Recombinant herpesvirus of turkey vectors expressing antigens of avian pathogens and uses thereof

Abstract

To provide recombinant viral vectors for the insertion and expression of foreign genes for use in safe immunizations to protect poultry against a variety of pathogens.SOLUTION: Provided are: a recombinant Herpesvirus of turkey (HVT) genome, comprising one or more nucleotide sequences coding for one or more heterologous antigens inserted into an intergenic loci UL35 / UL36 in a unique long region of the HVT genome and one or more nucleotide sequences coding for one or more heterologous antigens inserted at a UL55 / Gene 3 site in the unique long (UL) region of the HVT genome; and a recombinant HVT having the genome.SELECTED DRAWING: Figure 21

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) to U.S. Provisional Application No. 62 / 898,651, filed September 11, 2019, which is incorporated herein by reference in its entirety.

[0002] The present invention relates to recombinant viral vectors for the insertion and expression of foreign genes for use in safe immunization to protect against a variety of pathogens. The present invention also relates to multivalent compositions or vaccines comprising one or more recombinant viral vectors for protection against a variety of pathogens. The present invention also relates to methods of making and using such recombinant viral vectors. [Background technology]

[0003] Marek's disease (MDV) is a highly contagious lymphoproliferative disease that primarily affects young chickens and is one of the most prevalent avian infectious diseases. It is caused by the Marek's disease virus (MDV). Marek's disease virus (MDV) is a herpesvirus, a member of the Mardivirus genus, and has three serotypes: MDV-1 (Gallid herpesvirus 2), MDV-2 (Gallid herpesvirus 3), and MDV-3 (Meleagrid herpesvirus 1, or herpesvirus of turkeys (HVT)). MDV-1 is the most virulent of the three serotypes and causes widespread disease in unvaccinated poultry. Birds infected with MDV-1 exhibit neurological, visceral, and cutaneous clinical signs, including paralysis of the legs, wings, and neck; eye lesions and visual impairment; weight loss; and cancerous tumors in many organs, including the thymus, heart, lungs, gonads, muscles, and feather follicles. Morbidity in affected birds ranges from 10-50%, and mortality can be up to 100%. Marek's disease can affect birds of any age, but acute Marek's disease causes death in large numbers of unvaccinated birds as young as 4-8 weeks of age. Marek's disease is spread by direct or indirect exposure to chicken dander from infected chickens, and the virus is taken up by inhalation. MDV-2 and MDV-3 represent avirulent virus strains and have been used in the preparation of vaccinations against the related, more virulent MDV-1.

[0004] In addition to Marek's disease, there are several other pathogens that affect poultry and pose a threat to poultry husbandry. Producers must rely on the immunity provided by vaccines to protect their flocks from viruses, bacteria, and other pathogens. Live, killed, and recombinant vaccines have been used to vaccinate avian species. Live vaccines have the advantage of providing strong, long-lasting immunity, but they must be handled with care because they can cause mild to severe reactions. Killed vaccines, on the other hand, are more stable and safer than live vaccines but generate weaker immune responses and require multiple doses. While both live and killed vaccines have proven safe and effective, there is still a need to develop and continually improve multivalent vaccines to provide protection against more than one pathogen with a single vaccination.

[0005] Recombinant vectored vaccines have been developed to provide immunity to multiple pathogens simultaneously. These vaccines are made by removing several non-essential gene segments in the host genome of a non-pathogenic organism and replacing them with one or more genes encoding antigens involved in generating an immune response against the pathogenic organism. The newly produced vector The vector is used to infect the host, where the virulent organism replicates and expresses antigens to elicit an immune response. Recombinant vectored vaccines combine the advantages of live and killed vaccines. Like live vaccines, recombinant vectors provide longer-lasting immunity, while like killed vaccines, they induce a milder reaction after vaccination. In addition, both vectors and inserted genes can provide immunity that protects birds against more than one disease.

[0006] Marek's disease viruses (MDVs) are one of the most effective vectors for multivalent vaccines to immunize poultry against disease because they induce lifelong protection with just a single vaccination. Additionally, because these viruses are restricted to the avian host, they pose no risk of infection to other animals or people working on poultry farms. Among the Marek's disease viruses (MDVs), herpesvirus of turkeys (HVT) has been more widely used, both as a live vaccine and as a recombinant vaccine vector against the more virulent MDV-1. HVT was first isolated from turkeys in 1969–1970 and was soon found to be protective against MDV and licensed as a vaccine in 1971. While HVT shares antigenic characteristics with Marek's disease virus (MDV-1), it is not pathogenic to chickens. Additionally, because HVT is not susceptible to maternally derived antibodies against MDV or HVT, live HVT vaccines have been used to effectively vaccinate against MDV-1 in ovo or at early pre-hatch ages. Additionally, the HVT genome has been used as a vaccine vector to carry foreign DNA sequences of other avian pathogens. Summary of the Invention

[0007] The present invention provides recombinant viral vectors for the insertion and expression of foreign genes for use in safe immunization to protect birds against various pathogens. The present invention also provides multivalent compositions or vaccines containing one or more recombinant HVT viral vectors for protection against various pathogens. In addition, the present invention provides methods for making and using recombinant viral vectors alone or in combination with other vaccines or pharmaceutical compositions.

[0008] In one aspect, the present invention provides a recombinant herpesvirus of turkeys (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted within the intergenic locus UL35 / UL36 in the unique long (UL) region of the HVT genome.

[0009] In one aspect, the present invention provides a recombinant herpesvirus of turkeys (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens or antigens inserted within the intergenic locus UL35 / UL36 in the unique long region of the HVT genome, and one or more nucleotide sequences or sequences encoding one or more heterologous antigens inserted into the UL55 / Gene3 site in the unique long region (UL) of the HVT genome.

[0010] In one or more embodiments, the present invention provides a recombinant HVT, wherein the one or more heterologous antigen(s) are protective against an avian pathogen or pathogens selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV).

[0011] In one or more embodiments, the present invention provides a recombinant HVT comprising one or more The heterologous antigen is selected from the group consisting of the VP2, VP3, or VP4 protein of infectious bursal disease virus (IBDV), the VP1 or VP2 protein of chicken anemia virus (CAV), the F / HN chimeric protein or the F, NP, P, M, HN, or L protein of Newcastle disease virus (NDV), the S1, S2, or M protein of infectious bronchitis virus (IBV), the gB, gC, gD, gE, gH, gI, or gL protein of infectious laryngotracheitis virus (ILTV), and the HA, NA, NP, or M protein of avian influenza virus (AIV).

[0012] In one or more embodiments, the recombinant HVT of the present invention is provided with the proviso that one or more heterologous antigens are protective against IBDV. In one embodiment, the recombinant HVT of the present invention is provided with the proviso that the one or more heterologous antigens is the VP2 protein of IBDV. In one embodiment, the recombinant HVT of the present invention is provided with the proviso that the VP2 protein sequence is encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:10. In one embodiment, the recombinant HVT of the present invention provides a VP2 protein encoded by a nucleotide sequence comprising either SEQ ID NO:5 or SEQ ID NO:10.

[0013] In one or more embodiments, the recombinant HVT of the present invention provides that the one or more heterologous antigens or antigens are protective against Newcastle Disease Virus (NDV). In one embodiment, the recombinant HVT of the present invention provides that the one or more heterologous antigens is the F protein of NDV. In one embodiment, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:3. In one embodiment, the F protein of the recombinant HVT NDV of the present invention is encoded by a nucleotide sequence comprising SEQ ID NO:3.

[0014] In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens are protective against NDV and IBDV, and in one or more embodiments, the recombinant HVT of the present invention provides that at least one heterologous antigen is the F protein of NDV and the VP2 protein of IBDV.

[0015] In one or more embodiments, the recombinant HVT of the present invention provides an NDV F protein encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:3, and an IBDV VP2 protein encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:10.

[0016] In one or more embodiments, the recombinant HVT of the present invention is provided such that the NDV F protein is encoded by a nucleotide sequence comprising SEQ ID NO: 3, and the IBDV VP2 protein is encoded by a nucleotide sequence comprising SEQ ID NO: 5 or SEQ ID NO: 10.

[0017] In one or more embodiments, the recombinant HVT of the present invention comprises a genome comprising one or more expression cassettes or cassettes comprising one or more nucleotide sequences or sequences encoding one or more heterologous antigens or antigens. In one embodiment, the recombinant HVT comprises a recombinant HVT genome comprising an expression cassette comprising a nucleotide sequence encoding a promoter operably linked to one or more nucleotides encoding the antigen to be expressed. In one embodiment, the expressed antigen comprises the F protein of NDV. In one embodiment, the expressed antigen comprises the VP2 protein of IBDV. In one embodiment, the expressed antigen comprises the F protein of NDV. The antigens displayed include both the F protein of NDV and the VP2 protein of IBDV.

[0018] In one embodiment, the recombinant HVT of the present invention is provided with one or more promoters selected from the group consisting of an immediate-early cytomegalovirus human (hCMV) promoter, a guinea pig immediate-early CMV promoter, a mouse immediate-early CMV promoter, a Pec promoter, a β-chicken actin promoter, an SV40 promoter, a pseudorabies virus promoter of glycoprotein X promoter, a herpes simplex virus-1 alpha 4 promoter, a Marek's disease virus promoter of glycoprotein gA, gC, gB, gE, or gI promoter, an infectious laryngotracheitis virus promoter of glycoprotein gB, gE, gI, or gD promoter, and a bovine herpesvirus 1 / 1 VP8 promoter. In one embodiment, the recombinant HVT comprises a human CMV promoter. In one embodiment, the recombinant HVT comprises a mouse CMV promoter. In one embodiment, the recombinant HVT comprises an hCMV promoter and an mCMV promoter.

[0019] In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a polyadenylation (polyA) signal. In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a polyA signal, selected from BGH polyA (SEQ ID NO: 6) or the SV40 polyA sequence (SEQ ID NO: 12). In one embodiment, the polyA signal is a BGH polyA signal. In one embodiment, the polyA signal is an SV40 polyA signal.

[0020] In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding a VP2 protein from IBDV, which further comprises a nucleotide sequence encoding a polyadenylation signal, and the entire VP2 expression cassette is inserted into a non-coding region of the HVT genome. In one embodiment, the CMV promoter comprises an hCMV promoter (SEQ ID NO: 1). In one embodiment, the nucleotide sequence encoding the VP2 protein of IBDV is selected from SEQ ID NO: 5 or SEQ ID NO: 10. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO: 5. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO: 10. In one embodiment, the polyadenylation signal comprises SEQ ID NO: 6. In one embodiment, the polyadenylation signal comprises SEQ ID NO: 12. In one embodiment, the promoter, the nucleotide sequence encoding the VP2 protein, and the polyA signal comprise an expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55 / gene3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35 / 36 site within the genome. In one embodiment, the expression cassette comprises SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:10 and SEQ ID NO:6, in order, inserted into the HVT genome at the UL55 / gene3 site.

[0021] In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding an NDV F protein, which further comprises a nucleotide sequence encoding a polyadenylation signal, and all parts of the NDV F cassette are inserted into a non-coding position within the HVT genome. In one embodiment, the CMV promoter comprises an mCMV (SEQ ID NO: 2) promoter. In one embodiment, a nucleotide sequence encoding an NDV F protein comprises SEQ ID NO: 3. In one embodiment, the polyadenylation signal is encoded by a nucleotide sequence comprising SEQ ID NO: 12. In one embodiment, the promoter, the nucleotide sequence encoding the F protein, and the polyA signal comprise an expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55 / gene3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35 / 36 site within the genome. In one embodiment, the expression cassette The kit contains, in order, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:12 inserted into the HVT genome at the UL55 / gene3 site.

[0022] In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding the VP2 protein of IBDV, which further comprises a nucleotide sequence encoding a polyadenylation signal, all of which, including the VP2 expression cassette, are inserted into a non-coding location within the HVT genome. In one embodiment, the recombinant HVT of the present invention further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein of NDV, which further comprises a nucleotide sequence encoding a polyadenylation signal as part of an NDV F expression cassette inserted at the same insertion site as the VP2 cassette. In one embodiment, the recombinant HVT further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein of NDV, which further comprises a nucleotide sequence encoding a polyadenylation signal as part of an NDV F expression cassette inserted at a site different from the VP2 cassette.

[0023] In one embodiment, the recombinant HVT of the present invention provides a VP2 expression cassette comprising, in order, a nucleotide sequence encoding an hCMV promoter (SEQ ID NO: 1), a nucleotide sequence encoding IBDV VP2 (selected from SEQ ID NO: 5 or SEQ ID NO: 10), and a nucleotide sequence encoding a BGH polyadenylation signal (SEQ ID NO: 6) inserted into the HVT genome in the UL35 / 36 non-coding region, and a nucleotide sequence encoding an mCMV promoter (SEQ ID NO: 2), a nucleotide sequence encoding the F protein from NDV (SEQ ID NO: 3), and a nucleotide sequence encoding an SV40 polyadenylation signal (SEQ ID NO: 12) inserted into the HVT genome in the UL55 / gene3 non-coding region. In one aspect, the recombinant HVT of the present invention comprises a promoter operably linked to a nucleotide sequence encoding an infectious laryngotracheitis virus antigen, which further comprises a nucleotide sequence encoding a polyadenylation signal. In one embodiment, the ILT antigen comprises one or more antigens selected from the group consisting of one or more chimeric proteins of gB, gC, gD, gE, gH, gI, gL, or ILT antigens of infectious laryngotracheitis virus (ILTV). In one embodiment, the recombinant HVT of the present invention further comprises a nucleotide sequence encoding one or more antigens selected from the group consisting of infectious bursal disease virus, chicken anemia virus, Newcastle disease virus, infectious bronchitis virus, and avian influenza virus. In one embodiment, the recombinant HVT of the present invention further provides a promoter operably linked to a nucleotide sequence encoding an antigen selected from the group consisting of the VP1, VP2, VP3, or VP4 antigen of infectious bursal disease virus (IBDV), the VP1 or VP2 protein of chicken anemia virus (CAV), the F / HN chimeric protein or F, NP, P, M, HN, or L protein of Newcastle disease virus (NDV), the S1, S2, or M protein of infectious bronchitis virus (IBV), and the HA, NA, NP, or M protein of avian influenza virus (AIV).

[0024] In one embodiment, the recombinant HVT of the present invention comprises one or more ILT antigens as part of an expression cassette comprising a promoter operably linked to nucleotides encoding the ILT antigens and further comprising a nucleotide sequence encoding a polyadenylation signal. In one embodiment, the recombinant HVT of the present invention consists of any of the following: VP1, VP2, VP3, or VP4 antigens of infectious bursal disease virus (IBDV), VP1 or VP2 protein of chicken anemia virus (CAV), F / HN chimeric protein or F, NP, P, M, HN, or L protein of Newcastle disease virus (NDV), S1, S2, or M protein of infectious bronchitis virus (IBV), and HA, NA, NP, or M protein of avian influenza virus (AIV). and second and third expression cassettes, each comprising a nucleotide sequence encoding a promoter operably linked to a nucleotide sequence encoding an avian antigen selected from the group, and further comprising a nucleotide sequence encoding a polyadenylation signal.

[0025] In one or more embodiments, the present invention provides recombinant DNA encoding the recombinant HVT genome of the present invention.

[0026] In one or more embodiments, the present invention provides an immunogenic composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0027] In one or more embodiments, the present invention provides a vaccine composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0028] In one embodiment, the vaccine of the present invention further comprises an additional Marek's Disease Virus (MDV) selected from the group consisting of naturally attenuated MDV-1 strain Rispens (CVI-988) or Gallid herpesvirus 3 strain SB-1 virus. In one embodiment, the vaccine of the present invention provides that the additional MDV comprises a recombinant genome. In one embodiment, the vaccine of the present invention provides that the additional recombinant MDV genome comprises one or more nucleotide sequences encoding one or more heterologous antigens that are protective against one or more avian pathogens.

[0029] In one embodiment, the vaccine of the present invention provides for use in vaccinating birds against one or more diseases caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides for use in protecting birds against clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides for use in protecting birds against clinical symptoms caused by Marek's disease virus and clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides for one or more avian pathogens selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the vaccine of the present invention provides for the one or more avian pathogens to include Newcastle disease virus. In one embodiment, the vaccine of the present invention provides for the one or more avian pathogens to include infectious bursal disease virus (IBDV). In one embodiment, the vaccine of the present invention provides that the one or more avian pathogens include Newcastle disease virus and infectious bursal disease virus.

[0030] In one or more embodiments, the vaccine of the present invention is provided for use in vaccinating birds, wherein the vaccine is administered by at least one or more doses of the vaccine by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, intranasal administration, or a combination thereof. In one embodiment, the vaccine of the present invention provides that the vaccine is administered by in ovo administration. In one embodiment, the vaccine of the present invention provides that the in ovo administration is administered in an embryonated egg between about 16 and 22 days from emergence. In one or more embodiments, the vaccine of the present invention provides that the in ovo administration is administered in an embryonated egg at about 18 days from emergence. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises in ovo administration followed by spray administration. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises spray administration.

[0031] In one aspect, the present invention provides a method for treating Marek's disease and other avian diseases caused by one or more avian pathogens.

[0010] The present invention provides a method for vaccinating birds to treat or prevent one or more avian diseases, the method comprising administering an effective amount of a vaccine composition of the present invention. In one embodiment, the method provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method provides that the one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method provides that the one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method provides that the one or more avian pathogens include infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV).

[0032] One aspect of the present invention provides a method for inducing an immune response in an avian animal against Marek's disease virus and one or more avian pathogens, the method comprising administering to the bird an effective amount of an immunogenic or vaccine composition of the present invention. In one embodiment, the method of the present invention provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). In one or more embodiments, the method provides that the administration is carried out by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, or nasal administration. In one embodiment, the method includes in ovo administration. In one embodiment, the method provides that the in ovo administration is carried out in embryonated eggs between about 16 and 22 days of development. In one or more embodiments, the method provides that the in ovo administration is carried out in embryonated eggs at about 18 days of development. In one or more embodiments, the method provides that the route of administration includes in ovo administration followed by spray administration. In one embodiment, the method provides that the route of administration includes spray administration. In one or more embodiments, the method provides that the bird is selected from the group consisting of chicken, turkey, goose, duck, pheasant, ostrich, pigeon, and quail. In one embodiment, the method provides that the bird includes a chicken.

[0033] One aspect of the present invention provides a vaccine composition comprising a recombinant HVT of the present invention comprising a nucleotide sequence encoding an F protein from Newcastle Disease Virus, further comprising a composition comprising an attenuated infectious bursal disease virus and an antibody that specifically binds to the infectious bursal disease virus. In one or more embodiments, the composition comprising IBDV is attenuated IBD strain 2512 and comprises the Bursaplex™ vaccine. In one or more embodiments, the composition comprising IBDV is attenuated IBD strain V877 and comprises the Magniplex™ vaccine. [Brief explanation of the drawings]

[0034] [Figure 1] 1 is a representation of a PCR reaction showing correct insertion of the gfp gene at the UL55 / Gene3 site of the HVT genome. [Figure 2] 1 is a representation of a PCR reaction showing the integration site of the gfp gene at the UL35 / 36 integration site of the HVT genome. [Figure 3A] 1 is a representation of a PCR reaction showing correct integration of the VP2 gene into the HVT genome for HVT IBD 1. [Figure 3B] 1 is a representation of a PCR reaction showing correct integration of the VP2 gene into the HVT genome for HVT IBD 1. [Figure 3C] 1 is a representation of a PCR reaction showing correct integration of the VP2 gene into the HVT genome for HVT IBD 1. [Figure 4A] Representation of transfected / infected JBJ-1 cells staining for IBDV VP2 (Panel A) and HVT infection for HVT IBD 5 (Panel B). [Figure 4B] Representation of transfected / infected JBJ-1 cells staining for IBDV VP2 (Panel A) and HVT infection for HVT IBD 5 (Panel B). [Figure 5]1 is a representation of a PCR reaction performed to confirm the correct orientation of the VP2 insert into the UL35 / 36 integration site of the HVT genome for HVT IBD 5. [Figure 6] 1 is a representation of a Western blot analysis of infected cell lysates using a monoclonal antibody against IBDVR63 showing a protein band of approximately 50 KD for HVT IBD 6a. [Figure 7] 1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL35 / 36 site in the HVT genome for HVT IBD 6a. [Figure 8A] 1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL55 / gene3 site in the HVT genome for HVT IBD 9. [Figure 8B] 1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL55 / gene3 site in the HVT genome for HVT IBD 9. [Figure 9A] 1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL55 / gene3 site in the HVT genome for HVT IBD 30. [Figure 9B] 1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL55 / gene3 site in the HVT genome for HVT IBD 30. [Figure 9C] 1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL55 / gene3 site in the HVT genome for HVT IBD 30. [Figure 10] 1 is a representation of Western blot analysis of transfected / infected cell lysates using a monoclonal antibody against IBDVR63 for HVT IBD 31. [Figure 11] 1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL35 / 36 integration site in the HVT genome for HVT IBD 31. [Figure 12]1 is a representation of a PCR reaction showing correct VP2 gene integration at the UL55 / gene3 integration site in the HVT genome for HVT IBD 34. [Figure 13] 1 is a graphical representation of IBDV serological responses of HVT-IBD1, 5, 9, and 15. [Figure 14] 1 is a graphical representation of the IBDV serological response of HVT-IBD6a, 30, and 31. [Figure 15A] 1 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT ND#38. [Figure 15B] 1 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT ND#38. [Figure 16A] 1 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT ND#39. [Figure 16B] 1 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT ND#39. [Figure 17-1] 10 is a representation of a PCR reaction showing the correct orientation of the NDV F insert for HVT ND#40. [Figure 17-2] 10 is a representation of a PCR reaction showing the correct orientation of the NDV F insert for HVT ND#40. [Figure 18] 1 is a representation of multiple PCR reactions showing the correct orientation of the NDVF insert for HVT NDV42. [Figure 19] 10 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT NDV45. [Figure 20A] 1 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT NDV46. [Figure 20B] 1 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT NDV46. [Figure 20C] 1 is a representation of a PCR reaction showing the correct orientation of the NDVF insert for HVT NDV46. [Figure 21]1 is a linear representation of the HVT genome showing insertion site A, UL35 (HVT043)-UL36 (HVT044). [Figure 22] 1 is a linear representation of the HVT genome showing insertion site B, UL55(HVT065)-Gene3(HVT066). [Figure 23] This is a representation of IBDV VP2 Faragher strain F52 / 70. [Figure 24] 1 is a representation of the synthesis of plasmid pHVT-IBD #30. [Figure 25] 1 is a representation of the synthesis of plasmid pHVT-ND#42. [Figure 26] 1 is a representation of the circular map of transfer plasmid pSiteA #30. [Figure 27] 1 is a representation of the circular map of transfer plasmid pSite B#42. [Figure 28] 1 is a representation of the production of intermediate recombinant HVT-ND#42. [Figure 29] Representation of the production of HVT-IBD#30-ND#42. [Figure 30] 1 is a representation of the construct characterization of HVT-IBD#30-ND#42 based on PCR and site A restriction endonuclease digestion. [Figure 31] 1 is a representation of the construct characterization of HVT-IBD#30-ND#42 based on PCR and site B restriction endonuclease digestion. [Figure 32] 1 is a representation of Western blot analysis of HVT-IBD#30-ND#42 target protein expression of IBD VP2. [Figure 33] 1 is a representation of Western blot analysis of NDV F target protein expression in HVT-IBD#30-ND#42.

[0035] A brief description of arrays [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] DETAILED DESCRIPTION OF THE INVENTION

[0036] The following is a detailed description provided to assist those skilled in the art, who may make modifications and variations in the embodiments described herein without departing from the spirit or scope of the present invention.

[0037] The present invention relates to vaccines for use in birds based on live recombinant avian herpesviruses, specifically Marek's disease virus (MDV), and more particularly HVT virus (herpesvirus of turkeys), into which has been inserted one or more nucleotide sequences encoding and expressing antigenic polypeptides of the pathogenic agent or agents under conditions that confer immunity that results in effective protection of the vaccinated animal against the pathogenic agent or agents. Marek's disease (MD) is a common lymphoproliferative disease of chickens caused by Marek's disease virus (MDV) and can cause significant losses in the poultry industry. Currently, MD is controlled in poultry using vaccines employing serotype 3 of MDV, a related herpesvirus of turkeys (HVT). By introducing genes from poultry viruses other than MDV into the HVT genome at specific gene locations, the inventors have been able to develop novel recombinant viral vaccines that allow for the simultaneous protection of poultry against MD and one or more additional diseases with a single viral vaccine administration.

[0038] The present invention provides recombinant viral vectors for the insertion and expression of foreign genes for use in safe immunization to protect birds against various pathogens. The present invention also provides multivalent compositions or vaccines containing one or more recombinant HVT viral vectors for protection against various pathogens. In addition, the present invention provides methods for making and using recombinant viral vectors alone or in combination with other vaccines or pharmaceutical compositions.

[0039] In one aspect, the present invention provides a recombinant herpesvirus of turkeys (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted within the intergenic locus UL35 / UL36 in the unique long (UL) region of the HVT genome. The present invention provides an HVT genome comprising:

[0040] In one aspect, the present invention provides a recombinant herpesvirus of turkeys (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens or antigens inserted within the intergenic locus UL35 / UL36 in the unique long region of the HVT genome, and one or more nucleotide sequences or sequences encoding one or more heterologous antigens inserted into the UL55 / Gene3 site in the unique long region (UL) of the HVT genome.

[0041] In one or more embodiments, the present invention provides a recombinant HVT, wherein the one or more heterologous antigen(s) are protective against an avian pathogen or pathogens selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV).

[0042] In one or more embodiments, the present invention provides a recombinant HVT, wherein the one or more heterologous antigens are selected from the group consisting of the VP2, VP3, or VP4 protein of infectious bursal disease virus (IBDV), the VP1 or VP2 protein of chicken anemia virus (CAV), the F / HN chimeric protein or the F, NP, P, M, HN, or L protein of Newcastle disease virus (NDV), the S1, S2, or M protein of infectious bronchitis virus (IBV), the gB, gC, gD, gE, gH, gI, or gL protein of infectious laryngotracheitis virus (ILTV), and the HA, NA, NP, or M protein of avian influenza virus (AIV).

[0043] In one or more embodiments, the recombinant HVT of the present invention is provided with the proviso that one or more heterologous antigens are protective against IBDV. In one embodiment, the recombinant HVT of the present invention is provided with the proviso that the one or more heterologous antigens is the VP2 protein of IBDV. In one embodiment, the recombinant HVT of the present invention is provided with the proviso that the VP2 protein sequence is encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:10. In one embodiment, the recombinant HVT of the present invention provides a VP2 protein encoded by a nucleotide sequence comprising either SEQ ID NO:5 or SEQ ID NO:10.

[0044] In one or more embodiments, the recombinant HVT of the present invention provides that the one or more heterologous antigens or antigens are protective against Newcastle Disease Virus (NDV). In one embodiment, the recombinant HVT of the present invention provides that the one or more heterologous antigens is the F protein of NDV. In one embodiment, the recombinant HVT of the present invention provides that the F protein of NDV is encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:3. In one embodiment, the F protein of the recombinant HVT NDV of the present invention is encoded by a nucleotide sequence comprising SEQ ID NO:3.

[0045] In one or more embodiments, the recombinant HVT of the present invention provides that one or more heterologous antigens are protective against NDV and IBDV, and in one or more embodiments, the recombinant HVT of the present invention provides that at least one heterologous antigen is the F protein of NDV and the VP2 protein of IBDV.

[0046] In one or more embodiments, the recombinant HVT of the present invention comprises a nucleotide sequence comprising SEQ ID NO:3. and an IBDV VP2 protein encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:10.

[0047] In one or more embodiments, the recombinant HVT of the present invention is provided such that the NDV F protein is encoded by a nucleotide sequence comprising SEQ ID NO: 3, and the IBDV VP2 protein is encoded by a nucleotide sequence comprising SEQ ID NO: 5 or SEQ ID NO: 10.

[0048] In one or more embodiments, the recombinant HVT of the present invention comprises a genome comprising one or more expression cassettes or cassettes comprising one or more nucleotide sequences or sequences encoding one or more heterologous antigens or antigens. In one embodiment, the recombinant HVT comprises a recombinant HVT genome comprising an expression cassette comprising a nucleotide sequence encoding a promoter operably linked to one or more nucleotides encoding the antigen to be expressed. In one embodiment, the expressed antigen comprises the NDV F protein. In one embodiment, the expressed antigen comprises the IBDV VP2 protein. In one embodiment, the expressed antigen comprises both the NDV F protein and the IBDV VP2 protein.

[0049] In one embodiment, the recombinant HVT of the present invention is provided with one or more promoters selected from the group consisting of an immediate-early cytomegalovirus human (hCMV) promoter, a guinea pig immediate-early CMV promoter, a mouse immediate-early CMV promoter, a Pec promoter, a β-chicken actin promoter, an SV40 promoter, a pseudorabies virus promoter of glycoprotein X promoter, a herpes simplex virus-1 alpha 4 promoter, a Marek's disease virus promoter of glycoprotein gA, gC, gB, gE, or gI promoter, an infectious laryngotracheitis virus promoter of glycoprotein gB, gE, gI, or gD promoter, and a bovine herpesvirus 1 / 1 VP8 promoter. In one embodiment, the recombinant HVT comprises a human CMV promoter. In one embodiment, the recombinant HVT comprises a mouse CMV promoter. In one embodiment, the recombinant HVT comprises an hCMV promoter and an mCMV promoter.

[0050] In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a polyadenylation (polyA) signal. In one or more embodiments, the recombinant HVT comprises a nucleotide sequence encoding a polyA signal, selected from BGH polyA (SEQ ID NO: 6) or the SV40 polyA sequence (SEQ ID NO: 12). In one embodiment, the polyA signal is a BGH polyA signal. In one embodiment, the polyA signal is an SV40 polyA signal.

[0051] In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding a VP2 protein from IBDV, which further comprises a nucleotide sequence encoding a polyadenylation signal, and all parts of the VP2 expression cassette are inserted into a non-coding region of the HVT genome. In one embodiment, the CMV promoter comprises an hCMV promoter (SEQ ID NO: 1). In one embodiment, the nucleotide sequence encoding the VP2 protein of IBDV is selected from SEQ ID NO: 5 or SEQ ID NO: 10. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO: 5. In one embodiment, the nucleotide sequence encoding the VP2 protein comprises SEQ ID NO: 10. In one embodiment, the polyadenylation signal comprises SEQ ID NO: 6. In one embodiment, the polyadenylation signal comprises SEQ ID NO: 12. In one embodiment, the promoter, the nucleotide sequence encoding the VP2 protein, and a polyA signal comprise the expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55 / gene3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35 / 36 site within the genome. In one embodiment, the expression cassette comprises SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:10 and SEQ ID NO:6, in order, inserted into the HVT genome at the UL55 / gene3 site.

[0052] In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding an NDV F protein, which further comprises a nucleotide sequence encoding a polyadenylation signal, and all portions of the NDV F cassette are inserted into a non-coding position within the HVT genome. In one embodiment, the CMV promoter comprises an mCMV (SEQ ID NO: 2) promoter. In one embodiment, a nucleotide sequence encoding an NDV F protein comprises SEQ ID NO: 3. In one embodiment, the polyadenylation signal is encoded by a nucleotide sequence comprising SEQ ID NO: 12. In one embodiment, the promoter, the nucleotide sequence encoding the F protein, and the polyA signal comprise an expression cassette. In one embodiment, the expression cassette is inserted into the HVT genome at the UL55 / gene3 site. In one embodiment, the expression cassette is inserted into the HVT genome at the UL35 / 36 site within the genome. In one embodiment, the expression cassette comprises SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 12, in order, inserted into the HVT genome at the UL55 / gene3 site.

[0053] In one aspect, the recombinant HVT of the present invention comprises a CMV promoter operably linked to a nucleotide sequence encoding the VP2 protein of IBDV, which further comprises a nucleotide sequence encoding a polyadenylation signal, all of which, including the VP2 expression cassette, are inserted into a non-coding location within the HVT genome. In one embodiment, the recombinant HVT of the present invention further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein of NDV, which further comprises a nucleotide sequence encoding a polyadenylation signal as part of an NDV F expression cassette inserted at the same insertion site as the VP2 cassette. In one embodiment, the recombinant HVT further comprises a CMV promoter operably linked to a nucleotide sequence encoding the F protein of NDV, which further comprises a nucleotide sequence encoding a polyadenylation signal as part of an NDV F expression cassette inserted at a site different from the VP2 cassette.

[0054] In one embodiment, the recombinant HVT of the present invention provides a VP2 expression cassette comprising, in order, a nucleotide sequence encoding an hCMV promoter (SEQ ID NO: 1), a nucleotide sequence encoding IBDV VP2 (selected from SEQ ID NO: 5 or SEQ ID NO: 10), and a nucleotide sequence encoding a BGH polyadenylation signal (SEQ ID NO: 6), all of which are inserted into the HVT genome in the UL35 / 36 non-coding region; and a nucleotide sequence encoding an mCMV promoter (SEQ ID NO: 2), a nucleotide sequence encoding the F protein from NDV (SEQ ID NO: 3), and a nucleotide sequence encoding an SV40 polyadenylation signal (SEQ ID NO: 12), all of which are inserted into the HVT genome in the UL55 / gene3 non-coding region. In one embodiment, the recombinant HVT of the present invention further comprises a nucleotide sequence encoding one or more antigens selected from the group consisting of infectious bursal disease virus, chicken anemia virus, Newcastle disease virus, infectious bronchitis virus, infectious laryngotracheitis virus, and avian influenza virus. In one embodiment, the recombinant HVT of the present invention is selected from the group consisting of VP1, VP2, VP3, or VP4 antigens of infectious bursal disease virus (IBDV), VP1 or VP2 proteins of chicken anemia virus (CAV), F / HN chimeric proteins or F, NP, P, M, HN, or L proteins of Newcastle disease virus (NDV), S1, S2, or M proteins of infectious bronchitis virus (IBV), and HA, NA, NP, or M proteins of avian influenza virus (AIV). Further provided is a promoter operably linked to a nucleotide sequence encoding an antigen selected from the group consisting of any of the following:

[0055] In one embodiment, the recombinant HVT of the present invention comprises one or more ILT antigens as part of an expression cassette comprising a promoter operably linked to nucleotides encoding the ILT antigens, and further comprising a nucleotide sequence encoding a polyadenylation signal. In one embodiment, the recombinant HVT of the present invention comprises second and third expression cassettes, each comprising a nucleotide sequence encoding a promoter operably linked to a nucleotide sequence encoding an avian antigen selected from the group consisting of the VP1, VP2, VP3, or VP4 antigen of infectious bursal disease virus (IBDV), the VP1 or VP2 protein of chicken anemia virus (CAV), the F / HN chimeric protein or the F, NP, P, M, HN, or L protein of Newcastle disease virus (NDV), the S1, S2, or M protein of infectious bronchitis virus (IBV), and the HA, NA, NP, or M protein of avian influenza virus (AIV), and further comprising a nucleotide sequence encoding a polyadenylation signal.

[0056] In one or more embodiments, the present invention provides recombinant DNA encoding the recombinant HVT genome of the present invention.

[0057] In one or more embodiments, the present invention provides an immunogenic composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0058] In one or more embodiments, the present invention provides a vaccine composition comprising a recombinant HVT of the present invention and further comprising a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0059] In one embodiment, the vaccine of the present invention further comprises an additional Marek's Disease Virus (MDV) selected from the group consisting of naturally attenuated MDV-1 strain Rispens (CVI-988) or Gallid herpesvirus 3 strain SB-1 virus. In one embodiment, the vaccine of the present invention provides that the additional MDV comprises a recombinant genome. In one embodiment, the vaccine of the present invention provides that the additional recombinant MDV genome comprises one or more nucleotide sequences encoding one or more heterologous antigens that are protective against one or more avian pathogens.

[0060] In one embodiment, the vaccine of the present invention provides for use in vaccinating birds against one or more diseases caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides for use in protecting birds against clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides for use in protecting birds against clinical symptoms caused by Marek's disease virus and clinical symptoms caused by one or more avian pathogens. In one or more embodiments, the vaccine of the present invention provides for one or more avian pathogens selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the vaccine of the present invention provides for the one or more avian pathogens to include Newcastle disease virus. In one embodiment, the vaccine of the present invention provides for the one or more avian pathogens to include infectious bursal disease virus (IBDV). In one embodiment, the vaccine of the present invention provides that the one or more avian pathogens include Newcastle disease virus and infectious bursal disease virus.

[0061] In one or more embodiments, the vaccine of the present invention is administered by spray, inhalation, or other means. The present invention provides for use in vaccinating birds, wherein the vaccine is administered by at least one or more doses of the vaccine via ovo administration, subcutaneous administration, intramuscular administration, oral administration, nasal administration, or a combination thereof. In one embodiment, the vaccine of the present invention provides that the vaccine is administered by in ovo administration. In one embodiment, the vaccine of the present invention provides that the in ovo administration is administered in embryonated eggs between about 16 and 22 days from emergence. In one or more embodiments, the vaccine of the present invention provides that the in ovo administration is administered in embryonated eggs at about 18 days from emergence. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises in ovo administration followed by spray administration. In one embodiment, the vaccine of the present invention provides that the administration of the vaccine comprises spray administration.

[0062] In one aspect, the present invention provides a method of vaccinating birds to treat or prevent Marek's disease and one or more avian diseases caused by one or more avian pathogens, the method comprising administering an effective amount of a vaccine composition of the present invention. In one embodiment, the method of the present invention provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV).

[0063] One aspect of the present invention provides a method for inducing an immune response in an avian animal against Marek's disease virus and one or more avian pathogens, the method comprising administering to the bird an effective amount of an immunogenic or vaccine composition of the present invention. In one embodiment, the method of the present invention provides that the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include infectious bursal disease virus (IBDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include Newcastle disease virus (NDV). In one embodiment, the method of the present invention provides that the one or more avian pathogens include infectious bursal disease virus (IBDV) and Newcastle disease virus (NDV). In one or more embodiments, the method provides that the administration is carried out by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, or nasal administration. In one embodiment, the method includes in ovo administration. In one embodiment, the method provides that the in ovo administration is carried out in embryonated eggs between about 16 and 22 days of development. In one or more embodiments, the method provides that the in ovo administration is carried out in embryonated eggs at about 18 days of development. In one or more embodiments, the method provides that the route of administration includes in ovo administration followed by spray administration. In one embodiment, the method provides that the route of administration includes spray administration. In one or more embodiments, the method provides that the bird is selected from the group consisting of chicken, turkey, goose, duck, pheasant, ostrich, pigeon, and quail. In one embodiment, the method provides that the bird includes a chicken.

[0064] General methodology: It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

[0065] Unless otherwise defined, scientific and technical terms used in connection with the present invention described herein shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the terminology and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are well known and commonly used in the art.

[0066] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transfection, well known to those skilled in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally carried out according to conventional methods well known in the art and as described, including but not limited to the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., MOLECULAR CLONING: LAB. MANUAL (3 rded.,Cold Spring Harbor Lab.Press,Cold Spring Harbor,N.Y.,2001)、およびAusubel et al.Current Protocols in Molecular Biology(New York:Greene Publishing Association JWiley Interscience),Oligonucleotide Synthesis(M.J.Gait,ed.,1984)、Methods in Molecular Biology,Humana Press、Cell Biology:A Laboratory Notebook(J.E.Cellis,ed.,1998)Academic Press;Animal Cell Culture(R.1.Freshney,ed.1987)、Introduction to Cell and Tissue Culture(1.P.Mather and P.E.Roberts,1998)Plenum Press、Cell and Tissue Culture:Laboratory Procedures(A.Doyle,J.B.Griffiths,and D.G.Newell,eds.,1993-1998)J.Wiley and Sons、Methods in Enzymology(Academic Press,Inc.)、Handbook of Experimental Immunology(D.M.Weir and C.C.Blackwell,eds.)、Gene Transfer Vectors for Mammalian Cells(J.M.Miller and M.P.Calos,eds.,1987)、Current Protocols in Molecular Biology(F.M.Ausubel et al.,eds.,1987)、PCR:The Polymerase Chain Reaction,(Mullis et al.,eds.,1994)、Current IRL Press, 1988-1989), Monoclonal antibodies: a practice See, for example, The I approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995), and Cancer: Principles and Practice of Oncology (YT DeVita et al., eds., J.B. Lippincott Company, 1993).

[0067] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about."

[0068] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application.

[0069] definition Before describing the present invention in detail, some terms used in the context of the present invention will be defined. In addition to these terms, other terms will be defined elsewhere in this specification as necessary. Unless expressly defined herein, technical terms used herein have their art-recognized meanings.

[0070] It should be noted that in this disclosure, the terms "comprises," "comprised," "comprising," "contains," "containing," "consisting of," "consisted of," "consisted essentially of," "includes," "included," and the like are defined in accordance with standard U.S. and international patent law practice.

[0071] The term "about" is used herein to indicate that a value includes the standard deviation of error for the device or method being used to determine the value. The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or where the alternatives are mutually exclusive; however, the present disclosure supports the definition that refers to alternatives only and "and / or." Unless used in conjunction with non-inclusive language in the claims or unless otherwise specifically noted, the words "a" and "an" refer to "one or more." Thus, the term "conferred by a transgene" encompasses, for example, one or more transgenes.

[0072] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. An amino acid analog refers to a compound that has the same basic chemical structure as a naturally occurring amino acid (i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium). Such analogs can also contain modified R groups (e.g., norleucine). or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

[0073] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may similarly be referred to by their commonly accepted single-letter codes. Macromolecular structures, such as polypeptide structures, can be described in terms of various levels of organization. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered three-dimensional structures within a polypeptide. These structures are commonly known as domains, such as enzymatic domains, extracellular domains, transmembrane domains, pore domains, or cytoplasmic tail domains. Domains are portions of polypeptides that form compact units of the polypeptide. Exemplary domains include domains with enzymatic activity. Domains may be composed of stretches of beta-sheet and smaller organized segments such as alpha helices. "Tertiary structure" refers to the complete three-dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three-dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropy terms are also known as energy terms.

[0074] As used herein, "antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. Recognized immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains can be classified as either kappa or lambda. Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes IgY, IgG, IgM, IgA, IgD, and IgE, respectively.

[0075] An exemplary immunoglobulin (antibody) structural unit may comprise a tetramer, each tetramer consisting of two identical pairs of polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each chain defines a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain and variable heavy chain refer to these light and heavy chains. Antibodies exist, for example, as intact immunoglobulins or as several well-characterized fragments produced by digestion with various peptidases. While various antibody fragments are defined in terms of digestion of intact antibodies, those skilled in the art will understand that such fragments can be synthesized de novo, either chemically or using recombinant DNA methodologies. Accordingly, the term "antibody," as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies or identified using other methods known in the art.

[0076] Many techniques known in the art can be used to prepare antibodies, e.g., recombinant antibodies, monoclonal antibodies, or polyclonal antibodies. Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells and used to produce recombinant monoclonal antibodies. Gene libraries encoding the heavy and light chains of monoclonal antibodies can also be used. Random combination of heavy and light chain gene products generates a large pool of antibodies with different antigen specificities. Techniques for producing single-chain or recombinant antibodies are found in the art and can be adapted to produce antibodies against the polypeptides of the present invention. Phage display technology can also be used to identify antibodies and heteromeric fragments that specifically bind to a selected antigen. Antibodies can also be bispecific. Monomeric (ie, capable of recognizing two different antigens) or heteroconjugates, such as two covalently joined antibodies, or immunotoxins, may also be made.

[0077] As used herein, "antigen" refers to a viral protein or polypeptide, such as a viral polypeptide, as well as a viral particle. In some embodiments, an antigen according to the present invention may also be a viral nucleic acid. An antigen is a molecule that can be recognized by the immune system and induce an immune response in a host organism. An antigen may include a whole organism, an attenuated organism, a dead organism, or a live organism, or a subunit or part of an organism. It may also be a piece or fragment of DNA, a polypeptide, an epitope, a hapten, or any combination thereof, capable of inducing an immune response.

[0078] As used herein, the term "bird" includes poultry, such as members of the order Galliformes, and more specifically, a class of birds of economic and / or agricultural interest, such as chickens, turkeys, geese, ducks, pheasants, ostriches, pigeons, and quails.

[0079] As used herein, a "biological sample" or "sample" may include blood and blood fractions, including, but not limited to, serum, plasma, platelets, or red blood cells, sputum, cloacal swabs, mucosa, tissues, cultured cells (including primary cultures), explants, and transformed cells, biological fluids, feces, and urine. Biological samples may also include sections of tissue, such as biopsy and autopsy samples, as well as frozen sections taken for histological purposes. Biological samples may be obtained from eukaryotic organisms, such as birds, including, but not limited to, birds from the order Galliformes, such as chickens, quail, and turkeys. Any tissue suitable for use in accordance with the present invention may be used, such as skin, brain, spinal cord, adrenal glands, pectoral muscle, lung, heart, liver, crop, proventriculus, gizzard, duodenum, small intestine, large intestine, cloaca, kidney, bursa of Fabricius, spleen, pancreas, adrenal glands, bone marrow, lumbosacral spinal cord, or blood.

[0080] The term "conservative amino acid substitution" refers to any amino acid substitution for a given amino acid residue, where the substituted residue is chemically so similar to that of the given residue that it does not result in a substantial loss of polypeptide function (e.g., enzymatic activity). Conservative amino acid substitutions are generally known in the art, and examples are described, for example, in U.S. Patent Nos. 6,790,639, 6,774,107, 6,194,167, or 5,350,576. In a preferred embodiment, the conservative amino acid substitution is any that occurs within one of the following six groups: • 1. Small aliphatic essentially nonpolar residues: Ala, Gly, Pro, Ser, and Thr; • 2. Large aliphatic non-polar residues: lie, Leu, and Val, Met; • 3. Polar negatively charged residues and their amides: Asp and Glu; • 4. Amides of polar negatively charged residues: Asn and Gin, His, • 5.Positively charged polar residues: Arg and Lys, His, and • 6. Large aromatic residues: Trp and Tyr, Phe.

[0081] In preferred embodiments, the conservative amino acid substitution is any one of the following listed as naturally occurring residue (conservative substitution) pairs: Ala (Ser); Arg (Lys); Asn (Gin; His); Asp (Glu); Gin (Asn); Glu (Asp); Gly (Pro); His (Asn; Gln); Lie (Leu; Val); Leu (Lie; Val); Lys (Arg; Gin; Glu); Met (Leu; Lie); Phe (Met; Leu; Tyr); Ser (Thr); Thr (Ser); Trp (Tyr); Tyr (Trp; Phe), and Val (Lie; Leu).

[0082] The phrase "functional effect" in the context of an assay for testing a compound that modulates the activity of a virus as described herein includes determining a parameter, such as a phenotypic or chemical effect, indirectly or directly under the influence of the virus. A "functional effect" may include in vitro, in vivo, and ex vivo activity, and may be measured by any means known to those skilled in the art, such as changes in the spectroscopic, shape, chromatographic, or solubility characteristics of a protein, measuring an inducible marker or transcriptional activation of a protein, measuring binding activity or binding assays, e.g., binding to an antibody, measuring changes in ligand or substrate binding activity, measuring viral replication, measuring cell surface marker expression, measuring changes in protein levels, measuring RNA stability, identifying downstream or reporter gene expression, e.g., via chemiluminescence, fluorescence, colorimetric reaction, antibody binding, and / or inducible markers.

[0083] The term "gene" refers to a component comprising viral DNA or RNA, cDNA, viral intron and exon DNA, artificial viral DNA polynucleotide, or other DNA encoding a viral peptide, polypeptide, protein, or RNA transcript, as well as genetic elements that may be adjacent to coding sequences involved in regulating expression, such as promoter regions, 5' leader regions, 3' untranslated regions, which may be present as a native gene or a transgene in the viral genome. A gene or fragment thereof can be subjected to polynucleotide sequencing methods to determine the order of nucleotides that comprise the gene.

[0084] The term "herpesvirus of turkeys (HVT)" is defined as a non-pathogenic virus of domestic turkeys and is classified as the third serotype within the Marek's disease virus group of antigenically and genetically related lymphophilic avian herpesviruses.

[0085] The term "heterologous," when used in reference to a portion of a nucleic acid, indicates that the nucleic acid contains two or more sequences that are not found in the same relationship to each other in nature. For example, a nucleic acid is typically produced recombinantly, with two or more sequences from unrelated genes arranged to create a new functional nucleic acid, such as a promoter from one source and a coding region from another source. Similarly, a heterologous protein indicates that the protein contains two or more sequences that are not found in the same relationship to each other in nature (e.g., a fusion protein). Heterologous can also refer to a viral sequence, such as a gene or transgene, or a portion thereof, inserted into a viral genome in which it is not typically found, or a gene introduced into an organism in which it is not typically found.

[0086] The term "host cell" refers to any cell of any organism that can be selected, modified, transformed, grown, or used or manipulated in any way to produce a substance by the cell, for example, by cellular expression of a gene, DNA or RNA sequence, protein, or enzyme. A host cell is intended to include any individual cell or cell culture that can be or has been a recipient for a vector or for incorporation of an exogenous nucleic acid molecule, polynucleotide, and / or protein. It is also intended to include the progeny of a single cell. The progeny may not necessarily be completely identical (in morphology, or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. The cell may be prokaryotic or eukaryotic.

[0087] As used herein, the terms "host," "subject," "patient," or "organism" may include animals, particularly birds, especially poultry. For veterinary applications, birds are from the order Galliformes, which includes chickens, quail, and turkeys. The term "living host" refers to the host described above or another living organism. The term may also refer to the entire host or organism, not simply a part (e.g., a brain or other organ) excised from a living host. These terms also include individuals in all stages of development, including embryonic and fetal stages.

[0088] The term "identical" or "percent identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or contain a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, as described below, or by manual alignment and visual inspection (see, for example, the NCBI website, such as at ncbi.nlm.nih.gov / BLAST / ). Such sequences are then referred to as "substantially identical." This definition also refers to or applies to the complementarity of a particular sequence. This definition also includes sequences that have deletions, additions, and / or substitutions.

[0089] For sequence comparison, one sequence usually serves as reference sequence, and other sequences are compared to it.When using sequence comparison algorithm, reference sequence and comparison sequence can be input into computer, and if desired, sequence algorithm program parameters are selected.Then, based on selected parameters, generate the sequence identity percentage for comparison sequence with reference sequence.An example of an algorithm that can be suitable for determining sequence identity percentage and sequence similarity is BLAST and BLAST 2.0 algorithm, which are respectively described in Altschul et al., (Nuc Acids Res 25:3389-3402,1977) and Altschul et al., (J Mol Biol 215:403-410,1990).BLAST and BLAST 2.0 are well known in the art and can be used to determine the sequence identity percentage of any nucleic acid or protein, such as those described herein.

[0090] As used herein, "immunogenic composition" or "pharmaceutical composition" or "vaccine" is meant to encompass a composition comprising an antigen suitable for administration to a subject, e.g., an avian subject. The composition generally elicits an immune response in the subject. The immune response can include a T cell response, a B cell response, or both a T cell and a B cell response. The composition can serve to sensitize the subject by presenting the antigen in association with MHC molecules on the cell surface. In addition, antigen-specific T lymphocytes or antibodies can be generated to allow for future protection of the immunized host. An "immunogenic composition" may contain a live, attenuated, or killed / inactivated vaccine comprising a whole organism or immunogenic portions thereof that induces either a cell-mediated (T cell) or antibody-mediated (B cell) immune response, or both, and may protect the animal from one or more symptoms associated with infection by the microorganism or from death resulting from infection by the microorganism. Generally, an "immunogenic composition" is sterile and preferably free of contaminants that may elicit an undesired response in a subject (e.g., the compound(s) in the immunogenic composition are of pharmaceutical grade). Immunogenic compositions can be designed for administration to a subject in need thereof via several different routes of administration, including in ovo, oral, intravenous, buccal, rectal, parenteral, intraperitoneal, intradermal, intratracheal, intramuscular, subcutaneous, inhalation, etc.

[0091] As used herein, the term "immunogenic" protein or peptide includes a polypeptide that is immunologically active, in the sense that, upon administration to a host, it is capable of eliciting a humoral and / or cellular immune response directed against the protein. Preferably, a protein fragment is such that it has substantially the same immunological activity as the full-length protein. Thus, a protein fragment of the present invention comprises, consists essentially of, or consists of at least one epitope or antigenic determinant. "Immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of a protein, an analog thereof, or an immunogenic fragment thereof. "Immunogenic fragment" refers to a fragment of a protein that contains one or more epitopes and thus elicits the aforementioned immune response.

[0092] The term "immunogenic protein or peptide" further contemplates deletions, additions, and substitutions to the sequence, so long as the polypeptide functions to generate an immune response as defined herein. The term "conservative mutation" refers to the replacement of an amino acid residue with another biologically similar residue, or the replacement of a nucleotide in a nucleic acid sequence so that the encoded amino acid residue remains unchanged or is replaced with another biologically similar residue. In this regard, particularly preferred substitutions are generally conservative in nature, i.e., substitutions that occur within a family of amino acids. For example, amino acids are generally divided into four families: (1) acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) nonpolar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative mutations include replacing one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, with another hydrophobic residue, or replacing one polar residue with another polar residue, such as replacing arginine with lysine, replacing glutamic acid with aspartic acid, or replacing glutamine with asparagine, or similar conservative replacement of amino acids with structurally related amino acids that do not significantly affect biological activity.Therefore, a protein that has substantially the same amino acid sequence as a reference molecule but has minor amino acid substitutions that do not substantially affect the immunogenicity of the protein is within the definition of a reference polypeptide.All polypeptides produced by these modifications are included herein.The term "conservative mutation" also includes using a substituted amino acid instead of an unsubstituted parent amino acid, provided that the antibody listed in the substituted polypeptide also immunoreacts with the unsubstituted polypeptide.

[0093] As used herein, an "immunologically effective amount" refers to an amount of antigen or vaccine sufficient to elicit an immune response, either a cellular (T cell) or humoral (B cell or antibody) response, as measured by standard assays known to those skilled in the art. For example, with respect to the present invention, an "immunologically effective amount" is the minimum protective dose (titer). The effectiveness of an antigen as an immunogen can be measured by proliferation assays, by cytolytic assays such as a chromium release assay, which measures the ability of T cells to lyse their specific target cells, or by measuring the level of B cell activity by measuring the level of circulating antibodies specific to the antigen in serum, or by any other assay known and used by those skilled in the art. Furthermore, the protective level of an immune response can be measured by challenge testing an immunized host with injected antigen. For example, if the antigen to which an immune response is desired is a virus or tumor cell, the level of protection induced by an "immunologically effective amount" of the antigen is measured by detecting the survival or mortality of animals after virus or tumor cell challenge.

[0094] Determining what an immunologically effective amount of a vaccine according to the present invention is can be done, for example, by monitoring the immunological response after vaccination or after challenge infection (e.g., by re-isolation of the pathogen). The administration of a vaccine according to the present invention to a target organism can be achieved by monitoring the response of the target organism, or by monitoring clinical signs or serological parameters of the target disease and comparing these with the responses seen in mock-vaccinated animals. The dosing scheme for administering the vaccine of the present invention to the target organism can be a single dose or multiple doses, given simultaneously or sequentially, in a manner compatible with the formulation of the vaccine and in such amounts as will be immunologically effective.

[0095] The terms "inhibitor," "activator," and "modulator" of viral nucleic acid and polypeptide sequences are used to refer to activating, inhibiting, or modulating molecules identified using in vitro and in vivo assays of viral nucleic acid and polypeptide sequences. Inhibitors are compounds that can bind to the virus, partially or completely block its activity, reduce, prevent, delay activation, inactivate, desensitize, or downregulate viral activity or expression. Activators refer to compounds that increase, release, activate, promote, enhance activation, sensitize, antagonize, or upregulate viral activity. Inhibitors, activators, or modulators also include genetically modified versions of the viruses described herein, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, substrates, antagonists, agonists, antibodies, peptides, cyclic peptides, nucleic acids, antisense molecules, ribozymes, small chemical molecules, and the like. Assays for inhibitors and activators include, for example, expressing a virus of the invention in vitro, intracellularly, or at a cell membrane, applying a putative regulatory compound, and then determining the functional effect on activity, as described herein.

[0096] To determine the degree of inhibition, a test sample or assay containing a virus of the invention treated with a potential activator, inhibitor, or modulator may be compared to a control sample lacking the inhibitor, activator, or modulator. The control sample to which the test sample or assay is compared may be assigned a relative protein activity value of 100%. Viral inhibition is achieved when the activity value of the test sample compared to the control sample is less than about 80%, including about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, and about 0%.

[0097] An intergenic locus, as used herein, is defined as a region of DNA sequence located between genes, including untranslated regions, 5' and 3' flanking regions, introns, etc. An intergenic region is a portion of non-coding DNA that may contain gene control elements such as promoters and enhancers.

[0098] The term "isolated" refers to a material that is substantially separated from or enriched relative to other materials with which it naturally occurs. An isolated material is typically at least about 80% by weight, at least 90% by weight pure, at least 98% by weight pure, or at least about 99% by weight pure.

[0099] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include: 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, or haptens, as well as proteins that can be made detectable by, for example, incorporating a radioactive label into the peptide or that can be used to detect antibodies specifically reactive with the peptide.

[0100] As used herein, "Marek's disease virus" or "MDV" refers to Marek's disease virus, including herpesvirus of turkeys (HVT), as described herein. The term "Marek's disease virus" refers to any alphaherpesvirus of the genus Marek's disease virus. In a specific embodiment, the present invention relates to Marek's disease virus, its genetic components, genes, and proteins produced thereby. As used herein, such viruses can include the genetic components of the virus, i.e., its genome and transcripts, proteins encoded by the genome (including structural and nonstructural proteins), and functional or nonfunctional virus particles. Polynucleotide and polypeptide sequences encoding such viruses are well known in the art and would be readily found by one of ordinary skill in the art.

[0101] The terms "mutant" and "mutation" refer to any detectable change in genetic material (e.g., DNA), or any process, mechanism, or result of such a change. This includes genetic mutations in which the structure of a gene (e.g., DNA sequence) is altered, any gene or DNA resulting from any mutational process, and any expression product (e.g., protein or enzyme) expressed by a modified gene or DNA sequence. The term "variant" can also be used to refer to modified or altered genes, DNA sequences, enzymes, cells, etc., i.e., any kind of mutant.

[0102] As used herein, the term "nucleic acid" refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. A "nucleic acid" may also optionally contain non-naturally occurring or altered nucleotide bases that allow for correct readthrough by a polymerase and do not reduce the expression of the polypeptide encoded by the nucleic acid. The term "nucleotide sequence" or "nucleic acid sequence" refers to both the sense and antisense strands of a nucleic acid, either as individual single strands or double strands. The term "ribonucleic acid" (RNA) includes RNAi (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (microRNA), tRNA (transfer RNA, whether charged or uncharged with corresponding acylated amino acids), and cRNA (complementary RNA). The terms "nucleic acid segment," "nucleotide sequence segment," or more generally, "segment" will be understood by those skilled in the art as a functional term that includes genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operon sequences, and smaller engineered nucleotide sequences that express, or can be adapted to express, proteins, polypeptides, or peptides. The nomenclature used herein is that required by Title 37 of the United States Code of Federal Regulations §1.822 and set forth in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0103] The term "operably linked" is used herein to refer to the arrangement of flanking sequences, such that the flanking sequences are configured or assembled to perform their normal function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription, and / or translation of the coding sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. A flanking sequence need not be contiguous with the coding sequence so long as it functions properly. Thus, for example, intervening untranslated but transcribed sequences can be present between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0104] The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is physiologically compatible for administration to a subject. Pharmaceutically acceptable carriers include, but are not limited to: These include, but are not limited to, buffers, excipients, stabilizers, adjuvants, preservatives, diluents, aqueous or non-aqueous vehicles, and other additives. Additionally, the term generally refers to components of immunogenic compositions or vaccines approved by federal, state, or other regulatory agencies, or listed in the United States Pharmacopoeia or other pharmacopeias generally recognized for use in both humans and non-human animals. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. If desired, the compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. The compositions can be formulated as suppositories with traditional binders and carriers such as triglycerides. Oral formulations can include standard pharmaceutical-grade carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should be compatible with the mode of administration.

[0105] As used herein, "poultry" refers to domestic or commercial birds raised for the eggs they produce, as well as their meat and feathers. In some embodiments, poultry may include birds from the order Galliformes, including chickens, quail, and turkeys, and may also include geese, ducks, swans, guinea fowl, pigeons, and the like.

[0106] The polynucleotides described herein can be complementary to all or a portion of a viral gene sequence, including promoters, introns, coding sequences, exons, 5' untranslated regions, and 3' untranslated regions.

[0107] A particular nucleic acid sequence may also encompass "splice variants." Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any proteins encoded by splice variants of that nucleic acid. Splice variants are the product of alternative splicing of a gene. After transcription, an initial nucleic acid transcript may be spliced ​​such that different (alternative) nucleic acid splice products encode different polypeptides. Mechanisms for producing splice variants vary but include alternative splicing of exons. Alternative polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition.

[0108] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0109] "Polyvalent vaccine," "Combination or combo vaccine," and "Multivalent vaccine" The term "multivalent vaccine" is used interchangeably to refer to a vaccine containing more than one antigen. A multivalent vaccine may contain two, three, four, or more antigens. A multivalent vaccine may contain a recombinant viral vector, an active virus, an attenuated virus, or a killed wild-type virus, or a mixture of a recombinant viral vector and a wild-type virus in an active, attenuated, or killed form.

[0110] As used herein, "promoter" refers to a DNA sequence that defines where transcription of a gene by RNA polymerase begins. Promoters are typically located upstream of the transcription start site. Promoters can also contain distal enhancer or repressor elements, which can be located thousands of nucleotides away from the transcription start site. Promoters define the direction of transcription and indicate which DNA strand is transcribed. Promoters can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. Promoters can constitutively or differentially regulate the expression of gene components with respect to the cell, tissue, or organ in which expression occurs, or with respect to the developmental stage in which expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers. Representative examples of promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, RSV-LTR promoter, CMV IE promoter, human CMV promoter, mouse CMV promoter, Pec promoter, β-chicken actin promoter, guinea pig CMV promoter, pseudorabies virus promoter, glycoprotein X promoter, herpes simplex virus-1 promoter, Marek's disease virus promoter, and SV40 promoter.

[0111] As used herein, the term "prophylactically treat" or "prophylactically treating" refers to the complete or partial prevention of a disease or its symptoms, and / or may be therapeutic in terms of a partial or complete cure of the disease and / or adverse effects resulting from the disease.

[0112] The term "recombinant," when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise aberrantly expressed, under-expressed, or not expressed at all. In some embodiments, a recombinant sequence can also include a nucleic acid, protein, or recombinant genome (e.g., a viral genome). The recombinant viral vectors described herein can contain a transgene operably linked to a heterologous promoter for transcription of the transgene.

[0113] The phrase "stringent hybridization conditions" refers to conditions under which a probe hybridizes to its target sequence, typically in a complex mixture of nucleic acids, but does not hybridize to other sequences. Stringent conditions may be sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Stringent conditions can be achieved by adding destabilizing agents such as formamide.

[0114] Suitable stringency conditions that promote DNA hybridization are well known to those of skill in the art and may include, for example, 6x sodium chloride / sodium citrate (SSC) at about 45° C., followed by a 2x SSC wash at 50° C. Salt concentrations in the wash steps range from low stringency, approximately 2x SSC at about 50° C., to high stringency, approximately 0.2x SSC at 50° C. The stringency of the wash step can be selected from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Temperature and / or salt conditions can be varied as appropriate for optimal results. In accordance with the present invention, a nucleic acid can exhibit at least about 80% to about 100% sequence identity, e.g., at least about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity, to one or more nucleic acid molecules described herein.

[0115] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This occurs, for example, when copies of nucleic acids are created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.

[0116] As used herein, the terms "therapeutically effective amount," "effective amount," or "therapeutically effective dose" refer to a dose that produces the effect for which it is administered. Such a dose or amount may also refer to the amount of an administered pharmaceutical embodiment that alleviates to some extent one or more of the symptoms of the disease, i.e., infection being treated, and / or an amount that prevents to some extent one or more of the symptoms of the disease (i.e., infection) that the treated host develops or is at risk of developing. The exact dose will vary depending on the purpose of the treatment, and one of skill in the art will be able to determine such a dose using techniques known in the art.

[0117] As used herein, "transgene" refers to a segment of DNA containing heterologous coding sequences or other genetic material for transfer from one organism to another. For example, in certain embodiments, a transgene of the present invention may include an antigen-coding sequence, such as a viral gene, or a sequence encoding a viral protein.

[0118] As used herein, the terms "treatment," "treating," and "treating" are defined as affecting a disease, disorder, or condition with an agent to reduce or ameliorate the pharmacological and / or physiological effects of the disease, disorder, or condition, and / or its symptoms. "Treatment," as used herein, encompasses any treatment of a disease in a subject or host (e.g., an animal for veterinary purposes), including (a) reducing the risk of disease development in a subject determined to be predisposed to the disease but not yet diagnosed as infected with the disease, (b) preventing the onset of the disease, and (c) alleviating the disease, i.e., causing the reversal of the disease and / or alleviating one or more disease symptoms. "Treatment" is also meant to encompass the delivery of an inhibitor to provide a pharmacological effect even in the absence of a disease or condition. For example, "treatment" encompasses the delivery of a disease or pathogen inhibitor that results in an enhanced or desired effect in a subject (e.g., a reduction in pathogen load, a reduction in disease symptoms, etc.).

[0119] The term "unit dosage form," as used herein, refers to a physically discrete unit suitable as a unitary dosage for an animal subject, each unit containing a predetermined amount of a compound (e.g., an antiviral compound as described herein) calculated in an amount sufficient to produce a desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage form depend on the particular compound used, the route and frequency of administration, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[0120] The terms "vaccine" or "vaccine composition" are used interchangeably herein to refer to a pharmaceutical composition comprising at least one immunogenic composition of the present invention that induces an immune response in a subject. A vaccine or vaccine composition can protect a subject from disease or potential death and contains one or more additional components that enhance the immune activity of the active component. The compositions of the present invention that induce a protective immune response may or may not contain a heterologous antigen encoding gene inserted into the HVT genome at the intergenic region UL35 / 36. In some embodiments, the compositions of the present invention comprise a recombinant HVT virus having one or more heterologous antigen encoding genes inserted into the HVT genome at UL35 / 36 and one or more antigen encoding genes inserted into the HVT genome at UL55. In some embodiments, the antigen-encoding genes are antigens derived from poultry pathogens such as Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, avian influenza virus, infectious laryngotracheitis virus, and / or chicken anemia virus. In some embodiments, the recombinant HVT is combined with another recombinant Marek's disease virus vaccine that induces a protective immune response in poultry. The vaccines or vaccine compositions of the present invention may additionally contain additional components typical of vaccines or vaccine compositions, including, for example, adjuvants or immunomodulators. The vaccine may contain one of the above elements, or may contain more than one simultaneously.

[0121] The vaccine of the present invention may further comprise a suitable pharmaceutical carrier. The term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents in the host. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a pharmaceutical composition is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. If desired, the compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Depending on the method of administration, the compositions can be formulated with traditional binders and carriers, such as triglycerides. Certain formulations can contain standard pharmaceutical-grade carriers, such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. The formulation should be compatible with the mode of administration. The appropriate carrier will be apparent to those skilled in the art and will, in large part, depend on the route of administration. Additional components that may be present in the present invention are adjuvants, preservatives, surfactants, chemical stabilizers, suspending agents or dispersing agents. Typically, stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target subject.

[0122] A "variant" peptide, as used herein, refers to a peptide that differs in amino acid sequence from a "parent" vaccine peptide amino acid sequence by the addition, deletion, and / or substitution of one or more amino acid residues in the parent peptide sequence, but retains at least one desired activity of the parent vaccine peptide. For example, a variant can contain at least one, e.g., about 1 to about 10, preferably about 2 to about 5 substitutions, in one or more amino acid sequences of a peptide used as part of a vaccine of the invention. Typically, a variant will have at least 50% amino acid sequence identity with the parent amino acid sequence, preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 100%. or have an amino acid sequence with at least 95% sequence identity. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical to the parent peptide residues after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Neither N-terminal, C-terminal, nor internal extensions, deletions, nor insertions into the peptide sequence should be construed as affecting sequence identity or homology. Variants retain the ability to elicit an immune response and preferably have a desired activity that is superior to the activity of the parent peptide.

[0123] Variant peptides may be fully functional or may lack function in one or more activities.Fully functional variants typically contain only conservative mutations or mutations in non-critical residues or non-critical regions.Functional variants may also contain similar amino acid substitutions that do not change or only slightly change function.Alternatively, such substitutions may have a positive or negative effect on function to some extent.Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions, or deletions in critical residues or critical regions.

[0124] In addition, polypeptides often contain amino acids other than the 20 "naturally occurring" amino acids. Moreover, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques which are well known in the art. Known modifications include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Such modifications are well known to those of skill in the art and are well described in the scientific literature. Some particularly common modifications, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation, are described in the most basic texts, e.g., Proteins - Structure and Molecular Properties (2nd ed., TECreighton, W.H. Freeman & Co., NY, 1993). Many detailed reviews are available on this subject, e.g., Wold, Posttranslational Covalent Modification of proteins, 1-12 (Johnson, ed., Academic Press, NY, 1983), Seifter et al. Meth. Enzymol. 626-46 (1990), and Rattan et al. 663 Ann. NY Acad. Sci. 48-62 (1992).

[0125] Thus, the peptides of the present invention also encompass derivatives or analogs in which the substituted amino acid residue is not encoded by the genetic code. Similarly, additions and substitutions in the amino acid sequence, as well as the mutations and modifications described herein, may be equally applicable to the amino acid sequence of an antigen and / or its epitope or peptide, and are therefore encompassed by the present invention.

[0126] A "variant" nucleic acid, as used herein, refers to a molecule that differs in sequence from a "parent" nucleic acid. Deviations in polynucleotide sequence can include deletions, substitutions, or additions of one or more nucleotides. Each of these changes may occur alone, or in combination, one or more times in a given sequence.

[0127] Just as a polypeptide can contain conservative amino acid substitutions, the polynucleotide can contain conservative codon substitutions. A codon substitution is considered conservative if, when expressed, it produces a conservative amino acid substitution as described above. Degenerate codon substitutions that do not result in amino acid substitutions are also useful in the polynucleotides of the present invention. Thus, for example, a polynucleotide encoding a selected polypeptide useful in one embodiment of the present invention can be mutated by degenerate codon substitutions to approximate the codon usage exhibited by an expression host cell transformed therewith, or to otherwise improve its expression.

[0128] As used herein, "vector" refers to a construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. A vector, as described herein, has an expression control sequence, which refers to a nucleic acid sequence that directs transcription of a nucleic acid. An expression control sequence can be a promoter, such as a constitutive promoter or an inducible promoter, or an enhancer. An expression control sequence is "operably linked" to the nucleic acid sequence to be transcribed. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0129] As used herein, "viral protein" or "viral polypeptide" refers to a protein encoded by a virus described herein, including structural and nonstructural proteins. Such proteins can include naturally occurring or non-naturally occurring viral proteins from MDV, NDV, and / or IBDV, including VP2, F, and / or HN, NP, P, M, or L proteins. Such proteins can also include any of naturally occurring or non-naturally occurring viral proteins from ILTV, e.g., gB, gC, gD, gE, gH, gI, or gL, S1, S2, or M proteins from infectious bronchitis virus (IBV), VP1 or VP2 proteins of chicken anemia virus (CAV), and / or HA, NA, NP, or M proteins of avian influenza virus (AIV).

[0130] In accordance with the present invention, the recombinant viral vectors described herein may allow poultry to be protected from two or more different viral pathogens by providing a recombinant viral vector that expresses genes from the viral pathogens. In some embodiments, the recombinant viral vectors of the present invention may be provided to poultry in an immunogenic composition as described herein. Suitable recombinant viral vectors for use with the recombinant viral vectors described herein include: Genes from any viral pathogen may be used. For example, in some embodiments, recombinant viral vectors may express genes from Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), avian influenza virus (AIV), chicken anemia virus (CAV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), and the like.

[0131] In accordance with the present invention, transgenes that confer protection or resistance to a particular virus or viruses may be inserted into the viral genome at specific locations. For example, in some embodiments, the transgenes described herein are inserted into the HVT unique long region of the genome. The transgene may be inserted into the viral genome in the intergenic region flanked by UL35 / UL36. In another embodiment of the invention, the transgene is described herein as including one or more heterologous genes inserted into the viral genome in the intergenic region flanked by HVT UL35 / UL36 of the HVT genome, in addition to the second site used, where the one or more heterologous genes are inserted into the UL55 site in the HVT genome. In other embodiments, more than one transgene may be inserted into one or both of these regions.

[0132] In some embodiments, a recombinant viral vector may express multiple genes from a single viral species, or may express genes from more than one viral species to confer resistance to multiple viruses. For example, in one embodiment, the present invention provides a recombinant viral vector that comprises an HVT genome and at least one transgene from a different viral pathogen, thereby providing protection against Marek's disease and at least one other viral disease in birds, such as poultry. For example, in one embodiment, a recombinant viral vector of the present invention may provide protection against MDV and NDV, or may provide protection against MDV and IBDV, or may provide protection against MDV, NDV, and IBDV in poultry.

[0133] Viral antigens for expression in poultry by the recombinant viral vectors of the present invention can be encoded by viral genes, such as those described herein. In this regard, those skilled in the art will understand that it may not be necessary to integrate the entirety of a particular viral gene to achieve the desired viral resistance. Rather, portions of such genes may be used. It may be desirable to select specific portions of a desired gene specific to any given targeted virus or viruses. Regardless of the length of the protein, optimization of the desired viral protein or the sequence encoding such a protein can be readily performed using methodologies known in the art suitable for use with the present invention. Those skilled in the art will understand that modifications can be made to a viral gene or genes, or the protein encoded thereby, to increase the activity of the viral protein when introduced into a subject. Modifications made to a viral gene or protein can increase or decrease the host's response to a particular virus.

[0134] In a specific embodiment, a recombinant Marek's disease virus or recombinant viral vector of the invention may have a transgene encoding an IBDV viral protein or gene product, such as an IBDV VP2 protein or gene product. In another embodiment, such a recombinant virus or viral vector may have a transgene encoding an NDV viral protein or gene product, such as an NDV F or HN protein or gene product. In another embodiment, such a recombinant virus or viral vector may have a transgene encoding an avian influenza virus (AIV) viral protein or gene product, such as an AIV HA or N protein or gene product. In another embodiment, such a recombinant virus or viral vector may have a transgene encoding an infectious bronchitis virus (IBV) viral protein or gene product, such as an IBV S1 or The transgenes of the invention may have transgenes encoding an S1 or S2 protein or gene product. Transgenes of the invention may have more than one gene, including gene fusion proteins or gene products, such as NDV F-HN fusion proteins, chimeras, or gene products. In some embodiments, the complete coding sequence of such genes may be used such that a full-length or fully functional protein or polypeptide is produced. Alternatively, portions or fragments of viral proteins or polypeptides may be sufficient to provide protection from or resistance to a particular virus or multiple viruses.

[0135] In certain embodiments, the recombinant Marek's disease virus or recombinant viral vector of the present invention may carry a transgene encoding an immunomodulator, such as a cytokine protein or gene product. According to the present invention, the cytokine may be an interleukin (IL), including, but not limited to, IL2, IL6, IL7, IL8, IL12, IL18, etc. Such a transgene encoding a cytokine may be inserted into one or both genomic sites described herein. In some embodiments, the encoding transgene may be inserted into one site described herein, and a transgene encoding a viral protein may be inserted into the other site. Other immunomodulators, such as interferons, chemokines, glucans, and granulocyte colony-stimulating factors, may also be useful, and oligodeoxynucleotides may also be used in accordance with the present invention.

[0136] Isolation of viral genes or proteins In embodiments of the present invention, the viral genes described herein can be isolated using nucleic acid probes and / or oligonucleotides under stringent hybridization conditions, PCR or microarrays, DNA library screening, or any other method known in the art. Those skilled in the art will readily understand how to isolate viral genes or proteins for use in accordance with the present invention. Alternatively, expression libraries can be used to clone viruses, their polymorphic variants, orthologs, or alleles by immunologically detecting homologs using antisera or purified antibodies directed against viruses from other species or parts thereof.

[0137] The method of making and screening cDNA library is well known in the art.For example, to make the cDNA library for cloning the viral gene expressed by genome, mRNA can be reverse transcribed into cDNA using reverse transcriptase.Then, cDNA can be ligated into a vector such as recombinant vector, and introduced into host cell or organism for propagation, screening and cloning.

[0138] In the case of a genomic library, DNA may be extracted from the desired tissue, digested using biological enzymes, or mechanically sheared. The resulting DNA fragments may then be isolated from undesired DNA fragments and constructed into appropriate vectors, which may then be packaged in vitro. Recombinant vectors may be analyzed by any method known in the art.

[0139] Methods such as polymerase chain reaction (PCR and RT-PCR) and ligase chain reaction (LCR) may be used to amplify nucleic acid sequences directly from mRNA, from cDNA, or from genomic or cDNA libraries. Degenerate oligonucleotides can be designed to amplify homologs using the sequences provided herein. Restriction endonuclease sites may be incorporated into primers. Polymerase chain reaction or other in vitro amplification methods are also useful for, for example, cloning nucleic acid sequences encoding expressed proteins, generating nucleic acids to be used as probes to detect the presence of targeted diseases (e.g., MDV, NDV, and / or IBDV), and for amplifying nucleic acid sequences. These may be useful for encoding mRNA in biological samples, for sequence determination, or for other purposes. Genes amplified by PCR can be purified from agarose and cloned into an appropriate vector.

[0140] Viral gene expression can also be analyzed by techniques known in the art, such as reverse transcription and amplification of mRNA, isolation of total RNA or polyA RNA, Northern blotting, dot blotting, in situ hybridization, RNase protection, high-density polynucleotide array technology, and the like.

[0141] The nucleic acid encoding the viral genome or protein can be used to identify the viral genes, orthologues, alleles, variants thereof, and polymorphic variants of the present invention using high-density oligonucleotide array technology (e.g., GeneChip™). The selected gene can be cloned into an intermediate vector before being transformed into a prokaryotic or eukaryotic cell for replication and / or expression. These intermediate vectors can be prokaryotic vectors, such as plasmids, or shuttle vectors.

[0142] Nucleic acid modification Any number of methods known to those skilled in the art can be used to isolate and manipulate DNA molecules. For example, polymerase chain reaction (PCR) technology can be used to amplify a specific starting DNA molecule and / or produce variants of the starting DNA molecule. DNA molecules or fragments thereof can also be obtained by any technique known in the art, including directly synthesizing fragments by chemical means. Thus, all or part of the nucleic acids described herein can be synthesized.

[0143] As used herein, the term "complementary nucleic acids" refers to two nucleic acid molecules that can specifically hybridize with each other, forming a non-parallel double-stranded nucleic acid structure. In this regard, a nucleic acid molecule is said to be the complement of another nucleic acid molecule if they exhibit complete complementarity. Two molecules are said to be "minimally complementary" if they can hybridize with each other with sufficient stability to allow them to remain annealed to each other under at least conventional low stringency conditions. Similarly, molecules are said to be complementary if they can hybridize with each other with sufficient stability to allow them to remain annealed to each other under conventional high and low stringency conditions. Conventional stringency conditions are described by Sambrook, et al. (1989) and Haymes et al. (1985).

[0144] Departures from perfect complementarity are permissible as long as the molecules retain the ability to form double-stranded structures. Thus, for a nucleic acid molecule or fragment of a nucleic acid molecule to function as a primer or probe, such molecule or fragment need only be sufficiently complementary in sequence so as to form a stable double-stranded structure in the particular solvent and salt concentration used.

[0145] As used herein, the terms "sequence identity," "sequence similarity," or "homology" are used to describe the sequence relationship between two or more nucleotide sequences. The percentage of "sequence identity" between two sequences is determined by comparing two optimally aligned sequences over a specific number of nucleotides, and portions of the sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence. Two sequences are said to be identical if the nucleotides are the same at all positions. A nucleotide sequence when viewed in the 5' to 3' direction is considered identical if the first nucleotide sequence exhibits complete complementarity with the second or reference sequence when viewed in the 3' to 5' direction. A nucleotide sequence that is complementary to a reference nucleotide sequence is said to be the "complement" of a second nucleotide sequence observed in the 3' to 5' direction, or is complementary to a second nucleotide sequence observed in the 3' to 5' direction. As used herein, a nucleic acid sequence molecule is said to exhibit "perfect complementarity" when every nucleotide in one of the 5' to 3' positions of a sequence read is complementary to every nucleotide in the other sequence when read from 3' to 5'. A nucleotide sequence that is complementary to a reference nucleotide sequence exhibits a sequence identical to the reverse complement sequence of the reference nucleotide sequence.

[0146] Recombinant Vectors and Host Cells Recombinant DNA vectors can be, for example, linear or circular plasmids. A vector system can be a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of a host cell. The recombinant vectors described herein can be, for example, expression vectors to enable production of a desired protein in a host cell, such as a bacterial cell. The nucleic acid molecules described herein, or their complements or fragments, can be inserted into a vector under the control of a suitable promoter that functions in one or more microbial hosts and drives expression of the linked coding sequence or other DNA sequence. Many vectors are available and known in the art for this purpose, and the selection of an appropriate vector depends on the nucleic acid to be inserted into the vector and the size of the host cell to be transformed with the vector. Each vector can contain various components depending on its function (e.g., DNA amplification or DNA expression) and the particular host cell with which it is compatible. Vector components for bacterial transformation generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more selectable marker genes, and an inducible promoter that enables expression of exogenous DNA.

[0147] As used herein, "recombinant Marek's Disease Virus" or "recombinant HVT" or "recombinant virus" refers to an infectious virus or virus particle that has been genetically modified by incorporation into the viral genome of one or more heterologous nucleic acid sequences (i.e., DNA encoding viral genes or fragments or portions thereof that are not identical to the nucleic acid sequences of genes naturally occurring in the virus). Upon infection of a cell with the recombinant Marek's Disease Virus, the recombinant virus expresses the heterologous gene in the form of a heterologous polypeptide.

[0148] As used herein, "recombinant viral vector" or "viral vector" refers to a recombinant construct that is inserted into a virus for introduction into a host cell. Such vectors of the present invention can be derived from any HVT strain. Where appropriate, viral gene or protein coding sequences can be incorporated into such recombinant viral vectors described herein for introduction into chickens or other poultry for protection against one or more viral diseases.

[0149] As used herein, "insertion site" refers to a region in the viral genome into which a transgene or exogenous DNA is inserted. The insertion site of the present invention may be an intergenic region. The intergenic region of the present invention may be flanked by HVT UL35 and HVT UL36 in the unique long region of the genome. In some embodiments of the present invention, one or more heterologous nucleotides encoding antigens may also be inserted into the region defined by the UL55 locus of the HVT genome. In some embodiments, the insertion site of the present invention may include all or part of the flanking genes on either side of the intergenic region. Insertion of one or more transgenes into one of these regions allows for the production of a recombinant viral vector, which can then be introduced into chickens or other poultry for protection against one or more diseases.

[0150] As used herein, the term "operably linked," when used in reference to a regulatory sequence and a nucleotide sequence, means that the regulatory sequence is operably linked to the linked structural nucleotide sequence. The term "regulatory sequence," "regulatory element," or "control element" refers to a nucleotide sequence located upstream (5' sequences), within, or downstream (3' sequences) of a structural nucleotide sequence. Such sequences influence the timing and level or amount of transcription, RNA processing or stability, or translation of the associated structural nucleotide sequence. Regulatory sequences can include, but are not limited to, promoters, leader sequences, introns, enhancers, stem-loop structures, repressor binding sequences, and polyadenylation recognition sequences, including, but not limited to, bovine growth hormone polyA signal, simian virus 40 (SV40) polyA signal, Autographa californica nuclear polyhedrosis virus (AcNPV) 1629 polyA signal, and the like. Examples of promoters and / or regulatory elements include the ORF poly(A) signal, and the herpes simplex virus (HSV) thymidine kinase (TK) poly(A) signal. Those skilled in the art will recognize that different combinations of promoters and / or regulatory elements can be used to increase or decrease expression of the transgenes described herein.

[0151] Promoters that function in different species are also well known in the art. Useful promoters for expressing polypeptides include inducible, viral, synthetic, or constitutive promoters, and / or tissue-specific, temporally regulated, spatially regulated, and spatially and temporally regulated promoters. For example, promoters useful according to the present invention may include, but are not limited to, the immediate early (IE) cytomegalovirus human (CMV) promoter, the guinea pig CMV promoter, the SV40 promoter, pseudorabies virus promoters, such as the pseudorabies virus promoter of glycoprotein X promoter, herpes simplex virus-1, such as the alpha 4 promoter, Marek's disease virus promoter (including any isolate or strain of MDV, such as MDV-1, MDV-2, etc.), and promoters controlling the expression of HVT, such as glycoproteins, for example, gC, gB, gE, or gI, infectious laryngotracheitis virus promoters, such as the infectious laryngotracheitis virus promoter of glycoprotein gB, gE, gI, gD genes, or any other suitable promoter. Those of skill in the art will know how to identify promoters useful in accordance with the present invention.

[0152] According to the present invention, the recombinant Marek's disease virus or recombinant viral vector described herein can contain one or more transgenes operably linked to one or more promoters for expression of one or more viral proteins or peptides, or fragments or portions thereof. In some embodiments, a single transgene may be operably linked to a single promoter, or more than one transgene may be operably linked to a single promoter. In other embodiments, more than one transgene may be present in the recombinant vector, with a first transgene operably linked to a first promoter and a second transgene operably linked to a second promoter.

[0153] Vector construction and selection Construction of vectors containing one or more of the components described herein, useful for inserting a gene or transgene, or a portion thereof, into a target site, is known to those of skill in the art and may employ standard recombinant DNA techniques. The recombinant DNA vector or construct may include a selectable marker that confers a selectable phenotype on the cell. Selectable markers may also be used to select cells containing exogenous nucleic acids encoding the polypeptides or proteins described herein. Such markers may encode, for example, biocide resistance or antibiotic resistance (e.g., kanamycin, G418, bleomycin, hygromycin, etc.). Selectable markers are well known to those of skill in the art and may include any marker suitable for use in accordance with the present invention.

[0154] The recombinant vector or construct may also contain a screenable marker that can be used to monitor expression but that does not potentially cause cell death. Suitable screenable markers may include, for example, β-glucuronidase or uidA genes (GUS), one or more of various fluorescent protein genes such as green fluorescent protein (GFP), red fluorescent protein (RFP), or any one of a large family of proteins that fluoresce at characteristic wavelengths, genes encoding enzymes for which various chromogenic substrates are known, luciferase genes, xylE genes encoding catechol dioxygenases that convert chromogenic catechols, β-amylase genes, tyrosinase genes encoding enzymes that can oxidize tyrosine to DOPA and dopaquinone, which are then condensed into melanin, or α-galactosidases that catalyze the chromogenic α-galactose substrate.

[0155] Protein expression in host cells To obtain high-level expression of the cloned viral genes described herein, the nucleic acid may be subcloned into an expression vector containing a strong promoter to direct transcription and a transcription / translation terminator. For the encoded protein, a ribosome binding site for translation initiation may also be included. Suitable promoters for use in expression vectors are well known in the art, including bacterial promoters and viral promoters. Expression systems for protein expression are available for several prokaryotic and eukaryotic species known in the art. Commercially available kits for such expression systems are also readily available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

[0156] Selection of an appropriate promoter for directing expression of a heterologous nucleic acid will vary depending on the particular application. Such a promoter may be located at a distance from the heterologous transcription start site similar to that in its natural setting, although one of skill in the art will understand that some variation in this distance can be tolerated without loss of promoter function.

[0157] In addition to the promoter, an expression vector typically contains a transcription or expression cassette that contains all the elements necessary for the expression of nucleic acid in host cells. Any conventional vector known in the art that can be used for expression in eukaryotic or prokaryotic cells can be used to transport genetic information into cells. Thus, a typical expression cassette contains a promoter operably linked to the nucleic acid sequence encoding the selected nucleic acid, and the corresponding signals required for efficient processing (e.g., ribosome binding site, polyadenylation, and translation termination). Additional elements may include enhancers, and in the case of genomic DNA as a structural gene, introns with functional splice donor and acceptor sites.

[0158] In addition to a promoter sequence, such as the promoters described herein, the expression cassette may also contain a transcription termination region downstream of the structural gene to provide efficient termination of transcription. The termination region may be derived from the same gene as the promoter sequence or from a different gene. Markers such as fluorescent proteins, green or red fluorescent proteins, β-gal, CAT, etc., may be included in the vector as markers for vector transduction. Epitope or sequence tags may also be added to the recombinant protein to provide convenient isolation methods.

[0159] Expression vectors containing regulatory elements from eukaryotic viruses are typically used in eukaryotic expression vectors, such as SV40 vectors, papillomavirus vectors, retroviral vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and vectors derived from the CMV promoter, S Examples include any other vector that allows expression of a protein under the direction of the V40 early promoter, SV40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters known in the art that may be effective for expression in eukaryotic cells.

[0160] Protein expression from eukaryotic vectors can also be regulated using inducible promoters. In inducible promoters, the expression level is linked to the concentration of an inducer, such as tetracycline or ecdysone, by incorporating response elements for these drugs into the promoter. High levels of expression can be obtained from inducible promoters in the presence of an inducer. Some expression systems have markers, such as thymidine kinase and dihydrofolate reductase, that provide gene amplification.

[0161] The expression vector may also contain a replicon that functions in E. coli, an antibiotic resistance gene for selection of bacteria harboring the recombinant plasmid, and a unique restriction site in a non-essential region of the plasmid to allow for the insertion of eukaryotic sequences. Any antibiotic resistance gene suitable for use with the present invention may be used.

[0162] Standard transfection methods known in the art can be used to produce bacterial, mammalian, yeast, or insect cell lines that express large amounts of protein.These cell lines can then be purified using standard techniques known in the art, and prokaryotic and / or eukaryotic cells can be transformed according to any method known in the art for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells.Such methods can include, but are not limited to, plasmid or viral vectors, calcium phosphate transfection, protoplast fusion, electroporation, particle bombardment, liposomes, microinjection, or any method available in the art.

[0163] After the expression vector or transgene is introduced into the host cell, the cell can be cultured under optimal conditions for the expression of the desired protein, which can be recovered using standard techniques known in the art.The viral pathogen or viral protein, such as those described herein, can then be purified for use in diagnostic assays, for producing antibodies and immunogenic compositions, and for identifying antiviral compounds.Naturally occurring proteins can be purified from biological samples, such as tissue samples from birds infected with viruses as described herein, and recombinant proteins can be purified using any suitable method or expression system known in the art.

[0164] Several procedures for purifying recombinant proteins are available in the art. For example, a protein with established molecular adhesion properties can be reversibly fused with another protein. In addition, a specific protein can be selectively adsorbed to a purification column and then released from the column in a relatively pure form using an appropriate ligand or substrate. The fusion protein can then be removed by enzymatic activity. The protein can also be purified using an affinity column. Recombinant proteins can be purified from any suitable source.

[0165] Purification of proteins from recombinant bacteria Recombinant proteins can be expressed in large amounts by bacteria, for example, using inducible or constitutive promoters. Promoter induction using IPTG is an example of an inducible promoter system. Bacteria can be grown from fresh or frozen cultures according to standard procedures known in the art.

[0166] Proteins expressed in bacteria can form insoluble aggregates called inclusion bodies. Protocols suitable for purifying protein inclusion bodies are known in the art. Lysis of bacteria for recovery of expressed proteins can be carried out using any method known in the art, which may include the introduction of chemical buffers, sonication, mechanical disruption, etc. Inclusion bodies can also be solubilized, and the lysed cell suspension can be centrifuged to remove unwanted cell debris. Inclusion body proteins can be renatured by dilution or dialysis with an appropriate buffer.

[0167] Recombinant proteins can also be obtained from the bacterial periplasm. After lysis of the bacterial cells, the bacterial periplasmic fraction can be isolated by any method known in the art. The recombinant proteins present in the supernatant can be separated from host proteins by standard separation techniques well known to those skilled in the art.

[0168] Proteins can be separated using any technique known in the art, such as solubility fractionation or size differential filtration, which uses filtration through membranes with different pore sizes to isolate proteins based on molecular weight.Column chromatography can be used to isolate proteins from other proteins based on size, net surface charge, hydrophobicity, or affinity for ligands or substrates.In addition, antibodies against the protein of interest can be conjugated to a column, and the protein can be immunopurified.All of these methods are well known in the art.It will be clear to those skilled in the art that chromatography techniques can be performed at any scale and using any suitable commercial equipment.

[0169] antibody production Methods for producing polyclonal and monoclonal antibodies that specifically react with viral proteins, viral particles, and / or nucleic acids are known in the art. Such techniques can include antibody preparation by selection of antibodies from recombinant antibody libraries in phage or other vectors, and preparation of polyclonal and monoclonal antibodies by immunization of rabbits or mice.

[0170] Some antigens or antigenic regions, including viral proteins or portions thereof, viral particles, and / or nucleic acids, can be used to produce antibodies that specifically react with a desired viral pathogen. For example, recombinant viral proteins or antigenic fragments thereof can be isolated using any method described herein or known in the art. Recombinant proteins can be expressed in prokaryotic or eukaryotic cells and purified as described herein. Monoclonal and / or polyclonal antibodies can be produced using naturally occurring (in pure or impure form) or recombinant proteins using methods known in the art. Synthetic peptides derived from viral sequences can also be used to generate antibodies, and can be conjugated to carrier proteins and injected into animals capable of producing antibodies (e.g., rabbits).

[0171] Methods for producing polyclonal antibodies are known to those skilled in the art.For example, inbred strains of mice or rabbits can be immunized with a protein using standard adjuvants, such as those described herein, using standard immunization protocols known in the art.When a high titer of antibody against the protein is obtained, antisera can be prepared and enriched to obtain antibodies reactive with the protein.

[0172] Monoclonal antibodies can also be obtained by various methods known in the art. For example, spleen cells from an animal immunized with a desired antigen can be immortalized, typically by fusion with myeloma cells, or by transformation with Epstein-Barr virus (EBV), oncogenes, or retroviruses, or other methods known in the art. The immortalized cells may then be screened for production of antibodies of the desired specificity and affinity for the antigen. The yield of monoclonal antibodies produced by such cells may be enhanced by various techniques known in the art, for example, by intraperitoneal injection of a vertebrate host.

[0173] Monoclonal antibodies and polyclonal sera can be collected and titrated against the desired antigen or protein in an immunoassay, for example, a solid-phase immunoassay using a protein immobilized on a solid support. Antibodies specific for only a particular viral protein can be generated by subtracting out other cross-reactive proteins. In this way, antibodies that bind only to the selected protein can be obtained.

[0174] Once specific antibodies against a desired viral antigen, such as a protein, virus, and / or nucleic acid, are available, the desired antigen can be detected using a variety of immunoassay methods. The antibodies can also be used therapeutically.

[0175] Proteins associated with or distinct from the viral particles described herein can be detected and / or quantified using any of several well-recognized immunological binding assays. Viral particles may be detected based on epitopes defined by viral proteins displayed in the viral particle and / or epitopes defined by viral proteins separated from the viral particle (e.g., present in infected cells). Immunological assays may use antibodies that specifically bind to a selected protein or antigen. Antibodies may be produced by any of several methods well known to those skilled in the art. Immunoassays may also use a labeling agent to specifically bind to the complex formed by the antibody and antigen for detection purposes. The labeling agent may itself be one of the moieties comprising the antibody / antigen complex. Thus, the labeling agent may be a labeled viral protein nucleic acid or a labeled antiviral antibody. Alternatively, the labeling agent may be a third moiety, such as a secondary antibody, that specifically binds to the antibody / antigen complex. The secondary antibody may be specific for antibodies of the species from which the first antibody is derived. The labeling agent may be modified with a detectable moiety, such as biotin, to which another molecule (e.g., streptavidin) can specifically bind. A variety of detectable moieties are well known to those of skill in the art.

[0176] Immunoassays for detecting viral proteins, viruses, and / or nucleic acids in a sample are well known in the art. Such assays may be either competitive or non-competitive, and may be either quantitative or non-quantitative. Non-competitive immunoassays are assays in which antigens can be directly detected, and in some cases, the amount of antigen is directly measured. In competitive assays, viral antigens present in a sample are indirectly detected by a detectable signal associated with a known added (exogenous) viral antigen displaced from the anti-viral antigen antibody by the viral antigen present in the sample. In this way, such assays can also be adapted to provide an indirect measurement of the amount of viral antigen present in a sample. Competitive binding immunoassays can also be used to determine cross-reactivity, allowing any cross-reacting antibodies to be removed from pooled antisera. Additional assay formats, including, but not limited to, Western blots or liposome immunoassays, can also be used in accordance with the present invention.

[0177] Those skilled in the art will appreciate that it is often desirable to minimize nonspecific binding in immunoassays. In particular, when the assay involves an antigen or antibody immobilized on a solid substrate, it is desirable to minimize the amount of nonspecific binding to the substrate. Means for reducing such nonspecific binding are well known to those skilled in the art.

[0178] The assays described herein may contain a label or detectable group that does not significantly interfere with the specific binding of the antibody used in the assay. A detectable group may be any material having detectable physical or chemical properties. Such detectable labels are known in the art, and generally, any label useful in such methods may be applied to the present invention. Thus, as used herein, a "label" may be any composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Labels useful in the present invention include magnetic beads (e.g., DYNABEADS™), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), radioactive labels (e.g., 3 H, 125 1. 35 S, 14 C, or 32 P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and / or any others known in the art and used in ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

[0179] The labels of the present invention can be coupled directly or indirectly to the desired components of the assay according to methods well known in the art. As noted above, a wide variety of labels may be used, with the choice of label depending on sensitivity, ease of conjugation with the compound, stability requirements, available instrumentation, etc.

[0180] Non-radioactive labels can be attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand can then be bound to another molecule (e.g., streptavidin), which may be intrinsically detectable or covalently bound to a signal system such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. The ligand and its corresponding target can be used in any suitable combination with an antibody that recognizes a viral antigen or a secondary antibody that recognizes an antiviral antigen. The molecule can also be directly conjugated to a signal-generating compound, for example, by conjugation to an enzyme or fluorophore. The enzyme of interest used as a label can be, for example, a hydrolase such as phosphatase, esterase, and glycosidase, or an oxidase such as peroxidase. Fluorescent compounds can include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds may include luciferin, 2,3-dihydrophthalazinediones, such as luminol, or other compounds known in the art.

[0181] Means for detecting labels are well known to those skilled in the art and depend on the type of label used. For example, autoradiography may be used to detect radioactive labels, or fluorescent dyes may be used to detect fluorescent labels. Fluorescence may be detected visually, for example, by electronic detectors such as charge-coupled devices (CCDs) or photomultipliers. Similarly, enzymatic labels may be detected by providing an appropriate substrate for the enzyme and detecting the resulting reaction product. Colorimetric or chemiluminescent labels may be detected by observing the color associated with the particular label. In some embodiments, the assay format may not require the use of labeled components, but rather may be detected by simple visual inspection.

[0182] Pharmaceutical / Immunogenic Compositions and Their Administration In some embodiments, a recombinant vector comprising one or more transgenes expressing one or more viral proteins or peptides, or fragments thereof, described herein can be used as a pharmaceutical or immunogenic composition for administration to a subject, such as a chicken or other poultry, to provide protection from one or more viruses. For example, an immunogenic composition described herein comprises a recombinant vector having one or more transgenes described herein inserted into the viral genome in an intergenic region flanked by, for example, the intergenic locus UL35 / UL36 in the unique long (UL) region of the HVT genome. In one embodiment, The present invention provides a recombinant herpesvirus of turkeys (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted within the intergenic locus UL35 / UL36 in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL55 site in the unique long region (UL) of the HVT genome.

[0183] In other aspects, proteins or peptides, their immunogenic fragments, and / or polynucleotides, as well as anti-viral antibodies and / or T cells, can be incorporated into pharmaceutical or immunogenic compositions (e.g., vaccines). In another embodiment, the immunogenic compositions of the present invention can include at least a third transgene, a fourth transgene, etc., that can encode additional viral proteins. In such a manner, it is possible to provide an immunogenic composition to a subject, such as poultry, that provides protection from any desired number of viruses. Whole-virus vaccines (live, and attenuated, or replication-incompetent, or killed) or subunit vaccines, such as structural or nonstructural viral proteins or immunogenic fragments thereof, can be used to treat or prevent viral infections by eliciting an immune response in a subject. Alternatively, the pharmaceutical composition can include antigen-presenting cells transfected with a viral polynucleotide such that the antigen-presenting cells express viral peptides.

[0184] Immunogenic compositions of the present invention can be designed to generate antibody and / or cellular immunity in a subject. Such compositions can include one or more such compounds together with a non-naturally occurring pharmaceutically acceptable carrier. In other embodiments, immunogenic compositions of the present invention can include more than one adjuvant or pharmaceutically acceptable carrier, such that at least one is non-naturally occurring. A pharmaceutically acceptable carrier or adjuvant can be any substance that enhances the immune response in a subject to an exogenous antigen, including, but not limited to, adjuvants, liposomes, and biodegradable microspheres. A pharmaceutically acceptable carrier or adjuvant can also contain substances designed to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, or stimulators of the immune response, such as proteins derived from Bortadella pertussis or Mycobacterium tuberculosis. Commercially available adjuvants may include, for example, Freund's incomplete and complete adjuvant, Merck Adjuvant 65, aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate, CpG oligonucleotides, calcium, iron, or zinc salts, insoluble suspensions of acylated tyrosine, acylated sugars, cationic or anionic derivatized polysaccharides, polyphosphazenes, biodegradable microspheres, and monophosphoryl lipid A. One of ordinary skill in the art will be able to identify appropriate pharmaceutically acceptable carriers for use with the present invention.

[0185] Pharmaceutical or immunogenic compositions and / or vaccines within the scope of the present invention may also contain other compounds, which may be biologically active or inactive. For example, one or more immunogenic portions of other antigens may be incorporated into a fusion polypeptide or may be present in a composition or vaccine of the present invention as a separate compound. In some embodiments, polypeptides useful in the present invention may be conjugated to other macromolecules. Pharmaceutical or immunogenic compositions and vaccines may generally be used for prophylactic and / or therapeutic purposes. For example, in accordance with the present invention, the compositions described herein may be provided to a subject, such as a bird, prior to infection or exposure to one or more viruses to provide protection against infection or the development of symptoms of infection with one or more viruses. In other embodiments, such compositions may be provided to a subject, such as a bird, after infection or exposure to one or more viruses to provide treatment of the virus in the subject, e.g., by reducing or eliminating the infection in the subject.

[0186] Nucleic acid vaccines encoding the genome, structural or nonstructural proteins, or fragments thereof, of the viruses described herein may also be used to induce an immune response to treat or prevent viral infection. Numerous gene delivery techniques are well known in the art. An appropriate nucleic acid expression system may contain the DNA sequences necessary for expression in a subject (such as a suitable promoter and termination signal). In some embodiments, the DNA described herein may be introduced using a viral expression system (e.g., Marek's disease virus or HVT), which may involve the use of a non-pathogenic, replication-competent virus.

[0187] Pharmaceutical compositions or immunogenic compositions can be provided in single-dose or multi-dose containers, such as sealed ampoules or vials. Such containers can be sealed to maintain the sterility of the composition until use. Generally, the compositions described herein can be stored as a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, such compositions can be stored in a lyophilized state, requiring only the addition of a sterile liquid carrier immediately before use.

[0188] As described herein, the immunogenic composition may be combined with a pharmaceutically acceptable carrier. The selection of a suitable carrier may be determined in part by the particular composition to be administered (e.g., nucleic acid, protein, regulatory compound, or transduced cell), as well as the particular method used to administer the composition. Accordingly, a wide variety of suitable formulations of pharmaceutical or immunogenic compositions that can be used in the present invention are available. Administration may be by any convenient method, for example, injection, oral administration, inhalation, transdermal application, or rectal administration. Injections of the recombinant vectors or immunogenic compositions described herein may be provided to subjects, such as poultry, in a single administration or dose, or may be administered more than once, such as repeated administrations.

[0189] Formulations suitable for parenteral administration, such as, for example, intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal, in ovo, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes to render the formulation isotonic with the blood of the intended subject, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizing agents, thickeners, stabilizers, and preservatives. In the practice of the invention, compositions may be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically, or intrathecally.

[0190] Such compositions may also contain buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, or dextran), mannitol, proteins, polypeptides, or amino acids such as glycine, antioxidants, bacteriostatic agents, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic, or weakly hypertonic with the subject's blood, suspending agents, thickening agents, and / or preservatives. Alternatively, the compositions of the present invention may be formulated as lyophilizates. The compounds may also be encapsulated in liposomes using methods known in the art.

[0191] Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets as described herein. Cells transduced with nucleic acids for ex vivo therapy can be administered intravenously or parenterally as described above. The injections described herein can contain suspensions of one or more of the killed, inactivated, attenuated, or otherwise non-toxic virus cultures described herein, purified or non-purified solutions of viral proteins, or nucleic acids. The injection solutions can also contain pharmaceutically acceptable carriers as described herein.

[0192] Formulations suitable for oral administration include (a) liquid solutions, e.g., solutions containing diluents such as water, saline, etc. or PEG 400, (b) capsules or tablets each containing a predetermined amount of the active ingredient as a liquid, solid, granules, or gelatin; (c) a suspension in a suitable liquid; or (d) a suitable emulsion. Tablet forms may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavorings, dyes, disintegrants, and pharmaceutically compatible carriers. Lozenge forms may include the active ingredient in a flavor (e.g., sucrose), and pastilles containing the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia emulsion, gel, etc., containing the active ingredient in addition to carriers known in the art.

[0193] The compound of choice, alone or in combination with other suitable components, can be made into an aerosol formulation to be administered via inhalation. The aerosol formulation can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0194] The dose administered to a subject in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the subject over time. The dose is determined by the efficacy of the particular vector used, the condition of the subject, and the weight and / or surface area of ​​the patient being treated. The size of the dose may also be determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular vector or transduced cell type in a particular patient. For compositions containing the vectors described herein, the effective amount of the administered vector may be determined in part based on the circulating plasma level of the vector, vector toxicity, the health of the subject, and the production of anti-vector antibodies.

[0195] With respect to administration, the compounds of the present invention and transduced cells can be administered at a rate determined by the LD50 of the inhibitor, vector, or transduced cell type, as applied to the subject's weight and overall health, and the side effects of the inhibitor, vector, or cell type at various concentrations. Administration can be achieved by single, multiple, or divided doses.

[0196] Immunological detection of polypeptides and nucleic acids Immunoassays can be used to detect viral proteins, viral particles, and / or nucleic acids. Such assays can be useful for therapeutic and / or diagnostic applications, such as those described herein. Immunoassays are well known in the art and can be used to qualitatively or quantitatively analyze proteins, viral particles, and / or nucleic acids.

[0197] Assays for antibodies to viral proteins and antigens In one embodiment of the present invention, the presence of a virus, viral nucleic acid, or viral protein described herein in a sample can be determined by immunoassay. Enzyme-mediated immunoassays, such as immunofluorescence assays (IFA), enzyme-linked immunosorbent assays (ELISA), capture assays, microagglutination tests, and immunoblotting assays (e.g., Western blots), can be easily adapted to achieve virus or viral protein detection. ELISA methods can be effective for detecting the viruses or viral proteins described herein. Such ELISAs can include, for example, (1) binding an antiviral antibody or antigen to a substrate; (2) contacting the bound receptor with a biological sample containing a virus, viral antigen, viral protein, or antibody against a virus; and (3) irradiating the biological sample with a detectable moiety (e.g., horseradish peroxidase). The method may include (1) contacting the biological sample with an antibody conjugated to an enzyme (e.g., an oxidase or alkaline phosphatase enzyme); (2) contacting the biological sample with a substrate for the enzyme; (3) contacting the biological sample with a detection reagent, such as a color reagent; and (4) observing a detectable result. In some embodiments, a biological sample suitable for use in such an ELISA may be blood or other fluid. In another embodiment, the ELISA described herein may detect viruses or viral proteins in a tissue sample. Such methods may be readily modified by one skilled in the art to detect the presence of anti-viral antibodies, or specific viral proteins, as well as viruses, in a sample. In certain embodiments, the ELISA of the present invention may detect the presence of anti-viral antibodies.

[0198] The ELISA assays described herein may include nitrocellulose strips impregnated with the viral proteins described herein. The nitrocellulose strips may produce a visual result when contacted with a test sample containing anti-viral nucleoprotein antibodies. Such tests may identify subjects who already have antibodies to the viral proteins, thereby indicating that the subjects are immune to the virus. Administration of an immunogenic composition to prevent viral infection as described herein may be unnecessary in such subjects; therefore, identifying subjects who already have immunogenic antibodies may prevent unnecessary administration of immunogenic compounds to such subjects. In this regard, one embodiment of the present invention may involve using an assay described herein, such as an ELISA assay, to identify subjects lacking anti-viral antibodies, and then providing the subjects with an immunogenic composition described herein to prevent viral infection. In another embodiment, nitrocellulose strips for use in the ELISAs of the present invention may be impregnated with antibodies, such as anti-viral antibodies, and may produce a visual result when contacted with a test sample containing viral proteins. Such tests may identify subjects infected with the viruses described herein.

[0199] Another immunological technique that can be useful for detecting viruses is the competitive inhibition assay. Such assays utilize monoclonal antibodies (MABs) that react with specific viruses. A biological fluid from a subject (e.g., blood) may be contacted with a first antibody bound to a substrate, and a labeled monoclonal antibody may be contacted with the first antibody-virus complex. The amount of inhibition of monoclonal antibody binding is measured relative to a control.

[0200] As those skilled in the art will readily understand, the biological sample used in the above assay may be directly collected from a subject or may be in a partially purified form.An antibody specific to a particular virus reacts by binding to the virus as a primary reaction.Subsequently, to enhance the detection of the primary reaction, a secondary reaction with an antibody conjugated or labeled with a detectable moiety may also be added.Generally, in the secondary reaction, antibodies or other ligands that are specifically or nonspecifically reactive with different binding sites (epitopes) of the virus are selected for their ability to react with multiple sites on the antibody-virus complex.Thus, for example, several molecules of the antibody in the secondary reaction can react with each complex formed by the primary reaction, making the primary reaction more detectable.

[0201] The detectable moiety can allow for visual detection of a precipitate or color change, visual detection by microscopy, or automated detection by spectrometry, radioactivity measurement, etc. Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for light or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy), and alkaline phosphatase (for biochemical detection by color change). The detection methods and moieties used can be selected from, for example, any of those disclosed herein or available in the art.

[0202] Detecting the presence of viral nucleic acid In some embodiments, viral infections described herein can be detected based on the level of specific RNA or DNA in a biological sample. Primers from specific viruses or viral pathogens can be used to detect, diagnose, and determine the presence of viruses. Any suitable primers can be used to detect genomic DNA or any sequence, open reading frame, gene, or selected protein therein using any suitable method known in the art. Suitable nucleic acid sequences, as may be present in a biological sample, can be used as single- or double-stranded probes or primers to detect viral mRNA or cDNA generated therefrom. The viral polynucleotides described herein can be used to generate antisense oligonucleotides or as triplex-forming oligonucleotides to generate additional copies of the polynucleotide. For example, two oligonucleotide primers can be used in a PCR-based assay to amplify a portion of viral cDNA from a biological sample, where at least one of the oligonucleotide primers is specific for (i.e., hybridizes to) the viral polynucleotide. Such primers may be of any length sufficient to hybridize to and allow amplification of the viral nucleic acids described herein, including at least or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 nucleotides, or from about 12 to about 50 nucleotides in length, from 15 to 30 nucleotides in length, from 15 to 25 nucleotides in length, or from 20 to 30 nucleotides in length.DNA primers suitable for use with the present invention may be any of the primers described herein, for example, those set forth in SEQ ID NOs: 40-157.

[0203] The amplified nucleotides, e.g., cDNA, may then be separated and detected using techniques well known in the art, such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to viral polynucleotides may be used in hybridization assays to detect the presence of viral polynucleotides in biological samples.

[0204] Nucleic acid probes or primers specific to the viruses described herein can be generated using the polynucleotide sequences disclosed herein. The probes are preferably at least about 12, 15, 16, 18, 20, 22, 24, or 25 nucleotide fragments or other polynucleotide sequences encoding viral nucleic acids or polypeptides. Nucleic acid probes can be less than about 200 bp, 150 bp, 100 bp, 75 bp, 50 bp, 60 bp, 40 bp, 30 bp, 25 bp, 2 kb, 1.5 kb, 1 kb, 0.5 kb, 0.25 kb, 0.1 kb, or 0.05 kb in length. Probes can be generated, for example, by chemical synthesis, PCR amplification, generation from longer polynucleotides using restriction enzymes, or other methods well known in the art. The polynucleotides described herein can also be used in methods or assays involving the use of solid substrates, such as arrays. Such arrays can have one or more different polynucleotides immobilized on the array using methods known in the art.

[0205] In some embodiments, the polynucleotides of the present invention can be detectably labeled. Detectable labels include, but are not limited to, radiolabels, fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin, 6-carboxyfluorescein (6-FAM), 2',7'-dimethoxy-4 fluorescent dyes, including ',5'-dichloro-6-carboxyfluorescein, 6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein (5-FAM), or N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); 32 P, 35 S, and 3 In some embodiments, detectable labels may involve multiple steps (e.g., biotin-avidin, hapten-anti-hapten antibody, etc.).

[0206] According to the present invention, any suitable qualitative or quantitative method known in the art for detecting specific viral nucleic acids (e.g., RNA or DNA) may be used. The viral nucleic acids described herein can be detected, for example, by in situ hybridization in tissue sections, using methods that detect single base pair differences between hybridizing nucleic acids, by reverse transcriptase PCR, or in Northern blots containing polyA mRNA, or by other methods known in the art. For the detection of viral polynucleotides in blood or blood-derived samples, methods that allow the detection of single base pair mismatches can be used.

[0207] Because viral nucleic acid sequences may be present at relatively low levels in biological samples obtained from infected individuals, amplification techniques known in the art (e.g., PCR) may be used to amplify the sequences before performing a hybridization assay.

[0208] Nucleic acid probes can be prepared using the viral genomes described herein. Such probes can contain at least about 8 nucleotides or more and can be prepared synthetically or by excision from a recombinant polynucleotide. The probes described herein can hybridize with viral nucleic acids, and thus, such probes can be useful for detecting specific viruses in biological samples. The probes described herein can also be useful for identifying infected subjects and further characterizing viral genomes. Probes for detecting viral polynucleotides (natural or derived) can be of a specific length or have a sequence that allows for detection of unique viral sequences by hybridization. While sequences of about 6-8 nucleotides can be useful, longer sequences, such as sequences of about 10-12 nucleotides or about 20 or more nucleotides, may be preferred. Those skilled in the art will recognize how to make and use the probes described herein.

[0209] Nucleic acid probes can be prepared using conventional methods, including, but not limited to, automated oligonucleotide synthesis methods. Sequences useful for preparing such probes can include complements to any unique portion of a viral genome, such as a portion of a viral genome that allows a specific virus to be distinguished from other viruses that may be present in a sample. The probes described herein may have perfect complementarity to the target sequence of interest or may have one or more mismatches. Probes useful according to the present invention that have one of many mismatches will still hybridize to the target sequence of interest. To use such probes as diagnostics, the biological sample to be analyzed may be treated, if desired, prior to analysis to extract the nucleic acids contained therein. The resulting nucleic acids from the sample may be subjected to gel electrophoresis or other size separation techniques. The probes may be labeled with a detectable label as described herein. Suitable labels and methods for labeling probes are known in the art and may include any of the labels described herein or other labels useful in the present invention.

[0210] The probes may be perfectly complementary to the viral genome or a portion thereof (e.g., all or part of the sequence encoding a viral protein described herein). High stringency conditions may be desirable to prevent or at least minimize these effects. The stringency of hybridization can be determined by several factors during hybridization and washing, including temperature, ionic strength, length of time, and concentration of reagents. For such assays, probes or nucleic acids from a sample can be provided in solution or attached to a support (e.g., a solid or semi-solid support). Examples of supports that can be used include, but are not limited to, nitrocellulose (e.g., in membrane or microtiter well form), polyvinyl chloride (e.g., sheets or microtiter wells), polystyrene latex (e.g., beads or microtiter plates, polyvinylidine fluoride, diazotized paper, nylon membranes, activated beads, and protein A beads).

[0211] In one embodiment, probe or sample nucleic acids may be provided on an array for detection. Arrays can be created, for example, by spotting polynucleotide probes onto a substrate (e.g., glass, nitrocellulose, etc.) in a two-dimensional matrix or array. The probes may be bound to the substrate either by covalent bonds or nonspecific interactions, such as hydrophobic interactions. The polynucleotide sample can be detectably labeled (e.g., using a radioactive or fluorescent label) and then hybridized to the probe. Double-stranded polynucleotides, including labeled sample polynucleotides bound to probe polynucleotides, can be detected once the unbound portion of the sample is removed. Techniques for constructing arrays and methods for using these arrays are known in the art. Arrays can be used for a single sample to be analyzed for the presence of two or more nucleic acid target regions. In such cases, probes for each target region, as well as controls (both positive and negative), can be provided on a single array. Thus, arrays facilitate rapid and convenient analysis.

[0212] Diagnostic Tests and Kits The present invention further provides diagnostic reagents and kits containing one or more such reagents for use in various diagnostic assays, including, for example, immunoassays such as ELISAs and "sandwich"-type immunoassays, and nucleic acid assays (e.g., PCR assays). In related embodiments, the assays may be performed in a flow-through or strip test format, with the binding agent immobilized on a membrane such as nitrocellulose. Such kits may preferably include at least a first peptide, or a first antibody or antigen-binding fragment of the present invention, a functional fragment thereof, or a cocktail thereof, or a first oligonucleotide pair, and a signal-generating means. In some embodiments, the kit may include an immunogenic composition such as a recombinant virus described herein. Other compounds, such as reagents and a pharmaceutically acceptable carrier, may be included in the kit. When provided in such a kit, the immunogenic composition may be in solution, such as a pre-measured dose or amount, or may be a dry composition, such as a dried or lyophilized form suitable for rehydration or resuspension. The components of the kit may be pre-attached to a solid support or may be applied to the surface of a solid support when the kit is to be used. The signal generating means may be pre-associated with the antibody or nucleic acid of the invention, or may require combination with one or more components, e.g., buffers, nucleic acids, antibody-enzyme conjugates, enzyme substrates, etc., prior to use.

[0213] The kit may also include additional reagents, such as blocking reagents to reduce non-specific binding to the solid surface, washing reagents, enzyme substrates, enzymes, etc. The solid surface may be in the form of a microtiter plate, microspheres, or other material suitable for immobilizing nucleic acids, proteins, peptides, or polypeptides. Enzymes that catalyze the formation of chemiluminescent or chromogenic products or the reduction of chemiluminescent or chromogenic substrates are one such component of the signal generating means. Such enzymes are well known in the art. Radiolabels, chromogenic labels, fluorogenic, or When other types of detectable labels or detection means are included in the kit, the labeling agent may be provided in the same container as the diagnostic or therapeutic composition itself, or alternatively, may be placed in a second, separate container into which this second composition may be placed and appropriately aliquoted. Alternatively, the detection reagent and label may be prepared in a single container means.

[0214] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodology described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application.

[0215] The present invention is further illustrated and supported by the following examples. However, these examples should in no way be considered to further limit the scope of the present invention. On the contrary, those skilled in the art will readily understand that there are other embodiments, modifications, and equivalents of the present invention without departing from the spirit of the present invention and / or the scope of the appended claims. [Example]

[0216] Example 1 Construction of HVT-gfp plasmid HVT-green fluorescent protein (gfp)-B transfer plasmid construction The HVT-gfp-B transfer plasmid (SEQ ID NO: 18) was chemically synthesized by GeneArt (ThermoFisher). 2.5 μg of the above-mentioned plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. After approximately 4-6 hours, the transfected cells were transfected at 0.006 moi (1.5 x 10 4 pfu / 2.5x10 6 cells) were infected with HVT. Three days later, the cells were transferred to T75 (1x10) cells containing fresh CEF. 7The virus was passaged at 1:15 onto 100 cells / T75. The cells were then plated 3 days later at 1:50 onto 24-well plates. Cells from wells containing green fluorescent foci were plated onto 96-well plates at 1:200, 1:500, and 1:1000 dilutions with fresh cells (6x10^4 cells / well). Wells containing a single green focus were purified three times by limiting dilution using 96-well plates. The purified virus was expanded using CEF cells and frozen stocks were made. This was designated "HVT-gfp-B."

[0217] PCR analysis of the three purified clones using primers just outside the integration site of UL55-Gene3 (top primer: SEQ ID NO: 49, bottom primer: 5'-SEQ ID NO: 50) gave a 1.893 kb band, as expected. HVT gave a 0.15 kb band, as expected. See Figure 1.

[0218] HVT-gfp-A modified transfer plasmid construction The modified transfer plasmid HVT-gfp-A (SEQ ID NO: 16) was generated by site-directed mutagenesis using two pairs of primers (upper primer pair: SEQ ID NO: 40 and SEQ ID NO: 41 for generating SbfI upstream of the gfp gene, lower primer: SEQ ID NO: 42 for generating SbfI downstream of the gfp gene, and SEQ ID NO: 43 for the original transfer plasmid HVT-gfp-A (SEQ ID NO: 17) chemically synthesized by GeneArt, ThermoFisher). 0.01 μg of the modified transfer plasmid HVT-gfp-A was co-transfected with 2.5 μg of HVT DNA using 7.5 μL of PEI (polyethyleneimine) into secondary CEF cells on a 6-well plate. Green fluorescent foci were evident at the first passage. After three rounds of purification by limiting dilution, one clone of HVT-gfp-A was obtained. The line was further expanded and frozen stocks were prepared.

[0219] PCR analysis of the purified clone (left lane) using primers just outside the UL35-UL36 integration site (upper primer: SEQ ID NO: 44, lower primer: SEQ ID NO: 45) gave the expected 1.922 kb band (Figure 4). DNA of the modified transfer plasmid HVT-gfp-A was used as a control (right lane). See Figure 2.

[0220] Example 2 HVT-IBD construction Construction of HVT-IBD #1 HVT-IBD #1 transfer plasmid (SEQ ID NO: 20) was chemically synthesized by GeneArt (ThermoFisher). 2.5 μg of the above-mentioned plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. Approximately 4-6 hours later, the transfected cells were infected with HVT at 0.055 moi. Three days later, the cells were transferred to a T75 (1x10) containing fresh CEFs. 7 The cells were passaged at a ratio of 1:7.5 to 1000 cells / T75. The cells were then plated in 10 96-well plates, and duplicate plates were made three days later. One set of plates was fixed and stained with anti-IBDV chicken serum. Two wells containing foci that stained positive for IBD were identified. The corresponding wells containing positively stained foci were purified three times by limiting dilution using 96-well plates. The purified virus was expanded using CEF cells and a frozen stock was made. This was designated "HVT-IBD #1."

[0221] PCR analysis of different clones using primers immediately outside the integration site of UL55-Gene3 (top primer: SEQ ID NO: 46, bottom primer: SEQ ID NO: 47, panel A) yielded a 2.414 kb band, while the PCR band for the original vector was 1.922 kb. Correct integration was further confirmed by using primers surrounding the junction downstream of the insert (top primer SEQ ID NO: 48, located within the IBDV VP2 coding region; bottom primer SEQ ID NO: 49, located downstream of the transfer plasmid; panel B). As expected, a 1.118 kb PCR band was obtained. Correct integration of the upstream integration site was confirmed using primers surrounding the junction upstream of the insert (top primer SEQ ID NO: 50, located upstream of the transfer plasmid; bottom primer SEQ ID NO: 51, within the IBDV VP2 coding region; panel C). As expected, a 1.428 kb PCR band was obtained. See Figures 3A, B, and C.

[0222] Construction of HVT-IBD #5 The HVT-IBD #5 transfer plasmid (SEQ ID NO: 21) was chemically synthesized by GeneArt (ThermoFisher). JBJ-1 cells (chicken fibroblast cell line) in 6-well plates were transfected with 2.5 μg of the above-mentioned plasmid using LTX transfection reagent (Invitrogen). Approximately 5 hours after transfection, the transfected cells were infected with HVT at 0.05 moi. The transfected / infected cells were amplified by serial passage (1:4 to 1:10), and then aliquots were seeded into 96-well plates at limiting dilution. IBDV VP2 antigen expression was assessed by antibody staining of live cell monolayers without fixation (see Figures 4A and 4B). Stained foci were harvested by trypsinization of the cells using a cloning cylinder placed around the positive focus. This "live cell staining" followed by subsequent passage through the cloning cylinder was repeated four times to obtain pure VP2-positive cultures. This culture was amplified by serial passage on JBJ-1 cells. Final amplification was performed on primary CEF cells in roller bottles. The harvested CEF cells were used to generate a frozen cell stock, designated "HVT-IBD #5."

[0223] PCR analysis of clone #7 using two sets of primers to confirm integration of the insert across both insertion sites. In PCR A, the upper primer (SEQ ID NO: 52) binds to the IBDV VP2 coding region, while the lower primer (SEQ ID NO: 53) binds downstream of the UL35-UL36 integration site. This primer set yielded a 1.244 kb PCR band, as expected. In PCR B, the upper primer (SEQ ID NO: 54) binds upstream of the UL35-UL36 insertion site, while the lower primer (SEQ ID NO: 55) binds within the human CMV promoter of the insert, yielding a 0.926 kb PCR band, as expected. See Figure 5.

[0224] Construction of HVT-IBD #6a HVT-IBD #6a transfer plasmid (SEQ ID NO: 22) was chemically synthesized by BioBasic Inc. 0.1 μg and 0.01 μg of linearized transfer plasmid (digested with EcoR1 and HindIII) were cotransfected with 2.5 μg of HVT-gfp-A digested with Sbf1 in secondary CEF cells using PEI (polyethyleneimine) transfection reagent in a 6-well plate. Four days after transfection, four non-green foci were observed with 0.01 μg of transfer plasmid, three with 0.1 μg of transfer plasmid, and no foci were observed with HVT-gfp-A digested with Sbf1 alone. Two non-green foci were purified three times by limiting dilution using a 96-well plate. The purified virus was expanded using CEF cells and frozen stocks were generated. This was named "HVT-IBD #6a."

[0225] Infected cell lysates were prepared and subjected to Western blot analysis using a monoclonal antibody against IBDV R63. A protein band of approximately 50 KD was observed in all lanes except for the lane containing the HVT-gfp-A vector lysate (see Figure 6).

[0226] To confirm correct integration, PCR analysis of clones was performed using two sets of primers. The first primer set targeted the upstream integration site: the upper primer 5'-SEQ ID NO:56, located upstream of the UL35-UL36 integration site, and the lower primer SEQ ID NO:57, located within the Pec promoter. This primer set yielded a 0.911 kb PCR band, as expected. The second primer set targeted the downstream integration site: the upper primer 5'-SEQ ID NO:58, located within the IBDV VP2 coding region, and the lower primer 5'-SEQ ID NO:59, located downstream of the UL35-UL36 insertion site. As expected, a 1.244 kb PCR band was obtained. See Figures 7A and 7B.

[0227] Construction of HVT-IBD #9 The HVT-IBD #9 transfer plasmid (SEQ ID NO: 23) was chemically synthesized by GeneArt (ThermoFisher). 2.5 μg of the above-mentioned plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. After approximately 4-6 hours, the transfected cells were infected with HVT-gfp-B at 0.075 moi. Three days later, the cells were passaged three times at 1:10 in a T75 containing fresh CEF (1x10^7 cells / T75). The cells were then plated into 10 of 96-well plates, yielding 90 non-green foci. Three of them were incubated with anti-IBDV chicken serum. The virus stained positively with IgG. Two clones were purified three times by limiting dilution using 96-well plates. The purified virus was expanded using CEF cells and a frozen stock was made. This was designated "HVT-IBD #9."

[0228] PCR analysis of the different clones using primers immediately outside the integration site of UL55-Gene3 (top primer: SEQ ID NO: 60, bottom primer: SEQ ID NO: 61, panel A) yielded a 2.536 kb band, while the PCR band for the original vector HVT-gfp-B was 1.922 kb. Correct integration was further confirmed using primers surrounding the junction upstream of the insert (top primer SEQ ID NO: 62, located upstream of the UL55-Gene3 insertion site; bottom primer SEQ ID NO: 63, located within the IBDV VP2 coding region). As expected, a PCR band of 1.482 kb was obtained. Correct integration for the downstream site was confirmed using primers surrounding the junction downstream of the insert (top primer SEQ ID NO: 64, located within the IBDV VP2 sequence; bottom primer SEQ ID NO: 65, located downstream of the UL55-Gene3 insertion site). As expected, a PCR band of 1.166 kb was obtained. See Figures 8A and B.

[0229] Construction of HVT-IBD #30 The HVT-IBD #30 transfer plasmid (SEQ ID NO: 24) was chemically synthesized by GeneArt, ThermoFisher. Secondary CEF cells were cotransfected with 0.1 μg of the above-mentioned plasmid and 2.5 μg of HVT using PEI (polyethyleneimine) transfection reagent in a 6-well plate. After 3 days, the cells were passaged 1:12 onto fresh CEF cells. IBD VP2-expressing foci were visualized by staining unfixed cultures with chicken polyclonal serum against IBDV, and these foci were marked using a fluorescent microscope. A total of 16 positive foci were passaged onto fresh CEF cells by trypsinization using a cloning cylinder, and the foci were separated from those not expressing VP2. Four of these cultures were cloned three times using the same procedure and then expanded onto primary CEF cells in roller bottles. A frozen cell stock was established and designated "HVT-IBD #30."

[0230] PCR analysis of four different clones using primers for the upstream region of the UL55-Gene3 integration site (top primer: SEQ ID NO: 66, bottom primer: SEQ ID NO: 67, panel A) yielded a 1.673 kb band. Correct integration was further confirmed by using primers surrounding the 3' junction of the insertion (top primer SEQ ID NO: 68, located within the IBDV VP2 coding region; bottom primer SEQ ID NO: 69, located downstream of the UL55-Gene3 insertion site, panel B). As expected, a 1.082 kb PCR band was obtained. Correct integration for the downstream site was further confirmed by using primers outside the expression cassette (top primer SEQ ID NO: 70, bottom primer SEQ ID NO: 71, panel C). As expected, a 2.558 kb PCR band was obtained. See Figures 9A-C.

[0231] Construction of HVT-IBD #31 HVT-IBD #31 transfer plasmid (SEQ ID NO: 25) was chemically synthesized by GeneArt, ThermoFisher. 0.01 μg of linearized transfer plasmid (digested with EcoR1 and HindIII) was co-transfected with 2.5 μg of HVT-gfp-A digested with Sbf1 in secondary CEF cells using PEI (polyethyleneimine) transfection reagent in a 6-well plate. Four days after transfection, one non-green focus was observed, whereas no focus was observed with HVT-gfp-A digested with Sbf1 alone. After passage, two non-green foci were isolated by limiting dilution analysis using a 96-well plate. The purified virus was expanded in CEF cells and a frozen stock was prepared, which was designated "HVT-IBD #31."

[0232] Infected cell lysates were prepared and subjected to Western blot analysis using a monoclonal antibody against IBDV R63. A protein band of approximately 50 KD was observed in all lanes without mAb against IBDV R63 and with anti-IBDV chicken serum as the only probe (see Figures 10A and B).

[0233] To confirm correct integration, PCR analysis of the clones was performed using two sets of primers. The first primer set targeted the upstream integration site: the upper primer SEQ ID NO:72, which was located upstream of the UL35-UL36 integration site, and the lower primer SEQ ID NO:73, which was located within the chicken beta-actin promoter. As expected, this primer set yielded a PCR band of 0.835 kb. The second primer set targeted the downstream integration site: the upper primer SEQ ID NO:76, which was located within the IBDV VP2 coding region, and the lower primer SEQ ID NO:77, which was located downstream of the UL35-UL36 insertion site. As expected, a PCR band of 1.248 kb was obtained. See Figures 11A and B.

[0234] Construction of HVT-IBD #34 HVT-IBD #34 transfer plasmid (SEQ ID NO: 28) was chemically synthesized by GeneArt, ThermoFisher. 2.5 μg of the above-mentioned plasmid was transfected into secondary CEF cells using LTX transfection reagent (Invitrogen) in a 6-well plate. After approximately 4-6 hours, the transfected cells were infected with HVT-gfp-B at 0.05 moi. Three days later, the cells were transferred to a T75 (1x10) containing fresh CEFs. 7The infected cells were passaged at 1:15 relative to the total number of infected cells (T75 cells). The infected cells were plated onto 10 x 96-well plates, grown for 3 days, and then passaged in duplicate 96-well plates. One duplicate was fixed and stained with anti-IBDV chicken serum to identify three wells containing foci that stained positive for IBDV. Corresponding wells from the live cell duplicate plate were purified by three rounds of limiting dilution cloning using 96-well plates. One of the purified viruses was expanded using CEF cells and a frozen stock was made. This was designated "HVT-IBD #34."

[0235] PCR analysis of three different clones using primers for the region upstream of the Gene3-UL55 integration site (top primer: SEQ ID NO: 78, bottom primer: SEQ ID NO: 79, located within the chicken beta-actin promoter; Panel A) yielded a 0.815 kb band, as expected. Correct integration was further confirmed by using primers surrounding the junction downstream of the insertion (top primer: SEQ ID NO: 80, located within the IBDV VP2 coding region; bottom primer: SEQ ID NO: 81, located downstream of the Gene3-UL55 insertion site; Panel B). As expected, a 1.296 kb PCR band was obtained. Correct constructs were further confirmed by using primers outside the expression cassette (top primer: SEQ ID NO: 82, bottom primer: SEQ ID NO: 83; Panel C). As expected, a 3.001 kb PCR band was obtained. See Figures 12A-C.

[0236] Construction of HVT-ND #38 HVT-IBD #38 transfer plasmid (SEQ ID NO: 29) was chemically synthesized by BioBasic Inc. The HindIII and ApoI digested transfer plasmid for HVT-ND #38 was transformed into Sbf1 digested plasmid using PEI (polyethyleneimine, 7.5 uL) in a 6-well plate containing secondary CEF cells. The cells were co-transfected with HVT-gfp-A DNA. Six days after transfection, the transfected cells were plated on a 96-well plate and stained for viability with NDV chicken serum. Seven wells containing positively stained foci were purified three times by limiting dilution. One of the purified viruses was expanded using CEF cells to create a frozen stock, which was designated "HVT-ND #38."

[0237] PCR analysis of five different clones using primers for the region upstream of the UL35-UL36 integration site (top primer: SEQ ID NO: 84, bottom primer: SEQ ID NO: 85, located within the NDV F coding region, panel A) yielded a 2.122 kb band. Correct integration was further confirmed by using primers surrounding the 3' junction of the insertion (top primer SEQ ID NO: 86, located within the NDV F coding region, bottom primer SEQ ID NO: 87, located downstream of the UL35-UL36 insertion site, panel B). As expected, a 1.127 kb PCR band was obtained. Correct constructs were further confirmed by using primers outside the expression cassette (top primer SEQ ID NO: 88, bottom primer SEQ ID NO: 89, panel C). As expected, a 3.657 kb PCR band was obtained. See Figures 15A and B.

[0238] Construction of HVT-ND #39 HVT-IBD #39 transfer plasmid (SEQ ID NO: 30) was chemically synthesized by BioBasic Inc. Secondary CEF cells were cotransfected with 0.01 μg of the above-mentioned plasmid and 2.5 μg of HVT using PEI (polyethyleneimine) transfection reagent in a 6-well plate. Six days later, the cells were passaged 1:24 onto fresh CEF cells. Three days after passage, unfixed cultures were stained with anti-NDV-specific chicken polyclonal serum to visualize foci expressing NDV F protein, and these foci were marked using a fluorescent microscope. A total of four positive foci were passaged onto fresh CEF cells by trypsinization using a cloning cylinder, and the foci were separated from those not expressing F protein. Four of these cultures were cloned three times using the same procedure and then expanded on primary CEF cells in roller bottles. A frozen cell stock (clone 2 in the figure) was established and designated "HVT-ND #39."

[0239] PCR analysis of three different clones using primers for the region upstream of the UL35-UL36 integration site (top primer: SEQ ID NO: 90, bottom primer: SEQ ID NO: 91, located within the chicken beta-actin promoter; Panel A) yielded a 0.835 kb band. Correct integration was further confirmed by using primers surrounding the junction downstream of the insert (top primer SEQ ID NO: 92, located within the polyA tract; bottom primer SEQ ID NO: 93, located downstream of the UL35-UL36 insertion site; Panel B). As expected, a 0.856 kb PCR band was obtained. Correct constructs were further confirmed by using primers outside the expression cassette (top primer SEQ ID NO: 94, bottom primer SEQ ID NO: 95; Panel C). As expected, a 3.449 kb PCR band was obtained. See Figures 16A and B.

[0240] Construction of HVT-ND #40 HVT-IBD #40 transfer plasmid (SEQ ID NO: 31) was chemically synthesized by BioBasic Inc. HindIII-digested transfer plasmid for HVT-ND #40 was co-transfected with Sbf1-digested HVT-gfp-A DNA using PEI (polyethyleneimine, 7.5 uL) in a 6-well plate containing secondary CEF cells. Seven days after transfection, the transfected cells were plated on a 24-well plate and infected with NDV chicken blood. The supernatant was stained for live cells. Four wells containing positively stained foci were purified three times by limiting dilution. One of the purified viruses was expanded using CEF cells to create a frozen stock, which was designated "HVT-ND #40."

[0241] PCR analysis of four different clones using primers for the region upstream of the UL35-UL36 integration site (top primer: SEQ ID NO: 96, bottom primer: SEQ ID NO: 97, located within the chicken beta-actin promoter; panel A) yielded a 0.835 kb band. Correct integration was further confirmed by using primers surrounding the junction downstream of the insertion (top primer SEQ ID NO: 98, located within the NDV F coding region; bottom primer SEQ ID NO: 99, located downstream of the UL35-UL36 insertion site; panel B). As expected, a 0.856 kb PCR band was obtained. Correct constructs were further confirmed by using primers outside the expression cassette (top primer SEQ ID NO: 100, bottom primer SEQ ID NO: 101; panel C). As expected, a 3.449 kb PCR band was obtained. See Figures 17A-C.

[0242] Construction of HVT-ND #42 The initial transfer plasmid HVT-ND #42 (SEQ ID NO: 33) was chemically synthesized by BioBasic Inc. The cloning plasmid was chemically synthesized by DNA2.0. PCR amplification of the NDV F gene expression cassette of the HVT-ND #42 transfer plasmid by using the following primers: upper primer, SEQ ID NO: 102, lower primer, 5'-SEQ ID NO: 103. The amplified PCR fragment was cloned into the AscI and NheI sites of UL55 / gene3 to generate the final transfer plasmid HVT-ND #42 (SEQ ID NO: 35).

[0243] The transfer plasmid was transfected into HVT-infected CEF cells in a 6-well plate using Lipofectamine LTX. Three days after transfection, the transfected / infected cells were plated in duplicate into 6-well plates and then into a 96-well plate to screen for ND-expressing foci by staining with NDV antiserum. Wells containing ND-expressing foci were purified three times by limiting dilution. One of the purified viruses was expanded using CEF cells and a frozen stock was created. This was designated "HVT-ND #42."

[0244] PCR analysis of one final clone using primers outside the expression cassette (primer set 1: upper primer SEQ ID NO: 104, lower primer SEQ ID NO: 105) yielded a 3.597 kb band, as expected. Four sets of primers for the upstream integration region of UL55-Gene3, with all upper primers located upstream and outside the expression cassette and all lower primers located within the ND F coding region, were used. Primer set 2: upper primer: SEQ ID NO: 106, lower primer: SEQ ID NO: 107, yielded a 2.243 kb band. Primer set 3: upper primer: SEQ ID NO: 108, lower primer: SEQ ID NO: 109, yielded a 2.356 kb PCR band. Primer set 4: upper primer: SEQ ID NO: 110, lower primer: SEQ ID NO: 111, yielded a 2.424 kb PCR band. Primer set 5: upper primer: SEQ ID NO: 112, lower primer: SEQ ID NO: 113, yielded a 2.170 kb PCR band. Correct integration was further confirmed by using primers surrounding the junction downstream of the insertion (primer set 6: upper primer SEQ ID NO: 114 located within the NDV F gene coding sequence, lower primer SEQ ID NO: 115 located downstream of the UL55-Gene3 insertion site, panel C). As expected, a 0.971 kb PCR band was obtained. See Figure 18.

[0245] Construction of HVT-ND #44 HVT-IBD #44 transfer plasmid (SEQ ID NO: 36) was chemically synthesized by BioBasic Inc. Transfer plasmid #44 was digested with restriction enzymes EcoRI and HindIII to release the insert from the plasmid sequence, and the resulting digested DNA (10 ng) was used together with 2.5 μg of HVT-gfpB DNA to co-transfect secondary cells using PEI (polyethyleneimine). Four days after transfection, the transfected cells were passaged 1:6 with fresh secondary cells and stained for viability with chicken anti-NDV polyclonal serum. NDV-expressing foci were identified 3–4 days after passage. Three positively stained foci were harvested by trypsinization using a cloning kit. The harvested cells were serially diluted and plated onto fresh secondary CEF cells. This process was repeated every 3–4 days until NDV staining was uniform, followed by four subsequent cloning rounds. The cloned cultures were then expanded and frozen stocks were prepared. The frozen stock was designated "HVT-ND #44."

[0246] PCR analysis of one final clone using primers for the region upstream of the UL55-Gene3 integration site (top primer: SEQ ID NO: 116, located upstream of UL55; bottom primer: SEQ ID NO: 117, located within the chicken beta-actin promoter; Panel A) yielded a 0.71 kb band. A similarly localized primer pair, top primer: SEQ ID NO: 118; bottom primer: SEQ ID NO: 119 (Panel B), yielded a 0.965 kb PCR band. Correct integration was further confirmed by using primers surrounding the junction downstream of the insertion (top primer SEQ ID NO: 120, located within the NDV F gene coding sequence; bottom primer SEQ ID NO: 121, located downstream of the UL55-Gene3 insertion site; Panel C). As expected, a 0.971 kb PCR band was obtained. The correct construct was further confirmed by using primers outside the expression cassette (top primer SEQ ID NO: 122, bottom primer SEQ ID NO: 123 (Panel D)). As expected, a 3.438 kb PCR band was obtained.

[0247] Construction of HVT-ND #45 The HVT-IBD #45 transfer plasmid (SEQ ID NO: 1) was purchased from BioBasic The virus was chemically synthesized by Biosciences, Inc. This plasmid was transfected into CEF cells infected with HVT-GFP-B in a 6-well plate using Lipofectamine LTX. Three days after transfection, the transfected / infected cells were plated onto a 96-well plate for screening of GFP-negative foci. Wells containing GFP-negative foci were purified three times by limiting dilution. The purified virus was IFA stained with chicken NDV serum to confirm NDV F gene expression. One of the purified viruses was expanded using CEF cells to create a frozen stock. This was designated "HVT-ND #45."

[0248] PCR analysis of one final clone using primers outside the expression cassette (primer set 1: top primer SEQ ID NO: 124, bottom primer SEQ ID NO: 125) yielded a 2.830 kb band, as expected. Two sets of primers for the upstream integration region of Gene3-UL55, with both top primers located upstream and outside the expression cassette and both bottom primers located within the ND F coding region, primer set 2: top primer: SEQ ID NO: 126, bottom primer: SEQ ID NO: 127, yielded a 1.635 kb band. Primer set 3: top primer: SEQ ID NO: 128, bottom primer: SEQ ID NO: 129, yielded a 1.588 kb PCR band. Correct integration was confirmed by using two sets of primers around the junction downstream of the insertion (primer set 4: top primer SEQ ID NO: 130 located within the NDV F gene coding sequence, bottom primer SEQ ID NO: 131 located downstream of the Gene3-UL55 insertion site). , and further confirmed. As expected, a PCR band of 0.993 kb was obtained. Primer set 5: upper primer: SEQ ID NO: 132, lower primer: SEQ ID NO: 133, as expected, a PCR band of 1.137 kb was obtained. See Figure 19.

[0249] Construction of HVT-ND #46 The HVT-ND #46 transfer plasmid (SEQ ID NO: 38) was chemically synthesized by BioBasic Inc. This plasmid was transfected into CEF cells together with HVT-GFP-B viral DNA using PEI (polyethyleneimine, 7.5 uL) in a 6-well plate. Four days after transfection, the transfected cells were plated on a 96-well plate for screening of GFP-negative foci. Wells containing GFP-negative foci were purified three times by limiting dilution. The purified virus was IFA stained with chicken NDV serum to confirm NDV F gene expression. One of the purified viruses was expanded using CEF cells to create a frozen stock. This was designated "HVT-ND #46."

[0250] PCR analysis of one final clone using primers outside the expression cassette (top primer SEQ ID NO:134, bottom primer SEQ ID NO:135) yielded a 3.597 kb band, as expected. One set of primers for the upstream integration region of Gene3-UL55, with the top primer located upstream and outside the expression cassette (SEQ ID NO:136) and the bottom primer located within the mouse CMV promoter (SEQ ID NO:137), yielded a 1.107 kb PCR band, as expected. Correct integration was further confirmed by using four sets of primers surrounding the junction downstream of the insertion: P1: top primer SEQ ID NO:138, located within the NDV F gene coding sequence; bottom primer SEQ ID NO:139, located downstream and outside the expression cassette. A 1.003 kb PCR band was obtained, as expected. P2: top primer SEQ ID NO:140, bottom primer SEQ ID NO:141, yielded a 1.147 kb PCR band, as expected. P3: Upper primer: SEQ ID NO: 142, Lower primer: SEQ ID NO: 143. As expected, a PCR band of 1.019 kb was obtained. P4: Upper primer: SEQ ID NO: 144, Lower primer: SEQ ID NO: 145. As expected, a PCR band of 1.018 kb was obtained. See Figures 20A-C.

[0251] Construction of HVT-ND #48 The linear transfer plasmid for HVT-ND #48 (SEQ ID NO: 39) was co-transfected with HVT-gfp-B DNA using PEI (polyethyleneimine, 7.5 uL) in a 6-well plate containing secondary CEF cells. Four days after transfection, the transfected cells were plated on a 96-well plate and stained for viability with NDV chicken serum. Four non-green foci were found, two of which were positively stained using NDV chicken serum. See Figures 33A and B. Two clones were purified three times by limiting dilution. The purified virus was expanded using CEF cells and frozen stocks were made. This was designated "HVT-ND #48."

[0252] HVT-IBD #48 transfer plasmid was chemically synthesized by BioBasic Inc. This transfer plasmid was digested with EcoRI and HindIII to release the insert from the plasmid sequence, and the resulting digested DNA (10 ng) was used together with 2.5 μg of HVT-gfp-B DNA to co-transfect secondary cells using PEI (polyethyleneimine). Three days after transfection, the transfected cells were passaged 1:6 with fresh secondary cells and stained for live cells with NDV chicken polyclonal serum to identify NDV-expressing foci four days after passage. Three positively stained foci were isolated using a cloning cylinder. The cells were harvested by trypsinization. The harvested cells were serially diluted and plated onto fresh secondary CEF cells. This process was repeated every 3-4 days until NDV staining was uniform, followed by four subsequent clonings. The cloned cultures were then removed from the 6-well plates and plated onto 75cm plates. 2 Flask length: 225cm 2 After expansion into flasks, primary CEF cells were used to culture 850cm 2 Final amplification was performed in roller bottles. The final culture was harvested and designated "HVT-ND #48."

[0253] PCR analysis of one final clone using primers for the region upstream of the Gene3-UL55 integration site (top primer: SEQ ID NO:146, bottom primer: SEQ ID NO:147, located within the chicken beta-actin promoter; Panel A) yielded a 0.815 kb band, as expected. Correct integration was further confirmed by using primers surrounding the junction downstream of the insertion (top primer: SEQ ID NO:148, located within the NDV F coding region; bottom primer: SEQ ID NO:149, located downstream of the Gene3-UL55 insertion site; Panel B). As expected, a 1.003 kb PCR band was obtained. Another similarly localized primer: top primer: SEQ ID NO:150, bottom primer: SEQ ID NO:151 (Panel C) yielded a 1.147 kb PCR band, as expected. Correct construct was further confirmed by using primers outside the expression cassette (top primer: SEQ ID NO:152, bottom primer: SEQ ID NO:153; Panel D). As expected, a 3.430 kb PCR band was obtained.

[0254] Example 3 In vivo IBDV efficacy test of HVT-IBD#9 and #34 in SPF birds Two HVT-IBD recombinants, HVT-IBD#9 and #34, were tested in SPF birds for their in vivo efficacy against a highly virulent IBDV challenge (STD strain, provided by the USDA). In this study, the commercially available vaccine Vaxxitek (Merial) was used as a positive control. 1500 pfu of each recombinant virus was injected in ovo at E18. The reciprocal titer of each vaccine virus was also determined for each recombinant after vaccination. While 100% of HVT-IBD#9 express the IBDV VP2 antigen, we found that only 96% of HVT-IBD#34 express this antigen. IBDV STD challenge was performed on day 28 in accordance with USDA instructions. All birds were necropsied 5 days after challenge. We observed 100% protection for HVT-IBD#9 and 90% protection for HVT-IBD#34, while our positive control Vaxxitek conferred 97% protection. [Table 2]

[0255] Example 4 In vivo IBDV efficacy testing of HVT-IBD#1, #5, #6a, #9, #30, and #34 in SPF birds Six HVT-IBD recombinants, HVT-IBD#1, #5, #6a, #9, #30, and #34, were tested in SPF birds for their in vivo efficacy against a highly virulent IBDV challenge (STD strain, provided by the USDA). In this study, a positive control, the commercially available vaccine Vaxxitek (Merial), was used. 1500 pfu of each recombinant virus was injected in ovo at E18. The reciprocal titer of each vaccine virus was also determined for each recombinant after vaccination. All recombinants were found to express 100% of the IBDV VP2 antigen. IBDV STD challenge was performed on day 28 according to USDA instructions. All birds were necropsied 5 days after challenge. We observed 100% protection for HVT-IBD#9, 96% protection for HVT-IBD#1, #30, #34, and 92% protection for HVT-IBD#6a, while our positive control Vaxxitek conferred 92% protection. [Table 3]

[0256] Example 5: IBDV serological responses of commercial broiler chickens HVT-IBD#1, #5, #9, and #15 Serological responses to IBDV antigens were assessed using the commercially available Elisa kit ProFlok ND 1500 pfu (0.2 mL) of each recombinant (HVT-IBD#1, #5, #9, #15) was injected subcutaneously (SC) into 1-day-old chicks. Serum samples were isolated on days 12, 19, 26, 33, 39, 47, and 54 and can be seen in Table 3 below. The percentage of positive samples for each construct over time is shown in Figure 13. [Table 4]

[0257] Example 6: IBDV serological responses of commercial broiler chickens HVT-IBD#6a, #30, and #31 The serological response to IBDV antigens was measured using a commercially available ELISA kit, ProFlok IBD plus. 1500 pfu (0.2 mL) of each recombinant (HVT-IBD#61, #30, #31) was injected subcutaneously (SC) into 1-day-old chicks. Serum samples were isolated on days 12, 19, 26, 33, 39, 47, and 54, and are shown in Table 4 below. The percentage of positive samples for each construct over time is shown in Figure 14. [Table 5]

[0258] Example 7: In vivo efficacy test of HVT-ND#38, #39, #44, and #48 in SPF birds Four HVT-ND recombinants, HVT-IBD#38, #39, #44, and #48, were tested in SPF birds for their in vivo efficacy against a highly virulent NDV challenge (Texas GB strain, provided by the USDA). In this study, the commercially available vaccine, Vectormune ND (Ceva), was used as a positive control. 1500 pfu of each recombinant virus was injected in ovo at E18. The inverse titer of the vaccine virus was also determined for each recombinant after vaccination. While 100% of HVT-ND#38 and #48 expressed the NDV F antigen, we found that only 95-96% of HVT-IBD#39 and #44 expressed this antigen. NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were observed two weeks after challenge. We observed 90% protection for HVT-ND#38 and #48, 50% protection for HVT-ND#39, and 60% protection for HVT-ND#44, but not for our positive control Vectormune ND gave 90% protection. See Table 5 below. [Table 6]

[0259] The antibody response to various HVT-ND vaccine candidates was assayed by using the ProFlok ND plus kit (Zoetis LLC). All titers were included without using the cutoff value (345) recommended by this kit. The percentage of birds with positive ND titers is shown in Table 6 below. [Table 7]

[0260] Example 8: In vivo NDV efficacy test of HVT-ND#40, #42, #45, and #46 in SPF birds Four HVT-ND recombinants, HVT-IBD#40, #42, #45, and #46, were tested in SPF birds for their in vivo efficacy against a highly virulent NDV challenge (Texas GB strain, provided by the USDA). In this test, the commercially available vaccine, Vectormune ND (Ceva), was used as a positive control. 1500 pfu of each recombinant virus was injected in ovo at E18. The inverse titer of the vaccine virus was determined for each recombinant after vaccination. While 100% of HVT-ND#42, #45, and #46 express the NDV F antigen, we found that only 94-99% of HVT-IBD#40 expressed this antigen. NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were challenged. The results were observed two weeks after the first dose. We observed 95% protection for HVT-ND#42 and #45, 80% protection for HVT-ND#46, and 55% protection for HVT-ND#40, while the positive control Vectormune ND provided 90% protection. See Table 7 below. [Table 8]

[0261] The antibody response to various HVT-ND vaccine candidates was assayed by using the ProFlok ND plus kit (Zoetis LLC). All titers were included without using the cutoff value (345) recommended by this kit. The percentage of birds with positive ND titers is shown in Table 8 below. [Table 9]

[0262] Example 9 In vivo MDV efficacy test of HVT-ND#38, #42, and #45 in SFP birds Three HVT-ND recombinants, HVT-IBD#38, #42, and #45, were tested for their in vivo efficacy against a highly virulent MDV challenge (GA22) in SPF birds. In this test, the commercial vaccine Vectormune ND (Ceva) was used as a positive control. 1500 pfu of each recombinant virus was injected in ovo at E18. The reciprocal titer of the vaccine virus was determined for each recombinant after vaccination. MDV GA22 challenge was performed on day 5 according to USDA instructions. All birds were , and observed 54 days after challenge. We observed 69% protection for HVT-ND#42 and #45 and 46% protection for HVT-ND#38, while the positive control Vectormune ND provided 62% protection. See Table 9 below. [Table 10]

[0263] Example 10 In vivo ND efficacy test of HVT-ND#38, #42, and #45 in broiler chickens Three HVT-ND recombinants, HVT-IBD#38, #42, and #45, were tested in broiler chickens for their in vivo efficacy against a highly virulent NDV challenge (Texas GB strain, provided by the USDA). In this study, the commercially available vaccine, Vectormune ND (Ceva), was used as a positive control. 4000 pfu of each recombinant virus was injected in ovo at E18. The reciprocal titer of the vaccine virus was determined for each recombinant after vaccination. NDV Texas GB challenge was performed on day 28 according to USDA instructions. All birds were observed two weeks after challenge. We observed 100% protection for HVT-ND#42 and #45 and 37% protection for HVT-ND#38, while our positive control, Vectormune ND, provided 50% protection. The reciprocal titer of Vectormune ND was 0. [Table 11]

[0264] Antibody responses to various HVT-ND vaccine candidates were assayed using the ProFlok ND plus kit (Zoetis LLC). All titers were included without using the cutoff value (345) recommended by this kit.

[0265] Example 11 In vivo NDV efficacy test of HVT-ND#38, #42, and #45 in SFP birds on day 20 of challenge Three HVT-ND recombinants, HVT-IBD#38, #42, and #45, were tested for their in vivo efficacy against a highly virulent NDV challenge (Texas GB strain, provided by the USDA) in SPF birds on day 20 post-challenge. In this study, the commercially available vaccine, Vectormune ND (Ceva), was used as a positive control. 2000 pfu of each recombinant virus was injected in ovo at E18. The reciprocal titer of the vaccine virus was determined for each recombinant after vaccination. NDV Texas GB challenge was performed on day 20 according to USDA instructions. All birds were observed two weeks after challenge. We observed 87.5% protection for HVT-ND#42, 70% protection for HVT-ND#45, and 65% protection for HVT-ND#38, while our positive control, Vectormune ND, provided 87.5% protection. See Table 11 below. [Table 12]

[0266] Example 12 In vivo NDV efficacy test of HVT-ND (#42, MSV+5) in SPF birds on days 17, 18, and 19 of challenge Three HVT-ND recombinants, HVT-ND (#42, MSV+5), were tested for their in vivo efficacy against a highly virulent NDV challenge (Texas GB strain) in SPF birds on challenge days 17, 18, and 19. All birds were observed two weeks after challenge. The inventors observed that for in ovo vaccination, 100% (40 / 40), 88% (35 / 40), and 98% (39 / 40) protection was observed for NDV challenge on challenge days 17, 18, and 19, respectively. For subcutaneous vaccination on the day of hatch, 75% (30 / 40), 88% (35 / 40), and 93% (37 / 40) protection was observed. See Table 12 below. [Table 13]

[0267] Example 13 In vivo NDV efficacy testing of HVT-ND (#42, MSV+5) in SPF birds on days 16 and 19 of challenge Three HVT-ND recombinants, HVT-ND (#42, MSV+5), were tested for their in vivo efficacy against a highly virulent NDV challenge (Texas GB strain) in SPF birds on days 16 and 19 after challenge. All birds were observed two weeks after challenge. The inventors observed that for in ovo vaccination, 85% (37 / 40) and 93% (37 / 40) protection was observed for NDV challenge on days 16 and 19. For subcutaneous vaccination on the day of hatch, 70% (28 / 40) and 95% (38 / 40) protection was observed. See Table 13 below. [Table 14]

[0268] Example 14 Duration of immunity of SPF birds against HVT-ND (#42, MSV+5) up to 63 days after challenge Three HVT-ND recombinants, HVT-ND (#42, MSV+5), were tested for the duration of immunity against a highly virulent NDV challenge (Texas GB strain) in SPF birds on day 63 of challenge. All birds were observed two weeks after challenge. We observed 100% (30 / 30) protection for both in ovo vaccination or subcutaneous vaccination on the day of hatch. See Table 14 below. [Table 15]

[0269] Example 15 ND immunogenicity test of HVT-ND (#42, MSV+5) in SPF birds Three HVT-ND recombinants, HVT-ND (#42, MSV+5), were tested for immunogenicity against a highly virulent NDV challenge (Texas GB strain, provided by USDA) in SPF birds on day 28. All birds were tested 2 weeks after challenge. We observed 100% (30 / 30) protection for both in ovo or subcutaneous vaccination on the day of hatch. See Table 15 below. [Table 16]

[0270] Example 16 MD immunogenicity test of HVT-ND (#42, MSV+5) in SPF birds Three types of HVT-ND recombinants, HVT-ND (#42, MSV+5), were tested for immunogenicity against a highly virulent MDV challenge (GA22 strain) on day 5 in SPF birds. All birds were observed 54 days after challenge. We observed 100% (30 / 30) protection for both in ovo vaccination or subcutaneous vaccination on the day of hatch. See Table 16 below. [Table 17]

[0271] Example 17 In vitro growth experiments In vitro growth experiments were carried out on HVT-ND#38, #42, and #45. 2 5 x 10 roller bottles 8 HVT-ND#38, #42, and #45 were inoculated into roller bottles at three different MOIs: 0.001, 0.003, and 0.008. The infected cells were harvested 48 hours after infection and titrated on CEF cells. Both HVT-ND#42 and #45 grew well, with 2.86x10 primary CEF cells, respectively. 6 and 2.97x10 6pfu / mL. HVT-ND#38 has a titer of 1.67x10 6 pfu / mL. See Table 17 below. [Table 18]

[0272] Example 18 Construction of HVT-IBD-ND #42-#30LP C2 Generation of transfer plasmid #42 The initial transfer plasmid HVT-ND #42 was chemically synthesized by BioBasic Inc. The cloning plasmid UL55 / gene3 was chemically synthesized by DNA2.0 as described above. PCR amplification of the NDV F gene expression cassette in the HVT-ND #42 transfer plasmid by using the following primers: upper primer SEQ ID NO: 154, lower primer SEQ ID NO: 155.

[0273] The amplified PCR product was cloned into the AscI and NheI sites of UL55 / gene3 to generate the final transfer plasmid #42, which was used for transfection / infection to generate HVT-ND#42.

[0274] Generation of transfer plasmid-#30 The initial transfer plasmid HVT-IBD #30 was chemically synthesized by BioBasic Inc. The cloning plasmid was chemically synthesized by DNA2.0. The IBD gene expression cassette of the plasmid #30 plasmid was PCR amplified using the following primers: upper primer SEQ ID NO: 156, lower primer SEQ ID NO: 157. The amplified PCR product was cloned into the AgeI and NpnI sites of UL35 / 36 to generate the final transfer plasmid #30. This plasmid was used for transfection / infection to generate HVT-IBD-ND #42-#30 LP C2.

[0275] Construction of HVT-ND #42: Co-infection / transfection: CEF cells were seeded in 6-well plates, and the next day, transfection with HVT working seed (140 ul) + plasmid-#42 (linearized by SpeI + SbfI digestion) was performed using Lipofectamine™ LTX Reagent (ThermoFisher). Two days after transfection, the transfected cells were harvested. Screened positive foci in 6 wells were plated by IFA using chicken anti-NDV polyclonal antibody (live cell staining, approximately 1:250 dilution), and then further purified once (by viability staining) in 96-well plates by limiting dilution to obtain single clones. The purified clones were passaged twice in 6-well plates in duplicate to obtain clonal purity. The purity was confirmed by IFA (fixation and staining). This 6-well harvest was used for the construction of HVT-IBD-ND #42-#30.

[0276] Construction of HVT-IBD-ND #42-#30 Co-infection / transfection: CEF cells were seeded in 6-well plates, and the following day, HVT-ND #42 infection + plasmid-#30 (linearized by SbfI digestion) transfection was performed using Lipofectamine™ LTX Reagent (ThermoFisher). Three days after transfection, transfected cells were harvested. Screened positive foci in 6 wells were plated by IFA using chicken anti-IBD polyclonal antibody (viability staining, approximately 1:250 dilution) and then further purified once (by viability staining) in 96-well plates by limiting dilution to obtain single clones. Two purified clones were picked and passaged in duplicate in 6-well plates, and the purity of the clones was confirmed by IFA (fixation and staining). The clones were sequentially scaled up in T-75 flasks, T-150 flasks, T-225 flasks, and 850 ml roller bottles. The recombinant virus was harvested, aliquoted into 1 mL / vial, frozen at -80°C overnight, and then transferred to an LN tank.

[0277] Example 19: In vivo NDV efficacy test of HVT-IBD-ND#42-#30, #42-#32, and #104 in SPF birds Seven HVT-IBD-ND recombinants, HVT-IBD-ND #42-#30 (3 clones), #42-#32 (2 clones), and #104 (2 clones), were tested for their in vivo efficacy against highly virulent NDV challenge in SPF birds. NDV Texas GB challenge was performed on day 28. Approximately 1500 PFU of each recombinant virus was injected in ovo at E18. All birds were observed two weeks after challenge. See Table 18 below. [Table 19]

[0278] Example 20: In vivo IBD efficacy test of HVT-IBD-ND#42-#30, #42-#32, and #104 in SPF birds Seven HVT-IBD-ND recombinants, HVT-IBD-ND #42-#30 (3 clones), #42-#32 (2 clones), and #104 (2 clones), were tested for their in vivo efficacy against highly virulent IBDV challenge in SPF birds on days 14 and 21, respectively. Approximately 2000 PFU of each recombinant virus was injected in ovo at E18. All birds were necropsied 5 days after challenge. See Table 19 below. [Table 20]

[0279] Example 21 In vivo MDV efficacy test of HVT-IBD-ND #42-#30 (4 clones) in SFP birds Three HVT-IBD-ND recombinants #42-#30 (4 clones) were tested for their in vivo efficacy against a highly virulent MDV challenge (GA22) in SPF birds. Approximately 1500 PFU of each recombinant virus was injected in ovo at E18. MDV GA22 challenge was performed on day 5. All birds were observed 54 days after challenge. See Table 20 below. [Table 21]

[0280] Example 22 In vivo vvIBD efficacy test of HVT-IBD-ND#42-#30, #42-#32, and #104 in SPF birds Three HVT-IBD-ND recombinants, #42-#30 (2 clones), #42-#32 (2 clones), and #104, were tested for their in vivo efficacy against highly virulent IBDV challenge in SPF birds. Approximately 1500 PFU of each recombinant virus was injected in ovo at E18. vvIBDV challenge was performed on days 14 and 21. All birds were observed 10 days after challenge. At the end of the study, histological examination of the bursa was performed for each bird. See Table 21 below. [Table 22]

[0281] Example 23 Duration of immune IBD in SPF birds with HVT-IBD-ND (#42-#30, X+5) up to 63 days after challenge HVT-IBD-ND recombinants #42-#30 (MSV+5) were tested for duration of immunity against a highly virulent classical IBDV challenge in SPF birds on day 63 post-challenge. All birds were observed 4 days post-challenge and then necropsied. See Table 22. [Table 23]

[0282] Example 24 ND immunogenicity test of HVT-IBD-ND (#42-#30, MSV+5) in SPF birds HVT-IBD-ND recombinants #42-#30 (MSV+5) were tested for immunogenicity against a highly virulent NDV challenge (Texas GB strain) in SPF birds on day 28. All birds were observed 2 weeks after challenge. See Table 23. [Table 24]

[0283] Example 25 IBD immunogenicity test of HVT-IBD-ND (#42-#30, MSV+5) in SPF birds HVT-IBD-ND recombinants #42-#30 (MSV+5) were tested for immunogenicity against a highly virulent IBDV challenge in SPF birds on day 34. All birds were observed 4 days after challenge and then necropsied for cystic lesions. See Table 24 below. [Table 25]

[0284] Example 26 MD immunogenicity test of HVT-IBD-ND (#42-#30, MSV+5) in SPF birds Three HVT-IBD-ND recombinants #42-#30 (MSV+5) were tested for immunogenicity against a highly virulent MDV challenge (GA22 strain) in SPF birds on day 5. All birds were observed 54 days after challenge. See Table 25 below. [Table 26]

[0285] Example 27 ND immunogenicity test of HVT-IBD-ND (#42-#30, MSV+5) in SPF birds against the EU challenge strain HVT-IBD-ND recombinants #42-#30 (MSV+5) were tested for immunogenicity against a highly virulent NDV Europe challenge (Herts Weybridge 33 / 56) in SPF birds on day 21. All birds were observed 2 weeks after challenge. See Table 26 below. [Table 27]

[0286] Example 28 Compatibility of Bursaplex with HVT-ND IBDV efficacy BURSAPLEX™ (Zoetis, US5871748, incorporated herein by reference) is a vaccine against infectious bursal disease (IBD) containing a vaccine conjugate consisting of a live attenuated infectious bursal disease (IBD) strain 2512 bound to a virus and the neutralizing antibody BDA. Bursaplex generates active immunity against IBD in poultry, particularly chickens. E18 eggs were injected in ovo with either a control or test vaccine (HVT-ND containing Bursaplex at a 1:1 ratio) and transferred, along with uninjected eggs, to assigned incubators as designated by Biometrics. On the day of hatch, T04 birds were subcutaneously vaccinated. Blood samples were collected on days 5, 12, 19, 26, and 33 for IBDV serology. On day 34, designated birds were challenged with a classical, highly virulent IBDV, and on day 38, all birds were challenged with a virulent classical IBDV. The chickens were necropsied for the presence of cystic lesions. Chickens in the T01 negative group developed no gross observable lesions, and 100% of chickens in the T02 challenge control group developed gross observable lesions. T03 (HVT-ND + Bursaplex, in ovo) and T04 (HVT-ND + Bursaplex, SC) were both 100% protected. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Bursaplex are compatible when administered together and remain effective against IBDV challenge when administered either in ovo or subcutaneously.

[0287] NDV efficacy E18 eggs were injected in ovo with either the control or test vaccine (HVT-ND with Bursaplex at a 1:1 ratio) and, along with uninjected eggs, transferred to assigned incubators as specified by Biometrics. T04 birds were vaccinated subcutaneously on the day of hatch. Blood samples were collected on days 6, 13, 20, and 27 for NDV serology. On day 28, designated birds were challenged with velogenic NDV, and on day 42, all surviving birds were killed. Chickens in the T01-negative group developed no clinical signs, and 100% of chickens in the T02-challenged control group developed clinical signs of Newcastle disease, including mortality. T03 (HVT-ND + Bursaplex, in ovo) and T04 (HVT-ND + Bursaplex, SC) were 92.5% and 95% protected, respectively. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Bursaplex are compatible when administered together and remain effective against NDV challenge when administered either in ovo or subcutaneously.

[0288] Example 29 Compatibility of HVT-ND with Magniplex IBD Efficacy MAGNIPLEX™ (Zoetis) is a vaccine against infectious bursal disease (IBD) containing a vaccine conjugate consisting of a live attenuated infectious bursal disease (IBD) strain V877 bound to the virus and the neutralizing antibody BDA. Bursaplex generates active immunity against IBD in poultry, particularly chickens. E18 eggs were injected in ovo with either a control or test vaccine (HVT-ND pre-license serial containing Magniplex at a 1:1 ratio) and transferred, along with uninjected eggs, to assigned incubators as designated by Biometrics. T04 birds were vaccinated subcutaneously on the day of hatch. Blood samples were collected on days 5, 12, 19, 26, and 33 for IBDV serology. On day 34, designated birds were challenged with a classical, highly virulent IBDV. On day 38, all birds were necropsied for the presence of cystic lesions. Chickens in the T01 negative group developed no significant observable lesions, and 100% of chickens in the T02 challenge control group developed significant observable lesions. T03 (HVT-ND + Magniplex, in ovo) and T04 (HVT-ND + Magniplex, SC) were both 100% protected. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Magniplex are compatible when administered together and remain effective against IBDV challenge when administered either in ovo or subcutaneously. [Table 28] [Table 29]

[0289] ND Efficacy E18 eggs were injected in ovo with either the control or test vaccine (HVT-ND with Magniplex at a 1:1 ratio) and, along with uninjected eggs, were transferred to assigned incubators as designated by Biometrics. T04 birds were vaccinated subcutaneously on the day of hatch. Blood samples were collected on days 6, 13, 20, and 27 for NDV serology. On day 28, designated birds were challenged with velogenic NDV, and on day 42, all surviving birds were killed. Chickens in the T01-negative group developed no clinical signs, and 100% of chickens in the T02-challenged control group developed clinical signs of Newcastle disease, including mortality. T03 (HVT-ND + Magniplex, in ovo) and T04 (HVT-ND + Magniplex, SC) were 92.5% and 95% protected, respectively. It can be concluded that Poulvac Procerta HVT-ND and Poulvac Magniplex are compatible when administered together and remain effective against NDV challenge when administered either in ovo or subcutaneously. [Table 30] [Table 31]

Claims

1. A recombinant herpesvirus of turkeys (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted within the intergenic locus UL35 / UL36 in the unique long (UL) region of the HVT genome.

2. 1. A recombinant herpesvirus of turkeys (HVT) genome comprising one or more nucleotide sequences encoding one or more heterologous antigens inserted within the intergenic locus UL35 / UL36 in the unique long region of the HVT genome, and one or more nucleotide sequences encoding one or more heterologous antigens inserted into the UL55 / Gene3 site in the unique long region (UL) of the HVT genome.

3. The recombinant HVT of claim 1 or 2, wherein the one or more heterologous antigens are protective against an avian pathogen selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV).

4. One or more heterologous antigens a) the VP2, VP3 or VP4 protein of the infectious bursal disease virus (IBDV); b) the VP1 or VP2 protein of said Chicken Anemia Virus (CAV); c) the F / HN chimeric protein, or the F, NP, P, M, HN, or L protein of the Newcastle Disease Virus (NDV); d) the S1, S2 or M protein of infectious bronchitis virus (IBV); e) the gB, gC, gD, gE, gH, gI or gL protein of the infectious laryngotracheitis virus (ILTV), and f) A recombinant HVT according to any one of the preceding claims, selected from the group consisting of any of the HA, NA, NP or M proteins of said avian influenza virus (AIV).

5. 2. The recombinant HVT of any one of the preceding claims, wherein said one or more heterologous antigens are protective against IBDV.

6. The recombinant HVT of claim 5, wherein the one or more heterologous antigens is the VP2 protein of IBDV.

7. 7. The recombinant HVT of claim 6, wherein the VP2 protein sequence is encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:

10.

8. The recombinant HVT of claim 6, wherein the VP2 protein is encoded by a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:

10.

9. The recombinant HVT according to any one of claims 1 to 4, wherein the one or more heterologous antigens or antigens are protective against Newcastle Disease Virus (NDV).

10. The recombinant HVT of claim 9, wherein the one or more heterologous antigens comprise the F protein of NDV.

11. The recombinant HVT of claim 10, wherein the F protein of NDV is encoded by a nucleotide sequence comprising at least 80% sequence identity to a sequence comprising SEQ ID NO:

3.

12. The recombinant HVT of claim 10, wherein the F protein of NDV is encoded by a nucleotide sequence comprising SEQ ID NO:

3.

13. 2. The recombinant HVT according to any one of the preceding claims, wherein said one or more heterologous antigens are protective against NDV and IBDV.

14. The recombinant HVT of claim 13, wherein the at least one heterologous antigen comprises the F protein of NDV and the VP2 protein of IBDV.

15. The recombinant HVT of claim 14, wherein the F protein of NDV is encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:3, and the VP2 protein of IBDV is encoded by a nucleotide sequence comprising at least 80% sequence identity to a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:

10.

16. The recombinant HVT of claim 14 or 15, wherein the F protein of NDV is encoded by a nucleotide sequence comprising SEQ ID NO:3, and the VP2 protein of IBDV is encoded by a nucleotide sequence comprising SEQ ID NO:5 or SEQ ID NO:

10.

17. 2. A recombinant HVT according to any one of the preceding claims, comprising a genome comprising one or more expression cassettes comprising one or more nucleotide sequences encoding one or more heterologous antigens.

18. 18. The recombinant HVT genome of claim 17, wherein the expression cassette comprises one or more promoters.

19. 19. The recombinant HVT of claim 18, wherein the one or more promoters are selected from the group consisting of an immediate early cytomegalovirus human (hCMV) promoter, a guinea pig immediate early CMV promoter, a mouse immediate early CMV promoter, a Pec promoter, a β-chicken actin promoter, an SV40 promoter, a pseudorabies virus promoter of glycoprotein X promoter, a herpes simplex virus-1 alpha 4 promoter, a Marek's disease virus promoter of glycoprotein gA, gC, gB, gE, or gI promoter, an infectious laryngotracheitis virus promoter of glycoprotein gB, gE, gI, gD promoter, and a bovine herpesvirus 1 / 1 VP8 promoter.

20. 20. The recombinant HVT of claim 19, wherein the one or more promoters include the human CMV promoter.

21. 20. The recombinant HVT of claim 19, wherein the one or more promoters include the mouse CMV promoter.

22. The recombinant HVT of claim 19, wherein the one or more promoters include the hCMV promoter and the mCMV promoter.

23. An isolated DNA encoding a recombinant HVT genome according to any one of claims 1 to 22.

24. An immunogenic composition comprising a recombinant HVT according to any one of claims 1 to 23, further comprising a pharma- ceutically acceptable carrier, excipient, or adjuvant.

25. A vaccine composition comprising the recombinant HVT of any one of claims 1 to 23, further comprising a pharma- ceutically acceptable carrier, excipient, or adjuvant.

26. 26. The vaccine of claim 25, further comprising an additional Marek's Disease Virus (MDV) selected from the group consisting of naturally attenuated MDV-1 strain Rispens (CVI-988), or Gallid herpesvirus 3 strain SB-1 virus.

27. 27. The vaccine of claim 26, wherein the additional MDV comprises a recombinant genome.

28. 28. The vaccine of claim 27, wherein the recombinant MDV genome comprises one or more nucleotide sequences encoding one or more heterologous antigens that are protective against one or more avian pathogens.

29. 29. The vaccine of any one of claims 25 to 28 for use in vaccinating birds against one or more diseases caused by one or more avian pathogens.

30. A vaccine according to any one of claims 25 to 28 for use in the protection of birds against clinical symptoms caused by one or more avian pathogens.

31. 29. The vaccine of any one of claims 25 to 28 for use in the protection of birds against clinical symptoms caused by Marek's Disease Virus and clinical symptoms caused by one or more avian pathogens.

32. 32. The vaccine of any one of claims 29 to 31, wherein the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV).

33. 33. The vaccine of claim 32, wherein the one or more avian pathogens comprises the Newcastle Disease Virus.

34. 33. The vaccine of claim 32, wherein the one or more avian pathogens comprises the infectious bursal disease virus (IBDV).

35. 33. The vaccine of claim 32, wherein the one or more avian pathogens include the Newcastle Disease Virus and the Infectious Bursal Disease Virus.

36. 36. The vaccine of any one of claims 25 to 35 for use in vaccinating birds, wherein the vaccine is administered by at least one or more administrations of the vaccine by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration, intranasal administration, or a combination thereof.

37. 37. The vaccine of claim 36, wherein the vaccine is administered by in ovo administration.

38. The in ovo administration is carried out in embryonated eggs between about 16 and 22 days of development.

38. The vaccine of claim 37, wherein the vaccine is administered in a guinea pig.

39. 39. The vaccine of any one of claims 36 to 38, wherein the in ovo administration occurs in embryonated eggs at about day 18 of development.

40. 40. The vaccine of any one of claims 36 to 39, wherein the administration of the vaccine comprises an in ovo administration followed by a spray administration.

41. 37. The vaccine of claim 36, wherein the administration of the vaccine comprises spray administration.

42. 36. A method of vaccinating birds to treat or prevent Marek's disease and one or more avian diseases caused by one or more avian pathogens, comprising administering an effective amount of a vaccine composition according to any one of claims 25 to 35.

43. 43. The method of claim 42, wherein the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV).

44. 44. The method of claim 43, wherein the one or more avian pathogens comprises the infectious bursal disease virus (IBDV).

45. 44. The method of claim 43, wherein the one or more avian pathogens comprises the Newcastle Disease Virus (NDV).

46. 44. The method of claim 43, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV) and the Newcastle disease virus (NDV).

47. A method for inducing an immune response in an avian animal against Marek's Disease Virus and one or more avian pathogens, comprising administering to the bird an effective amount of an immunogenic or vaccine composition according to any one of claims 24 to 35.

48. 48. The method of claim 47, wherein the one or more avian pathogens are selected from the group consisting of infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), chicken anemia virus (CAV), and avian influenza virus (AIV).

49. 49. The method of claim 48, wherein the one or more avian pathogens comprises the infectious bursal disease virus (IBDV).

50. 49. The method of claim 48, wherein the one or more avian pathogens comprises the Newcastle Disease Virus (NDV).

51. 49. The method of claim 48, wherein the one or more avian pathogens include the infectious bursal disease virus (IBDV) and the Newcastle disease virus (NDV).

52. 52. The method of any one of claims 42 to 51, wherein said administration is performed by spray administration, in ovo administration, subcutaneous administration, intramuscular administration, oral administration or nasal administration.

53. 53. The method of claim 52, wherein the route of administration comprises in ovo administration.

54. 54. The method of claim 53, wherein said in ovo administration occurs in embryonated eggs between about 16-22 days of development.

55. 55. The method of any one of claims 52-54, wherein the in ovo administration occurs in embryonated eggs at about day 18 of development.

56. 56. The method of any one of claims 52 to 55, wherein the route of administration comprises in ovo administration followed by spray administration.

57. 53. The method of claim 52, wherein the route of administration comprises spray administration.

58. The method of any one of claims 44 to 57, wherein the bird is selected from the group consisting of chickens, turkeys, geese, ducks, pheasants, ostriches, pigeons and quails.

59. 59. The method of claim 58, wherein the bird comprises a chicken.

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