866). This region includes HVT BamHI fragments F', L, P, N1, E, and D, and the 2,092 base pair fragment B.

[0151] Subgenomic clone 172-07.BA2 Plasmid 172-07.BA2 contains a 25,931 base pair region of genomic HVT DNA. It was constructed by cloning the HVT BamHI B fragment [positions 37,663 to 63,593; Afonso et al., 2001, supra; Accession #AF291866] into plasmid pSP64 (Promega, Madison, WI).

[0152] Subgenomic clone 407-32.5G6 Cosmid 407-32.5G6 contains a 39,404 base pair region of genomic HVT DNA [positions 61,852 to 101,255; Afonso et al., 2001, supra; Acc. #AF291866]. This region includes HVT BamHI fragments H, C, Q, K1, M, and K2, as well as the 1,742 base pair fragment B and the 3,880 base pair fragment J.

[0153] Subgenomic clone 407-32.1C1 Cosmid 407-32.1C1 contains a 37,444 base pair region of genomic HVT DNA [positions 96,095 to 133,538; Afonso et al., 2001, supra; Acc. #AF291866]. This region includes HVT BamHI fragments J, G, I, F, and O, as well as the 1,281 base pair fragment K2 and the 6,691 base pair fragment A.

[0154] Subgenomic clone 378-50 Plasmid 378-50 contains a 28,897 base pair region of genomic HVT DNA [see Figure 8 of US Pat. No. 5,853,733]. This region includes the HVT BamHI fragment A. It was constructed by cloning the HVT BamHI A fragment [positions 126848 to 155744; Afonso et al., 2001, supra; Accession #AF291866] into cosmid pWE15.

[0155] Additional inserts to generate HVT / IBDV / ILTV / NDV 670-14.1-1A1 or A2 (see Figure 1) Subgenomic clone 484-1050-2641-10859 Insertion plasmid 484-1050-2641-10859 contains an 8636 base pair region of genomic HVT DNA derived from the unique long region [positions 109489 to 118124; Afonso et al., 2001, supra; Accession #AF291866] cloned into a derivative of plasmid pNEB193 (AatII-PvuII deletion). It is flanked by AscI sites and includes HVT BamHI fragments I and S, the 1337 base pair fragment G, and the 1177 base pair fragment F. Two elements were inserted within the XhoI site in the HVT UL54.5 open reading frame [positions 111240 / 111241, Afonso et al., 2001, supra; Acc. #AF291866, between amino acid residues 21 and 22]: an expression cassette consisting of the HCMV IE promoter, IBDV classical type F52 / 70, Faragher strain, viral protein 2 gene (VP2), and SV40 polyadenylation signal, followed by a 3563-base pair SalI-HindIII fragment from ILTV, NVSL challenge strain, Lot #83-2 [positions 10532-14094; Wild et al., Virus Genes 1999], encoding the full-length genes for glycoprotein D (gD) and glycoprotein I (gI), and a partial coding region from glycoprotein E (amino acids 1-101), and ORF5 (amino acids 734-985). 12:104-116 (1996); Acc. #U28832]. The IBDV VP2, ILTV gD, and ILTV gI genes are transcribed in the opposite direction to the HVT UL54.5 gene.

[0156] Subgenomic clone 672-01.A40 Plasmid 672-01.A40 contains a 14,731 base pair region of genomic HVT DNA derived from the unique long region [positions 96095 to 110825; Afonso et al., 2001, supra; Acc. #AF291866] cloned into a derivative of plasmid pNEB193. This region includes HVT BamHI fragments G, J, and the 1281 base pair K2.

[0157] Subgenomic clone 672-07.C40 Plasmid 672-07.C40 contains a 12,520 base pair region of genomic HVT DNA derived from the unique long region [positions 116948 to 129467; Afonso et al., 2001, supra; Acc. #AF291866] cloned into a derivative of plasmid pNEB193. This region includes the HVT BamHI fragments F, O, and 2620 base pairs of A.

[0158] Subgenomic clone 1322-48.1 Insertion plasmid 1322-48.1 contains a 7311-base pair EcoRI fragment of the HVT unique short region [positions 126880 to 144190; Afonso et al., 2001, supra; Accession #AF291866] cloned into plasmid pSP64 (Promega, Madison, WI). An expression cassette consisting of the HCMV IE promoter, NDV, clone 30 strain, fusion gene (F), and transcription termination sequence from the HCMV IE gene is cloned into the unique StuI site within the HVT US2 gene [positions 140540 / 140541; Afonso et al., 2001, supra; Accession #AF291866, between amino acid residues 124 and 125]. The NDV F gene is transcribed in the opposite direction to the HVT US2 gene.

[0159] Subgenomic clone pSY640 Plasmid pSY640 contains an approximately 13,600 base pair region [126848-140540; Afonso et al., 2001, supra; Acc. #AF291866] of genomic HVT DNA derived from BamHI fragment A. To generate this plasmid, a region of DNA located upstream of the US2 gene, starting from the StuI site located within the US2 gene and continuing to the end of the BamHI A fragment, was cloned into plasmid pSP64 (Promega, Madison, WI).

[0160] Subgenomic clone 556-60.6 Plasmid 556-60.6 contains an approximately 12,500 base pair region of genomic HVT DNA [approximately positions 143300 to 155744, Afonso et al., 2001, supra; Acc. #AF291866] derived from BamHI fragment A. To generate this plasmid, a region of DNA downstream of the US2 gene (starting from the StuI site located within the US2 gene and continuing to the end of the BamHI A fragment) was cloned into pSP64 (Promega, Madison, WI), then treated with exonuclease to "shatter" approximately 150 bp from the StuI site, and recloned into the pBR322 plasmid vector.

[0161] Standard CaCl2 transfection method Secondary CEFs were seeded onto 6-well culture plates and incubated at 38°C and 5% CO2 for 24 hours to form confluent monolayers. For each well, a total of 0.5 μg of cosmid and plasmid DNA was mixed in Hepes buffer, and 125 mM CaCl2 was added dropwise until precipitation was imminent. This mixture was added to the CEF cell monolayer and incubated for 2-3 hours. The supernatant was removed, and a 15% glycerol overlay was added and left on the cells for 1 minute. This was then removed, washed with phosphate-buffered saline (PBS), fresh medium was added, and the cells were incubated for 2 days. The cells were then harvested by trypsinization and plated onto larger plates, first onto 6 cm plates and then onto 10 cm plates (after 3 days). The infection was expanded twice until 50-90% CPE was achieved. The amplified transfected cells were then harvested by trypsinization and plated onto 10 cm plates. -3 ~10 -4 The dilutions were plated onto a 6 cm plate with a CEF monolayer and incubated. The next day, the plate was covered with agar and numerous individual plaques of HVT / IBDV / ILTV / NDV were isolated and amplified on CEF. A confluent monolayer of CEF on a 6 cm plate was incubated with 10 -4 ~10 -5Each virus stock was plaque-purified again by infecting cells with a diluted first-round purified stock and incubating the cells. The next day, plates were overlaid with agar, and numerous individual plaques of HVT / IBDV / ILTV / NDV were isolated and amplified on CEF.

[0162] Example 2 Recombinant HVT / IBDV / ILTV / NDV virus stocks are phenotypically stable with respect to the expression of the IBDV, ILTV, and NDV proteins after serial passage in tissue culture. Two plaque-purified isolates of HVT / IBDV / ILTV / NDV, each from a separate cotransfection stock, were serially passaged 15 times on secondary CEF cells and evaluated in immunofluorescence assays for expression of the inserted ILTV, NDV, and IBDV genes.

[0163] Preparation of tissue culture passage stocks For each tissue culture passage, confluent secondary CEF monolayers were inoculated with 50–100 μL of virus stock and incubated at 38°C, 5% CO2 for 2–5 days until CPE was evident. Cells were then harvested by trypsinization (passage 1 (P1)). This process was repeated to prepare further passage stocks (P2–P15).

[0164] Phenotypic stability analysis Secondary CEF monolayers were seeded in 6-well plates. The cells were inoculated with virus stocks recovered at various passage levels (P0–P15 or diluent only). Plates were inoculated at multiple dilutions to obtain countable plaque numbers per well and incubated at 38°C and 5% CO2. After 5 days of incubation, the supernatant was decanted, and the CEF monolayers were fixed with 70% acetone for approximately 20 minutes at 15–30°C. The acetone solution was decanted, and the cells were air-dried before staining with ILTV gD (polyclonal rabbit anti-ILTV gD), ILTV gI (polyclonal rabbit anti-ILTV gI), NDV F (Mab#57), and IBDV VP2 (MCA GDV-R63) primary antibodies. After a 1.5-hour blocking step, 5% goat serum + 0.5% Triton-X 100 in PBS (2 mL per well) was added to the dish and incubated at 36-39°C in a humidified incubator. Primary antibody was diluted appropriately and added at 2 mL per well and incubated at 36-39°C in a humidified incubator for 1.3 hours.

[0165] After antibody incubation, plates were washed three times with PBS + 0.5% Triton-X100. FITC-labeled secondary antibody solution (rabbit anti-mouse or goat anti-rabbit) was prepared at 1:50, and 2 mL was added to each well. After incubation, plates were washed three times with PBS + 0.5% Triton-X100 and examined under a fluorescent scope, and plaques were scored as positive or negative for fluorescent staining. Plates were then examined under a white light microscope, and plaques were re-counted. The percentage of fluorescent plaques at each passage level is shown in Table 1 below. Both isolates maintained acceptable expression levels for all four antigens (over 90%) at passage level 15. [Table 2]

[0166] Example 3 Recombinant HVT / IBDV / ILTV / NDV virus stocks are phenotypically stable with respect to expression of the ILTV, NDV and IBDV proteins after vaccination and recovery from birds Vaccines were prepared from two isolates of HVT / IBDV / ILTV / NDV 670-14.1-1 (isolates A1 and A2) and used to inoculate two groups of 21-day-old chickens by the subcutaneous route. A third group of birds was vaccinated with diluent alone to serve as a negative control. Pooled spleen samples from three birds were collected twice weekly for four weeks postvaccination and processed for virus isolation on chicken embryo fibroblasts (CEF). When cytopathic effects were clearly visible, monolayers were fixed, and plaques were analyzed for expression of IBDV VP2, ILTV gD and gI, and NDV F protein by immunofluorescence assay (IFA) using antibodies specific for each protein.

[0167] Phenotypic stability analysis Secondary CEF monolayers were seeded in 6-well plates. 5 x 10 splenocytes were inoculated onto the cells and incubated at 38°C, 5% CO2. After 5 days of incubation, the supernatant was decanted, and the CEF monolayers were fixed with 70% acetone for approximately 20 minutes at 15-30°C. The acetone solution was decanted, and the cells were air-dried before staining with ILTV gD (polyclonal rabbit anti-ILTV gD), ILTV gI (polyclonal rabbit anti-ILTV gI), NDV F (Mab #57), and IBDV VP2 (MCA GDV-R63) primary antibodies. After a blocking step of approximately 0.5 hours, 5% goat serum + 0.5% Triton-X 100 in PBS (2 mL per well) was added to the dishes, which were then incubated at 36-39°C in a humidified incubator. The primary antibody was diluted appropriately and added at 2 mL per well, followed by incubation for 1 hour at 36–39°C in a humidified incubator. After antibody incubation, the plates were washed three times with PBS + 0.5% Triton-X 100. FITC-labeled secondary antibody solution (rabbit anti-mouse or goat anti-rabbit) was prepared at a 1:50 ratio, and 2 mL was added to each well. The plates were incubated for 1 hour at 36–39°C in a humidified incubator. After incubation, the plates were washed three times with PBS + 0.5% Triton-X 100 and examined under a fluorescent microscope. Plaques were scored as positive (+) or negative (-) for fluorescent staining. The plates were then examined under a white light microscope, and the plaques were recounted. The percentage of fluorescent plaques at each passage level is shown in Table 2 below. [Table 3]

[0168] Example 4 HVT / IBDV / ILTV / NDV efficacy data for two isolates of each construct The following four studies were performed to demonstrate the efficacy of the single construct HVT / IBDV / ILTV / NDV 670-14 as a vaccine candidate to protect against challenge with virulent infectious laryngotracheitis virus (ILTV), virulent infectious bursal disease virus (IBDV), or virulent Marek's disease virus (MDV).

[0169] In the first study, one-day-old specific pathogen-free (SPF) chicks were inoculated with the HVT / IBDV / ILTV / NDV 670-14.1-1A2 vaccine candidate. Controls included a second group of unvaccinated chicks. On day 28 postvaccination, the vaccinated chicks and unvaccinated control chicks were challenged with virulent ILTV / USDA lot LT 96-3 via the intratracheal (IT) route. The birds were then observed for clinical signs of disease for 10 days. The results in Table 3 indicate that the 670-14.1-1A2 vaccine provided partial protection from challenge. A second study was conducted using the second clone of the vaccine, 670-14.1-1A1. The results in Table 4 demonstrate a significant improvement in protection. Therefore, two subsequent studies were conducted using the 670-14.1-1A1 vaccine candidate. These results provide evidence that HVT / IBDV / ILTV / NDV can be stable and effective. [Table 4]

[0170] In a third study, one-day-old specific pathogen-free (SPF) chicks were vaccinated with the HVT / IBDV / ILTV / NDV 670-14.1-1A1 vaccine. Controls included a second unvaccinated group. On day 28 postvaccination, vaccinated chicks and unvaccinated control chicks were challenged with the virulent IBDV / CS89 strain via the intraocular (IO) route. Birds were then observed for 10 days for clinical signs of disease, and bursae were harvested, examined histologically for gross lesions consistent with IBDV, and evaluated according to European Pharmacopoeia 9.0 (04 / 2013:0587). The results in Table 5 demonstrate that the 670-14.1-1A1 vaccine provided 100% protection from challenge. [Table 5]

[0171] In the fourth study, 1-day-old specific pathogen-free (SPF) chicks were vaccinated with the HVT / IBD / ILTV / NDV 670-14.1-1A1 vaccine. Controls included a second group of unvaccinated chicks. Five days after vaccination, the vaccinated chicks and unvaccinated control chicks were challenged with a virulent MDV / GA strain via the intraperitoneal (IA) route. The birds were then observed for clinical signs of disease for 50 days and, upon death or euthanasia, were examined for gross lesions consistent with MDV. The results in Table 6 indicate that the 670-14.1-1A1 vaccine provided 95% protection from challenge. In summary, these results indicate that HVT / IBD / ILTV / NDV vaccines can be stable and effective. It also follows that confidence regarding the upper limit of foreign antigens encoded in the multivalent HVT construct has not been achieved for a stable and effective multivalent HVT vaccine. [Table 6]

[0172] Example 5 Additional HVT / IBDV / ILTV / NDV constructs Construction of HVT / IBDV / ILTV / NDV#2 The triple recombinant HVT vector virus HVT / IBDV / ILTV / NDV#2 contains an NDV expression cassette inserted into the HVT UL54.5 site and an IBDV / ILTV expression cassette inserted into the HVT US2 site. Cosmid regeneration of HVT / IBDV / ILTV / NDV#2 was performed essentially as described in US Pat. No. 5,853,733 [e.g., Figure 8 of US Pat. No. 5,853,733]. To allow integration into the UL54.5 region of the FC126 HVT genome, the region covered by cosmid number 407-31.1C1 in US Pat. No. 5,853,733 was transfected, this time with three smaller plasmids [672-01.A40 and 672-07.C40], and one transfer plasmid (654-45:325341) overlapping these two and containing an NDV expression cassette within the UL54.5 locus. To allow integration into the US region of the FC126 HVT genome, the region covered by cosmid numbers 378-50 in US 5,853,733 was obtained this time from three smaller plasmids: pSY640 and 556-60.6, and one transfer plasmid (228509-ILT-435Vec6) that overlaps these two and contains an IBDV / ILTV expression cassette within the US2 locus.

[0173] The entire set of nine linearized constructs (two cosmids and seven plasmids) was transfected together into chicken embryo fibroblasts (CEFs) using standard CaCl transfection methods, and the resulting virus stock was plaque-purified twice.

[0174] Construction of HVT / IBDV / ILTV / NDV#3 The triple recombinant HVT vector virus, HVT / IBDV / ILTV / NDV#3, contains an IBDV expression cassette inserted into the HVT UL54.5 site and an ILTV / NDV expression cassette inserted into the HVT US2 site. Cosmid regeneration of HVT / IBDV / ILTV / NDV#3 was performed essentially as described in US Pat. No. 5,853,733 (e.g., Figure 8 of US Pat. No. 5,853,733). To allow integration into the UL54.5 region of the FC126 HVT genome, the region covered by cosmid number 407-31.1C1 in US Pat. No. 5,853,733 was obtained, this time from three smaller plasmids: 672-01.A40 and 672-07.C40, and one transfer plasmid (VP2 / 1C1#8) that overlaps these two and contains an IBDV expression cassette within the UL54.5 locus. To allow integration into the US region of the FC126 HVT genome, the region covered by cosmid number 378-50 in US 5,853,733 was derived, this time from three smaller plasmids: pSY640 and 556-60.6, and one transfer plasmid (1332-47.A2) that overlaps these two and contains an ILTV / NDV expression cassette within the US2 locus. The set of nine linearized constructs (two cosmids and seven plasmids) was transfected together into chicken embryo fibroblasts (CEFs) using standard CaCl transfection methods, and the resulting virus stock was plaque-purified twice.

[0175] Construction of HVT / IBDV / ILTV / NDV#4 The triple recombinant HVT vector virus, HVT / IBDV / ILTV / NDV#4, contains an ILTV expression cassette inserted into the HVT UL54.5 site and an IBDV / NDV expression cassette inserted into the HVT US2 site. Cosmid regeneration of HVT / IBDV / ILTV / NDV#4 was performed essentially as described in US Pat. No. 5,853,733 (e.g., Figure 8 of US Pat. No. 5,853,733). To allow integration into the UL54.5 region of the FC126 HVT genome, the region covered by cosmid number 407-31.1C1 in US Pat. No. 5,853,733 was obtained, this time from three smaller plasmids: 672-01.A40 and 672-07.C40, and one transfer plasmid (1332-23.7) that overlaps these two and contains an ILTV expression cassette within the UL54.5 locus. To allow integration into the US region of the FC126 HVT genome, the region covered by cosmid number 378-50 in US 5,853,733 was derived, this time from three smaller plasmids: pSY640 and 556-60.6, and one transfer plasmid (435Vec60) that overlaps these two and contains an IBDV / NDV expression cassette within the US2 locus. The set of nine linearized constructs (two cosmids and seven plasmids) was transfected together into chicken embryo fibroblasts (CEFs) using standard CaCl transfection methods, and the resulting virus stock was plaque-purified twice.

[0176] Construction of HVT / IBDV / ILTV / NDV#5 The triple recombinant HVT vector virus, HVT / IBDV / ILTV / NDV#5, contains an ILTV / NDV expression cassette inserted into the HVT UL54.5 site and an IBDV expression cassette inserted into the HVT US2 site. Cosmid regeneration of HVT / IBDV / ILTV / NDV#5 was performed essentially as described in US Pat. No. 5,853,733 (e.g., Figure 8 of US Pat. No. 5,853,733). To allow integration into the UL54.5 region of the FC126 HVT genome, the region covered by cosmid number 407-31.1C1 in US Pat. No. 5,853,733 was obtained, this time from three smaller plasmids: 672-01.A40 and 672-07.C40, and one transfer plasmid (1332-29.4) that overlaps with these two and contains the ILTV / NDV expression cassette within the UL54.5 locus. To allow integration into the US region of the FC126 HVT genome, the region covered by cosmid number 378-50 in US 5,853,733 was obtained, this time from three smaller plasmids: pSY640 and 556-60.6, and one transfer plasmid (435Vec60) that overlaps these two and contains an IBDV expression cassette within the US2 locus. The set of nine linearized constructs (two cosmids and seven plasmids) was transfected together into chicken embryo fibroblasts (CEFs) using standard CaCl transfection methods, and the resulting virus stock was plaque-purified twice.

[0177] Additional inserts to generate HVT / IBDV / ILTV / NDV#2 (see Figure 2) Subgenomic clone 654-45:325341 IE-F / 1C1 Insertion plasmid 654-45:325341 IE-F / 1C1 contains an 8636-bp region of the genomic HVT unique long region (positions 109489 to 118124; Afonso et al., 2001, supra; Accession No. AF291866) cloned into a derivative of the plasmid pNEB193 (AatII-PvuII deletion). It is flanked by AscI sites and contains the HVT BamHI fragments I and S, the 1337-bp fragment G, and the 1177-bp fragment F. An expression cassette consisting of the HCMV IE promoter, NDV clone 30 strain, the fusion gene (F), and the transcription termination sequence from the HCMV IE gene was inserted into the XhoI site within the HVT UL54.5 open reading frame (positions 111240 / 111241; Afonso et al., 2001, supra; Accession No. AF291866, between amino acid residues 21 and 22). The NDV F gene is transcribed in the opposite direction to the HVT UL54.5 gene.

[0178] Subgenomic clone 228509-ILT-435Vec6 [see International Application PCT / EP2017 / 064662] Insertion plasmid 228509-ILT-435Vec6 contains a 7311 base pair EcoRI fragment of the HVT unique short region [positions 126880 to 144190; Afonso et al., 2001, supra; Acc. #AF291866] cloned into plasmid pSP64 (Promega, Madison, WI). Within the unique StuI site in the HVT US2 gene [positions 140540 / 140541, Afonso et al., 2001, supra; Acc. #AF291866, between amino acid residues 124 and 125], the following two elements were inserted: an expression cassette consisting of the MCMV IE promoter, the IBDV classical type F52 / 70, Faragher strain, viral protein 2 gene (VP2), and the SV40 polyadenylation signal, followed by a 3563 base pair SalI-HindIII fragment from ILTV, NVSL challenge strain, Lot #83-2 [positions 10532-14094; Wild et al., Virus Genes 1999], encoding the full-length genes for glycoprotein D (gD) and glycoprotein I (gI), and a partial coding region from glycoprotein E (amino acids 1-101), and ORF5 (amino acids 734-985). 12:104-116 (1996); Acc. #U28832]. The IBDV VP2, ILTV gD, and ILTV gI genes are transcribed in the opposite direction to the HVT US2 gene.

[0179] Additional inserts to generate HVT / IBDV / ILTV / NDV#3 (see Figure 3) Subgenomic clone VP2 / 1C1#8 Insertion plasmid VP2 / 1C1#8 contains an 8636 base pair region of the genomic HVT unique long region [positions 109489 to 118124; Afonso et al., 2001, supra; Acc. #AF291866] cloned into a derivative of plasmid pNEB193 (AatII-PvuII deletion). It is flanked by AscI sites and includes HVT BamHI fragments I and S, the 1337 base pair fragment G, and the 1177 base pair fragment F. An expression cassette consisting of the MCMV IE promoter, IBDV classical type F52 / 70 (Faragher strain), viral protein 2 gene (VP2), and SV40 polyadenylation signal was inserted into the XhoI site of the HVT UL54.5 open reading frame (OPF) [positions 111240 / 111241, Afonso et al., 2001, supra; Acc. #AF291866, between amino acid residues 21 and 22]. The IBDV VP2 gene is transcribed in the opposite direction to the HVT UL54.5 gene.

[0180] Subgenomic clone 1332-47.A2 Insertion plasmid 1332-47.A2 contains a 7311 base pair EcoRI fragment of the HVT unique short region [positions 126880 to 144190; Afonso et al., 2001, supra; Acc. #AF291866] cloned into plasmid pSP64 (Promega, Madison WI). Within the unique StuI site in the HVT US2 gene [positions 140540 / 140541, Afonso et al., 2001, supra; Acc. #AF291866, between amino acid residues 124 and 125], two elements were inserted: a 3563 base pair SalI-HindIII fragment from ILTV, the NVSL challenge strain, Lot #83-2 [positions 10532 to 14094; Wild et al., Virus Genes 12:104-116 (1996); Acc. #U28832], encoding the full-length genes for glycoprotein D (gD) and glycoprotein I (gI), and a partial coding region from glycoprotein E (amino acids 1 to 101), and ORF5 (amino acids 734 to 985); followed by the HCMV IE promoter, the NDV, clone 30, fusion gene (F), and the HCMV. An expression cassette consisting of the transcription termination sequence from the IE gene has been inserted. The ILTV gD, ILTV gI and NDV F genes are transcribed in the opposite direction to the HVT US2 gene.

[0181] Additional inserts to generate HVT / IBDV / ILTV / NDV#4 (see Figure 4) Subgenomic clone 1332-23.7 Insertion plasmid 1332-23.7 contains an 8636 base pair region of the genomic HVT unique long region [positions 109489 to 118124; Afonso et al., 2001, supra; Acc. #AF291866] cloned into a derivative of plasmid pNEB193 (AatII-PvuII deletion). It is flanked by AscI sites and includes HVT BamHI fragments I and S, the 1337 base pair fragment G, and the 1177 base pair fragment F. A 3563-bp SalI-HindIII fragment from ILTV, the NVSL challenge strain, Lot #83-2 [positions 10532 to 14094; Wild et al., Virus Genes 12:104-116 (1996); Acc. #U28832], encoding the full-length genes for glycoprotein D (gD) and glycoprotein I (gI), as well as a partial coding region from glycoprotein E (amino acids 1 to 101), and ORF5 (amino acids 734 to 985), was inserted into the XhoI site within the HVT UL54.5 open reading frame [positions 111240 / 111241; Afonso et al., 2001, supra; Acc. #AF291866, between amino acid residues 21 and 22]. The ILTV gD and ILTV gI genes are transcribed in the opposite direction to the HVT UL54.5 gene.

[0182] Subgenomic clone 435Vec60 The insertion plasmid 435Vec60 contains a 7311 base pair EcoRI fragment of the HVT unique short region [positions 126880 to 144190; Afonso et al., 2001, supra; Acc. #AF291866] cloned into the plasmid pSP64 (Promega, Madison WI). Two elements were inserted into the unique StuI site within the HVT US2 gene [positions 140540 / 140541, Afonso et al., 2001, supra; Acc. #AF291866, between amino acid residues 124 and 125]: an expression cassette consisting of the MCMV IE promoter, the IBDV classical F52 / 70 (Faragher) strain viral protein 2 gene (VP2), and the SV40 polyadenylation signal, followed by an expression cassette consisting of the HCMV IE promoter, the NDV (clone 30) strain fusion gene (F), and the transcription termination sequence from the HCMV IE gene. Both the IBDV VP2 gene and the NDV F gene are transcribed in the opposite direction to the HVT US2 gene.

[0183] Additional inserts to generate HVT / IBDV / ILTV / NDV#5 (see Figure 5) Subgenomic clone 1332-29.4 [see US 9,409,954 B2] Insertion plasmid 1332-29.4 contains an 8636 base pair region of the genomic HVT unique long region [positions 109489 to 118124; Afonso et al., 2001, supra; Acc. #AF291866] cloned into a derivative of plasmid pNEB193 (AatII-PvuII deletion). It is flanked by AscI sites and includes HVT BamHI fragments I and S, the 1337 base pair fragment G, and the 1177 base pair fragment F. Two elements were inserted within the XhoI site in the HVT UL54.5 open reading frame [positions 111240 / 111241; Afonso et al., 2001, supra; Acc. #AF291866, between amino acid residues 21 and 22]: a 3563 base pair SalI-HindIII fragment from ILTV, NVSL challenge strain, Lot #83-2 [positions 10532 to 14094; Wild et al., Virus Genes 12:104-116 (1996); Acc. #U28832], encoding the full-length genes for glycoprotein D (gD) and glycoprotein I (gI), and a partial coding region from glycoprotein E (amino acids 1 to 101), and ORF5 (amino acids 734 to 985), followed by the HCMV IE promoter, the NDV, clone 30, fusion gene (F), and the HCMV. An expression cassette consisting of a transcription termination sequence from the IE gene. The ILTV gD, ILTV gI, and NDV F genes are transcribed in the opposite direction to the HVT UL54.5 gene.

[0184] Subgenomic clone 435Vec6 The insertion plasmid 435Vec6 contains a 7311-base pair EcoRI fragment of the HVT unique short region (positions 126880-144190; Afonso et al., 2001, supra; Accession No. AF291866) cloned into the plasmid pSP64 (Promega, Madison, WI). An expression cassette consisting of the MCMV IE promoter, the IBDV classical type F52 / 70 (Faragher strain) viral protein 2 gene (VP2), and the SV40 polyadenylation signal has been inserted into the unique StuI site within the HVT US2 gene (positions 140540 / 140541; Afonso et al., 2001, supra; Accession No. AF291866, between amino acid residues 124 and 125). The IBDV VP2 gene is transcribed in the opposite direction to the HVT US2 gene.

[0185] The sequences used in the HVT / ILTV / IBDV / NDV viral vectors disclosed in this Example are shown as SEQ ID NOs: 23 and 26 to 32 in Example 7 below.

[0186] Example 6 Failed constructs Recombinant vector vaccine viruses, by definition, are designed to carry and express foreign genes. If the transcription and expression of these foreign genes confers a growth disadvantage on the recombinant virus compared to the parent virus, these genes may be lost during vaccine production. For this reason, vaccine candidates must be tested for both genetic and phenotypic stability.

[0187] The standards of protection used are those established by the USDA and set forth in Title 9 Code of Federal Regulations, part 113 (9 CFR 113), "Standard requirements for Animal Products." To be licensed, live virus vaccines must provide at least 90% protection from disease-associated clinical signs or lesions in the case of NDV, IBDV, and ILTV, and at least 80% protection in the case of MDV.

[0188] Genetic stability of viral constructs was determined by Southern blot analysis at the highest expected vaccine production level and after a predetermined number of passages in tissue culture, and compared with DNA from the original isolate. DNA extracted from viral stocks was digested with restriction enzymes, transferred to membranes, and hybridized with probes designed to detect the presence of inserted foreign genes. Genetic stability can also be determined by PCR analysis. Using PCR primers designed to anneal to DNA within or adjacent to the foreign DNA, it may be possible to amplify fragments of known size from the viral DNA template both before and after passage in tissue culture.

[0189] The phenotypic stability of the viral constructs was determined by immunological staining of individual viral plaques with antibodies against the protein products of these inserted foreign genes. The protection provided by these recombinant vaccines is based on the expression of these protein products to stimulate the animal's immune system. In most cases, if the percentage of virus staining positive for foreign protein expression is below 90%, it is likely to be detrimental to the virus's ability to grow in tissue culture and is therefore unsuitable as a vaccine candidate.

[0190] As can be easily seen from Tables 7A and 7B below, most rMDVnp constructs do not meet these two criteria, i.e., stability with relatively strong antigen expression and / or efficacy. Table 7A shows a series of recombinant HVT constructs containing multiple heterologous inserts, one of which encodes an IBDV antigen. As the results show, none of the constructs in Table 7A met the criteria of stability and / or efficacy with relatively strong antigen expression. [Table 7]

[0191] Table 7B below shows a series of 11 recombinant HVT constructs and one single NAHV construct, each of which contains multiple heterologous inserts, with at least one of the heterologous inserts encoding either an NDV or ILTV antigen. 1 ( 1 (The data in Table 7B was submitted to the U.S. Patent and Trademark Office during prosecution of US 8,932,604 B2 in a declaration signed by one of the co-inventors of the present application.) As the results show, all of the constructs in Table 7B failed to meet the criteria for stability and / or efficacy accompanied by relatively strong antigen expression. [Table 8]

[0192] Example 7 array The following sequences are used in the exemplary rHVT constructs: The coding sequences shown below contain individual stop codons, which can be easily replaced with alternative stop codons without altering the properties of the protein antigen that the coding sequence encodes. [Table 9] TIFF0007721269000011.tif234167TIFF0007721269000012.tif235166TIFF0007721269000013.tif236165 TIFF0007721269000014.tif241167 TIFF0007721269000015.tif236164 TIFF0007721269000016.tif243165 TIFF0007721269000017.tif100163 TIFF0007721269000018.tif247165 TIFF0007721269000019.tif233163 TIFF0007721269000020.tif242165 TIFF0007721269000021.tif243164 TIFF0007721269000022.tif243164 TIFF0007721269000023.tif244165 TIFF0007721269000024.tif244164 TIFF0007721269000025.tif242164 TIFF0007721269000026.tif241165 TIFF0007721269000027.tif242163 TIFF0007721269000028.tif242164 TIFF0007721269000029.tif242164 TIFF0007721269000030.tif242165 TIFF0007721269000031.tif243165 TIFF0007721269000032.tif243165 TIFF0007721269000033.tif243166 TIFF0007721269000034.tif242164 TIFF0007721269000035.tif243165 TIFF0007721269000036.tif243163 TIFF0007721269000037.tif244166 TIFF0007721269000038.tif242165 TIFF0007721269000039.tif242163 TIFF0007721269000040.tif242164 TIFF0007721269000041.tif243164 TIFF0007721269000042.tif242166 TIFF0007721269000043.tif243167 TIFF0007721269000044.tif241164 TIFF0007721269000045.tif243164 TIFF0007721269000046.tif243165 TIFF0007721269000047.tif242165 TIFF0007721269000048.tif242164 TIFF0007721269000049.tif242164 TIFF0007721269000050.tif244165 TIFF0007721269000051.tif242165 TIFF0007721269000052.tif243168 TIFF0007721269000053.tif242165 TIFF0007721269000054.tif244165 TIFF0007721269000055.tif243165 TIFF0007721269000056.tif242166 TIFF0007721269000057.tif246165 TIFF0007721269000058.tif244166 TIFF0007721269000059.tif243164 TIFF0007721269000060.tif242166 TIFF0007721269000061.tif241165 TIFF0007721269000062.tif245165 TIFF0007721269000063.tif248164 TIFF0007721269000064.tif243164TIFF0007721269000065.tif242165 TIFF0007721269000066.tif244163TIFF0007721269000067.tif243167 TIFF0007721269000068.tif241165 TIFF0007721269000069.tif243165 TIFF0007721269000070.tif243166 TIFF0007721269000071.tif242164 TIFF0007721269000072.tif242163 TIFF0007721269000073.tif241164 TIFF0007721269000074.tif243163 TIFF0007721269000075.tif243163 TIFF0007721269000076.tif245165 TIFF0007721269000077.tif242164 TIFF0007721269000078.tif245165 TIFF0007721269000079.tif244165 TIFF0007721269000080.tif243165 TIFF0007721269000081.tif242166 TIFF0007721269000082.tif242166 TIFF0007721269000083.tif245167 TIFF0007721269000084.tif243166 TIFF0007721269000085.tif244165TIFF0007721269000086.tif244166 TIFF0007721269000087.tif243165 TIFF0007721269000088.tif242164 TIFF0007721269000089.tif243164 TIFF0007721269000090.tif231165 TIFF0007721269000091.tif242164 TIFF0007721269000092.tif243163 TIFF0007721269000093.tif244162 TIFF0007721269000094.tif243164 TIFF0007721269000095.tif245165 TIFF0007721269000096.tif242165 TIFF0007721269000097.tif240162 TIFF0007721269000098.tif248165 TIFF0007721269000099.tif243165 TIFF0007721269000100.tif242163 TIFF0007721269000101.tif243164 TIFF0007721269000102.tif246166 TIFF0007721269000103.tif244164 TIFF0007721269000104.tif205166

[0193] The present invention is not limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

[0194] Furthermore, it should be understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values given for nucleic acids or polypeptides are approximate and are given for illustrative purposes.

Claims

1. A recombinant herpesvirus of turkeys (rHVT) comprising at least three heterologous nucleotide sequences, wherein: a first heterologous nucleotide sequence is comprised by the first heterologous nucleic acid or the second heterologous nucleic acid, a second heterologous nucleotide sequence is comprised by the first heterologous nucleic acid or the second heterologous nucleic acid, and a third heterologous nucleotide sequence is comprised by the first heterologous nucleic acid or the second heterologous nucleic acid; a first heterologous nucleic acid located at a first non-essential site within the herpesvirus of turkeys (HVT) genome, and a second heterologous nucleic acid located at a second non-essential site within the HVT genome; the first non-essential site and the second non-essential site are different and are selected from the group consisting of a US2 site and a UL54.5 site; a first heterologous nucleotide sequence encoding one or more antigens from a first chicken pathogen, a second heterologous nucleotide sequence encoding one or more antigens from a second chicken pathogen, and a third heterologous nucleotide sequence encoding one or more antigens from a third chicken pathogen; A recombinant herpesvirus of turkeys (rHVT) wherein the first chicken pathogen is infectious bursal disease virus (IBDV), the second chicken pathogen is infectious laryngotracheitis virus (ILTV), and the third chicken pathogen is Newcastle disease virus (NDV).

2. The rHVT of claim 1, wherein the first heterologous nucleotide sequence comprises a coding sequence for infectious bursal disease virus viral protein 2 (IBDV VP2), the second heterologous nucleotide sequence comprises a coding sequence for infectious laryngotracheitis virus glycoprotein D (ILTV gD) and a coding sequence for infectious laryngotracheitis virus glycoprotein I (ILTV gI), and the third heterologous nucleotide sequence comprises a coding sequence for Newcastle disease virus fusion protein (NDV F).

3. 3. The rHVT of claim 2, wherein the first heterologous nucleic acid comprises a coding sequence for ILTV gD, a coding sequence for ILTV gI, and a coding sequence for NDV F, and the second heterologous nucleic acid comprises a coding sequence for IBDV VP2.

4. The rHVT of claim 3, wherein the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:28 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:27, or the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:31 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:

32.

5. the first heterologous nucleic acid comprises a coding sequence for IBDV VP2 and a coding sequence for NDV F; The rHVT of claim 2, wherein the second heterologous nucleic acid comprises a coding sequence for ILTV gD and a coding sequence for ILTV gI.

6. The rHVT of claim 5, wherein the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:30 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:

29.

7. 3. The rHVT of claim 2, wherein the first heterologous nucleic acid comprises a coding sequence for IBDV VP2, a coding sequence for ILTV gD, and a coding sequence for ILTV gI, and the second heterologous nucleic acid comprises a coding sequence for NDV F.

8. The rHVT of claim 3, wherein the first heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:23 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:26, or the first heterologous nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:21 and the second heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:

22.

9. The rHVT of claim 2, 3, 5 or 7, wherein ILTV gD comprises an amino acid sequence having greater than 95% identity to the amino acid sequence of SEQ ID NO:2, ILTV gI comprises an amino acid sequence having greater than 95% identity to the amino acid sequence of SEQ ID NO:4, IBDV VP2 comprises an amino acid sequence having greater than 95% identity to the amino acid sequence of SEQ ID NO:6, and NDV F comprises an amino acid sequence having greater than 95% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:

10.

10. 10. The rHVT of claim 2, 3, 5, 7, or 9, wherein the coding sequence for the IBDV VP2 protein of the first heterologous nucleotide sequence is operably under the control of the murine cytomegalovirus immediate-early 1 gene (mCMV IE1) promoter, the coding sequence for the ILTV gD protein of the second heterologous nucleotide sequence is operably under the control of the endogenous ILTV gD promoter, the coding sequence for the ILTV gI protein of the second heterologous nucleotide sequence is operably under the control of the endogenous ILTV gI promoter, and the coding sequence for NDV F is operably under the control of the human cytomegalovirus immediate-early 1 gene (hCMV IE1) promoter.

11. 11. The rHVT of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein the first non-essential site is a US2 site and the second non-essential site is a UL54.5 site.

12. 11. The rHVT of claim 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, wherein the first non-essential site is a UL54.5 site and the second non-essential site is a US2 site.

13. An immunogenic composition comprising the rHVT of any one of claims 1 to 12.

14. A vaccine comprising the immunogenic composition of claim 13.

15. 15. A method for helping to protect chickens against a virus selected from the group consisting of NDV, ILTV, IBDV, MDV and any combination thereof, comprising administering the vaccine of claim 14.

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